APPA2014 Jeju

Transcription

APPA2014 Jeju

APPA2014 Jeju
The 4th Asia Pacific Protein Association (APPA) Conference
Date : May 17 (Sat) – 20 (Tue), 2014
Place : ICC Jeju, Korea
Program & Abstracts
Organizer: Asia Pacific Protein Association
The Protein Society
Host:
Korean Society for Protein Science
Korean Magnetic Resonance Society
Contents
Welcome Address
1
Program at a Glance
4
Conference Map
5
General Information
6
APPA conference Committee Members
11
APPA Council Members
13
Exhibitors
14
Message from Editor-in-chief of Protein Science
15
Conference Programs
16
Nobel, Keynote & Plenary Lectures
17
Symposia
23
Young Scientist Talk
38
Abstract
40
Oral presentations
40
Poster presentations
163
Guide to Local Activity
204
Author/Invited Speaker Index
214
Welcome Address
Message from Prof. Young Kee Kang
Chair of APPA2014 Local Organizing Committee
Greetings,
On behalf of the Korean Society for Protein Science (KSPS), we give you a warm welcome
to the 4th Conference of the Asia Pacific Protein Association (APPA2014) in Jeju.
APPA was founded to advance protein science and share friendship between countries in the Asia Pacific
region. Currently, council members from 13 countries actively participate in APPA. It organizes a triennial
International Conference that provides an international forum to promote communication, cooperation and
collaboration in all aspects of protein science. After the successful conferences held in Yokohama, Japan
(2004), Caims, Australia (2008) and Shanghai, China (2011), the 4th Conference will be held in Jeju Island,
Korea during May 17-20, 2014.
Jeju Island is famous for three abundant things of stones, wind, and fishermen's wives, three rare things of
beggars, thieves, and house gates, and three treasury things of sea, Halla volcanic mountain, and dialect. In
particular, Jeju Island has been recognized as the beautiful and admirable World Natural Heritage Site with
volcanic island and molten lava tubes.
We invite you to explore the current aspects of protein science and simultaneously the exotic landscape in
this wonderful island. We hope you will enjoy the scientific and social programs and have a great time in
Jeju Island.
Young Kee Kang (Chair)
Kyou-Hoon Han, Weontae Lee (Co-chair)
Organizing committee of APPA2014 Jeju
1
Welcome Address
Message from Prof. Zengyi Chang
The President of Asia Pacific Protein Association (APPA)
Dear Colleagues,
On behalf of the APPA Council, I would like to warmly welcome you to the 4th APPA
Conference here on Jeju Island, Korea!!
The objectives of APPA are to promote protein research and education among Asia Pacific nations and
regions. The first three APPA Conferences were held in Japan (Yokohama, April 14-18, 2004), Australia
(Cairns, June 22-26, 2008), and China (Shanghai, May 6-9, 2011), respectively.
As a key type of biological molecule, proteins are involved in almost all life processes. Genome sequencing
studies revealed that about half of the proteins encoded by the genomes of various model organisms have
never been touched by us. Our understanding on how proteins work in the living cells is still very limited
and many more surprising discoveries are still awaiting us. We, as scientists from the Asia Pacific region,
should make our due contributions in this tremendously important field of life sciences.
The Protein Society Council has decided, at its council meeting held on Feb 1st, 2014 in Los Angeles, to
have its 31st conference in Shanghai, China in 2017 (exact dates to be decided). I hope to see you all there
three years from now.
Last, but not least, as the president of the Chinese Protein Society and a council member of the Protein
Society, I would like to congratulate our Korea colleagues for bringing about this event here on Jeju Island!
I hope you will enjoy this meeting and this beautiful island!
My warmest regards,
Zengyi Chang, Ph.D.
Professor of Biochemistry and Molecular Biology,
Peking University, Beijing, China
E-mail: changzy@pku.edu.cn
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3
Program at a Glance
4th APPA Conference
May 17 (Saturday), 2014
12:00 - 13:20
Registration
13:20 - 14:20
Young Scientists Talk (YST-1): 6 speakers
14:20 - 14:30
14:30 - 16:15
Coffee/Tea break
Symposium 1A
Symposium 1B
Symposium 1C
Symposium 1D
Proteins in
emerging fields I
Proteins in disease I
Protein folding and
dynamics
Structural targetomics for
drug discovery
16:15 - 16:30
Coffee/Tea break
16:30 - 16:40
Opening Ceremony (Room Yeongju A, ICC)
16:40 - 17:30
Nobel Lecture (NL): Prof. Roger Kornberg
17:30 - 18:20
Keynote Lecture (KL-1): Prof. Sunghoon Kim
18:20 - 20:40
Welcome Reception (Ocean View Hall, ICC)
20:40 -
Free evening
May 18 (Sunday), 2014
09:00 - 10:30
Symposium 2A
Symposium 2B
Symposium 2C
Symposium 2D
Protein catabolism
and trafficking
Proteins in disease II
Protein analysis
techniques I
Proteogenomics
10:30 - 10:50
Coffee/Tea break
10:50 - 11:30
Plenary Lecture (PL-1): George Fu Gao, Ph.D.
11:30 - 12:10
Plenary Lecture (PL-2): Haruki Nakamura, Ph.D.
12:10 - 14:20
Lunch & Lunch Workshop
(12:50 - 14:20)
Poster Presentations (Odd Numbers; Event Hall, ICC)
14:20 - 15:00
Plenary Lecture (PL-3): Ming-Daw Tsai, Ph.D.
15:00 - 15:10
15:10 - 16:40
Coffee/Tea break
Symposium 3A
Symposium 3B
Symposium 3C
Symposium 3D
Protein modification
Proteins as therapeutics I
Protein analysis
techniques II
Intriscilly disordered
protein
16:40 - 17:00
17:00 - 18:30
Coffee/Tea break
Symposium 4A
Symposium 4B
Symposium 4C
Symposium 4D
Proteins in
emerging fields II
Proteins as therapeutics II
Protein design and
engineering
Structure-function of GPCR
Free evening
18:30 -
APPA Council Meeting (Chalotte room, Lotte Hotel Jeju)
(18:30 - 20:30)
May 19 (Monday), 2014
09:00 - 10:30
10:30 - 12:15
Poster Presentations (Even Numbers; Event Hall, ICC)
Symposium 5A
Symposium 5B
Symposium 5C
Symposium 5D
Protein anabolism
Proteins as drug targets
Proteins in
nanobiotechnology
by proteomics and glycoproteomics
Cancer biomarker discovery
Lunch & Free time
12:15 - 19:00
(13:00-18:50)
Excursion
19:00 - 22:30
Conference Banquet (Crystal ballroom, Lotte Hotel Jeju)
May 20 (Tuesday), 2014
09:00 - 10:45
Symposium 6A
Symposium 6B
Symposium 6C
Symposium 6D
Proteins in membranes
Proteins and
drug discovery
Protein bioinformatics
Fronteirs in protein sciences
10:45 - 11:00
Coffee/Tea break
11:00 - 11:40
Plenary Lecture (PL-4): Hong Wanjin, Ph.D.
11:40 - 12:40
Young Scientists Talk (YST-2): 6 speakers
12:40 - 13:00
Closing Ceremony & Awards (Room Yeongju A, ICC)
4
APPA2014 Jeju Conference Rooms Map
5
General Information
Meeting dates and places
The meeting will be held in the 1st floor of ICC (International Convention Center), Jungmun, Seogwipo,
Jeju, May 17-20, 2014.
<Registration desk>
The registration desk is located in the hallway close to the room “Yeongju A”.
<Opening ceremony>
The opening and closing ceremonies will be held in the room “Yeongju A”.
<Oral presentation>
All oral presentations will be held in the room “Yeongju A,B and Baekrok A, B”.
<Poster>
Poster will be displayed in the event hall.
<Exhibition>
The conference exhibits are located in the hallway in front of the rooms „Yeongju A and Baekrok A”.
<Conference office>
The conference office is located at the “Event Hall”.
<Preview room>
The preview room is located in “Event Hall”
6
Registration
The registration desk will open at 12:00 pm on Saturday, May 17 (refer to hours below). Registration
includes admission to all scientific and poster session, exhibits, abstract book and one bag. Registration
also includes the first day welcome reception, light breakfast (donuts, Tteok, Tea and coffee), snack and
coffee in coffee break.
<Hours>
Saturday, May 17
12:00 pm – 19:00 pm
Sunday, May 18
9:00 pm – 19:00 pm
Monday, May 19
9:00 pm – 13:00 pm
Tuesday, May 19
9:00 pm – 13:00 pm
<Registration fee – on site registration>
Full registration $450
Student registration $165
<Name badge>
Name badges are required for entry in the conference zone (hallway and rooms) and welcome
reception.
Meals
<Welcome reception>
The welcome reception will be provided in Ocean View Hall, 5th floor of ICC.
<Breakfast>
The light breakfast will be provided from 8:30 AM to 9:00 AM from Sunday to Tuesday. Breakfast
includes doughnuts, tteok (Korean rice cake), tea and coffee. It is possible that the foods will be run out
earlier.
<Lunch>
Saturday, May 17: No service
Sunday, May 18: Lunch will be provided during workshop session.
Monday, May 19 & Tuesday, May 20: Lunch coupon will be available in the registration desk with 3,000
Korean Won. 100 meals will be prepared per each day. Coupon can be used in the cafeteria which is
located in the 1st floor of ICC.
<Conference banquet>
The conference banquet will be held on the evening of Monday 19th May at the Crystal Ballroom of the
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Lotte Hotel. This banquet is open to those who purchased the ticket. The banquet ticket will be
available on site by Sunday in the reception desk.
(Lotte Hotel phone number - +82 64 731 4343)
<Restaurants>
The restaurants within a working distance from ICC are listed. Maps and detailed menus are listed in
the “Guide to local activity (page 204)”.

DELIZIA in ICC - Western & Korean cuisine

Sinwoosung town – Korean, Japanese & Jeju local cuisine

Jeju MAWON – Pork, Beef, and Sea-food dishes

Garam Dolsotbap – Korean dishes

Heukdon Maeul – Pork dishes.
Accommodation
The following hotels are arranged in the organizing committee with discounted prices

Kensington Hotel - http://www.kensingtonjeju.com, +82 64 735 8900

Hyatt Regency Hotel - http://www.jeju.regency.hyatt.kr, + 82 64 733 1234

Silla Hotel - http://www.shilla.net/jeju, +82 64 735 8495

Hana Hotel - http://www.hotelhana.co.kr/, +82 64 738 7001-11

Corea Condo - http://www.coreacondo.co.kr/, +82 64 738 9101

Bareve Hotel - http://www.barevehotel.com/, +82 64 735 8899
.
Internet
The free wifi and wireless internet service will be available in the conference zone of ICC Jeju. A
password will be arranged for access to the internet. Computers close to the preview room will be used
for free e-mail and internet access.
Telephone numbers
Conference manager (Mr. Evan Ahn) 010 4939 5263
Conference security general (Prof. Yeon Gyu Yu) 010 8792 1121
Emergency/Fire 119
ICC
Information Desk – +82 64 735 1072
Duty Free Shop - +82 64 780 7600
DELIZIA restaurant - +82 64 738 6400
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Presentation
<Oral presentation>
It is recommended to copy your presentation file to the computer in the preview room at least 1/2
hour prior to the start time of the session. Presenters can also copy their presentation files to the
computer in the session room or bring their own computers. However, it is strongly recommended to
test the function of the computer and projects in advance. Your presentation file can be checked in the
preview room which is open during the conference period. For the presentation, only Powerpoint 2007
and 2010 operated in Window OS will be available. If you need to use programs in Mac OS, you need
to bring your own computer, connector and OS.
<Poster presentation>
Posters must be displayed in the event hall from Saturday to Tuesday. The size of poster board is 120
cm (width) x 150 cm (height). Poster board will available from 1:00 pm on the 17 th of May till the
closing day. Please be present at your poster at the designated time on the day to which you are
assigned and remove your poster before 1:00 pm on the 20th of May.
Posters with odd number: 12:50 PM – 14:20 PM, Sunday, May 18
Posters with even number: 9:00 AM – 10:30 AM, Monday, May 19
Lunch Workshop
Title: Advances in Circular-Dichroism (CD) Dynamic multi-mode spectroscopy, Automated CircularDichroism and Linear-Dichroism (LD) spectroscopy
Presentor: Geut Hoshen (Applied Photophysics Ltd., Leatherhead, UK)
12:10 – 13:00 PM, Sunday, May 18
Room Baekrok A
Organized by Maestor Korea
Free lunch will be available
Contact: Ms. Miran Suh, 02 2636 0369, mastor@mastor.co.kr
Tourist Information
The sightseeing places close to ICC are listed in the end of abstract book (page 206-215). The
group tour will be arranged by Yeha Tour (www.yehatour.com, +82 64 713 5505). Information
regarding tours will be available in the registration desk. For additional information on the
sights in Jeju, visit the city‟s website (http://english.jeju.go.kr/)
Pre-arranged half tour

East Tour Course – Seongsan Ilchulbong Peak [UNESCO World Heritage], Seongeup Folk Village

West Tour Course – Spirited Garden, Suweolbong Trekking, Cheonjeyeon Waterfall
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(Custom tour will be arranged by Yeha Tour)
Transportation
<Airport to ICC>
From Jeju International Airport
From Jeju Internation Airport, you will reach ICC Jeju within approximately 40-50 minutes by car
through Pyeonghwa-Ro Road. Limousine buses are also available every 15 minutes at the airport.
Guide to the airport limousines
(Jeju International Airport ↔ Jeju International Convention Center)

Place
Limousine bus stop at the left side of the front gate (Samyoung Traffic No. 600)

First departure
06:20 at the airport; last departure: after the arrival of the last airplane

Service route
Airport → T.H.E Hotel and Vegas Casino Jeju → Entrance to the Yeomiji Botanical Garden →
Hyatt Hotel → Shilla Hotel → Lotte Hotel → Hankook Condominium → Jeju International
Convention Center → New Gyeongnam Hotel → Seogwipo KAL Hotel
Taxi guide (Jungmun - Jeju International Airport)

Select the distance (long-distance or short-distance) at the taxi stop.

When you go to the Center, it is all right to take a taxi at the long-distance stop.

Since the taxi fare is fixed, please confirm the distance before taking a taxi

Fare (Korean won) : About 30,000 won; Distance: 40km; Duration: 40-45 minutes
<Hotel to ICC>
Limousine bus stops by each major hotel in Jungmun Area
Bus route: Jeju City (Jeju Airport)  Junmung Area (Kensington Hotel  Hyatt Hotel 
Silla Hotel  Suite Hotel  Hana Hotel  Lotte Hotel  Corea Condo  ICC **Bareve Hotel.
**Hotel shuttle from Bareve hotel to ICC will be available (~15 minutes)
Walking distance from hotel to ICC
Hyatt Regency Hotel: 2.5 km
Kensington: 2.2 km
Silla Hotel: 2.5 km
Hana Hotel: 2.4 km
Corea Condo: 2.2km.
Bareve Hotel: 9km
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The 4th Asia Pacific Protein Association (APPA)
Conference Committee Members
APPA Jeju 2014 Local Organizing Committee
Chair: Young Kee Kang, Chungbuk National University (Korea), ykkang@chungbuk.ac.kr
Co-chair1: Kyou-Hoon Han, Korea Research Institute of Bioscience and Biotechnology (Korea), khhan600@kribb.re.kr
Co-chair2: Weontae Lee, Yonsei University (Korea), wlee@spin.yonsei.ac.kr
Secretary General: Yeon Gyu Yu, Kookmin University (Korea), ygyu@kookmin.ac.kr
Scientific Program Committee
Chair : Kyeong Kyu Kim, Sungkyunkwan University (Korea), kyeongkyu@skku.edu
Vice Chair : Hyung-Sik Won, Konkuk University (Korea), wonhs@kku.ac.kr
Members : Yun-Ru (Ruby) Chen, Academia Sinica (Chinese Taipei), yrchen@gate.sinica.edu.tw
Yuji Goto, Osaka University (Japan), ygoto@protein.osaka-u.ac.jp
Kurt L. Krause, University of Otago (New Zealand), kurt.krause@otago.ac.nz
James R. Ketudat-Cairns, Suranaree University of Technology (Thailand), cairns@sut.ac.th, jrkcairns@yahoo.com
Sunghoon Kim, Seoul National University (Korea), sungkim@snu.ac.kr
Alex Law, Nanyang Technological University (Singapore), alaw@ntu.edu.sg
Ming Lei, National Center for Protein Science (China), leim@sibcb.ac.cn
Raja Noor Zaliha Raja Abd. Rahman, Enzyme and microbial technology research center (Malaysia), rnzaliha@upm.edu.my
Byung-Ha Oh, KAIST (Korea), bhoh@kaist.ac.kr
Euksu Oh, Ewha University (Korea), OhES@ewha.ac.kr
Yeon-Kyun Shin, Iowa State University (USA), colishin@iastate.edu
Richard Simpson, La Trobe University (Australia), Richard.simpson@latrobe.edu.au
Byeong Doo Song, Scripps Korea (Korea), bdsong@skai.or.kr
Financial Committee
Chair: Han Bin Oh, Sogang University (Korea), hanbinoh@sogang.ac.kr
Members: Sunshin Cha (KIOST)
Jungshin Lee (Kangwon National University)
Youngpil Kim (Hanyang University)
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Publication Committee
Chair: Sang Taek Jung, Kookmin University (Korea), sjung@kookmin.ac.kr
Vice Chair: Hugh I. Kim, Pohang University of Science and Technology (Korea), hughkim@postech.edu
Members: Won-Ki Huh, Seoul National University (Korea), wkh@snu.ac.kr
Min-Duk Seo, Ajou University (Korea), mdseo@ajou.ac.kr
Public Relations Committee
Chair: Sihyun Ham, Sookmyung Women's University (Korea), sihyun@sm.ac.kr
Vice-chair: Seong Soo A. An, Gachon University (Korea), seongaan@kyungwon.ac.kr
Members: Hae Sook Park, Cheju Halla University (Korea), phs1127kr@hanmail.net
Cheol-Won Lee, Chonnam National University (Korea), cwlee@chonnam.ac.kr
Jae-Hoon Kim, Jeju National University (Korea), kimjh@jeju.ac.kr
Jinhyuk Lee, KRIBB (Korea), jinhyuk@kribb.re.kr
12
APPA Council Members
President
Zengyi Chang, Peking University (China), changzy@pku.edu.cn
Australia
Raymond S. Norton, Monash University, Ray.Norton@monash.edu
Richard J. Simpson*, Ludwig Institute for Cancer Research Inc.
China
Ruiming Xu, Institute of Biophysics (CAS), rmxu@ibp.ac.cn
Ming Lei, National Center of Protein Research (Shanghai), leim@sibcb.ac.cn
Chinese Taipei
Po-Huang Liang, Academia Sinica, phliang@gate.sinica.edu.tw
Yun-Ru Ruby Chen*, Academia Sinica, yrchen@gate.sinica.edu.tw
India
Mohan Rao, Centre for Cellular & Molecular Biology, mohan@ccmb.res.in
Dinakar Salunke*, National Institute of Immunology, dinakar@nii.res.in
Indonesia
Arief Witarto, Indonesian Institute of Sciences, No-email
Bambang Sugiharto*, University of Jember, No-email
Japan
Haruki Nakamura, Osaka University, harukin@protein.osaka-u.ac.jp
Fumio Arisaka*, Tokyo Institute of Technology, farisaka@bio.titech.ac.jp
Korea
Young Kee Kang, Chungbuk National University, ykkang@chungbuk.ac.kr
Kyou-Hoon Han*, Korea Res. Inst. of Biosci. & Biotech. (KRIBB), khhan600@kribb.re.kr
Malaysia
Raja Noor Zaliha Raja Abd. Rahman, Universiti Putra, rnzaliha@biotech.upm.edu.my
New Zealand
Kurt L Krause, Otago University, kurt.krause@otago.ac.nz
Juliet A. Gerrard*, University of Canterbury, juliet.gerrard@canterbury.ac.nz
Philippines
Lemmuel Tayo, Mapua Institute of Technology, No-email
Singapore
Jianxing Song, National University of Singapore, bchsj@nus.edu.sg
Alex Law*, Nanyang Technological University, ALAW@ntu.edu.sg
Thailand
Jisnuson Svasti, Mahidol University, jisnuson.sva@mahidol.ac.th
James R. Ketudat-Cairns*, Suranaree University of Technology, cairns@sut.ac.th
Vietnam
Phan Van Chi, Vietnam Acad. of Sci. & Tech. (VAST), chi@ibt.ac.vn
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Exhibitors
1. Bioneer, 손은혜 팀장, 042-930-8690, ehson@bioneer.co.kr
2. Bion Life Science Co. Ltd., 고창욱 사장, 010-6280-5600, changwookgoh@gmail.com
3. Hucom Systems, 정해식 부장, 010-5654-5440, okhucom@naver.com
4. Malvern Instruments, 홍명희 과장, 031-786-0843, info.korea@malvern.com
5. DS&G, 김근희 차장, 02-6309-1530, ghkim@dsngsystem.co.kr
6. Waters Korea, 배상철 부장, 010-4954-7904, Marcelino_Bae@Waters.com,
7. The 7th Asian-Oceania HUPO Conference, Bangkok.
8. Maestor Korea, 서미란 대리, 02-2636-0369 mastor@mastor.co.kr
14
15
Scientific Program
4th Asian Pacific Protein Association Conference
Jeju, Korea
17-20, May 2014
16
 Nobel Laureate Lecture
The Molecular Basis of Eukaryotic Transcription
May 17 (Sat), 16:40-17:30, ROOM: YEONGJU A
Chair: Zengyi Chang, Ph. D. (APPA President, China)
Roger D. Kornberg, Ph.D.
Stanford University School of Medicine, USA
2006 Nobel Laureate in Chemistry
Roger Kornberg is Winzer Professor in Medicine in the Department of Structural Biology at Stanford
University. In his doctoral research, he demonstrated the diffusional motions of lipids in membranes,
termed flip-flop and lateral diffusion. He was a postdoctoral fellow and member of the scientific staff at the
Laboratory of Molecular Biology in Cambridge, England from 1972-5, where he discovered the nucleosome,
the basic unit of DNA coiling in chromosomes. He moved to his present position in 1978, where his
research has focused on the mechanism and regulation of eukaryotic gene transcription. Notable findings
include the demonstration of the role of nucleosomes in transcriptional regulation, the establishment of a
yeast RNA polymerase II transcription system and the isolation of all the proteins involved, the discovery of
the Mediator of transcriptional regulation, the development of two-dimensional protein crystallization and its
application to transcription proteins, and the atomic structure determination of an RNA polymerase II
transcribing complex.
17
 Keynote Lecture
Keynote Lecture
Funtionome of Human tRNA Synthetases for New Biology and Medicine
Chair: Young Kee Kang, Ph.D. (APPA2014 Chair & KSPS former president, Korea)
Sunghoon Kim, Ph.D.
Seoul National University, Korea
Professor Kim earned his Ph.D. in Biology and Medicine from Brown University, USA. At present, he is the
director of the Medicinal Bioconvergence Research Center at Seoul National University, Korea. Dr.
Sunghoon Kim received his bachelor's degree at Seoul National University College of Pharmacy, master's
degree at Korea Advanced Institute of Science and Technology and PhD degree at Brown University
Division of Biology and Medicine.
He has been studying novel functions of human aminoacyl-tRNA synthetases(ARSs) and searching for their
pathophysiological connections to human diseases with about 120 research publications (PNAS 105:11043,
2008; Nat Rev Cancer 11:708, 2011 for recent review). More specifically, he has identified potent novel
tumor suppressors such as AIMP2/p38 (Nat Genet 34:330, 2003), AIMP3/p18(Cell, 120:209, 2005). Besides,
he has also investigated novel extracellular activities of ARSs and associated factors such as lysyl-tRNA
synthetase (KRS, PNAS 102, 6356, 2005), tryptophanyl-tRNA synthetase(WRS)(Nat Struct Mol Biol 11:149,
2004) and AIMP1/p43(PNAS 103:14913, 2006). He also discovered the oncogenic variant of AIMP2,
designated AIMP2-DX2, as one of the critical factors that determines the survival of lung cancer patients
(Plos Genet 7:e1001351, 2011). More recently, he found that leucyl-tRNA synthetase (LRS) serves as an
amino acid sensor for mTOR signal pathway (Cell 149:410, 2012).
In summary, his research is unveiling novel regulatory network mediated by human aminoacyl-tRNA
synthetases that have been regarded as housekeeping machinery for protein synthesis. The regulatory
roles and implications of these proteins in human diseases have been largely overlooked for decades. His
series of the discoveries on the new function, pathology and medicine of ARSs are rapidly opening a
research area that throws new insights into the central dogma of life and human diseases.
18

Plenary Lectures
Plenary Lectures I
Influenza A virus "host jump": structural determinant
May 18 (Sun), 10:50-11:30, ROOM: YEONGJU A
Chair: Yuji Goto (APPA former president, Japan)
George Fu Gao, Ph.D.
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing, China
Professor George F. Gao obtained his Ph.D (DPhil) degree from Oxford University, UK and did his postdoc
work in both Oxford University and Harvard University (with a brief stay in Calgary University).
His research interests include enveloped viruses and molecular immunology. His group research is focusing
on the enveloped virus entry and release, esp. influenza virus interspecies transmission (host jump),
structure-based drug-design and structural immunology.
He has published more than 260 refereed papers, 10 books or book chapters and has applied and obtained
more than 25 UK, US and Chinese patents. His recent work on HA/receptor binding and structural basis of
both H7N9 and H5N1 influenza viruses provided novel insights into the molecular mechanism of avian-flu
“host jump” and the work on MERS-CoV entry delineated the molecular mechanism of receptor-viral
protein interaction (Science, 2013a, 2013b; Lancet, 2013; Nature Communications, 2014; Nature, 2013).
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Plenary Lectures II
New Approach to Electrostatic Properties of Proteins and Protein-Protein Interactions
Chair: Kyou-Hoon Han, Ph.D. (APPA2014 co-chair & KSPS president, Korea)
Haruki Nakamura, Ph.D.
Institute for Protein Research, Osaka University, Japan
Professor Nakamura obtained his Ph.D. from University of Tokyo, Japan. His research fields include
biophysics, protein science, and structural bioinformatics. From 2012, he is the Advisor to Osaka University
Trustees.
Haruki Nakamura is a Professor of Laboratory of Protein Informatics, Institute for Protein Research, Osaka
University. He is also a Head of Protein Data Bank Japan (PDBj, http://pdbj.org/), one of the four members
of the wwPDB (http://wwpdb.org/) as the international organization to look after PDB. He was born on 7
April 1952, at Tokyo, Japan. He graduated Department of Physics, Faculty of Science, the University of
Tokyo, in March 1975, and he took the Doctor of Science in March 1980, at Department of Physics, Faculty
of Science, The University of Tokyo, supervised by Dr. Akiyoshi Wada.
His research experiences are as follows: April 1980 - July 1987, Research Associate at Department of
Applied Physics, Faculty of Engineering, the University of Tokyo. August 1987 - March 1996, Protein
Engineering Research Institute, Osaka. April 1996 - March 1999, Biomolecular Engineering Research
Institute, Osaka. April 1999 -, Professor, Laboratory of Protein Informatics, Institute for Protein Research,
Osaka University. April 2012 -, Advisor to Osaka University Trustees. June 2001 -, Head of PDBj.
His research fields are structural bioinformatics, biophysical studies about protein architecture,
electrostatic properties and enzymatic functions, protein modeling, protein design, structure guided drug
development, and molecular and electronic simulation. He is an associate Editor of BREV (Biophysical
Reviews), and an editorial board member of PEDS (Protein Engineering Design and Selection), J. Struct.
Funct. Genomics, and Biophysics.
Since 2012, he has been a Council member of the Protein Society until December 2014, and a President of
Protein Science Society of Japan until March 2014. He is also a Council member of APPA.
20
Plenary Lectures III
How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick
incorporation

Chair: Myeong-Hee Yu, Ph.D. (Korea Institute of Science and Technology, Korea)
Ming-Daw Tsai, Ph.D.
Institute of Biological Chemistry, Academia Sinica, Chinese Taipei
Professor Tsai obtained his Ph.D. in Biochemistry and Medicinal Chemistry from Purdue University, USA. At
present, he is the director of the Institute of Biological Chemistry, Academia Sinica, Chinese Taipei.
Ming-Daw Tsai received a B.S. degree in chemistry from the National Taiwan University in 1972, a Ph.D. in
medicinal chemistry from Purdue University in 1978, and joined the faculty of The Ohio State University in
1981. He established the Chemistry-Biology Interface Training Program of OSU in 1996 and served as its
director through 2003. He has also directed OSU’s Office of Research Campus Chemical Instrument Center
for 14 years (1995-2007). From 2004-2008 Tsai served as Director of the Functional Proteomics Division of
the Genomics Research Center of Academia Sinica, Chinese Taipei. He also served as Director of the
National Core Facilities Office during 2004-2010. In 2006 Tsai took the directorship of the Institute of
Biological Chemistry of Academia Sinica.
His honour includes an Alfred P. Sloan Fellowship (1983-1985), the Camille and Henry Dreyfus TeacherScholar Award (1985-1990), the Distinguished Scholar Award of The Ohio State University (1992), an
Elected Fellow of the American Association for the Advancement of Science (1992), the Kimberly Professor
of Chemistry at The Ohio State University (2003-2007), and Distinguished Alumnus Award (Purdue College
of Pharmacy). He also serves as an Associate Editor of Biochemistry since 2010. He was elected to
Academician of Academia Sinica in 2012.
Tsai’s research interests include mechanistic enzymology of phosphoryl transfer enzymes particularly DNA
polymerases and kinases, and structure-function relationship of proteins in DNA damage response and
cancer signaling, particularly ankyrin repeat proteins and FHA domain containing proteins. He uses
structural biology approaches including NMR, X-ray, and mass spectrometry to study mechanistic problems.
Tsai has published over 250 articles in chemical and biological journals. His h index is 45.
21
Plenary Lectures IV
Membrane trafficking in mammalian cells and beyond
May 20 (Tue), 11:00-11:40, ROOM: YEONGJU A
Chair: Wontae Lee, Ph.D. (APPA2014 co-chair, Korea)
Hong Wanjin, Ph.D.
Institute of Molecular & Cell Biology, A*STAR, Singapore
After graduating from Xiamen University in 1982, Wanjin Hong was one of a few hundred Chinese students
chosen for further graduate training in the United States via the CUSBEA program. He received his PhD
from the State University of New York (SUNY Buffalo), and was a postdoctoral fellow there before he
joined IMCB as a principal investigator in 1989.
In Singapore, his research group has published over 200 papers in international journals including Science,
Nature, Nature Cell Biology, Developmental Cell, EMBO J, JCB, MBC, JCS and JBC. His work in the early
1990s identified the Golgi-targeting motifs for TGN38 and Golgi sugar transferases and defined the
trafficking pathway of KDEL receptor in mammalian cells. Among the 40 or so SNAREs in mammalian cells
involved in vesicle docking and fusion, about half of them were independently identified and functionally
characterized by his lab. His lab also showed that endobrevin (VAMP8) is a major v-SNARE responsible for
regulated exocytosis in exocrine cells and other secretory cells. His group also discovered that the PX (phox)
domain is a novel structural module capable of interacting with phosphoinositides, Arl1 GTPase regulates
Golgi targeting of the GRIP domain-containing proteins Golgin-97 and Golgin-245, and Rab7 and Rab34
share a common downstream effector (RILP). He also worked on COPII and COG complex.
His recent work has uncovered that TAZ (WWTR1) is an oncogene and TAZ interacts with TEAD
transcriptional factors to drive oncogenic process. Wbp2 and Amot were identified as positive and negative
regulator, respectively, of TAZ/YAP. TAZ and YAP are inhibited by the emerging Hippo tumor suppressor
pathway.
He was the recipient of National Science Award in 1999. Presently, he is a Professor and Executive
Director of IMCB. He serves as the Editor-in-Chief of Bioscience Reports and is on the editorial board of
several Journals such as PLOS One and TRAFFIC.
22
 Symposia
May 17 (Sat.)
Symposium 1A (14:30 - 16:15; ROOM: YEONGJU A)
Proteins in emerging fields I
14:30 - 14:52
Nei-Li Chan
Chinese Taipei
14:52 - 15:14
Shan-Ho Chou
Chinese Taipei
15:14 - 15:36
Jeong-Sun Kim
Korea
15:36 - 15:58
Linwoo Kang
Korea
15:58 - 16:15
Rui Wang
China
Symposium 1B (14:30 - 16:15; ROOM: YEONGJU B)
Proteins in disease I
14:30 - 14:51
Cong Liu
China
14:51 - 15:12
Yao Cong
China
15:12 - 15:33
SangYun Kim
Korea
15:33 - 15:54
SeungJae Lee
Korea
15:54 - 16:15
David Churchill
Korea
Symposium 1C (14:30 - 16:15; ROOM: BAEKROK A)
Protein folding and dynamics
14:30 - 14:52 Kunihiro Kuwajima
Japan
14:52 - 15:14 Jayant B. Udgaonkar
India
15:14 - 15:36
Wei Wang
China
15:36 - 15:58
Jooyoung Lee
Korea
15:58 - 16:15
Matthias Buck
USA
Symposium 1D (14:30 - 16:15; ROOM: BAEKROK B)
Structural targetomics for drug discovery
14:30 - 14:56
Jeong-Kyu Bang
Korea
14:56 - 15:22 Kwang Yeon Hwang
Korea
15:22 - 15:48
Yunje Cho
Korea
15:48 - 16:15
Young Ho Jeon
Korea
May 18 (Sun.)
Symposium 2A (09:00-10:30; ROOM: YEONGJU A)
Protein catabolism and trafficking
09:00 - 09:18
Nobuo N. Noda
Japan
09:18 - 09:36
Koji Okamoto
Japan
09:36 - 10:54 M. Sakoh-Nakatogawa
Japan
10:54 - 10:12
Taijoon Chung
Korea
10:12 - 10:30
Hyun Kyu Song
Korea
Symposium 2B (09:00-10:30; ROOM: YEONGJU B)
Proteins in disease II
09:00 - 09:22
Senyon Choe
USA
09:22 - 09:45
Joon Kim
Korea
09:45 - 10:07
Chinpan Chen
Chinese Taipei
10:07 - 10:30
Feng Shao
China
23
Symposium 2C (09:00-10:30; ROOM: BAEKROK A)
Protein analysis techniques I
09:00 - 09:23
Hong-Wei Wang
China
09:23 - 09:46
Kenji Sugase
Japan
09:46 - 10:00
Tae-Young Yoon
Korea
10:00 - 10:15 Jose M.M. Caaverio
Japan
10:15 - 10:30
Hajin Kim
Korea
Symposium 2D (09:00-10:30; ROOM: BAEKROK B)
Proteogenomics
09:00 - 09:22
Daehee Hwang
Korea
09:22 - 09:45
Myeong-Hee Yu
Korea
09:45 - 10:07
Youngsoo Kim
Korea
10:07 - 10:20
Eunok Paek
Korea
15:10 - 15:34
Ho Chul Kang
Korea
15:34 - 15:58
Yong Chen
China
15:58 - 16:22
Ji-Hong Lim
Korea
16:22 - 16:40
Tadashi Satoh
Japan
Proteins as therapeutics I
15:10 - 15:33
Kouhei Tsumoto
Japan
15:33 - 15:55
Junho Chung
Korea
15:55 - 16:18
Tse Wen Chang
Chinese Taipei
16:18 - 16:40
Sang Taek Jung
Korea
Protein analysis techniques II
15:10
15:34
15:58
16:22
-
15:34
15:58
16:22
16:40
Kozo Kaibuchi
Won Do Heo
Rahul Roy
Willem M. Albers
Japan
Korea
India
Finland
15:10
15:15
15:32
15:49
Intrinsically disordered protein
Kyou-Hoon Han (Session Overview)
- 15:15
- 15:32
Carmay Lim
Chinese Taipei
- 15:49
Hong-Yu Hu
China
- 16:06
Hugh I. Kim
Korea
16:06 - 16:23
16:23 - 16:40
Gary Daughdrill
Mamoru Sato
USA
Japan
Proteins in emerging fields II
17:00 - 17:22
Injae Shin
Korea
17:22 - 17:45 Andrew H.-J. Wang Chinese Taipei
17:45 - 18:07
Hyun-Suk Lim
Korea
18:07 - 18:30 James Ketudat Cairns
Thailand
Proteins as therapeutics II
17:00 - 17:22
Zhou Songyang
China
17:22 - 17:45
Ruibao Ren
China
17:45 - 18:07
Sangyong Jon
Korea
18:07 - 18:30 Byeong Doo Song
Korea
Protein design and engineering
17:00 - 17:20
Seung-Goo Lee
Korea
17:20 - 17:40 Katsumi Maenaka
Japan
17:40 - 18:00 Abu Bakar Salleh
Malaysia
18:00 - 18:15
Guan Siyu
Singapore
18:15 - 18:30
Madan Bharat
Korea
Structure-function of GPCR
17:00 - 17:20
Wei Liu
USA
17:20 - 17:40 Jae Young Seong
Korea
17:50 - 18:10
Han Suk Choe
Korea
18:10 - 18:30
Art Cho
Korea
24
May 19 (Mon.)
Symposium 5A (10:30-12:15; Rm. YEONGJU A)
Protein anabolism
10:30 - 10:58
Weiping Han
Singapore
10:58 - 11:26
Lu Lei
Singapore
11:26 - 11:54
Han-Jung Chae
Korea
11:54 - 12:15
Jinsong Liu
China
Symposium 5B (10:30-12:15; Rm. YEONGJU B)
10:30 - 10:56
Sung-Jean Park
Korea
10:56 - 11:22 Toshiyuki Shimizu
Japan
11:22 - 11:48
Beili Wu
China
11:48 - 12:15
Xin Xie
China
Proteins in nanobiotechnology
10:30 - 11:05
Haesik Yang
Korea
11:05 - 11:40
Fan-Gang Tseng
USA
11:40 - 12:15
Ryuji Yokokawa
Japan
Cancer biomarker (proteomics and glycoproteomics)
10:30 - 11:05
Jong Shin Yoo
Korea
11:05 - 11:40
Je Yeol Cho
Korea
11:40 - 12:15
Hyun Joo An
Korea
May 20 (Tue.)
09:00 - 09:28
Yeon-Kyun Shin
USA
09:28 - 09:56
Tae-Joon Jeon
Korea
09:56 - 10:24 Masaki Yamamoto
Japan
10:24 - 10:45 Atsushi Nakagawa
Japan
Proteins and drug discovery
09:00 - 09:22
Po-Huang Liang
Chinese Taipei
09:22 - 09:44
Koichi Kato
Japan
09:44 - 10:06
Sun Choi
Korea
10:06 - 10:28 Raymond S. Norton
Australia
10:28 - 10:45
Qian Wang
China
09:00 - 09:26
Motonori Ota
Japan
09:26 - 09:52 Kwang-Hyun Cho
Korea
09:52 - 10:18
Nozomi Nagano
Japan
10:18 - 10:45
Keun Woo Lee
Korea
Frontiers in protein sciences
09:00 - 09:20
Kurt L. Krause
New Zealand
09:20 - 09:40
Peter Czabotar
Australia
09:40 - 10:00
Hyung-Sik Won
Korea
10:00 - 10:15
Yi Liang
China
10:15 - 10:30
James Torres
Singapore
10:30 - 10:45
Young Mee Jung
Korea
25
Symposium 1A: Proteins in emerging fields I





Nei-Li Chan, Ph.D. (National Taiwan University, Chinese Taipei)
- Structural Analysis of a Metal-Activated Transcriptional Regulator from Bacillus subtilis
Shan-Ho Chou, Ph.D. (National Chung Hsing University, Chinese Taipei)
- Structure and function of a novel bacterial c-GMP binding protein
Jeong-Sun Kim, Ph.D. (Chonnam National University, Korea)
- Structural features of two Cmr proteins in the CRISPR RNA-mediated bacterial Iimmunity
Linwoo Kang, Ph.D. (Konkuk University, Korea)
- PLP cofactor-based catalysis mechanism
Rui Wang (Peking University, China)
- DegP primarily functions as a protease for the biogenesis of β-barrel outer membrane proteins in the
Gram-negative bacterium Escherichia coli
The most recent topics in “Structural and/or functional studies of hot proteins in various fields including
enzyme, microbial & plant proteins” will be discussed.
Organizer and Chair 1: Shan-Ho Chou, Ph.D. (National Chung Hsing University, Chinese Taipei)
Organizer and Chair 2: Sangkee Rhee, Ph.D.(Seoul National University, Korea)
Symposium 1B: Proteins in disease I
May 17 (Sat), 14:30-16:15, ROOM: YEONGJU B





Cong Liu, Ph.D. (Shanghai Institute of Organic Chemistry, China)
- Atomic view of amyloids
Yao Cong, Ph.D. (Institute of Biochemistry and Cell Biology, SIBS, CAS, China)
- Cryo-EM study on asymmetric and dynamic macromolecular machines
SangYun Kim, Ph.D. (Medical school on Alzheimer's disease, Seoul National University, Korea)
- New concepts of Alzheimer’s disease: diagnosis and treatment
SeungJae Lee, Ph.D. (Medical Graduate School on Cellular transmission of alpha-synuclein, KonKuk
University, Korea)
- Lysosomal function is the key determinant of propagation of synucleinopathy
David Churchill, Ph.D. (Department. of Chemistry, KAIST, Korea)
- Small molecules in the context of phosphorylated PD proteins
The most recent topics in “Protein folding diseases” will be discussed.
Organizer and Chair 1: Yao Cong, Ph.D. (Institute of Biochemistry and Cell Biology, SIBS, CAS, China)
Organizer and Chair 2: Sungsoo Ahn, Ph.D.(Gachon University, Korea)
26
Symposium 1C: Protein folding and dynamics
May 17 (Sat), 14:30-16:15, ROOM: BAEKROK A





Kunihiro Kuwajima, Ph.D. (The Graduate University for Advanced Studies, Hayama, Kanagawa, Japan)
- The problem of protein folding and its relationship to bioscience
Jayant B. Udgaonkar, Ph.D. (National Centre for Biological Sciences, Tata Institute of Fundamental
Research, India)
- Unfolding of a small protein proceeds via dry and wet globules and a solvated transition state
Wei Wang, Ph.D. (School of Physics, Nanjing University, China)
- Metal ion binding induced folding and allosteric motions of proteins
Jooyoung Lee, Ph.D. (Korea Institute for Advanced Study Seoul, Korea)
- The atomistic mechanism of conformational transition of adenylate kinase investigated by
Lorentzian structure-based potential
Matthias Buck, Ph.D. (Case Western Reserve University, USA)
- Molecular Simulation and Experimental Study of Transitions and Dissociation Events in a Dynamic
Protein Complex
The most recent topics in “Theory and experiments for protein folding, unfolding, and dynamics etc.” will
be discussed.
Organizer and Chair 1: Satoshi Takahashi, Ph.D. (Institute of Multidisciplinary Research for Advanced
Materials, Tohoku University, Japan)
Organizer and Chair 2: Jooyoung Lee, Ph.D. (Korea Institute for Advanced Study, Korea)
Symposium 1D: Structural targetomics for drug discovery
May 17 (Sat), 14:30-16:15, ROOM: BAEKROK B




Jeong-Kyu Bang, Ph.D. (Division of Magnetic Resonance, Korea Basic Science Institute, Korea)
- Design and synthesis of small molecule inhibitors targeting the polo-box domain of polo-like kinase
kinase 1
Kwang Yeon Hwang, Ph.D. (College of Life Sciences & Biotechnology, Korea University, Korea)
-Structural and mechanistic studies for the interaction of prolyl-tRNA synthetase and halofuginone,
the anti-cancer and -fibrotic compound
Yunje Cho, Ph.D. (Department of Life Science, POSTECH, Korea)
-Regulation of cellular metabolism and signaling through an ordered assembly of amino acyl tRNA
synthetase
Young Ho Jeon, Ph.D. (College of Pharmacy, Korea University, Korea)
-Pro-metastatic interaction between the two translational components and its therapeutic potential
The most recent topics in “Structural targetomics for drug discovery” will be discussed.
Organizer and Chair: Young-Ho Jeon, Ph.D. (Korea University, Korea)
27
Symposium 2A: Protein catabolism and trafficking
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



Nobuo N. Noda, Ph.D. (Institute of Microbial Chemistry, Japan)
- Structural basis of autophagy initiation by starvation
Koji Okamoto, Ph. D. (Graduate School of Frontier Biosciences, Osaka University, Japan)
- Targeting autophagy for mitochondrial clearance
Machiko Sakoh-Nakatogawa, Ph. D. (Frontier Research Center, Tokyo Institute of Technology, Japan)
- Regulation of lipidation of the ubiquitin-like protein Atg8 that drives autophagosome formation
Taijoon Chung, Ph.D. (Department of Biological Sciences, Pusan National University, Korea)
- Autophagy of peroxisomes in plant cells
Hyun Kyu Song, Ph.D. (Division of Life Sciences, Korea University, Korea)
- Structural basis of autophagosome maturation by swapping interaction partners of Atg5
The most recent topics in “Protein degradation, autophagy, proteolytic enzymes, ubquitination-dependent
proteolysis” will be discussed.
Organizer and Chair 1: Nobuo N. Noda, Ph.D. (Institute of Microbial Chemistry, Japan)
Organizer and Chair 2: Hyun Kyu Song, Ph.D. (Korea University, Korea)
Symposium 2B: Proteins in disease II
May 18 (Sun), 09:00-10:30, ROOM: YEONGJU B




Senyon Choe, Ph.D. (Salk Institute for Biological Studies, La Jolla, USA)
- Drug discovery collaboratory by CNDY and RASCH
Joon Kim, Ph.D. (Division of Life Sciences, Korea University, Korea)
- Ribosomal proteins play important roles for the determination of cell fate under stress conditions
Chinpan Chen, Ph.D. (Institute of Biomedical Sciences, Academia Sinica, Chinese Taipei)
- Structure of the activated full-length PmrA response regulator in complex with DNA from Klebsiella
pneumoniae
Feng Shao, Ph.D. (National Institute of Biological Sciences, Beijing, China)
- Biochemical dissection of bacterial virulence and macrophage innate immunity
The most recent topics in “Pathogenesis-related proteins” will be discussed.
Organizer and Chair 1: Tai-Huang Huang, Ph.D. (Institute of Biomedical Sciences, Academia Sinica, Chinese
Taipei)
Organizer and Chair 2: Myung Hee Kim, Ph.D. (Korea Research Institute of Bioscience and Biotechnology,
Korea)
28
Symposium 2C: Protein analysis techniques I
May 18 (Sun), 09:00-10:30, ROOM: BAEKROK A





Hongwei Wang, Ph.D. (Tsinghua University, China)
- Visualization of distinct substrate recruitment pathways in the yeast exosome by electron
microscopy
Kenji Sugase, Ph.D. (Suntory Institute for Bioorganic Research , Japan)
- Dynamics of nuclear proteins analyzed by relaxation NMR spectroscopy
Tae-Young Yoon, Ph.D. (Korea Advanced Institute of Science and Technology, Korea)
- Personalized diagnosis of cancers at the protein-protein interaction level with real-time singlemolecule co-IP analysis
Jose M.M. Caaverio, Ph.D. (University of Tokyo, Japan )
- Thermodynamic Tools in the Early Stages of Drug Discovery
Hajin Kim (Ulsan National Institute of Science and Technology, Korea)
- Protein-guided RNA dynamics during early ribosome assembly
The most recent topics in “Novel technologies in protein analysis - in vitro” will be discussed.
Organizer and Chair 1: Kouhei Tsumoto, Ph.D. (University of Tokyo, Japan)
Organizer and Chair 2: Tae-Young Yoon, Ph.D. (Korea Advanced Institute of Science and Technology, Korea)
Symposium 2D: Proteogenomics
May 18 (Sun), 09:00-10:30, ROOM: BAEKROK B
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


Daehee Hwang, Ph.D. (Department of New Biology, DGIST, Korea)
- An integrative proteogenomics approach in gastric cancer
Myeong-Hee Yu, Ph.D. (Korea Institute of Science and Technology, Korea)
- How to achieve the goal of quantitative proteomics: 1000 immino-MRM assay program
Youngsoo Kim, Ph.D. (Seoul National University, Korea)
- International effort to develop analytically validated multiple reaction monitoring (MRM)-based
assays to breast cancer cell proteins
Eunok Paek, Ph.D. (Hanyang University, Korea)
- ExonGraph: Discovery of novel transcripts using nucleotide-based splice graphs
The most recent topics in “Proteogenomics” will be discussed.
Organizer and Chair 1: Eun Gyeong Yang, Ph.D. (Korean Institute of Science and Technology, Korea)
Organizer and Chair 2: Cheolju Lee, Ph.D. (Korean Institute of Science and Technology, Korea)
29
Symposium 3A: Protein modification
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
Ho Chul Kang, Ph.D. (Department of Physiology, Ajou University School of Medicine, Korea)
- Structural and functional roles of both Poly(ADP-ribose) and Poly(ADP-ribosyl)ation
Yong Chen, Ph.D. (National Center for Protein Science Shanghai, China)
- The structural basis for activity regulation of MLL-family histone methyltransferase
Ji-Hong Lim, Ph.D. (Biomedical Chemistry, Konkuk University, Korea)
- PKA (Protein Kinase A) phosphorylates and activates SIRT1
Tadashi Satoh, Ph.D. (Nagoya City University, Japan)
- Structural insight into substrate recognition mechanism of glycoprotein processing enzyme ER
glucosidase II
The most recent topics in “Protein modification, ubiquitination, sumolylation, phosphorylation, acetylation,
methylation etc.” will be discussed.
Organizer and Chair 1: Yong Chen, Ph.D. (National Center for Protein Science Shanghai, China)
Organizer and Chair 2: Young Jun Kim, Ph.D. (Konkuk University, Korea)
Symposium 3B: Proteins as therapeutics I
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Kouhei Tsumoto, Ph.D. (Department of Bioengineering, Graduate School of Engineering, Japan
Institute of Medical Science, The University of Tokyo, Japan)
- How we can improve affinity of antibodies for the targets for therapeutics
Junho Chung, Ph.D. (Department of Biochemistry and Molecular Biology & Cancer Research Institute
Seoul National University College of Medicine, Korea)
- In vitro and in vivo application of anti-cotinine antibody and cotinine-conjugated compounds
Tse Wen Chang, Ph.D. (Genomics Research Center Academia Sinica Taipei, Chinese Taipei)
- Rational drug design: antibodies for treating severe asthma and allergy
Sang Taek Jung, Ph.D. (Department of Bio and Nano Chemistry, Kookmin University, Korea)
- Tailoring Aglycosylated Antibodies for a New Class of Next –Generation Immunotherapeutics
The most recent topics in “Antibodies and vaccines” will be discussed.
Organizer and Chair 1: Tse-Wen Chang, Ph.D. (Genomics Research Center, Academia Sinica, Chinese Taipei)
Organizer and Chair 2: Junho Jung, Ph.D.(Seoul National University, Korea)
30
Symposium 3C: Protein analysis techniques II
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Kozo Kaibuchi, Ph.D. (Department of Cell Pharmacology, Nagaya Ubiversity, Graduate School of
Medicine, Japan)
- Protein phosphorylation remains a black box in signal transduction: developing new methods to
search for substrates of specific kinases including Rho-kinase
Won Do Heo, Ph.D. (Department of Biological Sciences , KAIST, Korea)
- Optical control of cell signaling in mammalian cells
Rahul Roy, Ph.D. (Indian Institute of Science, India)
- Spatial organization and dynamics of eukaryotic gene regulation
Willem M. Albers, Ph.D. (BioNavis Ltd, Finland)
- Multi-Parametric Surface Plasmon Resonance (MP-SPR): new possibilities for characterization of
biomolecular layers
The most recent topics in “Novel technologies in protein analysis - in vivo” will be discussed.
Organizer and Chair 1: Kozo Kaibuchi, Ph.D. (Nagoya University, Graduate School of Medicine, Japan)
Organizer and Chair 2: Sungchul Hohng, Ph.D. (Seoul National University, Korea)
Symposium 3D: Intrinsically disordered protein
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
Kyou-Hoon Han, Ph.D. (Korea Research Institute of Bioscience and Biotechnology, Korea)
- Session Introduction: A brief overview on IDPs (No abstract available)
Carmay Lim, Ph.D. (Institute of Biomed. Sci., Academia Sinica, Chinese Taipei)
- Two potential therapeutic antibodies bind to a peptide segment of membrane-bound IgE in different
conformations
Hong-Yu Hu, Ph.D. (Shanghai Institutes for Biological Sciences, China)
- Ordered in disordered: structural transformation of the amyloidogenic core in the C-terminal part of
TDP-43
Hugh I. Kim, Ph.D. (Pohang University of Science and Technology, Korea)
- Structural transitions of intrinsically disordered -synuclein to helix by hydrophobic interaction with
neutral lipid membrane
Gary Daughdrill, Ph.D. (University of South Florida, USA)
- Evolution of transient helical secondary structure in intrinsically disordered proteins
Mamoru Sato, Ph.D. (Yokohama City University, Japan)
- Protein flexibility investigated by SAXS and MD simulation
The most recent topics in “Intrinsically Disordered Protein” will be discussed.
Organizer and Chair 1: Jin-Hyun Ahn, Ph.D. (Sungkyunkwan University, Korea)
Organizer and Chair 2: Mi-Hee Lim, Ph.D. (Ulsan National Institute of Science and Technology, Korea)
31
Symposium 4A: Proteins in emerging fields II
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Injae Shin, Ph.D. (Department of Chemistry, Yonsei University, Korea)
- Glycan microarrays as a powerful tool for studies of glycan-protein interactions
Andrew H.-J. Wang, Ph.D. (Institute of Biological Chemistry, Academia Sinica, Chinese Taipei)
- DNA mimic proteins: a new paradigm for regulation of DNA functions
Hyun-Suk Lim, Ph.D. (Department of Chemistry, Pohang University of Science and Technology, Korea)
-Targeting protein-protein interactions using proteomimetics
James Ketudat Cairns, Ph.D. (Department of Biochemistry Suranaree University of Technology,
Nakhon Ratchasima, Thailand)
-Protein-carbohydrate interactions leading to specificity in plant glycoside hydrolase family 1 enzymes
The most recent topics in “Structural and/or functional studies of hot proteins in various fields including
glycobiology, protein-protein or protein-DNA interactions etc.” will be discussed.
Organizer and Chair 1: Andrew H.-J. Wang, Ph.D. (Institute of Biological Chemistry, Academia Sinica,
Chinese Taipei)
Organizer and Chair 2: Injae Shin, Ph.D. (Yonsei University, Korea)
Symposium 4B: Proteins as therapeutics II
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
Zhou Songyang, Ph.D. (Sun Yat-Sen University School of Life Sciences, China)
- Telomere signaling networks
Ruibao Ren, Ph.D. (Shanghai Jiao-Tong University School of Medicine, China)
- Targeting the RAS signaling network
Sangyong Jon, Ph.D. (Department of Biological Sciences, KAIST, Korea)
- Biomedical applications of aptides
Sangyong Jon, Ph.D. (Department of Biological Sciences, KAIST, Korea)
- Protein combination enables facile construction of spatially addressed antibody library for functional
screening
The most recent topics in “Hormones, growth factors, cytokines, peptides, and chimeric proteins” will be
discussed.
Organizer and Chair 1: Ruibao Ren, Ph.D. (Shanghai Jiatong University Medical School, Ruijing Hospital,
China)
Organizer and Chair 2: Yong Sung Kim, Ph.D. (Ajou University, Korea)
32
Symposium 4C: Protein design and engineering





Seung-Goo Lee, Ph.D. (KRIBB, Korea)
- Genetic devices for protein engineering and synthetic biology
Katsumi Maenaka, Ph.D. (Hokkaido University, Japan)
- Protein expression systems for cell surface receptors
Abu Bakar Salleh, Ph.D. (Department of Biochemistry, University Putra Malaysia, Malaysia)
- Miniaturization of proteins for possible catalytic functions
Guan Siyu, Ph.D. (Nanyang Technology University, Singapore)
- Characterization of Single Amino Acid Substitutions in the β2 Integrin Subunits of Patients with
Leukocyte Adhesion Deficiency (LAD)-1
Madan Bharat (Pusan National University, Korea)
- Modulation of intracellular protein activity at level of protein folding by beta-turn engineering
The most recent topics in “Protein design, engineering and synthetic biology” will be discussed.
Organizer and Chair 1: Katsumi Maenaka, Ph.D. (Hokkaidou University, Faculty of Pharmaceutical Science,
Japan)
Organizer and Chair 2: Hak-Sung Kim, Ph.D. (Korea Advanced Institute of Science and Technology, Korea)
Symposium 4D: Structure-function of GPCR




Wei Liu, Ph.D. (Scripps Research Institute, USA)
- Serial femtosecond crystallography of G protein-coupled receptors
Jae Young Seong, Ph.D. (Korea University, Korea)
- Ligand binding pocket formed by evolutionarily conserved residues in the GLP1 receptor core
domain
Han Choe, Ph.D. (University of Ulsan, Korea)
- Homology modeling of GPCRs
Art Cho, Ph.D. (Korea University, Korea)
- Targeting GPCR’s
The most recent topics in “Structure-function of GPCR ” will be discussed.
Organizer and Chair 1: Hyun-Soo Cho, Ph.D. (Yonsei University, Korea)
Organizer and Chair 2: Wontae Lee, Ph.D. (Yonsei University, Korea)
33
Symposium 5A: Protein anabolism
May 19 (Mon), 10:30-12:15, ROOM: YEONGJU A




Weiping Han, Ph.D. (Department of Biochemistry, National University of Singapore, Singapore)
- Regulated exocytosis and diabetes.
Lu Lei, Ph.D. (School of Biological Sciences, Nanyang Technological University, Singapore)
- A novel imaging method for systematic super-localizations of Golgi proteins.
Han-Jung Chae, Ph.D. (Department of Pharmacology, Chonbuk National University Medical School,
Korea)
- BAX Inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation
Jinsong Liu, Ph.D. (Guangzhou Institutes of Biomedicine and Health, China)
- Structural Studies on Sorting Nexins
The most recent topics in “protein synthesis, folding, translation, and trafficking” will be discussed.
Organizer and Chair 1: Wanjin Hong, Ph.D. (Institute of Molecular and Cell Biology, Singapore)
Organizer and Chair 2: Kyung Tae Chung, Ph.D. (Dong-Eui University, Korea)
Symposium 5B: Proteins as drug targets
May 19 (Mon), 10:30-12:15, ROOM: YEONGJU B




Sung-Jean Park, Ph.D. (Gachon University, Korea)
- Discovery of a novel compound acting on the C-terminal domain of HSP90: Knowledge-based
approach
Toshiyuki Shimizu, Ph.D. (University of Tokyo, Japan)
- Structural study of TLR8 sensing single stranded RNA in innate immune system
Beili Wu, Ph.D. (Shanghai Institute of Materia Medica, China)
- Structural studies of HIV-1 co-receptors CXCR4 and CCR5
Xin Xie, Ph.D. (Shanghai Institute of Materia Medica, China)
- Targeting G protein-coupled receptors for the treatment of autoimmune diseases.
The most recent topics in “Target identification and validation” will be discussed.
Organizer and Chair 1: Xin Xie, Ph.D. (Shanghai Institute of Materia Medica, China)
Organizer and Chair 2: Jun-Goo Jee, Ph.D. (Kyungpook National University, Korea)
34
Symposium 5C: Proteins in nanobiotechnology
May 19 (Mon), 10:30-12:15, ROOM: BAEKROK A



Haesik Yang, Ph.D. (Pusan National University of Chemistry, Korea)
- Ultrasensitive immunosensors using redox cycling combined with enzymatic amplification
Fan-Gang Tseng, Ph.D. (UCLA of Engineering and System Science, USA)
- High throughput 3-in-1 protein chip toward utra-sensitive single protein nano array
Ryuji Yokokawa, Ph.D. (Kyoto University of Micro Engineering, Japan)
- Nano system integration using micro/nano fabrications and motor proteins
The most recent topics in “Proteins for nanobiotechnology application including sensors and drug delivery”
will be discussed.
Organizer and Chair 1: Ryuji Yokokawa, Ph.D. (Kyoto University, Japan)
Organizer and Chair 2: Sangyong Jon, Ph.D. (Korea Advanced Institute of Science and Technology, Korea)
Symposium: 5D
glycoproteomics
Cancer
biomarker
discovery
by
proteomics
and
May 19 (Mon), 10:30-12:15, ROOM: BAEKROK B



Jong Shin Yoo, Ph.D. (Korea Basic Science Institute, Korea)
- The Advent of High throughput Glycoproteomics and Application to Biomarker Discovery in Human
Plasma
Je Yeol Cho, Ph.D. (Seoul National University, Korea)
- Lung Cancer Proteome Biomarkers: Discovery, Validation and Clinical Assay Development
Hyun Joo An, Ph.D. (Chungnam National University, Korea)
- The sweet spot of post-translational modifications – understanding the role of the glycosylation in
diseases and health
The most recent topics in “Cancer biomarker discovery by proteomics and glycoproteomics” will be
discussed.
Organizer and Chair: Jong Shin Yoo, Ph.D. (Korea Basic Science Institute, Korea)
35
Symposium 6A: Proteins in membranes




Yeon-Kyun Shin, Ph.D. (Iowa State University, USA)
- Reconstructing synaptic membrane fusion
Tae-Joon Jeon, Ph.D. (Inha University, Korea)
- Biomimetic membranes for ion channel studies and engineered sensor applications
Masaki Yamamoto, Ph.D. (Advanced Photon Technology of RIKEN SPring-8 Center, Japan)
- Macromolecular crystallography at SPring-8 and SACLA
Atsushi Nakagawa, Ph.D. (Osaka University, Japan)
- Crystal Structure of Voltage-gated Proton Channel, Hv1/VSOP
The most recent topics in “Structure, function, and/or production of membrane-bound proteins” will be
discussed.
Organizer and Chair 1: Che (Alex) Ma, Ph.D. (Genomics Research Center, Academia Sinica, Chinese Taipei)
Organizer and Chair 2: Yeon-Kyun Shin, Ph.D. (Iowa State University, USA)
Symposium 6B: Proteins and drug discovery
May 20 (Tue), 09:00-10:45, ROOM: YEONGJU B





Po-Huang Liang, Ph.D. (Academia Sinica, Chinese Taipei)
- Protein-protein interaction inhibitors against drug-resistant cancers
Koichi Kato, Ph.D. (Okazaki Institute for Integrative Bioscience, National Institutes of Natural
Sciences, Japan)
- Structural views of glycosylation as potential drug target
Sun Choi, Ph.D. (Ewha Womans University, Korea)
- Computer-aided drug discovery of adenosine receptor modulators using multiple receptor
conformation and network analysis
Raymond S. Norton, Ph.D. (Monash University, Australia )
- Developing new anti-malarial agents using fragment-based drug discovery
Qian Wang (Peking University, China)
- Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia Coli D3-Phosphoglycerate Dehydrogenase
The most recent topics in “Protein-targeted drug discovery and protein-drug interaction” will be discussed.
Organizer and Chair 1: Jia Li, Ph.D. (Shanghai Institute of Materia Medica, China)
Organizer and Chair 2: Sun Choi, Ph.D. (Ewha Womans University, Korea)
36
Symposium 6C: Protein bioinformatics
May 20 (Tue), 09:00-10:45, ROOM: BAEKROK A




Motonori Ota, Ph.D. (Nagoya University, Japan)
- How an intrinsically disordered region function: a case of CARMIL protein
Kwang-Hyun Cho, Ph.D. (Korea Advanced Institute of Science and Technology, Korea)
- Systems biology: How to understand the functional interaction network of proteins
Nozomi Nagano, Ph.D. (National Institute of Advanced Industrial Science and Technology, Japan)
- Biosynthetic mechanism prediction for a secondary metabolite, ustiloxin B, in Aspergillus flavus
Keun Woo Lee, Ph.D. (Gyeongsang National University, Korea)
- Computer-aided drug design/discovery (CADD) via systems biology
The most recent topics in “Database, Systems biology, Modeling, etc.” will be discussed.
Organizer and Chair 1: Nozomi Nagano, Ph.D. (National Institute of Advanced Industrial Science and
Technology, Japan)
Organizer and Chair 2: Keun Woo Lee, Ph.D. (Gyeongsang National University, Korea)
Symposium 6D: Frontiers in protein sciences
May 20 (Tue), 09:00-10:45, ROOM: BAEKROK B






Kurt L. Krause, Ph.D. (Department of Biochemistry, University of Otago, New Zealand)
- Orf virus chemokine binding protein – structural basis of chemokine binding
Peter Czabotar, Ph.D. (The Walter and Eliza Hall Institute of Medical Research, Australia)
- Crystal structures of Bax and Bak reveal molecular events initiating apoptosis
Hyung-Sik Won, Ph.D. (Konkuk University, Korea)
- A protein allostery that discriminates cyclic nucleotide second messengers
Yi Liang, Ph.D. (Wuhan University, China)
- Amino acid sequence influences fibril formation of the recombinant full-length prion proteins
James Torres, Ph.D. (Nanynag Technological University, Singapore)
- Structural and functional aspects of viroporins in enveloped viruses
Young Mee Jung, Ph.D. (Kangwon University, Korea)
- 2D Correlation Analysis of Protein Denaturation
The most recent topics in “Frontier protein sciences” will be discussed.
Organizer and Chair: Cheol-Won Lee, Ph.D. (Department of Chemistry, Chonnam Nat’l University, Korea)
37
 Young Scientists Talk I






Winny Ariesandi (Academia Sinica, Chinese Taipei)
- Temperature-dependent structural changes of Parkinson’s alpha-synuclein reveal the role of preexisting oligomers in alpha-synuclein fibrillization
Xinmiao Fu (Peking University, China)
- In vivo substrate diversity and preference of small heat shock protein IbpB as revealed by using a
genetically incorporated photo-crosslinker
Tae Hun Kim (University of Toronto, Canada)
- Conformational dynamics in the regulation of β2-adrenergic receptor signaling
Yuxi Lin (Osaka University, Japan)
- Solubility and supersaturation-dependent protein misfolding revealed by ultrasonication
Yong-Yea Park (Ajou University School of Medicine, Korea)
- MARCH5-mediated quality control on acetylated Mfn1 facilitates mitochondrial homeostasis and cell
survival
Mayu S. Terakawa (Osaka University, Japan)
- Membrane Curvature Affects the Fibrillation of Amyloid β
Oral presentations by six eminent young scientists (Ph.D. students or Post-Doc) selected in the symposia
topics
Organizer and Chair 1: Raja Noor Zaliha Raja Abd. Rahman, Ph.D. (Enzyme and Microbial Technology
Research Center, Malaysia)
Organizer and Chair 2: Alex Law, Ph. D. (School of Biological Sciences, Nanyang Technological University
Singapore)
38
 Young Scientists Talk II






Bui Chi Bao (University of medicine and pharmacy Hochiminh city, Vietnam)
- Characteristics of oxidized LDL and technetium 99m for non-invasive SPECT/CT imaging of
atherosclerotic diseases
Tae Su Choi (Pohang University of Science and Technology, Korea)
- The effect of environmental factors on amyloid assembly of insulin
Khanit Ruangjaroon (Chulabhorn Graduate Institute, Thailand)
- Toxicoproteomics revealed neuronal projection and developmental impairment of human
neuroblastoma cells upon treatment with fipronil
Yuichi Yoshimura (Aarhus University, Denmark)
- Inhibition of α-synuclein oligomer toxicity by epigallocatechin gallate
Chen Yu (Peking University, China)
- Discovery of Compounds that Can Bind to Intrinsically Disordered Proteins
Li Zhang (National Institute of Biological Sciences, Beijing, China)
- Manipulation of host signal transduction by secreted effectors from enteropathogenic E. coli
Oral presentations by six eminent young scientists (Ph.D. students or Post-Doc) selected in the symposia
topics
Organizer and Chair 1: James R. Ketudat-Cairns, Ph.D. (Suranaree University of Technology, Thailand)
Organizer and Chair 2: Byeong Doo Song, Ph.D. (Scripps Korea Antibody Institute, Korea)
39
Abstract
(Oral presentaions)
4th Asian Pacific Protein Association Conference
Jeju, Korea
17-20, May 2014
40
Nobel Laureate Lecture
The molecule Basis of Eukaryotic Transcription
Roger D. Kornberg
Stanford University School of Medicine, USA
RNA polymerase II (pol II) assembles with general transcription factors (GTFs) and DNA in a giant 32-protein
initiation (PIC) complex prior to every round of transcription. The GTFs escort promoter DNA through the stages of
unwinding to create a transcription bubble, descent into the pol II cleft, RNA synthesis to a length of 25 residues, and
transition to a stable elongating complex. The topography and arrangement of subunits of a complete PIC was
determined by cryo-electron microscopy and a combination of chemical cross-linking and mass spectrometry.
Previous partial structures from X-ray and EM analysis could be docked to the complete PIC, providing crossvalidation and revealing many features in detail. The PIC showed a marked division between GTFs and pol II.
Promoter DNA was associated with the GTFs, suspended above the pol II active center cleft. Interaction with pol II
could not occur because the DNA must bend to enter the active center cleft. The PIC is a poised structure, awaiting
promoter melting for DNA bending and the transition to a transcriptionally active state.
41
Keynote Lecture
Metamorphosis of aminoacyl-tRNA synthetases:
Implications for human health and disease
Sunghoon Kim
Department of Molecular Medicine and Biopharmaceutical Sciences, Medicinal Bioconvergence Research Center,
College of Pharmacy and Graduate School of Convergence Technology, Seoul National University
E-mail: sungkim@snu.ac.kr
Aminoacyl-tRNA synthetases (ARSs) have been considered as "housekeeping proteins" dedicated to cellular protein
synthesis. However, it is becoming obvious that they are poly-functional proteins forming a functional network
coordinating protein synthesis with diverse cellular regulatory processes [1, 2]. Among many functional complexes
mediated by ARS, multi-tRNA synthetase complex consisting of nine different enzymes and three auxiliary factors is
most intriguing since many components of this complex serve distinct signaling roles outside of the complex. For
instance, leucyl-tRNA synthetase was recently shown to control mTOR signal [3] while lysyl-tRNA synthetase can
control transcription in nucleus [4] or cell migration in plasma membrane [5] As more functions are unveiled from
these proteins, the molecular mechanisms for their functional diversity have been emerging as important issues to
understand. As the tools for multifunctionality, many ARSs are post-translationally modified at specific sites, which
triggers significant conformational change. In some cases, the catalytic and non-canonical activities of ARSs are
controlled by the generation of splicing variants or peptide cleavage. With all of these mechanisms, ARSs form a
unique metamorphic functionome that serves a master coordinator for system control. This lecture will introduce some
recent studies on metamorphic changes of ARSs and their pathophysiological implications.
References
Essential nontranslational functions of tRNA synthetases, Guo et al. Nat Chem Biol. 9: 145-153, 2013
Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping, Kim et al. Nat Rev Cancer, 11:708-718,
2011
Leucyl-tRNA synthetae is an intracellular leucine sensor for the mTORC1-signaling pathway, Han et al. Cell 149:
410-424, 2012
Structural Switch of lysyl-tRNA synthetase between translation and transcription, Ofir-Birin et al. Mol Cell 49: 30-42,
2013
Chemical inhibition of promatastatic lysyl-tRNA synthetase-laminin receptor interaction, Kim et al. Nat Chem Biol
10: 29-34, 2014
42
Plenary Lecture I
Novel Influenza A (H7N9) Virus: Origin, ―Host jump‖ and Drug-resistance
George F. Gao
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101, China
Chinese Center for Disease Control and Prevention, Beijing 102207, China
E-mail: gaof@im.ac.cn
Since February 18, 2013, the discovery of the first case of H7N9 avian influenza virus (AIV) infected human, more
than 300 human infection cases of this novel reassortant AIV have emerged in China, and gained worldwide attentions.
We performed phylogenetic and coalescent analyses to extrapolate potential origins of the novel H7N9 AIVs. Our
results showed that this novel H7N9 virus originated from multiple reassortment events. The HA gene might come
from AIVs of duck origin, and the NA gene might come from AIVs from migratory birds along the East AsiaAustralian Flyway. The six internal genes of this virus likely originated from two different groups of chickens H9N2
AIVs. Conclusively, H7N9 AIVs of this outbreak has evolved from at least four origins, and the diversity among virus
isolates implies the virus has evolved to two different lineages. Then we evaluated the viral hemagglutinin (HA)
receptor binding properties from two human H7N9 isolates, SH-H7N9 (containing the avian-signature Q226) and AHH7N9 (containing the mammalian-signature L226). We found that SH-H7N9 HA preferentially binds the avian
receptor analog, whereas the AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9
mutant HA (L226Q) has dual receptor binding property, indicating that other amino acid substitutions contribute to the
receptor binding switch. Furthermore, we described the features of two distinct neuraminidases (NA) from the H7N9
viruses, and showed the structural basis for different drug-resistances of these two NAs.
43
Plenary Lecture II
New Approach to Electrostatic Properties of Proteins and Protein-Protein
Interactions
Haruki Nakamura
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
E-mail: harukin@protein.osaka-u.ac.jp
Electrostatic properties of proteins govern most of the biological phenomena, in particular, specific molecular
recognitions on protein interfaces, which controls biological signal transductions through protein-protein interactions
(PPIs) [1-3]. Appropriate treatment of the electrostatic interaction is critical for computational analyses of PPI in a
realistic manner. Since the potential function is long-range, it is not simple to handle the interactions in an effective
manner: high accuracy, low computational cost, ease of the implementation, and freedom from artifacts. We have
recently developed a novel algorithm, zero-multipole summation method, for evaluating the electrostatic energy of
charged particle systems [4-6]. Its simple pair wise form enables us to effectively apply the scheme to highperformance parallel computation with GPGPU systems [7]. Several applications to homogeneous and inhomogeneous
molecular systems have confirmed that it could replace the conventional Ewald method in order to perform rapid and
accurate molecular dynamics simulations [8-10]. This method is applied to simulate a protein-protein docking
procedure in an ab-initio manner, based on the computed free energy landscape [11, 12].
References
H. Nakamura, Roles of electrostatic interaction in proteins. Quart. Rev. Biophys. 29, 1-90 (1996).
E. Kanamori et al., Prediction of Protein-Protein Complex Structures. "Biomolecular Forms and Functions" (Eds. M.
Bansal & N. Srinivasan), pp. 160-172, World Scientific Publishing (2013)
Y. Murakami, K. Kinoshita, A. R. Kinjo, H. Nakamura, Exhaustive comparison and classification of ligand-binding
surfaces in proteins. Protein Science 22, 1379-1391 (2013)
I. Fukuda, Y. Yonezawa, H. Nakamura, Molecular dynamics scheme for precise estimation of electrostatic interaction
via zero-dipole summation principle. J. Chem. Phys. 134, 164107 (2011).
I. Fukuda, Zero-multipole summation method for efficiently estimating electrostatic interactions in molecular system. J.
Chem. Phys. 139, 174107 (2013)
I. Fukuda, H. Nakamura, Non-Ewald methods: Theory and applications to molecular systems. Biophys. Rev. 4, 161170 (2012)
T. Mashimo, Y. Fukunishi, N. Kamiya, Y. Takano, I. Fukuda, H. Nakamura, Molecular dynamics simulations
accelerated by GPU for biological macromolecules with a non-Ewald scheme for electrostatic interactions. J. Chem.
Theory Comput. 9, 5599-5609 (2013)
I. Fukuda, N. Kamiya, Y. Yonezawa, H. Nakamura, Simple and accurate scheme to compute electrostatic interaction:
Zero-dipole summation technique for molecular system and application to bulk water. J. Chem. Phys. 137, 054314
(2012)
N. Kamiya, I. Fukuda, H. Nakamura, Application of zero-dipole summation method to molecular dynamics
simulations of a membrane protein system. Chem. Phys. Lett. 568-569, 26-32 (2013)
T. Arakawa, N. Kamiya, H. Nakamura, I. Fukuda, Molecular dynamics simulations of a double-stranded DNA in an
explicit solvent model with zero-dipole summation method. PLos One 8, e76606 (2013)
J. Higo, J. Ikebe, N. Kamiya, H. Nakamura, Enhanced and effective conformational sampling of protein molecular
systems for their free energy landscapes. Biophys. Rev. 4, 27-44 (2012)
J. Higo, K. Umezawa, H. Nakamura, A virtual-system coupled multicanonical molecular dynamics simulation:
Principles and applications to free-energy landscape of protein–protein interaction with an all-atom model in explicit
solvent. J. Chem. Phys. 138, 184106 (2013)
44
Plenary Lecture III
How a low-fidelity DNA polymerase chooses non-Watson-Crick from
Watson-Crick incorporation
Ming-Daw Tsai
1
2
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
E-mail: mdtsai@gate.sinica.edu.tw
A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism
contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity
DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA
polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes
efficient dG:Dgtp incorporation in addition to correct repair. In this lecture I will report the use of solution structures
of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick
pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve
the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can pre-bind purine MgdNTP tightly
and undergo a specific conformational change in the absence of DNA. The pre-bound MgdGTP assumes an unusual
syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique
mechanism
, the pre-bound syn-dGTP forms a Hoogsteen base pair with the template
anti-dG. Interestingly, while Pol X pre-binds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed
in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but
not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA
polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a
low-fidelity DNA polymerase.
Reference:
The abstract is reproduced from J. Am. Chem. Soc. (2014), in press.
45
Plenary Lecture IV
Membrane trafficking in mammalian cells and beyond
Hong Wanjin
Institute of Molecular & Cell Biology, A*STAR
Over past two decades, we have seen the transformation on our knowledge about protein targeting/transport along the
endocytic and secretory pathways. Many targeting motifs/signals
were defined and many protein machineries were revealed. My lab has been extensively involved in defining the
targeting motifs and studying the molecular machineries involved in the transport system including SNAREs, small
GTPases, sorting nexins, coat proteins and tethering protein complexes. I will summarize our work in this area and
highlight some key findings.
46
Structural Basis of the Hg2+-Mediated Transcriptional
Regulation of mer Operon by the Dual-Function Regulator MerR
Nei-Li Chan1,2, Chih-Chiang Chang1, Li-Ying Lin1,2, Xiao-Wei Zou1,2, and Chieh-Chen Huang3
1
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
2
Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan.
3
Department of Life Sciences and Center of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
E-mail:nlchan@ntu.edu.tw
The mer operon confers bacterial resistance to environmental inorganic mercury (Hg2+) and organomercurial
compounds by encoding proteins involved in the sensing, transport, and detoxification of these cytotoxic agents.
Expression of the mer operon is tightly regulated by the dual-function transcriptional regulatory protein MerR.
Whereas in the absence of Hg2+, MerR binds to the operator/promoter region (O/P) of mer operon to supress
transcription, MerR is converted into a transcriptional activator upon Hg2+-binding to induce mer operon expression.
Sequence analysis suggests that the O/P of mer operon is pseudopalindromic with the -35 and -10 boxes being spaced
by 18~20 bps, different from the optimal of 17 bp. Therefore, a Hg2+-dependent DNA distortion by the MerR dimer,
which brings closer the distance and reorients the two polymerase binding sites, is required to activate transcription.
To understand the structural basis by which Hg2+-binding regulates the functional state of MerR, we have determined
the crystal structures of apo- and Hg2+-bound MerR dimer form gram-positive bacteria Bacillus megaterium MB1,
which correspond to the suppressor and activator conformation of MerR, respectively. Structural analysis not only
illustrated how the assembly of Hg2+-binding pocket is achieved, but also revealed pronounced and functionally
relevant tertiary and quaternary changes between the apo- and Hg2+-bound MerR dimer. More detailed analysis and
additional functional implications of these structures will be presented during the meeting.
47
Structure and Function of a Novel Bacterial
c-GMP Binding Protein
Shan-Ho Chou
Institute of Biochemistry, College of Life Sciences, National Chung Hsing University, Taichung, 40227, Taiwan
E-mail: shchou@nchu.edu.tw
cAMP is an important secondary messenger molecule widely distributed across all living kingdoms, whereas cGMP is
generally considered to be restricted to eukaryotes. Recently, solid evidences for cGMP signaling in Rhodospirillum
centenum have been provided, and it is proposed that cGMP could also be adapted to deliver messages to diverse
outputs via unknown mechanisms. While the structures and functions of binding between cAMP and its receptor
protein CRP have been well studied in the past, currently no structure of prokaryotic cGMP-binding protein complex
is known. Here we report the first determination of a cGMP-receptor crystal structure from the plant pathogen
Xanthomonas campestris (Xcc) to a resolution of 2.2 Å . The new cGMP receptor Xcc0249 is found to belong to the
CRP/FNR family protein containing both a cyclic-Nucleotide Binding Domain (cNBD) and a GGDEF domains, and
exhibits strong cGMP binding and diguanylate cyclase activities. Mutations of crucial amino acid residues responsible
for cGMP binding to Xcc0249 are found to significantly reduce the biofilm formation and virulence in Xcc.
Isothermal calorimetry (ITC) measurements demonstrate that Xcc0249 can bind preferentially to cGMP with a much
stronger affinity (KD: 2.93E-7) than cAMP (KD: 1.79E-5). cGMP binding to Xcc0249 is also found to enhance the
GGDEF diguanylate cyclase activity, implying a broader functional role of cGMP and a possible linkage between the
cGMP and c-di-GMP interaction networks in bacteria.
References
[1]
[2]
[3]
An S-Q, Chin K-H, Febrer M, McCarthy Y, Yang J-G, et al. Chou S-H.*, Ryan R. * (2013) A cyclic GMPdependent signalling pathway regulates bacterial phytopathogenesis. EMBO J 32: 2430-2438.
Gomelsky M, Galperin MY (2013) Bacterial second messengers, cGMP and c-di-GMP, in a quest for regulatory
dominance. EMBO J 32: 2421-2423.
Hofer U (2013) cGMP completes the cycle. Nature Review in Microbiology 11: 596.
48
Structural features of two Cmr proteins in the CRISPR RNA-mediated
bacterial immunity
Jiali Sun1, Jae-Hyun Jeon2, Minsang Shin3, Jeong-Hoh Park3, Ho-Chul Shin2, Byung-Ha Oh2 and Jeong-Sun Kim1,3
1
Interdisciplinary Graduate Program in Molecular Medicine, Chonnam National University,
Gwangju 501-746, Korea.
2
Department of Biological Sciences, KAIST Institute for the Biocentury,
Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
3
Department of Chemistry, Chonnam National University, Gwangju 500-757, Korea.
E-mail: jsunkim@chonnam.ac.kr
Many bacteria and archaea have an inheritable RNA-based immune defense mechanism for destroying invading
phages and plasmids. Central to this mechanism is the presence of clustered regularly interspaced short palindromic
repeats (CRISPRs) and CRISPR-associated (cas) genes on prokaryotic genomes. A multi-subunit ribonucleoprotein
complex, termed the Cmr RNA silencing complex, recognizes and destroys viral RNA by an as yet unclear mechanism
in the Type IIIB system. This complex in Archaeoglobus fulgidus and Pyrococcus furiosus consists of six subunits,
Cmr1-Cmr6. Cmr5 from P. furiosus forms a single globular structure and exists as a monomeric protein both in the
crystalline state and in solution. In vitro assays indicate that Cmr5 interacts with Cmr4. The crystal structure of Cmr1
from A. fulgidus reveals that the protein is composed of two tightly associated ferredoxin-like domains. An ensuing
mutational analysis identified a highly conserved basic surface patch that binds single-stranded nucleic acids
specifically, including the mature CRISPR RNA, but in a sequence-independent manner. In addition, this subunit was
found to cleave single-stranded RNA. Together, these structural and biophysical data might help in understanding the
complicated and not well-characterized Cmr effector complex.
References
[1] Hale C.R., Zhao P., Olson S., Duff M.O., Graveley B.R., Wells L., Terns R.M. and Terns, M.P. ―RNA-guided
RNA cleavage by a CRISPR RNA-Cas protein complex.‖, Cell, Vol. 139, (2009), pp 945-956.
[2] Park J.H., Sun J., Park S.Y., Hwang H.J., Shin M and Kim, J.S. ―Crystal structure of Cmr5 from Pyrococcus
furiosus and its functional implications‖, FEBS Letters, Vol. 587, No. 6, (2013), pp 562–568.
[3] Sun J., Jeon J.H., Shin M., Shin H.C., Oh B.H. and Kim J.S. ―Crystal structure and CRISPR RNA-binding site of
the Cmr1 subunit in the Cmr interference complex‖, Acta Crystallographica Section D: Biological
Crystallography D, Vol. 70, No. 2, (2014), 535-543.
49
PLP undergoes conformational changes during the course of an enzymatic
reaction
Thanh Thi Ngoc Doan1, Jin-Kwang Kim1, Myoung-Ki Hong1, Pedro Alexandrino Fernandes2,
Maria João Ramos2, Lin-Woo Kang1
1
Department of Biological Sciences, Konkuk University, 1 Hwayang dong, Gwangjin-gu, Seoul 143-701, Korea
2
Requimte/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto,
Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
E-mail: lkang@konkuk.ac.kr
Numerous enzymes, such as the pyridoxal 5‘-phosphate (PLP)-dependent enzymes, require cofactors for their activities.
By x-ray crystallography, we determined the structural snapshots of L-serine dehydratase catalytic reactions of a
bacterial PLP-dependent enzyme. In each structure, the dihedral angle between the pyridine ring and the Schiff base
linkage of PLP varied from 18° to 52°. We proposed the organic cofactor PLP directly catalyzes reactions with the
active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by highlevel quantum mechanical calculations. The conformational change was essential for the nucleophilic attack of the
substrate on PLP, for the concerted proton transfer from the substrate to the protein, and for the directing of the
carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions
like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying
the previously unknown catalytic roles of organic cofactors.
Fig. PLP dihedral angles in crystallographic intermediate structures (a) Superimposition between the active-sites. (b) Comparison
of the average dihedral angles. (c) Tetrahedral geometry of PLP and attacking and leaving amino groups in PGD. (d) Schematic
representations of chemical structures of Schiff-base-linked PLP.
References
[1] Ngo, H. P., Cerqueira, N. M., Kim, J. K., Hong, M. K., Fernandes, P. A., Ramos, M. J. & Kang, L. W. (2014).
Acta crystallographica. Section D, Biological crystallography 70, 596-606.
50
DegP primarily functions as a protease for the biogenesis of β-barrel outer
membrane proteins in the Gram-negative bacterium Escherichia coli
Xi Ge1,6, Rui Wang1,6, Jing Ma1,6, Yang Liu1, Anastasia N. Ezemaduka1, Peng R. Chen2,4,5,
Xinmiao Fu1,2,*, and Zengyi Chang1,2,3,*
1
State Key Laboratory of Protein and Plant Gene Research and School of Life Sciences, Peking University,
Beijing 100871, China
2
Center for Protein Science, Peking University, Beijing 100871, China
3
Center for the History and Philosophy of Science, Peking University, Beijing 100871, China
4
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China
5
Peking-Tsinghua Center for Life Sciences, Beijing 100871, China
6
These authors contributed equally to this work.
Email: Zengyi Chang (changzy@pku.edu.cn) or Xinmiao Fu (fuxinmiao@pku.edu.cn)
DegP (also designated as HtrA) and its homologs are found in prokaryotic cells and such eukaryotic organelles as
mitochondria and chloroplast. DegP has been found to be essential for the growth of Gram-negative bacteria under heat
shock conditions and is arguably considered to possess both protease and chaperone activities. The function of DegP
has not been clearly defined. Using genetically incorporated non-natural amino acid as photo-crosslinkers, here we
identified the β-barrel outer membrane proteins (OMPs) as the major natural substrates of DegP in Escherichia coli
cells. We also demonstrated that DegP primarily functions as a protease, at both low and high temperatures, to
eliminate unfolded OMPs, with hardly any appreciable chaperone activity in cells. We also found that toxic and cell
membrane-damaging misfolded OMPs would accumulate in DegP-lacking cells cultured under heat shock conditions.
Together, our study defines the primary function of DegP in OMP biogenesis and offers a mechanistic insight into the
essentiality of DegP for cell growth under heat shock conditions.
51
Atomic View of Amyloids
Cong Liu1, Chuchu Wang1, Man Zou1, Lin Jiang2 and David Eisenberg2
1
Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai, China
2
UCLA-DOE Institute for Genomics and Proteomics, Howard Hughes Medical Institute, Molecular
Biology Institute, University of California, Los Angeles, California 90095, United States
E-mail: liulab@sioc.ac.cn
A wide range of devastating amyloid diseases, including Alzheimer‘s, Parkinson‘s and the prion conditions, are
associated with aberrant aggregation from diverse amyloid proteins1. As the ultimate products of amyloid aggregation,
amyloid fibrils consisting of extended β-sheets structure were long suspected to be the pathogenic agents2. But a
variety of amyloid oligomers found on the pathways to fibril were recently identified to be more toxic than the fibrils3.4.
My research mainly focuses on (1) Understanding the molecular mechanism that determine how a normal functional
protein assembles into an abnormal, aggregated protein including both oligomeric and fibrillar states. (2) Combining a
variety of chemical, biophysical and biochemical approaches to pin down and characterize the etiologic agents –
amyloid oligomer in different amyloid diseases. (3) Structure-based design of chemical compounds to inhibit amyloid
toxicity.
References
[1] Caughey B., Lansbury P., Annu. Rev. Neurosci. (2003), 26, 267.
[2] Nelson R, et al., Nature. (2005), 435, 773.
[3] Laganowsky A, et al., Science. (2012), 335, 1228.
[4] Chiti, F and Dobson C, Annu. Rev. Biochem. (2006). 75, 333.
52
Cryo-EM Study on Asymmetric and Dynamic
Macromolecular Machines
Yao Cong
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, CAS, Shanghai 200031, China
Email: cong@sibcb.ac.cn
Electron cryomicroscopy (cryo-EM) is emerging as a powerful tool for determining the 3D structure of large
biomolecules and biomolecular assemblies in close to their native solution environment. With the resolvability still
improving (approaching 3 Å resolution for virus system), cryo-EM technique forms a bridge in structural biology that
bridging the gap from cell resolution to atomic resolution. In addition, it plays an irreplaceable role in resolving the
difficult asymmetric or dynamic systems such as ribosome and ATP synthase, which usually have important biological
functions. In this presentation, I will mainly focuses on my work on the asymmetric structure study of the eukaryotic
group II chaperonin TRiC/CCT in its ATP-driven conformational cycle by cryo-EM, as well as corresponding
computational methodology development. In addition, I will also introduce my current research on structural and
functional study of protein quality control macromolecular machines.
53
The Conceptual Changes in Diagnosis and Treatment of Alzheimer’s Disease
SangYun Kim
Department of Neurology. Seoul National University College of Medicine
Neurocognitive Behavior Center. Seoul National University Bundang Hospital
Alzheimer‘s disease (AD) is a complex, progressive, and eventually fatal neurodegenerative disease that currently
lacks effective treatment options. The worldwide national economic and caregiver‘s emotional burdens of this
cognitively destructive illness are continuously increasing without bounds. Without disease modifying measures of
AD, we are going to face huge impact of greater number of AD patients in this earth.
According to NINDS-ADRDA criteria, to diagnose a person as a patient with Alzheimer‘s disease, the patient should
be at the stage of dementia. But after two new diagnostic criteria of Alzheimer‘s disease published at 2011 by NIA-AA
and IWG criteria, we can diagnose a person as a patient with Alzheimer‘s disease, if this patient is in any stage of
cerebral amyloidopathy, despite of clinical severity. This is a big concept change in Alzheimer‘s disease. Because of
this change, Alzheimer‘s disease became a particular disorder based on a specific molecular pathomechanism from a
clinical syndrome. So now we can have a concrete ground which makes it possible to handle the state of cerebral
amyloidopathy as a basic pathologic state of Alzheimer‘s disease.
Current AD drugs can improve symptoms without alteration of disease progression. We need urgently some ways to
prevent, stop or slow down the progression of AD. However we don‘t have any measure to do that and also don‘t have
any appropriate clinical research method to find those kinds of compounds which can alter the progression of AD.
In this presentation, I‘d like introduce the preliminary results of our recent studies which can provide us new concepts
of Alzheimer‘s disease treatment. This is ―Oligomeric beta amyloid detection by multimer detection system in
Alzheimer‘s disease‖. This is a totally new blood based biomarker of AD having very high sensitivity and specificity,
which should be a reliable and simple, cost effective biomarker. Using this new biomarker, we could diagnose the
patient with AD in a very early clinical or preclinical stage. Using this method, we can find new compounds which can
reduce Aβ oligomer, like metformin or sulfonylurea for DM control. Although these substances cannot improve the
end organ function after impairment, they can prevent the progression of end organ damage by stopping the causative
state, hyperglycemia or hyperoligomeric-beta-amyloidosis. Oligomeric beta amyloid detection by MDS in
Alzheimer‘s disease must be the starting point of this new treatment paradigm.
I will also present the new concept of AD treatment, AD control. This can be an ideal and optimal treatment option,
but we‘ve never think about this kind of AD treatment because we don‘t have a reliable biomarker based on a simple
blood draw. If we can measure the oligomeric beta amyloid reliably and easily, and the long standing hyper-oligomeric
beta amyloidosis is a cause of Alzheimer‘s disease as a result of end organ impairment, and we can prevent
progression of Alzheimer‘s pathological changes, by reducing the oligomeric beta amyloid level of our body
54
Lysosomal function is the key determinant of propagation of synucleinopathy
Eun-Jin Bae1, Na-Young Yang1, Miyoung Song1, Cheol Soon Lee1, He-Jin Lee2, Eliezer Masliah3,
Sergio Pablo Sardi4, Seung-Jae Lee1
1
Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Korea
2
Department of Anatomy, School of Medicine, Konkuk University, Seoul 143-701, Korea
3
Departments of Pathology and Neurosciences, University of California, San Diego, La Jolla, CA
4
Genzyme, a Sanofi Company, Framingham, MA
E-mail: sjlee@konkuk.ac.kr
Deposition of -synuclein aggregates occurs widely in the central and peripheral nervous systems in Parkinson‘s
disease (PD). Although recent evidence has suggested that cell-to-cell transmission of -synuclein aggregates drives
the progression of PD, the mechanism by which -synuclein aggregates spread remains undefined. Here, we show that
-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates,
co-aggregation with endogenous -synuclein, and exocytosis of the co-aggregates. Moreover, we found that
glucocerebrosidase depletion, which has previously been strongly associated with PD and increased cognitive
impairment, promoted propagation of -synuclein aggregates. These studies define how -synuclein aggregates spread
among neuronal cells and explain how glucocerebrosidase mutations increase the risk of developing PD and other
synucleinopathies.
References
[1] Lee H-J, Bae E-J, Lee S-J. Extracelullar a-synuclein: A novel and crucial factor in Lewy body diseases. Nature
Rev. Neurol. (2014) , Published online 28 January 2014 doi:10.1038/nrneurol.2013.275
[2] Yang NY, Lee NY, Lee H-J, Kim YS, Lee S-J Glucocerebrosidase, a new player changing the old rules in Lewy
body diseases. Biol. Chem. (2013) 394, 807-818
[3] Lee S-J, Desplats P, Sigurdson C, Tsigelny I, Masliah E. Cell-Cell Transmission of Non-Prion Protein Aggregates,
Nature Review Neurology, (2010) 6, 702-706
55
Facile ―Stop Codon‖ Method for the Generation of Serine 87–, 129–, and 87/129–
Phosphorylated α–Synuclein: Isolation of Discrete Oligomers, Neuronal Toxicity
of S87p–α–Synuclein, and Oligomerization Inhibition with Vitamin B6
David G. Churchill,*,1 Yonghwang Ha,1,2 Ae–Rin Yang,1 Seyoung Lee,1 Kibong Kim,1 Hyunjeong Liew,3 Ju
Eun Lee,4 Hong–In Lee,4 Yoo–Hun Suh,*,3 Hee–Sung Park,*,1
1.
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373–1 Guseong–dong, Yuseong–
gu, Daejeon, 305–701, Republic of Korea. 2. Convergence Science & Technology Research Center (CSTRC), Jungwon
University, Goesan–gun, Chungbuk, 367–805, Republic of Korea. 3. Department of Pharmacology, College of Medicine, Seoul
National University, 28 Yeongeon–Dong, Jongno–Gu, Seoul, 110–799, Republic of Korea. 4. Department of Chemistry and
Green–Nano Materials Research Center, Kyungpook National University, Daegu 702–701, Republic of Korea.
Research into the etiology of neurodegenerative disease research continues unabated in the biological and
chemical fields with particular focus on certain proteins that compose disease–associated plaques/inclusions.
Herein, a stop codon “Sep” incorporation method [Park, H.–S. et al., Science, 2011,333, 1151.] has been
applied to a neurodegenerative disease–linked protein for the first time. Facile milligram–scale production of
discrete phosphorylated α–synuclein analogues: S87p–, S129p–, and S87/129p–α–synuclein, has been
verified with LC–MS/MS and western blotting. Oligomerization trends of phosphorylated α–synuclein
were similar with those for non–phosphorylated, in the presence of dopamine (DA) or DA/Cu2+. For
neuronal toxicity testing, phosphorylated α–synuclein oligomers showed higher toxicity than non–
phosphorylated α–synuclein (Non–P–α–syn), especially, for S87p–α–synuclein. In Non–P–α–syn, DA/Cu2+–
induced oligomers showed slightly higher toxicity than for DA–induced ones, especially over the monomer.
Pyridoxal 5‟–phosphate (PLP), the active form of Vitamin B6, showed very high inhibition effects on
oligomerization of non–phosphorylated and phosphorylated versions where aggregation was induced by DA
or DA/Cu2+. In oligomerization assays, DA or DA/Cu2+induction showed different trends. Anaerobically,
whereas DA induced minimal oligomerization of α–syn, DA/Cu2+ produced aerobic–level amounts. While
electrophoretic band positions of DA–induced oligomers were higher than those for untreated samples,
oligomers from DA/Cu2+ went to positions lower than those for DA–treated ones, especially in the cases for
protein dimers and trimers. Redox reactions were suspected more in multimers (NBT assay). Radical
species were detected through EPR, from co–incubation of α–Syn, DA and Cu2+. DA and Cu2+appear to
induce different mechanisms: cross–linking vs radical–mediated covalent modification.
KEYWORDS: α–synuclein; stop codon technique; serine phosphorylation; neurotoxicity; oligomerization;
pyridoxal 5‟–phosphate; copper; dopamine.
56
The problem of protein folding and its relationship to bioscience
Kunihiro Kuwajima
The Center for the Promotion for Integrated Sciences (CPIS), the Graduate University for Advanced Studies
(Sokendai), Shonan Village, Hayama, Kanagawa 240-0193, Japan
E-mail: kuwajima_kunihiro@soken.ac.jp
The problem of protein folding is an interdisciplinary problem between biological and physicochemical sciences, and
hence fundamental in biophysics. Anfinsen and his colleagues first raised the protein folding problem in the early
1960s. Since then a variety of experimental, theoretical and simulation studies on the protein folding problem have
been carried out for more than 50 years. In this talk, I would like to introduce these studies, mainly focusing on the
kinetic studies on protein folding reactions, in which I have been involved. Finally, I also would like to talk about
possible biological functions of non-folding states of proteins, because it is now increasingly evident that not only the
rigid native structures but also flexible structures like molten globules are biologically functional. I will talk about
molecular mechanisms of cytotoxicity of human -lactalbumin made lethal to tumor cells (HAMLET) and other
protein-oleic acid complexes.
References
[1] Kamagata, K., Arai, M. & Kuwajima, K. (2004). Unification of the folding mechanisms of non-two-state and twostate proteins. J. Mol. Biol. 339, 951-965.
[2] Kamagata, K. & Kuwajima, K. (2006). Surprisingly high correlation between early and late stages in non-twostate protein folding. J. Mol. Biol. 357, 1647-1654.
[3] Nakamura, T., Makabe, K., Tomoyori, K., Maki, K., Mukaiyama, A. & Kuwajima, K. (2010). Different folding
pathways taken by highly homologous proteins, goat -lactalbumin and canine milk lysozyme. J. Mol. Biol. 396,
1361-1378.
[4] Tomoyori, K., Nakamura, T., Makabe, K., Maki, K., Saeki, K. & Kuwajima, K. (2012). Sequential four-state
folding/unfolding of goat -lactalbumin and its N-terminal variants. Proteins 80, 2191-2206.
[5] Nakamura, T., Aizawa, T., Kariya, R., Okada, S., Demura, M., Kawano, K., Makabe, K. & Kuwajima, K. (2013).
Molecular mechanisms of the cytotoxicity of human -lactalbumin made lethal to tumor cells (HAMLET) and
other protein-oleic acid complexes. J. Biol. Chem. 288, 14408-14416.
57
Unfolding of a small protein proceeds via dry and wet globules and a solvated
transition state
Jayant B. Udgaonkar1, Saswata Sankar Sarkar2 and G. Krishnamoorthy2
1
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India
2
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India
Email: jayant@ncbs.res.in
Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that
maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when
such solvation occurs during unfolding. In this study, far-UV CD measurements suggest a simplistic two-state view of
the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. NearUV CD measurements show that unfolding commences with the loosening of tertiary interactions in a native-like
intermediate, N*. Fluorescence resonance energy transfer measurements show that N* then expands rapidly but
partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N* and IE
have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic
quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later
in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show
that tight packing around the core tryptophan is lost when IL forms. Importantly, the slowest step is unfolding of the
wet molten globule, and involves a solvated transition state.
References
[1] Sarkar, S.S., Udgaonkar, J.B. and Krishnamoorthy, G. Step-wise unfolding of a small protein proceeds via dry
and wet molten globule intermediates and a solvent-accessible transition state. Biophys. J. 105, (2013) 23922402.
58
Metal ion binding induced folding and allosteric motions of proteins
Wei Wang
National Laboratory of Solid State Microstructure and Department of Physics,
Nanjing University, Nanjing 210093, P. R. China
E-mail: wangwei@nju.edu.cn
Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors and can mediate the successful folding
and functioning of proteins. Binding of metal ions can reshape the energy landscapes of proteins, therefore modify the
folding and allosteric motions. However, how the local interactions involving the metal ion binding can induce the
global conformational motions of proteins remains elusive. Investigating such question requires multiple models with
different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we
have been developing such multiscale methods which can reasonably model the metal ion binding induced charge
transfer, protonation/deprotonation, and large conformational motions of proteins. We elucidated the zinc-binding
induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in
the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric
motions of calmodulin domain.
References
[1] Li W.F., Zhang J. Wang J. and Wang W. ―Metal-Coupled Folding of Cys2His2 Zinc Finger‖, J. Am. Chem. Soc.
130 (2008), pp 8920-900.
[2] W.F. Li, Zhang J., Su Y., Wang J., Qin M, Wang W. ―Effect of Zinc Binding on the Conformation Distribution of
the Amyloid-beta peptide based on Molecular Dynamics Simulation‖, J. Phys. Chem. B, 111 (2007) pp 1381413821.
[3] Li W.F., Terakawa T., Wang W. and Takada S. ―Energy Landscape and Multiroute Folding of Topologically
Complex Protein Adenylate Kinase and 2out-knot‖, Proc. Natl. Acad. Sci. USA 109 (2012), pp 17789-17794.
[4] Tan C., Li. W.F., Wang W. and Thirumalai D., ―Binding of Ca2+ and Allosteric Motion in Calmodulin‖,
unpublished.
59
The atomistic mechanism of conformational transition of adenylate kinase
investigated by Lorentzian structure-based potential
Juyong Lee, Keehyoung Joo, Jooyoung Lee∗,
School of Computational Sciences, Korea Institute for Advanced Study, Seoul, Korea
E-mail: jlee@kias.re.kr
For better understanding of conformational transitions of proteins, we have developed a new all-atom structure-based
approach by incorporating the structure-based potential term with the Lorentzian function. In this approach, native
contacts of a protein are stabilized by Lorentzian attractive terms instead of conventional harmonic terms. When
dealing with two alternative distance information, the smoothness and boundedness of the Lorentzian function enables
the generation of a differentiable double-well potential by an addition operation, which is simpler than existing mixing
schemes. To test the validity of this approach, we have investigated the conformational transition of Adenylate kinase
(AdK), which catalyzes the interconversion between ATP and ADP. We find that straight-forward molecular dynamics
simulations performed with the proposed approach reproduce experimental results more accurately than the
conventional harmonic model. In addition, our model allows us to observe numerous conformational transitions, over
1,000 times during 6 μs MD simulations, between open and closed states of AdK without introducing any biasing
forces. Such a large number of transitions enable us to perform quantitative analysis on the kinetics of conformational
transition of the enzyme in a statistically significant way. We find that the flexibility prediction of the native contact
based on the evolutionary information provide us accurate conformational ensemble of AdK. In this work, in addition
to the experimentally observed open and closed states, two transition states and one metastable state are observed and
their free energy differences are estimated. We propose that the motions of LID and NMP domains of AdK are based
on qualitatively different mechanisms.
60
Molecular Simulation and Experimental Study of
Transitions and Dissociation Events in a Dynamic Protein Complex.
Matthias Buck1, Susmita Borthakur1, SoonJeung Kim1, HyeongJu Lee1 and Liqun Zhang1
1
Department of Physiology and Biophysics, Case Western Reserve University,
School of Medicine, Cleveland, Ohio 44106, USA
E-mail: Matthias.buck@case.edu
It is now recognized that protein-protein interactions in solution are dynamic, especially if the binding affinities are
only moderately strong or weak. Dynamics originate in part from the population of alternative structures of the protein
complex, e.g. one bound state that is in equilibrium with one or several alternate configurations. Recently, through the
use of Anton and other computational platforms, it has become possible to carry out multi-microsecond molecular
dynamics simulations of such protein states. Here we present a protein complex that exhibits fluctuations on the
microsecond and submicrosecond timescale between different configurations: the EphA2: SHIP2 SAM: SAM
complex[1]. A number of techniques are used to shift the equilibrium and/or to demonstrate the population of alternate
complex configurations, including ion-pair swap mutagenesis and chemical shift perturbations in NMR spectra. The
origin of the alternate structures is revealed, particularly in the simulations, by the different patterns of sidechain
contacts that are possible in the complexes[2]. As expected some of the configurations of the protein complexes are
unstable and for the first time we show the protein dissociation process in all-atom unrestrained molecular dynamics
simulation. The protein separation is preceded by transitions to more weakly interacting protein surfaces – an
observation that informs on the reverse process: that of protein association. The possible functional significance of the
protein complex alternate states and their dynamics are discussed.
References
[1] Lee, HJ, Hota, P.K, Chugha, P., Miao, H., Zhang, L, Kim, SJ, Alviani, R.S, Stetzig, L., Wang, B. and Buck, M.
―NMR structure of a heterodimeric SAM: SAM complex: Characterization and manipulation of EphA2 binding
reveal new cellular functions of SHIP2‖. Structure Vol. 20 (2012), pp 41-55.
[2] Zhang, L., and Buck, M. "Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar
Interactions in Configurational Transitions" Biophysical. Journal Vol. 105 (2013), pp. 2412-2417
61
DESIGN AND SYNTHESIS OF SMALL MOLECULE INHIBITOR TARGETING THE
POLO-BOX-DOMAIN OF POLO-LIKE KINASE KINASE 1
Jeong Kyu Bang, Mija Ahn, Ga-hyang Lee
Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea
E-mail: bangjk@kbsi.re.kr
Over the years, a great deal of effort has been focused on the design and synthesis of potent linear peptide inhibitors
that target the polo-like kinase 1(Plk1) which is critically involved in multiple mitotic processes and has been
established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its
polo-box domain (PBD) and the inhibition of the Plk1 PBD has been considered as an approach to circumvent the
specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket.
Polo-box domain consists of two different binding regions such as unique broader pyrrolidine-binding pocket and the
conserved narrow Try-rich hydrophobic channel among the three Plk PBDs (Plks 1-3), respectively. Therefore, studies
that provide insights into the binding nature of the unique broader pyrrolidine-binding pocket might lead to the
development of selective Plk1-inhibitory compounds.
In an attempt to improve the selectivity and target the unique broader pyrrolidine-binding pocket, here for the first
time, a systematic approach was undertaken to examine the structure-activity relationship (SAR) of N-terminal
truncated PLHSpTM-derivatives, to apply site-directed ligand approach to restrict the conformation of the peptides
using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray
crystallographic studies1-2. We have identified new mode of binding interactions at the pyrrolidine-binding pocket with
improved binding affinity while retaining Plk1 PBD specificity. Furthermore, our data revealed that the pyrrolidinebinding pocket was very specific to recognize the short and bulky hydrophobic ligand like adamantane whereas the
Tyr-rich hydrophobic channel was turned out to be specific with lengthy and small hydrophobic groups.
Progress obtained using our site-directed ligands validate this approach to specifically direct the ligand into unique
pyrrolidine-binding region and extends its applicability of the strategy for discovering selective protein-protein
interaction inhibitors.
References
[1] Murugan., et al., ―Exploring the binding nature of pyrrolidine pocket dependent interactions in the polo-box
domain of polo-like kinase‖, PLoS One, Vol. 8, No. 11, (2013), e80043.
[2] Murugan., et al., ―Development of cyclic peptomer inhibitors targeting the polo-box domain of polo-like kinase 1‖,
Bioorganic. Med. Chem, Vol. 21, (2013), pp 2623-2634
62
Structural and mechanistic studies for the interaction of prolyl-tRNA
synthetase and halofuginone, the anti-cancer and -fibrotic compound
Jonghyeon Son1, Eun Hye Lee1 , Minyoung Park2,3, Jong Hyun Kim2,3, Junsoo Kim1, Sunghoon Kim2,3,
Young Ho Jeon4, Kwang Yeon Hwang1
1
Divison of Biotechnology, College of Life Sciences, Korea University, Seoul 136-701,
2
Medicinal Bioconvergence Research Center,
3
College of Pharmacy, Seoul National University, Seoul 151-742,4College of Pharmacy,
Korea University,Chungnam 136-713, Korea
E-mail: chahong@korea.ac.kr
Aminoacyl-tRNA synthetases recognize cognate amino acids and tRNAs from their noncognate counterparts and
catalyze the formation of aminoacyl-tRNAs. Halofuginone (HF), a coccidiostat used in veterinary medicine, exerts its
effects by acting as a high-affinity inhibitor of the enzyme glutamylprolyl- tRNA synthetase (EPRS). In order to
elucidate the precise molecular basis of this inhibition mechanism of human EPRS, the crystal structures of the prolyltRNA synthetase domain of human EPRS (hPRS) at 2.4 Å resolution (hPRSapo), of hPRS complexed with ATP and
the substrate proline at 2.3 Å resolution (hPRS-sub) and of hPRS complexed with HF at 2.62 Å resolution (hPRS-HF)
are presented. These structures show plainly that motif 1 functions as a cap in hPRS, which is loosely opened in hPRSapo, tightly closed in hPRSsub and incorrectly closed in hPRS-HF. In addition, the structural analyses are consistent
with more effective binding of hPRS to HF with ATP. Mutagenesis and biochemical analysis confirmed the key roles
of two residues, Phe1097 and Arg1152, in the HF inhibition mechanism. These structures will lead to the development
of more potent and selective hPRS inhibitors for promoting inflammatory resolution.
References
[1] Zhou, H., Sun, L., Yang, X.-L. and Schimmel, P. Nature, 494, (2013). 121–124.
[2] Kim, S., You, S. and Hwang, D. Nature Rev. Cancer, 11, (2011). 708–718.
[3] Keller, T. L., Zocco, D., Sundrud, M. S., Hendrick, M., Edenius, M., Yum, J., Kim, Y.-J., Lee, H.-K., Cortese, J.
F., Wirth, D. F., Dignam, J. D., Rao, A., Yeo, C.-Y., Mazitschek, R. and Whitman, M. Nature Chem. Biol. 8,
(2012). 311–317.
[4] Sundrud, M. S., Koralov, S. B., Feuerer, M., Calado, D. P., Kozhaya, A. E., Rhule-Smith, A., Lefebvre, R. E.,
Unutmaz, D., Mazitschek, R.,Waldner, H.,Whitman, M., Keller, T. and Rao, A. Science, 324, (2009). 1334–1338.
63
A multi-dimensional interactions between tRNA synthetases and cofactor:
Implications for translation and signaling
Yunje Cho
Department of Life Sciences, Pohang University of Life Science and Technology
Aminoacyl-tRNA synthethases (ARSs) catalyze the attachment of amino acids to their cognate tRNAs, the essential
step for the protein synthesis in all life. In higher eukyaryotes, ARSs have additional motifs or domains to their
catalytic domains and organized with auxiliary proteins to form a multisynthetase complex (MSC). MSC is composed
of nine ARSs and three accessory proteins (AIMPs) and can be grouped into three sub-complexes. One complex is
formed with RRS, QRS, and AIMP1/p43. Another complex is comprised of MRS, IRS, LRS, EPRS, DRS, and AIMP3.
The MSC is believed to facilitate the protein synthesis through channeling mechanism, to enhance ARS activity by
recruiting non-specific tRNA-binding domains, and to regulate the balance between translation and non-canonical
functions. Here, I will discuss the potential models of the MSC, and the interactions between components of the MSC
through integrative approaches.
64
Pro-metastatic interaction between the two translational components and its
therapeutic potential
Young Ho Jeon1, Hye Young Cho1, Ameeq Ul Mushtaq1, Jin Young Lee2, Dae Gyu Kim2, Sunghoon Kim2
1
2
College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong 339-700, Korea
Medicinal Bioconvergence Research Center, Seoul National University, Seoul 151-742, Republic of Korea.
E-mail: yhjeon@korea.ac.kr
Lysyl-tRNA synthetase (KRS) is the ARS ligating the lysine to tRNALys. Although human lysyl-tRNA synthetase
(KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological
implications have not been understood. KRS enhances the cell migration in membrane through the binding with 67kDa
laminin receptor (67LR), dimerized form of p40/37LRP which is one of the ribosomal components. 67LR was well
reported as a critical factor in cell migration and cancer metastasis. Here we present the structural study and the
binding analyses of KRS with the selected inhibitors of KRS-laminin receptor interaction. N-terminal 72 residues of
human KRS shows mostly unstructured except short helical structures between S19 and T52. Residues from A51 to
N57 shows interaction with a selected inhibitor BC-K1, which implies that T52 phosphorylation dependent interaction
change of this region may be affected by this compound. Another interaction site of KRS to BC-K1 is located on the
anticodon binding domain, which is identified to interact with the laminin receptor. Restrained docking study with
NMR-derived experimental data elucidates the binding mode of BC-K1 into the pocket of KRS anticodon binding
domain. These results suggest a mechanism of action to prevent cancer metastasis by modulating the interaction of
KRS with MSC and laminin receptor in cellular membrane.
References
[1] Kim D.G., et al., ―Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction‖,
Nature Chemical Biology, Vol. 10, No. 1, (2014), pp 29-34.
[2] Kim D.G., et al., ―Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma
membrane promotes laminin-dependent cell migration‖, FASEB Journal, Vol. 26, No. 10, (2014), pp 41424159.
65
Symposium 2A: Protein catabolism
Structural basis of autophagy initiation by starvation
Yuko Fujioka1, Sho W. Suzuki2, Hayashi Yamamoto2, Chika Kondo-Kakuta2, Yayoi Kimura3, Hisashi Hirano3,
Rinji Akada4, Fuyuhiko Inagaki5,6, Yoshinori Ohsumi2 and Nobuo N. Noda1,6
1
Institute of Microbial Chemistry (BIKAKEN), Tokyo, Tokyo 141-0021, Japan.
Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503, Japan.
3
Graduate School of Medical Life Science and Advanced Medical Research Center,
Yokohama City University, Yokohama 230-0045, Japan.
4
Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan.
5
Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
6
CREST, Japan Science and Technology Agency, Tokyo 102-0076, Japan.
2
E-mail: nn@bikaken.or.jp
Autophagy is an intracellular degradation system and contributes to the maintenance of cell homeostasis through
degradation of harmful materials such as aggregated proteins and damaged organelles. During autophagy, a double
membrane-bound structure called an autophagosome is generated, which sequesters degradation targets and delivers
them to the lytic compartment, lysosome or vacuole, for degradation. Starvation strongly induces autophagy, and 18
Atg proteins have been shown to mediate autophagosome formation upon starvation in yeast. Most of them are
localized to the pre-autophagosomal structure (PAS), from which autophagosomes are generated. PAS assembly
requires the formation of the Atg1-Atg13-Atg17-Atg29-Atg31 complex (Atg1 complex), for which starvationinduced dephosphorylation of Atg13 is required. However, the molecular details underlying these events have not
been established. Here we studied the interactions of Atg13 with Atg1 and Atg17 by X-ray crystallography. Atg13
binds to tandem microtubule interacting and transport domains in Atg1 using an elongated helix-loop-helix region.
Atg13 also binds to the N-terminal acidic pocket in Atg17 using a short basic region, thereby constructing the Atg1
complex. Biochemical and cell biological studies revealed that these interactions are accomplished only when
specific serine residues of Atg13 are dephosphorylated, which explains why starvation initiates the PAS assembly
and thus autophagy.
66
Targeting Autophagy for Mitochondrial Clearance
Koji Okamoto1
1
Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences,
Osaka University, Suita 565-0871, Japan.
E-mail: kokamoto@fbs.osaka-u.ac.jp
Recent numerous studies demonstrate that mitophagy, an autophagy-dependent mitochondria-specific degradation
pathway conserved from yeast to humans, serves to control the quality and quantity of mitochondria [1]. However,
the molecular details underlying this selective clearance process are poorly understood. We have previously
established that mitophagy in the yeast Saccharomyces cerevisiae requires Atg32, a transmembrane protein highly
expressed, likely in response to oxidative stress, and anchored on the surface of mitochondria [2-4]. Notably, Atg32
interacts with Atg8, a phosphatidylethanolamine (PE)-conjugated ubiquitin-like protein localized to autophagosomes.
Mitophagy is partially impaired when the Atg8-Atg32 interaction is reduced, suggesting that Atg8 contributes to
degradation of mitochondria via Atg32 [5].
Recently, we found that mitophagy is strongly impaired in cells lacking Opi3, a phospholipid methyltransferase acting
in conversion of PE to phosphatidylcholine (PC). In this mutant, starvation-induced bulk autophagy was only slightly
affected. In addition, mitochondria are degraded normally in cells lacking Cho2, a PE methyltransferase that catalyzes
the first step in conversion of PE to PC and generates phosphatidylmonomethylethanolamine (PME), a substrate for
Opi3, indicating that PE to PC biosynthesis itself is not important for mitophagy. Using phospholipid composition
assays, we established that PME is accumulated in cells lacking Opi3 under mitophagy conditions. Strikingly, the
levels of lipidated Atg8 were drastically increased in the absence of Opi3. Our mass spectrometry revealed that Atg8 is
conjugated to PME in the opi3 mutant. In vitro, Atg8 can be conjugated to PME, while Atg8-PME is not efficiently
delipidated by Atg4, a cysteine protease essential for all autophagy-related pathways. Finally, we found that Atg32
induction is strongly suppressed in cells lacking Opi3. Taken together, our data suggest that the regulation of Atg8
lipidation and recycling is critical for mitophagy, and that the induction of Atg32 is linked to phospholipid metabolism.
References
[1] Kondo-Okamoto N. and Okamoto K. (2012). Mitochondria and autophagy: critical interplay between the two
homeostats. Biochim. Biophys. Acta 1820, 595–600.
[2] Okamoto K., Kondo-Okamoto N. and Ohsumi Y. (2009). Mitochondria-anchored receptor Atg32 mediates
degradation of mitochondria via selective autophagy. Dev. Cell 17, 87–97.
[3] Okamoto K., Kondo-Okamoto N. and Ohsumi Y. (2009). A landmark protein essential for mitophagy. Autophagy,
5: 1203-1205.
[4] Eiyama A., Kondo-Okamoto N. and Okamoto K. (2013). Mitochondrial degradation during starvation is selective
and temporally distinct from bulk autophagy in yeast. FEBS Lett. 587, 1787-1792.
[5] Kondo-Okamoto N., Noda N.N., Suzuki S.W., Nakatogawa H., Takahashi I., Matsunami M., Hashimoto A.,
Inagaki F., Ohsumi Y. and Okamoto, K. (2012). Autophagy-related protein 32 acts as autophagic degron and
directly initiates mitophagy. J. Biol. Chem. 287, 10631–10638.
67
Regulation of lipidation of the ubiquitin-like protein Atg8 that drives
autophagosome formation
Machiko Sakoh-Nakatogawa1, Kazuaki Matoba2, Nobuo N. Noda2, Fuyuhiko Inagaki3,
Hitoshi Nakatogawa1 and Yoshinori Ohsumi1
1
Frontier Research Center, Tokyo Institute of Technology, Yokohama 226-8503, Japan.
2
Institute of Microbial Chemistry, Tokyo 141-0021, Japan.
3
Department of Structual Biology, Hokkaido University, Sapporo 001-0021, Japan.
E-mail: mnakatogawa@iri.titech.ac.jp
Autophagy is a bulk degradation system whereby cytoplasmic proteins and organelles are sequestered by doublemembrane vesicles called ―autophagosomes‖, and transported to the vacuole/lysosome for degradation. De novo
formation of the autophagosome requires two ubiquitin-like proteins, Atg8 and Atg12. Similar to protein
ubiquitination, via a series of enzymatic reactions with E1 and E2 enzyme, they are conjugated to the lipid molecule
phosphatidylethanolamine (PE) and Atg5, respectively. Intriguingly, the Atg12-Atg5 conjugate binds directly to Atg3,
which is an E2 enzyme for the Atg8-PE conjugation reaction, and facilitates the transfer of Atg8 from Atg3 to PE.
Thus, Atg12-Atg5 serves as an E3 enzyme in the reaction. However, the primary sequence and structure of Atg12Atg5 show no similarity to typical E3 enzymes.
Here, we show the mechanism of Atg3 activation by Atg12-Atg5 using Saccharomyces cerevisiae proteins. We
established a biochemical assay based on the structural information to determine the configuration of the catalytic
center of Atg3. This approach revealed that Atg12-Atg5 induces a conformational change in the catalytic center of
Atg3 to enhance its E2 activity. Moreover, mutational analyses indicated how the activity of Atg3 is suppressed in the
absence of Atg12-Atg5. We also obtained an Atg3 mutant, whose activity is increased even in the absence of Atg12Atg5. In cells expressing this mutant, Atg8-PE was produced without Atg12 or Atg5. However, in these cells, neither
Atg3 nor Atg8 localized to the pre-autophagosomal structure (PAS), and autophagy was impaired. These results
suggest that Atg12-Atg5 is important for the activation of Atg3 at the PAS to produce Atg8-PE on autophagy-related
membranes, which drives the formation of those membranes.1
1. Sakoh-Nakatogawa et al. Nat. Struct. Mol. Biol. 20: 433-439, 2013.
68
Autophagy of peroxisomes in plant cells
Jimi Kim1, Heeeun Lee1, Han Nim Lee1, Kwang Deok Shin1, and Taijoon Chung1
1
Department of Biological Sciences, Pusan National University, Busan 609-735, Korea
E-mail: taijoon@pusan.ac.kr
Plant peroxisomes have enzymes for fatty acid beta-oxidation and anti-oxidant system. Peroxisomes in germinating
oilseeds also contain enzymes for the glyoxylate cycle, by which acetyl-CoA is utilized for the biosynthesis of organic
acids and subsequently sugars via gluconeogenesis. When seedlings are exposed to light, chloroplasts are fully
developed and perform photosynthesis. Another important aspect of cell remodeling by light involves peroxisomes, in
which obsolete glyoxylate cycle enzymes are degraded and newly synthesized photorespiratory enzymes are imported.
Three possible mechanisms have been proposed for the degradation of the glyoxylate cycle enzymes: i) degradation by
resident matrix proteases; ii) retranslocation and ubiquitylation of the enzymes followed by proteasome-mediated
degradation; and iii) autophagic delivery of the peroxisomes to the vacuole for degradation. Autophagy is a conserved
pathway by which a variety of cytoplasmic materials are targeted to a lytic compartment. We recently presented
genetic and cell biological data supporting the autophagy of peroxisomes in model plant species, Arabidopsis thaliana
[1]. When compared to wild type, autophagy-deficient mutants showed over-accumulation of peroxisomes and the
glyoxylate cycle enzymes in hypocotyl cells during seedling growth. Degrading peroxisomes were occasionally
detected in the vacuole of wild-type but not of autophagy-deficient cells [1]. Pexophagy, or selective autophagy of
peroxisomes in plant cells was also demonstrated by other research groups [2-5]. Genetically tractable Arabidopsis can
provide a useful model for studying pexophagy in higher eukaryotes.
References
[1] Kim J., Lee H., Lee H.N., Kim S., Shin K.D., and Chung T. ―Autophagy-related proteins are required for
degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth‖, Plant Cell, Vol. 25, No. 12,
(2013), pp 4956–4966.
[2] Farmer L.M., Rinaldi M.A., Young P.G., Danan C.H., Burkhart S.E. and Bartel B. ―Disrupting autophagy restores
peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal
matrix protein degradation‖, Plant Cell, Vol. 25, No. 10, (2013), pp 4085–4100.
[3] Shibata M., Oikawa K., Yoshimoto K., Kondo M., Mano S., Yamada K., Hayashi M., Sakamoto W., Ohsumi Y.,
and Nishimura M. ―Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis‖, Plant
Cell, Vol. 25, No. 12, (2013), 4967–4983.
[4] Yoshimoto K, Shibata M., Kondo M., Oikawa K., Sato M., Toyooka K., Shirasu K., Nishimura M., and Ohsumi
Y. ―Organ-specific quality control of plant peroxisomes is mediated by autophagy‖, J. Cell Sci., Vol. 127, (2014),
pp 1161–1168.
[5] Goto-Yamada S., Mano S., Nakamori C., Kondo M, Yamawaki R., Kato A., and Nishimura M. ―Chaperone and
protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy‖, Plant
Cell Physiol., Vol. 55, No. 3, (2014), pp 482–496.
69
Structural basis of autophagosome maturation
by swapping interaction partners of Atg5
Jun Hoe Kim and Hyun Kyu Song
Division of Life Sciences, Korea University, Seoul 136-701, South Korea.
E-mail: hksong@korea.ac.kr
Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a
conserved mechanism in all eukaryotes. When autophagy is induced, cellular components are surrounded by an
autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the Atg12~Atg5
conjugate requires two different binding partners, Atg16L1 for autophagosome elongation and TECPR1 for lysosomal
fusion. In our current study, we describe the crystal structures of human Atg5 in complex with an N-terminal domain
of Atg16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar -helical structure
containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of C-terminal
unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell
biological analyses. These results provide new insights into the molecular details of the autophagosome maturation
process, from its elongation to its fusion with a lysosome.
70
Drug Discovery Collaboratory: CNDY and RASCH
Senyon Choe
Director, Joint Center for Biosciences, Incheon, Korea
Professor, Structural Biology Laboratory, Salk Institute, CA 92037, USA
www.salk.edu/faculty/choe.html
Drug Discovery Collaboratory (DDColl) is an international network to collaborate and to advocate open development
of biological technology. As part of its efforts, we have employed two platform technologies, CNDY and RASCH, to
speed up identifying potential drug leads.
1) We have devised CNDY (Cell-free, NMR spectroscopy, Molecular Dynamics) strategy to search for small molecule
leads, we. Nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs). A
major hurdle to study hIMPs, however, lies in methods of protein production for biochemical and structural
characterization. By a combination of Cell-Free (CF) expression and Combinatorial Dual isotope-Labeling (CDL)
strategy to facilitate high-speed structure determination of hIMPs by solution NMR spectroscopy (Klammt et al., 2012,
Nature Methods), the CNDY strategy can offer a significant discovery toolset specially when combined with
computational methods (Lindert et al., 2014).
2) We devised a RASCH (Random Assembly of Segmental Chimera and Heteromers) strategy to search for biologics.
The underlying hypothesis is that natural ligands can be systematically varied in their amino acid sequences. We
have applied RASCH to TGF-beta superfamily ligands. For this purpose, we used human BMP-2 and Activin A as two
parental sequences. Their sequences were divided into six segments, swapped and reassembled to create the AB2
library. One such AB2 chimera, AB204, shows remarkable osteogenic property that far exceeds the healing power of
BMP-2 (Yoon et al., 2014, J. Bone and Mineral Research). RASCH strategy will dramatically expand therapeutic
values and potentials of many natural signaling ligands.
71
Ribosomal proteins play important roles for
the determination of cell fate under stress conditions
Joon Kim
Lab of Biochemsitry, Division of Life Sciences Korea University, Seoul 136-701, Korea
Gcn4p from Saccharomyces cerevisiae is a very well-characterized transcriptional activator which has been known to
activate more than 500 genes under various stress conditions.
Previously, our results showed that the transcriptions of ribosomal protein (RP) genes were decreased under stress
conditions. This showed a novel model for a repressive role of yeast transcription factor Gcn4 protein. This also
revealed that Gcn4p plays simultaneous dual roles as a repressor for RP genes as well as an activator for other
biosynthetic genes in stress conditions. We also showed that a reduction in the gene transcription of RP during the
hyphae formation in a human fungal pathogen Candida albicans in several stress conditions. We also confirmed that
some stress conditions induce post-translational modifications of a ribosomal protein to cope with the stress. Our
current study also reveals a reduction in gene transcription of RP during the hyphae formation in a human fungal
pathogen Candida albicans. When it switches its morphology from yeast to hyphal form, the levels of polysomes and
monosomes were decreased by hyphal stimuli. This morphological transition from yeast to hyphal form renders C.
albicans virulent. Tor1 kinase appear to show a novel link between ribosome biogenesis and morphogenesis in C.
albicans .
Our lab also identified that human ribosomal proteins play critical roles not only in protein synthesis but also in DNA
repair, apoptosis and metastasis inhibition. Among them, we demonstrated that rpS3 is directly correlated with its
DNA repair endonuclease activity mediated by a kinase, PKCδ and a phosphatase, PP2A in the nucleus or by
migration of rpS3 protein into mitochondria for DNA repair. Moreover, we will present novel data that ribosomes are
involved in the crosstalks between cell survival and apoptosis via various kinds of post-translational modifications. In
conclusion, different post-translational modifications of RPs in eukaryotes contribute for the determination of the fate
of the cell not only in translation but also in DNA repair, apoptosis and cell cycle regulation.
72
Structure of the activated full-length PmrA response regulator in complex with
DNA from Klebsiella pneumoniae
Chinpan Chen, Yuan-Chao Lou and Yi-Fen Kao
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
E-mail: bmchinp@ibms.sinica.edu.tw
Gram-negative bacteria can avoid detection by host immune systems and resist being killed by antibiotics or
antimicrobial peptides by modifying lipopolysaccharide in their outer membrane. The PmrA/PmrB two-component
system (TCS) is the major regulator of genes for lipopolysaccharide modification in bacteria. A classical TCS typically
consists of a transmembrane sensor histidine kinase and a cytoplasmic response regulator (RR). After perceiving
external stimuli by the sensor histidine kinase, a phosphoryl group is transferred to the conserved Asp residue on its
cognate RR protein to elicit adaptive responses. The C-terminal effector domain of PmrA (PmrAC) recognizes tandem
imperfect repeat sequences on the promoters of genes to activate transcription of genes for lipopolysaccharide
modification after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). In Klebsiella
pneumoniae, the small basic connector protein PmrD can protect phospho-PmrA from dephosphorylation and prolongs
the expression of PmrA-activated genes. We have previously published complex structures of PmrAN-PmrD and
PmrAC-DNA [1-3]. To further gain insights, we apply X-ray and NMR methods to solve the structure of BeF3-activated full-length PmrA in complex with DNA, and the molecular basis for the formation of this complex that
precedes the assembly of RNA polymerase is discussed.
References
[1] Luo S.C., Lou Y.C., Cheng H.Y., Pan Y.R., Peng H.L. and Chen C. ―Solution structure and phospho-PmrA
recognition mode of PmrD from Klebsiella pneumoniae‖. Journal of Structural Biology, 172 (2010), pp 319-330.
[2] Luo S.C, Lou, Y.C., Rajasekaran M., Chang Y.W., Hsiao, C.D. and Chen, C. ―Structural basis of a physical
blockage mechanism for the interaction of response regulator PmrA with connector protein PmrD from Klebsiella
pneumoniae‖. J. Biol. Chem. 288 (2013), pp 25551-25561.
[3] Lou Y.C., Wang I., Rajasekaran M., Kao Y.F., Ho M.R., Hsu S.T.D., Chou S.H., Wu S.H. and Chen, C. ―Solution
structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae‖.
Nucleic Acids Research, (2014), DOI: 10.1093/nar/gkt1345.
73
Biochemical dissection of bacterial virulence and macrophage
Innate immunity
Feng Shao
National Institute of Biological Sciences, Beijing 102206, China.
E-mail: shaofeng@nibs.ac.cn
My laboratory studies molecular mechanisms of bacterial infection and host innate immune defense. Using pathogens
such as Shigella, Salmonella, enteropathogenic E. coli (EPEC), Legionella and Burkholderia as the model, we have
discovered several novel posttranslational modifications utilized by these pathogens to paralyze host innate immunity.
Specifically, the OspF effector from Shigella flexneri employs a MAPK phosphothreonine lyase activity to block host
MAPK signaling and IL-8 production. The CHBP/Cif family of effectors from Burkholderia pseudomallei and EPEC
catalyze deamidation of Gln-40 in ubiquitin and ubiquitin-like NEDD8, thereby inactivating host ubiquitin pathway
and related cellular processes. NleE from EPEC and Salmonella typhimurium carries out a novel cysteine methylation
modification on key ubiquitin-chain sensory proteins in host NF-κB pathway, blocking NF-κB-mediated inflammatory
responses during infection. NleB from EPEC instead catalyzes GlcNAc modification on an arginine residue in host
death domain proteins such FADD, TRADD and RIPK1 and thereby blocks multiple death receptor signaling in host
cells. On the host side, we are also interested in the role of the inflammasome pathway in macrophage immune defense
against bacterial infections. Macrophage senses many kinds of pathogen-derived molecular patterns and activates the
cytoplasmic inflammasome complex, leading to Il-1β production and pyroptosis. We are combining biochemical
reconstitution, cell biology and bacterial/mouse genetics to identify new sensors and components involved in
inflammasome signaling. I will also discuss our recent identification and characterization of the NAIP family of NODlike proteins (NLRs) that serves as inflammasome receptors for bacterial flagellin and also the type III secretion
apparatus, which plays an important role in restricting various bacterial infections.
74
Visualization of Distinct Substrate Recruitment Pathways in the Yeast
Exosome by Electron Microscopy
Jun-Jie Liu1,2, Matthew A. Bratkowski3, Xueqi Liu4, Chu-Ya Niu1, Ailong Ke3 & Hong-Wei Wang1
1
Ministry of Education Key Laboratory of Protein Science, Tsinghua Peking Joint Center For Life Sciences, Center
For Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
2
Joint Graduate Program of Peking-Tsinghua-NBIS, Tsinghua University, Beijing 100084, China;
3
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA;
4
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine,
New Haven, CT 06520, USA.
E-mail: hongweiwang@tsinghua.edu.cn
The eukaryotic exosome, responsible for the 3‘ end processing and degradation of RNA, is a multi-subunit complex
typically composed of a catalytically inactive core and the Rrp44 protein, which contains both 3‘ to 5‘ exo- and endoribonuclease activities. RNA substrates have been shown to be recruited through the core to reach Rrp44‘s
exoribonuclease (EXO) site. We provide biochemical evidence that an additional substrate recruitment pathway exists,
where the RNA reaches Rrp44‘s EXO site directly. Using single particle electron microscopy analysis, we provide
visual evidence for the two distinct RNA recruitment pathways. In the through-exosome route, channeling the single
stranded substrates from core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative
direct-access route, this conformational change does not take place and the RNA substrate is directly visualized
entering the Rrp44‘s EXO site directly. Our results provide mechanistic explanation for several RNA processing
scenarios by the eukaryotic exosome and indicate substrate specific modes of degradation by this complex.
75
Dynamics of nuclear proteins analyzed by relaxation NMR spectroscopy
Kenji Sugase1, Erisa Harada1 and Tsuyoshi Konuma1
1
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Osaka 618-8503, Osaka.
E-mail: sugase@sunbor.or.jp
Comprehensive structural predictions of proteins have shown that many nuclear proteins have long intrinsically
disordered regions or completely disordered (IDR/IDP). IDR/IDP is lacking stable tertiary structure when they are free
in solution, but they fold upon binding to their target molecules, such as proteins and nucleic acids. This phenomenon
is so-called coupled folding and binding. IDR is enriched especially in proteins related to cell cycle regulation, cell
signaling, and transcription. These biological processes are often associated with posttranslational modifications of
IDP, which can change the preference or binding affinity of IDP against target molecules. Some IDPs can
promiscuously bind to multiple targets with different conformations. For example, the C-terminal region of p53 forms
α-helix and β-strand upon binding to S100ββ and sirtuin, respectively, whereas it is coil structures in complex with
CBP and cyclin A2 [1]. By virtue of such conformational flexibility and adaptability, many IDPs function as hub
proteins in protein-protein interaction networks. Since the classical lock and key model cannot explain the binding
modes of IDPs, it is important to characterize conformational dynamics of IDPs to understand their functions.
We have been developing NMR methods to characterize conformational dynamics of proteins and applied them to
various proteins including IDPs. For the method development, we combined ligand titration experiments, relaxation
NMR spectroscopy (relaxation dispersion spectroscopy and CLEANEX-PM), and computational analyses [2-4].
Relaxation dispersion spectroscopy provides kinetics of proteins on the millisecond time scale and chemical shift
differences. It can probe low-populated minor states, which are invisible to most of biophysical methods. CLEANEXPM provides water-amide exchange rates on the millisecond to second time scales. During ligand titrations, the
fractional population of each state and binding kinetics change according to the concentration ratios between the
protein and ligand, and thus the parameters obtained by aforementioned NMR methods also changes. Some
biophysical parameters regarding ligand-protein interactions, such as kon and koff, are derived by analyzing the
concentration-dependent NMR parameters using the homemade program GLOVE [5]. Using the methods, we revealed
that there are different types of binding modes of IDPs. For example, the pKID domain of CREB folds after binding,
whereas the HMG domain of Sox2 is partially folded in the free state.
References
[1] Oldfield C.J., Meng J., Yang J.Y., Yang M.Q., Uversky V.N., Dunker A.K., ―Flexible nets: disorder and induced
fit in the associations of p53 and 14-3-3 with their partners‖, BMC Genomics. Vol. 9, Suppl. 1, (2008), S1.
[2] Sugase K., Dyson H.J. and Wright P.E., ―Mechanism of coupled folding and binding of an intrinsically disordered
protein‖, Nature, Vol. 447, No. 7147, (2007), pp 1021-1025.
[3] Sugase K., Lansing J.C., Dyson H.J. and Wright P.E., ―Tailoring relaxation dispersion experiments for fastassociating protein complexes‖, J. Am. Chem. Soc. Vol. 129, No. 44, (2007), pp 13406-13407.
[4] Sugase K., ―Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal
relaxation NMR spectroscopy‖ J. Biomol. NMR, Vol 56, No.3, (2011), pp 219-227.
[5] Sugase K., Konuma T., Lansing J.C. and Wright P.E., ―Fast and accurate fitting of relaxation dispersion data
using the flexible software package GLOVE‖, J. Biomol. NMR, Vol. 56, No. 3, (2013), pp 275-283.
76
Personalized diagnosis of cancers at the protein-protein interaction
level with real-time single-molecule co-IP analysis
Tae-Young Yoon1,2,3
1
National Creative Research Initiative Center for Single-Molecule Systems Biology,
2
Samsung Science and Technology Foundation and 3 Department of Physics,
KAIST, Daejeon 305-701, Korea
E-mail: tyyoon@kaist.ac.kr
The large-scale genome sequencing for individual cancer patients provides an unprecedented opportunity
to reveal molecular lesions in specific cancers. Owing to formidable complexity of the cell signaling
network, however, the genome sequencing data cannot be directly translated into real defects at the
protein-protein interaction level. We have recently demonstrated the real-time single-molecule coimmunoprecipitation analysis [1,2]. In this approach, the target cell-signaling proteins are pulled down
from a tumor tissue extract, onto the imaging plane of the total internal reflection microscope. After
thorough washing, one type of downstream proteins, tagged with fluorescent proteins, are introduced for
reaction. When the surface-immobilized proteins are hyperactivated, they show active binding events
with the downstream proteins, forming an increased number of diffraction-limited spots during real-time
imaging. Quantitative analysis of these fluorescence spikes reveals the signaling kinetics of cancerderived proteins with the single-molecule resolution. Our approach allows us to probe the previously
hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards
precision molecular diagnostics at the protein–protein interaction level.
References
[1] Hong-Won Lee et al., ―Real-time single-molecule co-immunoprecipitation analyses reveal cancerspecific Ras signaling dynamics‖, Nature Communications, Vol. 4, (2013), p 1505.
[2] Hong-Won Lee et al., ―Real-time single-molecule co-immunoprecipitation weak protein-protein
interactions‖, Nature Protocols, Vol. 8, (2013), p 2405.
77
Thermodynamic Tools in the Early Stages of Drug Discovery.
Jose M. M. Caaveiro1, Akihiro Kobe1, Shinya Tashiro1, Daisuke Kajihara2,
Masato Kikkawa2, Tomoya Mitani2 and Kouhei Tsumoto1,3
1
Department of Bioengineering, Graduate School of Engineering,
The University of Tokyo, Tokyo, 113-8656, Japan
2
Life Science Division, GE Healthcare Japan, Tokyo 169-0073, Japan
3
Laboratory of Medical Proteomics, Institute of Medical Science,
The University of Tokyo, Tokyo 169-0073, Japan
E-mail: jose@bioeng.t.u-tokyo.ac.jp
There is an increasing need to develop more precise methods in the process of drug discovery. In this regard,
fragment-based drug discovery (FBDD) has enjoyed increasing popularity as screening method in the early stages of
drug discovery. The small size of the fragments used in FBDD requires not only more sensitive instruments, but also
novel approaches to extract quality data using fewer resources. In this presentation I will introduce SITE (singleinjection thermal extinction), a thermodynamic methodology that selects high-quality hits early in FBDD [1]. SITE is
a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration
calorimetry but using significantly less time and materials. In this presentation I will describe the principles and
advantages of this methodology with practical examples.
References
[1] Kobe A., Caaveiro, J. M. M., Tashiro, S., Kajihara, D., Kikkawa, M., Mitani, T., and Tsumoto, K. ―Incorporation of
Rapid Thermodynamic Data in Fragment-Based Drug Discovery‖, Journal of Medicinal Chemistry, Vol. 56,
(2013), pp 2155-2159.
78
Protein-guided RNA dynamics during early ribosome assembly
Hajin Kim1, Sanjaya C. Abeysirigunawarden2, Ke Chen3,4, Megan Mayerle2,
Kaushik Ragunathan3, Zaida Luthey-Schulten3,4, Taekjip Ha3,5 and Sarah A. Woodson2
1
Department of Biomedical Engineering/Department of Physics,
Ulsan National Institute of Science and Technology, Ulsan, Korea
2
T. C. Jenkins Department of Biophysics, Johns Hopkins University,
3400 N. Charles Street, Baltimore, Maryland 21218, USA
3
Center for Biophysics and Computational Biology,
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
4
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
5
Howard Hughes Medical Institute, Urbana, Illinois 61801, USA
E-mail: hajinkim@unist.ac.kr
The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early
binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood.
Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters
between Escherichia coli ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly.
Dynamic initial S4–RNA complexes pass through a stable non-native intermediate before converting to the native
complex, showing that non-native structures can offer a low free-energy path to protein–RNA recognition. Threecolour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix
motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided
dynamics offer an alternative explanation for induced fit in RNA–protein complexes.
79
An integrative proteogenomics approach in gastric cancer
Daehee Hwang1, Harkyun Kim2, Sanghyuk Lee3, Sang-Won Lee4, Eunok Peak5, and Eunkyung Yang6
1
Department of New Biology, DGIST, Republic of Korea; 2National Cancer Center, Republic of Korea, 3Department of
Life Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea; 4Department of Chemistry, Korea
University, Republic of Korea; 5Department of Computer Science, Hanyang University, Republic of Korea; and
5
Korean Institute of Science and Technology, Republic of Korea.
Agreement between genomic and proteomic molecular signatures can be used to identify core disease-related
signatures. Recently, proteogenomics analyzing proteomic and genomic data for the same biological samples has
emerged to identify these core molecular signatures related to diseases. In this study, we performed mRNA- and
exome-sequencing and also global protein and phosphorylation profiling of the tissues collected from 50 gastric
cancer tissues and matched normal tissues. By integrating proteomic and genomic datasets, we analyzed agreements
between two datasets, identified subgroups of disease samples based on two datasets, and selected cellular pathways
represented by both proteomic and genomic datasets as core pathways of gastric cancer. Our results demonstrate the
power of analyzing collectively multiple global datasets in selecting the core disease-related pathways. Currently, the
clinical implications of the identified core gastric cancer pathways are being verified using independent samples by
multiple reaction monitoring technics.
80
How to achieve the goal of quantitative proteomics: 1000 Immino-MRM Assay
Program
1
Myeong-Hee Yu1, Cheolju Lee1 and Jin Young Kim2
Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Korea; 2Division of Mass
Spectrometry Research, Korea Basic Science Institute, Ochang 363-883, Korea
E-mail: mhyu@kist.re.kr
Among various approaches to find disease-related proteins, proteomics has been considered a revolutionary technology,
especially for discovering phenotype-based biomarker candidates for diagnostic tests and drug treatments. Despite
tremendous developments achieved recently in proteome separation and mass spectrometry, the current technology of
analyzing the whole proteome in a quantitative manner is very challenging. The MRM-MS technology has recently
been adapted to peptides (proteotypic peptides - protein surrogates) and being developed and applied for targeted
protein analysis. We have recently joined 1000 Immino-MRM Assay Program which is coordinated internationally
under the auspices of the National Cancer Institute at the National Institutes of Health of the U. S. A. The overall goal
of the program is to develop a community resource of quantitative mass spectrometry assays targeting protein networks
(two assays for each 500 unique gene products - 1000 immuno-MRM assays) expressed in human tissue and fluids. A
feasibility study targeting human proteins expressed in well-characterized human cell lines will be piloted, which
includes: i) Development of immuno-MRM peptide assays to panels of protein networks; ii) Production of immunoMRM reagents - monoclonal antibodies, proteotypic peptides, and assay parameters; and iii) Assessment of different
affinity reagents to peptide antigens, including alternative rabbit monoclonal antibody production methods. Among
several components for the program, the NCI will serve as the coordinating host of data sharing and QA/QC antibody
characterization (Antibody Portal, Immuno-MRM Assay Portal and Data Portal for the community). Each participating
labs will carry out the development of immuno-MRM assays using at least two synthetic, purified proteotypic peptides,
labeled with stable isotope(s) and available in accurately quantitated aliquots, for use as internal measurement
standards for quantitation of each protein. Our preliminary evaluation studies with a set of 37 peptides and monoclonal
antibodies showed that low-abundance peptides can be accurately quantified by immune-MRM.
81
International effort to develop analytically validated Multiple Reaction
Monitoring (MRM)-based assays to breast cancer cell proteins
Kyunggon Kim1, Susan E. Abbatiello2, Jacob J. Kennedy3, Jeffrey R. Whiteaker3, Jun Seok Kim4, Hophil Min1,
Youngju Lee4, Myeong-Hee Yu4, Eun Gyeong Yang4, Cheolju Lee4, Steven A. Carr2, Amanda G. Paulovich3,
Youngsoo Kim1
1
Department of Biomedical Engineering, Seoul National University College of Medicine, 28 Yongon-Dong, Seoul 110799 Republic of Korea. 2Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142. 3Fred
Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109. 4Center for Theragnosis, Korea
Institute of Science and Technology, Seoul 136-791, Republic of Korea
The successful application of multiple reaction monitoring (MRM) in cancer cell lines raises the exciting possibility
that assays can be configured to measure all human proteins, resulting in an assay resource for biomedical research.
We report the results of the proof-of–concept study designed to test the feasibility of a large-scale, international effort
in MRM assay generation. Across 3 international performance sites including SEOUL site (Seoul National University
and KIST), novel MRM assays expressed were configured, validated in human breast cancer cell lines. Assays were
multiplexed up to > 150 assays and deployed to quantify endogenous analyte in a panel of breast cancer-related cell
lines. Assay precision was measured, with high inter-laboratory correlation. Peptide measurements in breast cancer cell
lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer
proteome. These results establish the feasibility of a scaled, international effort including SEOUL, SEATTLE, and
BOSTON.
82
ExonGraph: Discovery of novel transcripts using nucleotide-based splice
graphs
Hyunwoo Kim1, Heejin Park2 and Eunok Paek2
1
Department of Electronics and Computer Engineering, Hanynag University, 222 Wangsimni-ro,
Seongdong-gu, Seoul 133-791, Korea.
2
Division of Computer Science and Engineering, Hanyang University, 222 Wangsimni-ro,
Seongdong-gu, Seoul 133-791, Korea.
E-mail: eunokpaek@hanyang.ac.kr
Proteogenomics has recently emerged as a new research field that combines knowledge and findings from proteomics
and genomics. One common practice is to identify peptides from mass spectrometry data and use such proteomic
findings to improve gene annotations.
The previous methods often involve construction of protein databases consisting of amino-acid sequences (most often
in FASTA format) that may well come from 6 frame translation of whole genome sequences[1-3], or translated splice
graphs[4] built from next-generation sequencing results[1, 3]. Representation of protein sequences in terms of amino
acids makes it difficult to add alternative splice and/or junction variation events because they may result in frame shifts
during translation, necessitating additional translation for each event. In our newly proposed method ExonGraph, a
splice graph is not translated into amino acid sequences but remains as nucleotide sequences. Therefore, it is much
easier to add hypothetical novel events such as exon skipping and junction variation than previous methods, achieved
by simple insertion of new nodes and/or edges to the splice graph.
Representing protein sequence database as a graph structure consisting of nucleotide sequences requires a proteomic
search tool that can conduct searches against nucleotide sequences instead of amino acid sequences. We have modified
MODa[5] (version 1.20) so that it can traverse a nucleotide splice graph while it interprets tandem mass spectra.
Searching a nucleotide-based splice graph has an additional advantage apart from the ease of representing novel
transcript events, that is, the reduced size of the database. It is known that translated amino acid databases are at least
one order of magnitude bigger than the original databases. For example, the recent report showed that translating a
splice graph into amino acid sequences resulted in a FASTA file of size at least twenty times bigger than the original
graph.
ExonGraph constructs a compact splice graph by using all transcripts of Ensembl database (version 71) and adding
hypothetical alternative splice and junction variation events. Then, peptides are identified by applying the modified
version of MODa against the nucleotide sequences in splice graph form. The identified peptides are mapped back to
the splice graph in order to confirm any putative transcript events included in the peptides. It is also possible to
visualize identified peptides in UCSC Genome Browser, by converting these peptides into general feature format
(GFF).
References
[1] Woo, S.; Cha, S. W.; Merrihew, G.; He, Y.; Castellana, N.; Guest, C.; Maccoss, M.; Bafna, V., Proteogenomic
Database Construction Driven from Large Scale RNA-seq Data. J. Proteome Res. (2013).
[2] Sheynkman, G. M.; Shortreed, M. R.; Frey, B. L.; Smith, L. M. Discovery and mass spectrometric analysis of
novel splice-junction peptides using RNA-Seq. Mol. Cell. Proteomics (2013), 12 (8), 2341−53.
[3] Castellana, N. E.; Payne, S. H.; Shen, Z.; Stanke, M.; Bafna, V.; Briggs, S. Discovery and revision of Arabidopsis
genes by protegenomics. P. Proc. Natl. Acad. Sci. U.S.A. (2008), 105, 21034−21038.
[4] Tanner, S.; Shen, Z.; Ng, J.; Florea, L.; Guigo, R.; Briggs, S. P.;Bafna, V., Improving gene annotation using
peptide mass spectrometry. Genome Res. (2007), 17, 231−239.
[5] Na, S.; Bandeira, N.; Paek, E. Fast multi-blind modification search through tandem mass spectrometry. Mol. Cell.
Proteomics (2012), 11 (4), M111 010199.
83
Structural and functional roles of both poly (ADP-ribose) and
Poly (ADP-ribosyl)ation.
Ho Chul Kang
Department of Physiology, Ajou University School of Medicine, Korea.
Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly
(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR
binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3
ligase. Iduna's E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate
Iduna's PAR binding and Iduna's E3 ligase activity. Iduna's E3 ligase activity also requires an intact really interesting
new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of
ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either
PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86,
XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna
ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna's PAR-dependent ubiquitination of PARP1
targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell
death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1
arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing
apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed
by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA
repair.
84
The structural basis for activity regulation of MLL-family histone
methyltransferase
Yong Chen1, Yanjing Li1, Fang Cao2, Yali Dou2 and Ming Lei1
1
National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
320 Yueyang Road, Shanghai 200031, China
2
Department of Pathology, University of Michigan Medical School, 1301 Catherine, Ann Arbor, MI 48109, USA
E-mail: yongchen@sibcb.ac.cn
The MLL-family histone methyltransferases play essentials role in the maintenance of the histone H3 Lysine 4 (H3K4)
methylation status for gene expression during differentiation and development1. In contrast to most SET-domain
containing methyltransferases, the methyltransferase activity of MLL by itself is severely compromised2. The optimal
activity of MLL requires three additional factors, WDR5, RbBP5 and Ash2L, which are shared core components of all
MLL complexes and also evolutionarily conserved from yeast to humans 2,3. However, the molecular mechanism of
activity regulation of MLL complexes remains elusive. Here we show that the RbBP5-Ash2L heterodimer is the
minimum unit to interact with and activate MLL proteins. WDR5 functions exclusively in the MLL1 complex as a
scaffold molecule to stably tether MLL1 to RbBP5-Ash2L. The crystal structures of the MLL3-RbBP5-Ash2L and
MLL1-RbBP5-Ash2L complexes reveal a two-step mechanism of MLL activation. Upon RbBP5-Ash2L binding, a
short activation segment of RbBP5 causes a rigid-body rotation of the SET-I motif in MLL SET domain towards the
substrate-binding groove, followed by a substrate-induced local rearrangement in the SET-I motif to enclose the target
lysine access channel for catalysis. These results provide the first structural model for complex assembly and activity
regulation of MLL-family histone methyltransferases.
References
[1] Ruthenburg, A. J., Allis, C. D. & Wysocka, J. Methylation of lysine 4 on histone H3: intricacy of writing and
reading a single epigenetic mark. Mol Cell 25, 15-30, (2007).
[2] Dou, Y. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat Struct Mol Biol
13, 713-719, (2006).
[3] Southall, S. M., Wong, P. S., Odho, Z., Roe, S. M. & Wilson, J. R. Structural basis for the requirement of
additional factors for MLL1 SET domain activity and recognition of epigenetic marks. Mol Cell 33, 181-191,
(2009)
85
SIRT1 is phosphorylated by cAMP/PKA signaling pathway
Lim Ji-Hong
Department of Biomedical Chemistry, Konkuk University (GLOCAL campus)
The NAD(+)-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that
mediates homeostatic responses to certain physiological stresses such as nutrient restriction. Previous reports have
implicated fluctuations in intracellular NAD(+) concentrations as the principal regulator of SIRT1 activity. However,
here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1
catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD(+) levels.
Attenuation of SIRT1 expression or the use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated
stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice
significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological
β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the
cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis
86
Structural insight into substrate recognition mechanism of glycoprotein
processing enzyme ER glucosidase II
Tadashi Satoh1,2, Takayasu Toshimori1, Takumi Yamaguchi1,3,4, Zhu Tong1,3,4 and Koichi Kato1,3,4,5,6
1
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
2
JST, PRESTO, Nagoya 467-8603, Japan.
3
Department of Functional Molecular Science, Graduate University for Advanced Studies,
Okazaki 444-8787, Japan.
4
Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes
of Natural Sciences, Okazaki 444-8787, Japan.
5
The Glycoscience Institute, Ochanomizu University, Tokyo 112-8610, Japan.
6
GLYENCE Co., Ltd., Nagoya 464-0858, Japan.
E-mail: tadashisatoh@phar.nagoya-cu.ac.jp
In eukaryotic cells, a series of N-linked oligosaccharide processing intermediates attached on the proteins operates as
specific tags for protein quality control system, dictating their fates, i.e. folding, intracellular transport, or degradation
[1]. In the ER quality control system, glucosidase II plays an essential role as a processing enzyme, which catalyzes
deglucosylation of non-reducing terminal glucose residue of the N-linked oligosaccharides. Glucosidase II is composed
of approximately 110 kDa catalytic α subunit (GIIα) belonging to a glycoside hydrolase family 31 (GH31) and 60 kDa
non-catalytic regulatory β subunit (GIIβ). It has been demonstrated that GIIα alone can only hydrolyze a small αglycosidase model substrate (pNP-glucose), whereas it cannot deglucosylate bulky high-mannose-type
oligosaccharides unless it associates with GIIβ.
In this study, we determined the 1.6-Å high-resolution crystal structure of GIIα alone and in complex with its
inhibitor 1-deoxynojirimycin. The crystal structure revealed that GIIα possesses a distinctive N-terminal segment in
comparison with the cognate glycoside hydrolase enzymes (GH31). Intriguingly, the N-terminal segment was
accommodated on the substrate-binding pocket. Our crystallographic data thus provide structural insights into the
substrate recognition mechanism whereby the N-terminal segment of GIIα undergoes structural rearrangement through
association with GIIβ, thereby promoting the substrate binding capacity for the bulky glucosylated high-mannose-type
oligosaccharides.
References
[1] Kamiya Y, Satoh T. and Kato K., ―Molecular and structural basis for N-glycan-dependent determination of
glycoprotein fates in cells‖, Biochim. Biophys Acta, 1820, (2012) 1327-1337.
87
How we can improve affinity of antibodies for the targets for therapeutics
Kouhei Tsumoto
School of Engineering, and Institute of Medical Science, The University of Tokyo, 108-8639 Tokyo, Japan
E-mail: tsumoto@ims.u-tokyo.ac.jp
Specific recognition of ligands by proteins is a fundamental biological phenomenon, and interaction between antigen
and antibody in the immune system is a typical example. Recent advances in physical biochemistry have enabled us to
describe what factors dominated the specificity and affinity of protein interactions. We have focused on several
antigen-antibody interactions, including those specific for model antigens, e.g. hen lysozyme, toxin, and EGFR, and
dissected the interactions from physicochemical viewpoints. Our conclusions could be summarized as follows. 1.
Specificity is dominated by only a couple of residues, named hot-spot; 2. Hydrophobic interaction and/or a couple of
hydrogen bonds are created by hot-spots, and cooperative binding of other paratope residues is induced via hot-spot
interactions; 3. Other paratope residues are tolerant to site-specific mutation, and make incremental contributions to the
interaction; 4. Variable domain interactions work as a cushion for fine-tuning of the paratope interface; 5. Interfacial
water molecules make enthalpic contribution to the interaction, which complements the imperfect interfaces of antigen
and antibody. 6. In principle, enthalpy change dominates the high affinity of antigen-antibody interaction, e.g. affinity
maturation of the antibody. On the basis of these conclusions, we could propose one strategic scheme on improvement
of antibody affinity for targets. These conclusions could be applied not only to improve the specificity and affinity of
an antibody, but also to screen and/or design of small molecules, which can control specific biomolecular interactions.
88
In vitro and in vivo application of anti-cotinine antibody
and cotinine-conjugated compounds
Junho Chung
Department of Biochemistry and Molecular Biology & Cancer Research Institute Seoul National University College of
Medicine, Korea
The combination of a high-affinity antibody to a hapten, and hapten-conjugated compounds, can provide an
alternative to the direct chemical cross-linking of the antibody and compounds. An optimal hapten for in vitro use is
one that is absent in biological systems. For in vivo applications, additional characteristics such as pharmacological
safety and physiological inertness would be beneficial. Additionally, methods for cross-linking the hapten to various
chemical compounds should be available. Cotinine, a major metabolite of nicotine, is considered advantageous in these
aspects. A high-affinity anti-cotinine recombinant antibody has recently become available, and can be converted into
various formats, including a bispecific antibody. The bispecific anti-cotinine antibody was successfully applied to
immunoblot, enzyme immunoassay, immunoaffinity purification, and pre-targeted in vivo radioimmunoimaging. The
anti-cotinine IgG molecule could be complexed with aptamers to form a novel affinity unit, and extended the in vivo
half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical
compounds.
89
Rational drug design: antibodies for treating severe asthma and allergy
Tse Wen Chang
Genomic Research Center, Academia sinica, Taipei 11529, Taiwan
E-mail: twchang@gate.sinica.edu.tw
In this lecture, the speaker will share his experience in creating a new therapeutic concept and bringing it through
laboratory research, drug optimization processes, clinical trials, and finally to broad patients‘ use. In the beginning of
this lengthy drug development program, IgE and membrane-bound IgE (mIgE) were recognized as therapeutic targets,
when other researchers did not think that these molecules are suitable targets or that they are targetable. Today, the
anti-IgE antibody, omalizumab (trade name Xolair) has been studied in 118 clinical trials for various allergic and some
non-allergic diseases and approved in more than 90 countries to treat patients with severe allergic asthma untreatable
with other medicine. New-generation drug candidates for targeting mIgE, which may have certain advantages over
omalizumab, have been developed and one of those is in Phase IIb clinical trial. The editor of Nature Biotechnology
commented that these programs were good examples of rational drug design approach.
Reference
[1]
[2]
[3]
[4]
[5]
Chang TW. Pharmacological basis of anti-IgE therapy (a review). Nature Biotechnology 18, 157-162 (2000).
Chang TW. and Shiung YY. Anti-IgE as a mast cell-stabilizing therapeutic agent. J Allergy Clin Immunol 117,
1203-12 (2006).
Chang TW, Wu PC, Hsu CL, and Hung AF. Anti-IgE antibodies for the treatment of IgE-mediated allergic
diseases. Adv. Immunol. 93, 63-119 (2007).
Chen JB, Wu PC, Hung AF, Chu CY, Tsai TF, Yu HM, Chang HY, and Chang TW. Unique epitopes on CεmX in
IgE-BCR are potentially applicable for targeting IgE-committed B cells. J. Immunol. 184, 1748-1756 (2010).
Chu, HM, Wright J, Chan YH, Lin CJ, Chang TW, and Lim C. Two potential therapeutic antibodies bind to a
peptide segment of membrane-bound IgE in different conformations. Nature Communications 5, article No.
3139 (2014).
90
Tailoring Aglycosylated Antibodies for a New Class of Next –
Generation Immunotherapeutics
Sang Taek Jung
Department of Bio and Nano Chemistry, Kookmin University, Seoul, Korea, 136-702
E-mail: sjung@kookmin.ac.kr
Aglycosylated antibodies are almost indistinguishable from glycosylated counterparts in terms of antigen binding,
stability at physiological temperature, pharmacokinetics, and biodistribution. Therefore, to circumvent drawbacks of
conventional clinically used glycosylated antibodies that require high capital investment for expensive mammalian
cell culture and complicate purification from heterogeneous IgG glycan variants, aglycosylated IgG antibodies have
been developed and several of them are under clinical trials. Although wild type aglycosylated antibodies exhibit
nearly complete loss of FcγR binding and immune effector cell activation, recent comprehensive engineering of
antibody Fc allowed isolation of a set of aglycosylated antibody Fc variants displaying unique FcγRs selectivities and
novel therapeutic effector functions. In this lecture, we will discuss recent engineering efforts to convert aglycosylated
antibodies into a new class of highly efficient immunotherapeutics.
References
[1] Jung ST, et al., Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell
killing by monocyte-dendritic cells (2010), Proc Natl Acad Sci USA 107(2), 604-609.
[2] Jung ST, Kang TH, & Georgiou G, Efficient expression and purification of human aglycosylated Fcγ receptors in
Escherichia coli (2010), Biotechnol Bioeng 107(1), 21-30.
[3] Jung ST, Kang TH, Kelton W, & Georgiou G, Bypassing glycosylation: engineering aglycosylated full-length IgG
antibodies for human therapy (2011), Curr Opin Biotechnol 22(6), 858-867.
[4] Jung ST, Kelton W, Kang TH, Ng DT, Andersen JT, Sandlie I, Sarkar CA, Georgiou G, Effective phagocytosis of
low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcγRIIa affinity and selectivity
(2013), ACS Chem Biol 15(8), 368-375.
91
Protein phosphorylation remains a black box in signal transduction:
developing new methods to search for substrates of specific kinases including
Rho-kinase
Mutsuki Amano1, and Kozo Kaibuchi1,
1
Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan
E-mail: m-amano@med.nagoya-u.ac.jp
There are about 500 putative protein kinase genes. Many protein kinases have been thought to be involved in the
various biological processes such as cell polarization. Nonetheless, knowledge about the downstream substrates for
each protein kinase is largely limited, and is far from the comprehensive understanding of molecular network beneath
the kinase to exert the physiological functions. Despite the importance of identifying substrates, efficient methods to
search for substrates remain poorly explored. We have recently developed the efficient screening method combined the
interactome analysis between kinase and substrates and shotgun liquid chromatography tandem-mass spectrometry
(LC-MS/MS) analysis, named kinase-interacting substrate screening (KISS) method. Using polarity-related kinases
including Rho-kinase, PAK, and aPKC as baits, we obtained the numerous candidate substrates from the rat brain
lysate, which included the proteins previously reported as their substrates. A lot of the candidates were phosphorylated
by these kinases in vitro. Moreover, we have developed the in vivo method, named phosphatase inhibitor and kinase
inhibitor substrate screening (PIKISS) method. Hela cells were treated with phosphatase and/or kinase inhibitors, and
then the cell lysates were subjected to 14-3-3-coated beads to enrich phosphorylated proteins, followed by the LCMS/MS analysis. These methods enables us to identify novel specific substrates for specific kinases including Rhokinase both in vitro and in vivo.
92
Optical Control of Cell Signaling in Mammalian Cells
Won Do Heo
Department of Biological Sciences, KAIST & Center for Cognition and Sociality, IBS
Signaling networks in mammalian cells are built from thousands of signaling proteins that carry out a variety of
cellular tasks including development, cell proliferation, cell migration, and cancer cell metastasis. In order to
understand complex cell signaling networks in mammalian cells we use live cell imaging approaches, including
visualizing behaviors and molecular interactions of signaling proteins. We have been developing new bioimaging
technologies for visualizing P-P interactions and technologies for controlling signaling proteins in live mammalian
cells. Recently, the advent of light responsive elements in cell biology has led us to design a rapid and reversible
control system to modulate protein functions by stimulating specific wavelength of light in a spatiotemporal manner.
We expect this versatile platform could specifically control the functions of endogenous target proteins in neuron and
provide a powerful way to control a broad range of signaling proteins in vivo system. I will discuss in the talk mostly
novel imaging technologies we have developed to control and visualize cell signaling and cell functions in mammalian
cells by using blue and green light receptors from plants.
93
Spatial organization and dynamics of eukaryotic gene regulation
Rahul Roy1,2, Ziqing Zhao2, J Christof Gebhardt2, David Suter2, Alex Chapman2 and Sunney Xie2
1
Department of Chemical Engineering and Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
2
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, USA
E-mail: rahulroy@chemeng.iisc.ernet.in
Accurate quantification of stoichiometry and dynamics of nuclear processes like transcription is challenging due to
limited signal-to-noise ratio or inadequate resolution of current imaging techniques. We developed reflected light-sheet
microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of
living mammalian cells. The thin light sheet allows for an increased signal-to-background ratio and enables imaging of
single fluorescent proteins with up to 100-Hz time resolution[1]. By measuring the DNA-bound fraction of
glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and
mutants of GR and estrogen receptor-α (ERα), we could resolve different modes of DNA binding by hormone
receptors. Furthermore, by combining reflected light-sheet illumination with stochastic localization based super
resolution microscopy[2], we probed the spatial organization of transcription by RNA polymerase II (RNAP II)
molecules and quantified their global extent of clustering inside the mammalian nucleus[3]. Spatiotemporal clustering
analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the
transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules
was detected within the length scale of the reported diameter of ―transcription factories‖. Colocalization measurements
of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution,
arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. We
demonstrate that combination of reflected light sheet microscopy with various imaging modalities provides powerful
avenues for studying molecular dynamics and stoichiometry in living cells.
References
[1] ZW Zhao*, R Roy*, JCM Gebhardt*, DM Suter*, AR Chapman and S Xie, "Spatial organization of RNA
polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet super-resolution microscopy"
PNAS (USA), 111(2):681-6 (2014) *equal contribution
[2] R Roy, "Next generation optical microscopy" Current Science, 105(11):1524-36 (2013)
[3] JCM Gebhardt, DM Suter, R Roy, ZW Zhao, AR Chapman, S Basu, T. Maniatis and S Xie, ―Single-molecule
imaging of transcription factor binding to DNA in live mammalian cells‖ Nature Methods, 10(5):421-6 (2013)
94
Multi-Parametric Surface Plasmon Resonance(MP-SPR):
new possibilitiesfor characterization of biomolecularlayers.
Willem M. Albers1, Tony Munter2, Inger Vikholm-Lundin3,
Janusz W. Sadowski1, Niko Granqvist1, Annika Jokinen1
1
BioNavis Ltd, Elopellontie 3.C, 33470 Ylöjärvi, Finland.
VTT Technical Research Centre of Finland, Process Chemistry and Environmental Engineering, Sinitaival 6, 33720
Tampere, Finland.
3
University of Tampere, Institute of Biomedical Technology (BioMediTech), Biokatu 6, 33014 University of Tampere,
Finland
2
Surface Plasmon Resonance is an established method for studies of molecular interactions now for 24 years. In recent
years, the Finnish National Research Institute (VTT) has developed ―Multi-Parametric Surface Plasmon Resonance‖
(MP-SPR). With MP-SPR not only studies of molecular interactions can be conducted, but also nanolayers can be
further characterized in terms of thickness, refractive index and adsorption coefficient. MP-SPR provides absolute
calibrated measurements of the whole SPR curve with maximum angle range and the measurements are possible in
various media ranging from air to ethanol. Therefore, MP-SPR cannot only be used in life sciences, but also in
materials development, gas sensing, analysis of electrodeposition processes etc.
Recent examples of application of MP-SPR to the characterization of biofunctional layers with engineered avidins and
in self-assembled layers of hydrophilic polymers and imprinted monomolecular layers are presented. Also some
examples are given of small molecule interactions with proteins.
95
Two potential therapeutic antibodies bind to a peptide
segment of membrane-bound igE in different conformations
Hsing-Mao Chu1, Jon Wright1,2, Yueh-Hsuan Chan3, Chien-Jen Lin1, Tse Wen Chang1 & Carmay Lim2,4
Institute of Biomed. Sci., Academia Sinica, Chinese Taipei
IgE mediates hypersensitivity reactions responsible for most allergic diseases, which affect 20–40% of the population
in developed countries. A 52-residue domain of membrane-bound IgE (mIgE) called CemX is currently a target for
developing therapeutic antibodies; however, its structure is unknown. Here we show that two antibodies with
therapeutic potential in IgE-mediated allergic diseases, which can cause cytolytic effects on mIgE-expressing Blymphocytes and downregulate IgE production, target different conformations of an intrinsically disordered region
(IDR) in the extracellular CemX domain. We provide an important example of antibodies targeting an extracellular
IDR of a receptor on the surface of intended target cells. We also provide fundamental structural characteristics unique
to human mIgE, which may stimulate further studies to investigate whether other monoclonal antibodies (mAbs)
targeting intrinsically disordered peptide segments or vaccine-like products targeting IDRs of a membrane protein can
be developed.
96
Ordered in Disordered: Structural Transformation of the
Amyloidogenic Core in the C-terminal Part of TDP-43
Hong-Yu Hu
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
E-mail: hyhu@sibcb.ac.cn
TAR DNA binding protein of 43 kDa (TDP-43) is a major deposited protein in amyotrophic lateral sclerosis and
frontotemporal lobar degeneration with ubiquitin. The TDP-43 protein is comprised of an N-terminal domain, two
RNA recognition motifs (RRMs), and a C-terminal glycine rich region (GRR, residues 262-414). Generally, the GRR
is a long unstructured part, which is thought to relate to the aggregation and proteinopathies of TDP-43. We identified
a hydrophobic patch (318 – 343) in the flexible C-terminal part that is the amyloidogenic core essential for TDP-43
aggregation. NMR and other biophysical studies demonstrated that the homologous peptide forms a helix-turn-helix
structure in solution, while it undergoes structural transformation from α-helix to β-sheet during aggregation, which
plays crucial roles in TDP-43 aggregation. Mutation or deletion of this core region significantly reduces the
aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural
transformation of the local ordered segment initiates the aggregation of the C-terminal disordered part and cytoplasmic
inclusion formation of TDP-43.
Keywords: TDP-43; Disordered structure; Helix-turn-helix; Aggregation; Inclusion.
References
[1] Neumann M., Sampathu D.M., Kwong L.K., Truax A.C., Micsenyi M.C., Chou T.T., Bruce J., Schuck T.,
Grossman M., Clark C. M., McCluskey L. F., Miller B. L., Masliah E., Mackenzie I.R., Feldman H., Feiden W.,
Kretzschmar H. A., Trojanowski J.Q., and Lee V.M. Ubiquitinated TDP-43 in frontotemporal lobar degeneration
and amyotrophic lateral sclerosis. Science, Vol. 314, No. 5796, (2006), pp 130-133.
[2] Che M.X., Jiang Y.J., Xie Y.Y., Jiang L.L., and Hu H.Y. Aggregation of the 35-kDa fragment of TDP-43 causes
formation of cytoplasmic inclusions and alteration of RNA processing. FASEB J, Vol. 25, No.7, (2011), pp 23442353.
[3] Jiang L.L., Che M.X., Zhao J., Zhou C.J., Xie M.Y., Li H.Y., He J.H., and Hu H.Y. Structural Transformation of
the Amyloidogenic Core Region of TDP-43 Initiates Its Aggregation and Cytoplasmic Inclusion. J Biol Chem,
Vol. 288, No. 27, (2013), pp 19614-19624.
97
Structural Transitions of Intrinsically Disordered -Synuclein to Helix by
Hydrophobic Interaction with Neutral Lipid Membrane
Hugh I. Kim1
1
Department of Chemistry and Division of Advanced Materials Science,
Pohnag University of Science and Technology, Pohang 790-784, Korea.
E-mail: hughkim@postech.edu
α-Synuclein is a small neural protein whose fibrillation is known to be associated with neurodegeration in
Parkinson‘s disease. The structures of -synuclein can be confined by intermolecular interactions with lipid
membrane which have attracted considerable attention due to its pathologic significant. Structural changes of
disordered -Synuclein (-Syn) to helix are analyzed using solution small angle X-ray scattering (SAXS) and
electrospray ionization ion mobility mass spectrometry (ESI-IM-MS). The application to probe the helix formation
of -Syn via association with large unilamellar vesicles as model lipid membrane systems is also demonstrated.
Details of regional interactions of -Syn with lipid vesicles and its structural change mechanisms with the different
polarity of the membrane surface are further discussed based on the hydrogen-deuterium exchange mass
spectrometry experiments. In addition to the conventionally believed helix formation mechanism of -Syn on
anionic lipid membranes via electrostatic interaction between the N-terminal region of the protein and the lipid head
groups, we observed that -Syn can adopt a helical structure through deep penetration of the central region in the
neutral lipid membrane via hydrophobic interactions.
References
[1] Lee, S. J. C.,; Lee, J. W.,; Choi, T. S.,; Jin, K. S.,; Lee, S.,; Ban, C.,; Kim, H. I. , "Probing Conformational
Change of Intrinsically Disordered α-Synuclein to Helical Structures by Distinctive Regional Interactions with
Lipid Membranes" Anal. Chem. 2014 , 86, 1909–1916.
98
Levels of disorder and residual helicity determine the p53-Mdm2 binding
affinity required for productive signaling in cells
Wade Borcherds1,2$, François Xavier Theillet3$, Andrea Katzer4$, Ana Finzel4, Katie M. Mishall1,2, Anne Powell1,2,
Hongwei Wu1,2, Wanda Manieri5, Christoph Dieterich6, Philipp Selenko3,*,
Alexander Loewer4,* and Gary W. Daughdrill1,2,*
1
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620,
USA
2
Center for Drug Discovery and Innovation, University of South Florida, Tampa, FL 33612, USA
3
Department of NMR-supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin),
13125 Berlin, Germany
4
Berlin Institute for Medical Systems Biology, Max-Delbrueck-Center, 13125 Berlin, Germany
5
Drug Discovery Department, Moffitt Cancer Center, University of South Florida, Tampa, FL 33612, USA
6
Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany
E-mail: gdaughdrill@usf.edu
The p53 transactivation domain (p53TAD) is an intrinsically disordered protein (IDP) domain that undergoes coupled
folding and binding when it interacts with partner proteins like the E3 ubiquitin ligase, Mdm2, and the 70 kDa subunit
of replication protein A, RPA70. The secondary structure and dynamics of six closely related mammalian orthologues
of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy1. Clustering analysis showed
that the divergence in transient helical secondary structure of the p53TAD orthologues is more extensive than the
amino acid sequence divergence. In contrast, strong correlations were observed between the backbone dynamics of the
orthologues and the sequence identity matrix, suggesting that the dynamic behavior of IDPs is under positive
evolutionary selection. Mutating conserved prolines that flank the Mdm2 binding site to Alanines doubled the level of
transient helical secondary structure in this region. This doubling of transient helical secondary structure increased the
in vitro binding affinity between p53TAD and Mdm2. The in vivo binding affinity between full-length p53 and Mdm2
was also increased in the proline mutants. This increase in binding affinity disrupted the expression of p53 target genes
and inhibited the ability of cells to arrest in G1 following radiation induced DNA damage. Taken together our results
demonstrate that the transient helical secondary structure of p53TAD has been finely tuned by evolution and disrupting
this structure has deleterious effects on target gene expression and cell fate decisions.
References
[1] Borcherds, W., Kashtanov, S., Wu, H. & Daughdrill, G.W. Structural divergence is more extensive than sequence
divergence for a family of intrinsically disordered proteins. Proteins 81, 1686-98 (2013).
99
Protein Flexibility Investigated by SAXS and MD Simulation
Mamoru Sato
Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho,
Tsurumi-ku, Yokohama 230-0045, Japan.
Recent development of protein crystallography allows us to understand protein function at atomic level. However,
protein is always in motion, thus knowledge of protein dynamics in solution being a prerequisite of understanding how
proteins function in cell. Small-angle X-ray scattering (SAXS) and all-atom molecular dynamics (MD) simulation can
provide us with such knowledge. Furthermore, the combination of SAXS experiments with MD simulations is
becoming a powerful tool to investigate protein dynamics in solution at an atomic resolution. In view of this
consideration, we have developed a combined method of all-atom MD simulations and SAXS (called MD-SAXS)
(Oroguchi et al., 2009). The method offers a link between the low-resolution solution structure obtained from SAXS
and the high-resolution crystal structure. In this conference, we show two research results on protein flexibility in
solution as revealed by MD-SAXS.
In the first research, we elucidated the intrinsic dynamics of a type II restriction endonuclease, EcoO109I using a 150ns MD simulation. The SAXS profile calculated from the simulation, in which X-ray scattering from explicit water
molecules hydrating proteins is explicitly evaluated, was in agreement with the experimentally obtained SAXS profile.
The detailed analysis of protein motions revealed that EcoO109I is intrinsically flexible in the functional movement
that cleaves ds-DNA specifically.
In the second research, we investigated the solution structure of the ε subunit of F1-ATPase, where the structure of the
ATP-free subunit is likely to be largely fluctuated. The ATP-free ε subunit of F1-ATPase was therefore subjected to
MD-SAXS analysis and shown to have an extremely flexible structure that differs significantly from the crystal
structure of the ATP-bound subunit.
MD-SAXS has been extended so that it can be applied to intrinsically disordered proteins (IDPs). IDPs are mostly
found in eukaryotes and interact with several target molecules, thus playing important roles as protein hubs in
intracellular protein networks.
Reference
[1]
Oroguchi, T., Hashimoto, H., Shimizu, T., Sato, M., & Ikeguchi, M. Biophys. J. 96, 2808-2822 (2009).
100
Glycan microarrays as a powerful tools for
studies of glycan-protein interactions
Injae Shin
Department of Chemistry, Yonsei University, Seoul 120-749, Korea
Carbohydrates compose a large group of biomolecules with diverse structures and are found largely in the form of
glycoconjugates inside or on the surface of cells. These glycan substances participate in many important cellular
processes, such as cell adhesion, signaling and trafficking, through interactions with proteins. In addition, glycanmediated biomolecular interactions also play key roles in various pathological processes. As a consequence,
understanding the molecular basis of glycan-protein interactions provides deep insights into glycan-mediated
biological processes. Conventional approaches have been employed to evaluate glycan-protein recognition events over
past decades. Although successfully applied for studying these biomolecular interactions, the conventional techniques
are labor intensive and often require large amounts of carbohydrate samples. As a high-throughput analytic tool for
glycan-protein interactions, carbohydrate microarrays have been developed. The notable advantage of the carbohydrate
microarray-based technology is this technology allows for simultaneous analysis of a number of glycan-protein
interactions using small amount of carbohydrate samples. The carbohydrate microarrays have been applied for
biological and biomedical research. In this presentation, I will discuss our recent advance in glycan microarrays for
studying carbohydrate-mediated biological processes.
References
[1] Sungjin Park, Jeffrey C. Gildersleeve, Ola Blixt, Injae Shin, ―Carbohydrate microarrays‖, Chem. Soc. Rev. Vol. 42,
(2013), pp 4310-4326.
[2] Xizhe Tian, Kyung-Hwa Baek, Injae Shin, ―Dual-labeled glycoclusters: synthesis and their application to
monitoring lectin-mediated endocytosis‖, Mol. BioSyst. Vol. 9, (2013), pp 978-986.
[3] Xizhe Tian, Jaeyoung Pai, Injae Shin, ―Analysis of density-dependent binding of glycans by lectins using
carbohydrate microarrays‖, Chem. Asian J. Vol. 7, (2012), pp 2052–2060.
[4] Xizhe Tian, Jaeyoung Pai, Kyung-Hwa Baek, Sung-Kyun Ko, Injae Shin, ―Fluorophore-labeled, Peptide-based
Glycoclusters: Synthesis, Binding Properties for Lectins and Detection of Carbohydrate-binding Proteins in Cells‖,
Chem. Asian J. Vol. 6, (2011), pp 2107-2113.
[5] Sungjin Park, Myung-Ryul Lee, Injae Shin, ―Fabrication of Carbohydrate Chips and Their Use to Probe ProteinCarbohydrate Interactions‖, Nat. Protoc. Vol. 2, (2007), pp 2747-2758.
[6] Myung-ryul Lee, Injae Shin, ―Fabrication of Chemical Microarrays by Efficient Immobilization of HydrazideLinked Substances on Epoxide-Coated Glass Surfaces‖, Angew. Chem. Int. Ed. Vol. 44, (2005), pp 2881-2884.
[7] Sungjin Park, Myung-ryul Lee, Soon-Jin Pyo, Injae Shin, ―Carbohydrate Chips for Studying High-throughput
Carbohydrate-protein Interactions‖, J. Am. Chem. Soc. Vol. 126, (2004), pp 4812-4819.
[8] Sungjin Park, Injae Shin, ―Fabrication of Carbohydrate Chips for Studying Protein-carbohydrate Interactions‖,
Angew. Chem. Int. Ed. Vol. 41, (2002), pp 3180-3182.
101
DNA mimic proteins: A new paradigm for regulation of DNA functions
Andrew H.-J. Wang
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
DNA mimic proteins (DMPs) occupy the DNA binding sites of DNA binding proteins, and prevent these sites from
being accessed by DNA. To date, only a few DMPs have been reported. Our work aims to extend our knowledge of
this novel kind of control factors. We searched and analyzed new DNA mimic proteins from selected species including
bacteriophages, animal virus and pathogenic bacteria. We have successfully identified five DNA mimic proteins by
using structural, proteomic, biochemical and bioinformatic approaches: (1) White spot syndrome virus ICP11: ICP11
formed a dimer with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate
groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins and interfered with nucleosome
assembly. (2) Neisseria DMP12: DMP12 interacts with the bacterial histone-like protein HU. Structural analysis of
DMP12 showed that the molecular and electrostatic surfaces of the DMP12 monomer are similar to those of bent
DNA and complementary to those of HU protein dimer. Since HUs participate in bacterial nucleoid formation,
recombination, gene regulation and DNA replication, the interaction between DMP12 and HU protein might play
important roles in these mechanisms. (3) Neisseria DMP19: DMP19 used its dsDNA-like surface to interact with
Neisseria transcription factor NHTF. Our in situ gene regulation assay provided evidence that NHTF is a repressor of
its down-stream genes and that DMP19 can neutralize this effect. We conclude that the interaction of DMP19 and
NHTF provides a novel gene regulation mechanism in Neisseria spps. (4) S. aureus uracil DNA glycosylase inhibitor
(SaUGI): We have analyzed the SaUGI by a combination of bioinformatics, structural and biochemical approaches.
Detailed interactions will be presented.
References
[1] Wang HC, et al. (2008) Proc. Natl. Acad. Sci. 105, 20758-20763. (2). Wang HC et al. (2013) Nucleic Acids Res.
41, 5127-38. (3). Wang HC et al. (2012) Nucleic Acids Res. 40, 5718-30. (4). Wang HC et al. (2014) Nucleic
Acids Res. 42, 1354-64.
102
Discovery of Pharmacological Modulators of Protein-Protein Interactions
Hyun-Suk Lim1
1
Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea
E-mail: hslim@postech.ac.kr
Protein-protein interactions (PPIs) play a critical role in a wide range of cellular functions, and their misregulation is
linked to numerous disease states. As such, there has been a great interest in developing molecules capable of
modulating specific PPIs, which can be valuable research tools to uncover molecular functions of target proteins and
be potential therapeutic candidates [1]. However, discovering such molecules is a daunting task mostly due to the fairly
large and flat protein interfaces involved in PPIs. Typical ‗drug-like‘ small molecules may not be suitable to
effectively cover such extended protein contact areas. Indeed, screens of the existing compound collections rarely
identify PPI modulators. Thus, there is an urgent need for the development of different types of molecules to target
protein interfaces (e.g., larger molecules with structural diversity or molecules that can mimic functional domains of
proteins such as well-defined protein secondary structures). Moreover, there are limited numbers of screening methods
for detecting PPI regulators [2]. In this talk, I will present our approach to address all these challenges. We designed a
novel class of molecules that can mimic protein surface structures and thus are suitable to target protein interfaces. And
we developed a simple high-throughput screening method that allows for rapid and efficient discovery of potential
pharmacological modulators. Finally, we successfully demonstrated the feasibility of our strategy by identifying
modulators of several disease-specific PPIs.
References
[1] Wells J.A. and McClendon C.L., ―Reaching for high-hanging fruit in drug discovery at protein-protein
interactions‖, Nature, 450 (2007), 1001-1009.
[2] Heeres J.T. and Hergenrother P.J., ―High-throughput screening for modulators of protein-protein interactions‖,
Chem. Soc. Rev., 40 (2011), 4398-4410.
103
Protein-Carbohydrate Interactions Leading to Specificity in Glycoside
Hydrolase I Enzymes.
James R. Ketudat-Cairns1,2, Salila Pengthaisong2, Anupong Tankrathok, Juthamath Komvongsa, and Jisnuson Svasti2
1
School of Biochemistry & Center for Biomolecular Structure, Function and Application, Institute of Science,
Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
2
Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand
E-mail: cairns@sut.ac.th
Protein-carbohydrate interactions are of primary importance for many processes, including signaling via cell surface
glycoproteins, modulation of the activities of bioactive compounds by glycosylation and deglycosylation and
enzymatic turnover of biomass for biofuel production. Glycoside hydrolase family 1 (GH1) serves as a good model
for studying protein-carbohydrate interactions due to the broad range of specificities for reactants and products among
closely related members of this family, especially in plants. Os3BGlu7 (also called rice BGlu1) has 6 subsites for
binding -1,4-linked glucosyl residues leading to efficient hydrolysis of cellooligosaccharides by Os3BGlu7 and their
synthesis by its glycosynthase mutant E376G. Mutagenesis of the cellooligosaccharide binding subsites, showed an
amazing ability to adapt to lose binding residues with limited effect on oligosaccharide binding and hydrolysis [1].
Protein crystallographic studies identified 3 different effective binding modes for cellooligosaccharides in the active
site cleft, confirming the high plasticity in this protein-carbohydrate interaction, and suggested that the overall shape of
the active site and bound water network facilitate celloligosaccharide binding. Comparison of Os3BGlu7 -glucosidase
and Os7BGlu26 -mannosidase binding of sugars and transition state mimic inhibitors indicated that both proteins can
hydrolyze both glucosides and mannosides due to their abilities to accommodate multiple transition state shapes in the
active site, but the differences leading to alternate preferences are subtle [2]. Both of these enzymes predominantly
catalyze hydrolysis, but can also catalyze transglycosylation. In contrast, Os9BGlu31 is a phenylpropanoid ester
transglucosidase with relatively little hydrolysis activity [3]. Probing the residues around the active site indicated that
removal of the tryptophan from the W243 position in the active site cleft leads to higher transglycosylation of hydroxyl
groups relative to carboxylates, but the enzyme was not converted to a hydrolase by single mutations to make the
acceptor site more hydrophilic. These and other studies have shown that the hydrogen bonding and hydrophobic
interactions, along with active site shape and its water network facilitate the diverse substrate specificities of GH1
enzymes with high adaptability.
References
[1] Pengthaisong S., Withers S.G., Kuaprasert B., Svasti J., Ketudat Cairns J.R. ―The role of the oligosaccharide
binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1
glycosynthase.‖ Protein Science Vol. 21, (2012), pp 362-372.
[2] Tankrathok A., Iglesias-Fernández J., Luang S., Robinson R., Kimura A., Rovira C., Hrmova M., Ketudat
Cairns J. ―Structural analysis and insights into glycon specificity of the rice GH1 Os7BGlu26 -d-mannosidase.‖
Acta Crystallographica Section D Biological Crystallography Vol. D69, No. 10, (2013), 2124-2135.
[3] Luang S., Cho J.-I., Mahong B., Opassiri R., Akiyama T., Phasai K., Komvongsa J., Sasaki N., Hua Y., Matsuba
Y., Ozeki Y., Jeon J.-S., Ketudat Cairns J.R. ―Os9BGlu31 is a transglucosidase with the capacity to equilibrate
phenolpropenoid, flavonoid and phytohormone glycoconjugates.‖ Journal of Biological Chemistry Vol. 288, No.
14, (2013), pp. 10111- 10123.
104
The role of DAXX and ATRX in telomere maintenance and cancer
Zhou Songyang1,2 and Mengfan Tang2
1,2
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030. USA.
2
School of Life Sciences, Sun Yat-sen University, Guangzhou, P.R. China, 510006
Email: songyang@bcm.edu
Mutations in the DAXX and ATRX have been found in human pancreatic neuroendocrine tumors and pediatric
glioblastoma, and correlate with the Alternative Lengthening of Telomeres (ALT) phenotype. DAXX has been shown
to interact with ATRX and mediate histone H3.3 deposition. However, the role of DAXX and ATRX in telomere
maintenance and development of ALT-type cancers remains unclear. We found that endogenous DAXX can localize to
Cajal bodies, associate with telomerase, and regulate telomerase assembly and targeting. Furthermore, disease
mutations located in different regions of DAXX differentially impacted its ability to interact with its binding partners,
and its targeting to Cajal bodies and telomeres. These findings support a DAXX-centric pathway for telomere
regulation. To further understand the function of DAXX and ATRX, we studied DAXX and ATRX protein complexes
by IP mass spec. Our data suggested that DAXX and ATRX have both overlaping and distinct functions in
controlling chromatin integrity, providing new insights into cellular mechanisms that regulate telomere stability and
ALT cancer.
References
[1] Heaphy CM et al., Altered telomeres in tumors with ATRX and DAXX mutations. Science. (2011) 333(6041):425.
[2] Schwartzentruber J. et al., Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric
glioblastoma. Nature. (2012) 482(7384): 226-31.
[3] Goldberg AD et al., Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell.
(2010) 140(5): 678-91.
105
Targeting the RAS signaling network in cancer
Ruibao Ren
State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology,
Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, China
RAS proteins are small GTPases that act as molecular switches, transducing signals from many activated receptors that
regulate cell proliferation, survival, and differentiation. Hyperactivation of RAS, either by oncogenic mutations of RAS
genes or by mutations of genes that regulate the RAS activity, is common in human cancers. Since the enzymatic
activity of RAS is used to turn itself off and is inactive in oncogenic RAS, RAS proteins are considered to be ―nontargetable‖ for developing cancer therapies. Identification of alternative targets that block RAS signaling is critical to
develop therapies for RAS-related cancer. We take both molecular biology and chemical biology approaches to
discover targeted therapies for RAS related cancer. Since the biological activity of RAS proteins relies upon posttranslational modifications (PTMs) that anchor RAS to cellular membranes, one potential approach to block RAS
function is to inhibit PTMs of RAS. We have previously found that expression of oncogenic NRAS induces chronic
myelomonocytic leukemia (CMML)-like or acute myeloid leukemia (AML)-like disease in mice. Using this in vivo
model, we examined the importance of PTMs in NRAS leukemogenesis and found for the first time that palmitoylation
is essential for NRAS leukemogenesis. This study demonstrates for the first time that palmitoylation is an essential
process for RAS leukemogenesis and suggests that targeting palmitoylation may be an effective therapy for RAS
related cancers. We are in the process of developing cancer therapies targeting RAS palmitoylation. In addition, we are
screening anti-RAS compounds that target the RAS signaling network.
106
Biomedical Applications of Aptides
Sangyong Jon
KAIST Institute for the BioCentury, Department of Biological Sciences,
Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Daejeon 305-701, Korea.
E-mail: syjon@kaist.ac.kr
Aptides, a novel class of high-affinity peptides, are designed to possess a ―tweezers-like‖ structure comprising a
unique structure-stabilizing scaffolding region and two high-affinity target-binding components.1 We have produced
aptides with nanomolar-range binding affinities for several targets using phage display selection, demonstrating the
utility of this technology. Indeed, peptides with high target affinity isolated from aptide libraries have offered the
potential for use as diagnostic or drug candidates. An aptide targeting the tumor biomarker fibronectin extradomain B
(EDB) showed remarkable in vivo accumulation at the tumor site. Thus we have utilized the anti-EDB aptide for
various biomedical applications, including targeted drug delivery, cancer imaging by MRI, and targeted protein
therapeutics.2-4 In this lecture, bio-inspired design, characterization, and a couple of biomedical applications of aptides
will be presented.
References
[1] Kim S, Kim D, Jung HH, Lee IH, Kim JI, Suh JY and Jon S*. ―Bio-inspired Design and Potential Biomedical
Applications of a Novel Class of High Affinity Peptides‖, Angew. Chem. Int. Ed., 51, (2012), pp 1890-1894.
[2] Park J, Kim S, Saw PE, Lee IH, Yu MK, Kim M, Lee K, Kim YC, Jeong YY* and Jon S*. ―Fibronectin extra
domain B-specific aptide conjugated nanoparticles for targeted cancer imaging‖, J. Control. Release, 163, (2012),
pp 111-118.
[3] Park J, Park S, Kim S, Lee IH, Saw PE, Lee K, Kim YC, Kim YJ, Farokhzad OC, Jeong YY* and Jon S*.
―HER2-specific aptide conjugated magneto-nanoclusters for potential breast cancer imaging and therapy‖, J.
Mater. Chem. B, 1, (2013), pp 4576 - 4583.
[4] Saw PE, Kim S, Lee IH, Park J, Yu M, Lee J, Kim JI and Jon S*. ―Aptide-conjugated liposome targeting tumorassociated fibronectin for glioma therapy‖, J. Mater. Chem. B, 1, (2013), pp 4723-4726.
107
Protein combination enables facile construction of spatially addressed
antibody library for functional screening
Byeong Doo Song
Scripps Korea Antibody Institute
Antibody has several features including diversity and selectivity that make antibody an excellent drug candidate.
Antibody has been exploited successfully in disease treatment and antibody therapeutics is growing fast with annual
growth of 12%. Currently candidate antibodies are obtained based on affinity for target proteins mostly from screening
a library of mixed antibodies. This type of screening requires preparation of a purified target protein, thus limiting its
accessibility to targets with simple structure, such as secreted or single pass membrane proteins. We developed a
method of protein combination enabling facile construction of spatially addressed antibody library, which allows
screening of individual antibodies based on function regardless of the structure of target proteins including multi-pass
membrane proteins such as GPCRs or ion channels. In addition, functional antibody screening technology (FAST) can
lead to discovery of antibodies with noble functions other than blocking.
108
Design and Evolutionary Engineering of Tyrosine Phenol-lyase Activity from a
Homologous Protein
Seung-Goo Lee, Eugene Rha, Kil-Kwang Kwon, and Haseong Kim
1
Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology,
Daejeon 305-806, Korea.
E-mail: sglee@kribb.re.kr
Tyrosine phenol-lyase (TPL) catalyzes the complicated reversible reaction converting L-tyrosine derivatives to phenol,
pyruvate, and ammonia. The reverse reaction of the ,-elimination has been used for the synthesis of ring-substituted
L-tyrosine derivatives which are useful for therapeutics and antibiotics developments. However, the catalytic turnover
of TPL was extremely low, 2 sec-1, and thermal stability was not good either. The quick loss of TPL activity during Ldopa synthesis was determined to be originated by dissociation of the cofactor, pyridoxal-5-phosphate, during the
reaction, and finally protein engineering of N-terminal in our laboratory could lessen it. Next, a simultaneous
evolution of catalytic rate and thermal stability of the enzyme was attempted by DNA shuffling, although the
flexibility and rigidity issues are difficult to be compatible with each other. After various combinations of mutations,
we concluded that the two issues can be co-evolved if they are managed on different moiety in the protein structure.
Last, we challenged to the evolution of a new TPL from a homologous protein, tryptophan indole-lyase. A rational
library with mutations in the active site was designed and was subjected to a high throughput screening based on flow
cytometry that was developed recently in our laboratory. The method enabled a quantitative analysis of million genes
in a day and we identified finally a new TPL that is different only in four residues from the mentioned homologue.
Thus, this study shows that the protein design and engineering will be more fruitful if quantitative and faster screening
methods are accessible.
References
[1] Choi SL et al., ―Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial
Genetic Circuits‖, ACS Synth Biol. Vol. 3 (2014), pp 163-171.
[2] Rha E et al., ―Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by
directed evolution‖, FEBS J. Vol. 276 (2009), pp 6187-94.
[3] Lee SG et al., ―Inactivation of tyrosine phenol-lyase by Pictet-Spengler reaction and alleviation by T15A mutation
on intertwined N-terminal arm‖, FEBS J. Vol. 273 (2006), pp 5564-73.
[4] Phillips RS, Demidkina TV, and Faleev NG., ―Structure and mechanism of tryptophan indole-lyase and tyrosine
phenol-lyase‖, Biochim Biophys Acta, Vol. 1647 (2003), pp167-72.
109
Protein expression systems for surface proteins
Katsumi Maenaka1,2,3
1
Laboratory of Biomolecular Science, and 2Center for Research and Education on Drug Discovery,
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
3
CREST, Japan Science and Technology Agency, Saitama, Japan.
e-mail: maenaka@pharm.hokudai.ac.jp
Cell surface receptors in eukaryotes as well as viral surface proteins are produced with various posttranslational
modifications and disulfide formation. The E. coli expression system cannot be directly applied to prepare all of these
proteins, but in some cases, we can successfully refold target proteins which do not require the modifications either by
dilution or dialysis method using inclusion bodies. However, these modifications are often necessary for surface
proteins. Therefore, insect and mammalian expression systems attract us to solve these problems. In our laboratory, we
have two main expression systems; (1) transient expression system for HEK293 cells, and (2) baculovirus BmNPVsilkworm expression system. In this talk, I will explain these expression systems using the examples, such as two
glycolipid-recognizing surface proteins, Mincle and CD1, for structural studies.
References
[1] Furukawa A., Kamishikiryo J., Mori D., Toyonaga K., Okabe Y., Toji A., Kanda R., Miyake Y., Ose T.,
Yamasaki S and Maenaka K., ―Structural analysis for glycolipid recognition by the C-type lectins Mincle and
MCL.‖, Proc Natl Acad Sci U S A. Vol. 110, No. 43, pp17438-43 (2013).
[2] Sasaki K., Kajikawa M., Kuroki K., Motohashi T., Shimojima T., Park E.Y., Kondo S., Yagi H., Kato K.,
Maenaka K., ―Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1.‖,
Biochem Biophys Res Commun. Vol. 387, No. 3, pp575-80 (2009).
[3] Hashiguchi T., Ose T., Kubota M., Maita N., Kamishikiryo J., Maenaka K., Yanagi Y., ―Structure of the measles
virus hemagglutinin bound to its cellular receptor SLAM.‖, Nat Struct Mol Biol. 18, No. 2, pp135-41 (2011).
110
Miniaturization of proteins for possible catalytic functions
Abu Bakar Salleh1,3,4, Noor Shartika Jusoh1, Arilla Sri Masayu Abd Rahim1,4 Nur Nadia Razali3,4 ,
Adam Leow Thean Chor2,4.
1 Department of Biochemistry, Faculty of Biotechnology and Biomolecular,
Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor. Malaysia
2Department of Cell Biology, Faculty of Biotechnology and Biomolecular,
Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor. Malaysia
3 Laboratory of Enzyme Technolgy, Institute of Bioscience,
Universiti Putra Malaysia, 43400 Serdang, Selangor Malaysia
4 Enzyme and Microbial Technology Research Center,
Universiti Putra Malaysia, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Enzyme is the preferred catalysts in many applications owing to its specificity of reaction. However, large protein
molecules are vulnerable to destabilizing agents particularly high temperature and organic solvents. One approach is to
look at the native molecule and try to minimize the size while retaining its specific function. Computer modeling was
used in a preliminary study, using templates selected from the database. The protein domains are predicated using the
homolog structure to retain the functionality of the enzyme. By selective splicing, mini proteins are produced. By
molecular dynamics simulations, analyzing the RMSD and RMSF data, radius of gyration and solvent accessible
surface area (SASA) the best model showing high protein folding and compactness. Docking study was carried out to
study the binding interaction between the mini proteins and substrates to select the mini protein with the highest
binding energy. We look at two enzymes: urate oxidase which has clinical applications and amine oxidase which can
be used to monitor fish product freshness.
111
Characterization of Single Amino Acid Substitutions in the β2 Integrin
Subunits of Patients with Leukocyte Adhesion Deficiency (LAD)-1
Siyu Guan1, Suet-Mien Tan1, Jaume Torres1, Gulbu Uzel2, and S.K. Alex Law1
1
School of Biological Sciences, Nanyang Technological University, Singapore.
Laboratory of Infectious Disease, National Institute of Allergy and Infectious Diseases, National Institute of Health, B
ethesda, Maryland, USA.
2
E-mail: syguan@ntu.edu.sg
Leukocyte Adhesion Deficiency 1 (LAD-1) syndrome is characterized by defect in the β2 integrins due to mu
tations in the ITGB2 gene. 18 missense mutations of the β2 integrin subunit were studied. 14 of these mutations
were not able to supportαLβ2, αMβ2, and αXβ2 surface expression: they are T44P, C62R, L105P, D128Y, D134N,
K174E, D238N, A239T, R257W, N282K, D300V, P302L, C557S, and C612R. The other four, namely G150D, S453N,
P648L, and G716A were able to support surface expression of the three β2 integrins. Integrins with the S453N and
P638L mutations have adhesion properties similar to the wild-type counterparts: i.e. αLβ2 with these mutations require
either Mg2+/EGTA or the activating mAb KIM185 for their adhesion to ICAM-1, and the presence of both reagents for
their adhesion to ICAM-3. αMβ2 and αXβ2 with these mutations showed low level of adhesion to denatured BSA but
can be boosted up by either Mn2+ or KIM185. Thus these two ―mutations‖ were probably mischaracterized but
represent rare polymorphisms of the β2 integrin subunit. Integrins with the G150D mutations do not support adhesion
under all conditions tested. It is possibly due to the aspartic acid locking the integrin into a resting state. Introduction of
the mutation into the β3 subunit also results in the expression of the αIIbβ3, which is not able to adhere to fibrinogen
with Mn2+ stimulation. The αLβ2 with the G716A mutation was in an intermediate activation state such that it is
constitutively active in adhesion to ICAM-1 but requires either Mg2+/EGTA or mAb KIM185 to adhere to ICAM-3.
αMβ2 and αXβ2 with this mutation also showed marginal higher level of adhesion to denature BSA. The G716A of
the β2 subunit faces the L1104 of the αL subunit. The αL:β2 residue pairs at this location were studied. Whereas the LF,
and AG combinations yielded active αLβ2; AF, and GF combinations yielded αLβ2 with wild-type like adhesion
properties. Hence, a small and a bulky residue combination at this site is required to maintain the αLβ2 in a resting
state.
112
Modulation of intracellular protein activity
at level of protein folding by beta-turn engineering
Bharat Madan1, and Sun-Gu Lee1
1
Department of Chemical Engineering, Pusan National University, Busan 609735, Korea.
E-mail: bharat@pusan.ac.kr
Control of the intracellular protein activity is very important in various biological studies and biotechnology. This has
generally been achieved at the transcription and translation levels. Although control of the intracellular activity at the
protein folding level is conceptually possible, but there have been few studies. The present study examined this
possibility by modulating the in vivo protein folding rate of green fluorescence protein (GFP) through beta-turn
engineering. A type II‘ two residue beta-turn in GFP was targeted to generate two sets of mutants. First, a switch-off
mutant was designed to stop the protein activity completely. The modulation mutants were then constructed to change
the rates of GFP folding. The design of mutants was based on the rationale that residues i+1 and i+2 of a beta-turn have
defined residue preferences, and their perturbation affects the rate of protein folding. The in vivo fluorescence activity
of the designed GFP variants was switched off and modulated as expected. The change in the in vivo folding patterns
of the mutants was confirmed by SDS-PAGE and found to be similar to the intracellular fluorescence activities of the
mutants. The in vitro refolding kinetics performed with purified variants showed correlations with the in vivo folding
patterns. These results showed that the beta-turns in a protein can be a target for modulating the in vivo protein folding
pattern and activity.
References
[1]
B. Madan, S. Seo , and S.-G. Lee (2014) Structural and sequence features of two residue turns in beta-hairpins. Proteins:
Struct. Funct. Bioinform. In press: doi: 10.1002/prot.24526.
[2]
B. Madan, and S.-G. Lee (2014) Modulation of intracellular protein activity at level of protein folding by beta-turn
engineering. Biotechnol. Bioproc. Eng (Submitted)
113
Serial Femtosecond Crystallography of G Protein-Coupled Receptors
Wei Liu1,2, and Vadim Cherezov1
1
The Scripps Research Institute, Department of Integrative Structural and Computational Biology, La Jolla, California
92037, USA
2
Marine Drug Research Institute, Huaihai Institute of Technology, Lianyungang 222005, China
E-mail: weiliu@scripps.edu
X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties
associated with growing sufficiently large crystals that withstand radiation damage and yield high- resolution data at
synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray
pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin
receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase.
Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two
orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal
motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a
cellular environment.
References
[1] Liu W, Wacker D, Gati C, Han GW, James D, Wang D, Nelson G, Weierstall U, Katritch V, Barty A, Zatsepin
NA, Li D, Messerschmidt M, Boutet S, Williams GJ, Koglin JE, Seibert MM, Wang C, Shah ST, Basu S, Fromme
R, Kupitz C, Rendek KN, Grotjohann I, Fromme P, Kirian RA, Beyerlein KR, White TA, Chapman HN, Caffrey
M, Spence JC, Stevens RC, Cherezov V., ―Serial femtosecond crystallography of G protein-coupled receptors‖,
Science 342: (2013) 1521-1524;
[2] Weierstall U, James D, Wang C, White TA, Wang D, Liu W, Spence JC, Bruce Doak R, Nelson G, Fromme P,
Fromme R, Grotjohann I, Kupitz C, Zatsepin NA, Liu H, Basu S, Wacker D, Han GW, Katritch V, Boutet S,
Messerschmidt M, Williams GJ, Koglin JE, Marvin Seibert M, Klinker M, Gati C, Shoeman RL, Barty A,
Chapman HN, Kirian RA, Beyerlein KR, Stevens RC, Li D, Shah ST, Howe N, Caffrey M, Cherezov V., ―Lipidic
cubic phase injector facilitates membrane protein serial femtosecond crystallography.‖, Nat Commun., (2014),
5:3309.
114
Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the
Glucagon-like Peptide-1 Receptor Core Domain
Jae Young Seong1, Han Choe2 and Mi Jin Moon1
1
Graduate School of Medicine, Korea University, Seoul 136-705, Republic of Korea.
Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul
138-736, Korea
2
E-mail: jyseong@korea.ac.kr
Glucagon-like peptide-1 (GLP1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Although the
crystal structure of ligand-bound extracellular domain of the GLP1R was explored, little is known about interaction
between GLP1 and the heptahelical core domain of GLP1R that confers ligand-induced receptor activation. Previously,
we demonstrated interactions of GLP1 His1 and Thr7 with GLP1R Ile196, Met233, and Asn302. In an extention of our
earlier study, using chimeric and point-mutated GLP1R, we further identified that Asp9 and Gly4 of GLP1 interact
with the evolutionarily conserved basic residue Arg380 flanked by hydrophobic Leu379 and Phe381 in extracellular
loop 3. Molecular modeling suggests that Arg380 may locally neutralize the negatively charged environment of the
binding pocket, allowing approach of Asp9 of GLP1 to this pocket. Leu379 and Phe381 may contribute to receptor
conformation that favors ligand binding although they do not have direct contacts with the ligand. The similar
interaction between Asp9 of peptide and Arg in ECL3 likely occurs glucagon/receptor and glucagon-related
peptide/receptor pairs. Together, this study may shed light on the mechanism underlying high affinity interaction
between GLP1 and the binding pocket that are formed by evolutionarily conserved residues in the GLP1R core domain.
In addition, this study may also explain how evolutionary pressure contributes to selective interactions between the
GLP1 family peptides and their cognate receptors.
References
[1] Moon M.J., Kim H.Y., Park S., Kim D.K., Cho E.B., Park C.R., You D.J., Hwang J.-I., Kim K., Choe H., and
Seong J.Y. ―Evolutionarily Conserved Residues at Glucagon-like Peptide-1 (GLP-1) Receptor Core Confer
Ligand-induced Receptor Activation‖ J Biol Chem 287 (2012), pp 3873-3884
[2] Moon M.J., Choe H., Park C.R., Park S., Kim D.K., Cho E.B., You D.J., Hwang J.-I. and Seong JY ―The ligand
binding pocket formed by evolutionarily conserved residues in the glucagon-like peptide-1 receptor core
domain‖. British Journal of Pharmacology (2014) in press
115
Homology modeling of GPCRs
Han Choe1
1
Department of Physiology, College of Medicine, University of Ulsan, Seoul 138-736, Korea.
E-mail: hchoe@ulsan.ac.kr
Recent technological developments have enabled structural elucidation of 26 GPCRs (113 PDB entries in total) so
far and more structures would be solved in the future. These triumphs provide us opportunities of homology modeling
of GPCRs and thereby understanding of their structure-function relationships (see References). It has been shown that
homology model of GPCR is amenable to structure-based drug design. One of the most critical steps in homology
modeling is the sequence alignment and GPCR is notorious for reliable sequence alignment. I have superimposed PDB
structures of GPCRs and translated it into a sequence alignment. I also have downloaded 370 human GPCR sequences
from the UniProt database excluding olfactory receptors and aligned them using PRALINE. The alignments was read
in EXCEL program and fine-tuned manually. This allows easy setup for homology modeling using MODELLER.
Several EXCEL macros were developed to semi-automate the job.
References
[1] Moon MJ, Kim HY, Park S, Kim DK, Cho EB, Park CR, You DJ, Hwang JI, Kim K, Choe H, Seong JY.,
―Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced
receptor activation‖, J Biol Chem, Vol. 287, No. 6, (2012), pp 3873-84.
[2] Oh DY, Yoon JM, Moon MJ, Hwang JI, Choe H, Lee JY, Kim JI, Kim S, Rhim H, O'Dell DK, Walker JM, Na HS,
Lee MG, Kwon HB, Kim K, Seong JY., ―Identification of farnesyl pyrophosphate and N-arachidonylglycine as
endogenous ligands for GPR92‖, J Biol Chem, Vol. 283, No. 30, (2008), pp 21054-64.
[3] Li JH, Choe H, Wang AF, Maiti K, Wang C, Salam A, Chun SY, Lee WK, Kim K, Kwon HB, Seong JY.,
―Extracellular loop 3 (EL3) and EL3-proximal transmembrane helix 7 of the mammalian type I and type II
gonadotropin-releasing hormone (GnRH) receptors determine differential ligand selectivity to GnRH-I and
GnRH-II‖, Mol Pharmacol, Vol. 67, No. 4, (2005), pp 1099-110.
[4] Acharjee S, Do-Rego JL, Oh DY, Ahn RS, Choe H, Vaudry H, Kim K, Seong JY, Kwon HB. ―Identification of
amino acid residues that direct differential ligand selectivity of mammalian and nonmammalian V1a type
receptors for arginine vasopressin and vasotocin. Insights into molecular coevolution of V1a type receptors and
their ligands‖, J Biol Chem, Vol. 279, No. 52, (2004), pp 54445-53
116
Targeting GPCR’s
Art Cho1
1
Department of Bioinformatics, Korea University, Sejong, Korea.
E-mail: artcho@korea.ac.kr
The study of GPCR‘s as drug targets have been intensified in recent days as more and more X-ray structures of GPCR
are solved. In fact, awarding of the 2012 Nobel prize in chemistry to Lefkowitz and Kobilka was just the beginning of
the race to the discovery of GPCR targeted drugs. Many research groups have been trying to use current-state docking
programs for screening of compound libraries against GPCR targets to varying degrees of success. There is a GPCR
docking competition that gives a hint where the state-of-the-art docking methods stand for GPCR targets. Docking to
GPCR targets is in a few ways quite different from docking to other targets. Binding sites of GPCR‘s are often open
and flexible. They can also encompass both water and lipid regions. One must consider these peculiarities for GPCR
docking. Over the years we have developed docking methodologies utilizing QM/MM calculations in order to take
into account of phenomena that cannot be described by conventional force fields. Recently we devised a docking
protocol based on these QM/MM docking methods, combined with induced fit docking and solvent calculations with
GPCR targets in mind. Application of this protocol to a set of known GPCR cocrystal structures reveals that when the
particularities of GPCR binding sites are carefully treated, the performance of docking to GPCR can be greatly
enhanced.
117
Symposium 5A: Protein anabolism and trafficking
Regulated exocytosis and diabetes
Weiping Han
Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Singapore
E-mail: weiping_han@sbic.a-star.edu.sg
Numerous proteins involved in neurotransmitter release are expressed in endocrine cells, including SNAREs and their
associated proteins. Interestingly, members of the active zone proteins, such as RIM2, Munc13-1 and ELKS are also
present in insulin-secreting cells and other endocrine/neuroendocrine cells. It is not clear, however, whether these
proteins perform similar functions in defining vesicle-plasma membrane fusion sites in endocrine and neuroendocrine
cells. To address this question, we generated pancreas-specific deletion of ELKS1/2 and performed physiology and
biochemical studies. Here I will present evidence that links ELKS to the regulation of insulin secretion.
118
A novel imaging method for systematic super-localizations of Golgi proteins
Tie Hieng Chiong1 and Lu Lei1
1
School of Biological Sciences, Nanyang Technological University, Singapore 637551
E-mail: lulei@ntu.edu.sg
The Golgi apparatus is an extremely complicated organelle consisting of a series of tightly stacked membrane sacs or
cisternae, which are artificially divided into four regions—cis, medial, trans and trans-Golgi network. In secretory
pathway, cargos enter at the cis-Golgi and exit at trans-Golgi or trans-Golgi network. The localization of a Golgi
protein along cis-trans Golgi axis is important in understanding the Golgi protein‘s function. However, such
localization information is usually qualitatively obtained by electron microscopy because the conventional light
microscopy is not able to resolve the cisternal organization of Golgi. We introduce here a novel method to
systematically and rapidly pinpoint the numerical super-localizations of Golgi proteins by conventional light
microscopy. Briefly, Golgi mini-stacks induced by microtubule depolymerizing drug are imaged and centers of
fluorescence masses of Golgi mini-stacks are calculated at nanometer resolution. With reference Golgi markers,
relative localizations of various Golgi proteins are obtained at sub-cisternal resolution along the cis-trans Golgi axis.
Using this method, we monitored the secretion of vesicular stomatitis virus G protein (VSVGtso45), a classical cargo
in probing secretory pathway, and successfully observed its entry at the cis, transition through the medial and exit at
the trans side of Golgi apparatus.
119
BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen
degradation in pulmonary fibrosis
Mi-Rin Leea, Geum-Hwa Leea, Hwa-Young Leea, Hyung-Ryong Kimb, Han-Jung Chaea*
a
Department of Pharmacology and Institute of Cardiovascular Research, Medical School, Chonbuk National
University, Jeonju, Chonbuk, Republic of Korea; bDepartment of Dental Pharmacology, School of Dentistry,
Wonkwang University, Iksan, Chonbuk, Republic of Korea
Endoplasmic reticulum (ER) stress is considered one of the pathological mechanisms of idiopathic pulmonary fibrosis
(IPF). Therefore, we examined whether an ER stress regulator, Bax inhibitor-1 (BI-1), regulates collagen accumulation,
which is both a marker of fibrosis and a pathological mechanism of fibrosis. The presence of BI-1 inhibited the
transforming growth factor (TGF)-β1-induced epithelial–mesenchymal transition of epithelial pulmonary cells and
bleomycin-induced pulmonary fibrosis in a mouse model by enhancing collagen degradation, most likely by enhanced
activation of the lysosomal V-ATPase through glycosylation. We also found a correlation between post-translational
glycosylation of the V-ATPase and its associated chaperone, calnexin, in BI-1-overexpressing cells. BI-1-induced
degradation of collagen through lysosomal V-ATPase glycosylation and the involvement of calnexin were confirmed
in a bleomycin-induced fibrosis mouse model. These results highlight the regulatory role of BI-1 in IPF, and reveal for
the first time the role of lysosomal V-ATPase glycosylation in IPF.
Keywords: idiopathic pulmonary fibrosis, BI-1, epithelial–mesenchymal transition, ER stress, lysosome, V-ATPase
glycosylation
120
Structural Studies on Sorting Nexins
Jinxin Xu1, Tingting Xu1, and Jinsong Liu1
1
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China 510530
E-mail: liu_jinsong@gibh.ac.cn
Sorting nexins (SNXs) are a family of proteins that involved in diverse intracellular endosomal trafficking pathways.
They all contain a PX (phox-homology) domain. The PX domain, via binding to certain phosphoinositide lipids, is
responsible for membrane attachment to organelles of the endosomal system. The PX domain mostly binds to
PtdIns3P, although other specificities have been reported for a few SNXs.
It was previously reported that over expression of SNX10 could induce the formation of giant vacuoles in mammalian
cells. Furthermore, an SNX10/V-ATPase regulated vesicular trafficking pathway was identified to be crucial during
early embryonic development. More recently, several mutations in SNX10 have been linked to osteopetrosis.
Meanwhile, SNX11, a close homologue of SNX10, was found to be able to inhibit SNX10-induced vacuolation. We
recently reported the crystal structure of SNX11 and proposed a novel extended PX domain. Our study on SNX10
confirmed this finding. We also observed different specificities for phosphoinositide lipids between SNX10 and
SNX11. These findings will further our understanding on SNXs‘ roles in endosomal trafficking.
References
[1] Xu, J., T. Xu, B. Wu, Y. Ye, X. You, X. Shu, D. Pei and J. Liu (2013). "Structure of sorting nexin 11 (SNX11)
reveals a novel extended phox homology (PX) domain critical for inhibition of SNX10-induced vacuolation." J
Biol Chem 288(23): 16598-16605.
121
Structural overview of client interaction in Hsp90
complex and application of inhibitor discovery
Sung Jean Park1, Jane H. Dyson2, Jun goo Jee3, and Dongyun Shin1
1
College of Pharmacy, Gachon University, 534-2 Yeonsu 3-dong, Yeonsu-gu, Incheon, 406-799, Korea
2
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037
3
College of Pharmacy, Kyung-pook University, Dea-gu, Korea
E-mail: psjnmr@gachon.ac.kr
The nature of the interactions between the chaperone Hsp90 and its client proteins remains unclear, although the
structure of Hsp90 has been extensively characterized by X-ray crystallography. We present a series of spectroscopic
studies that strongly suggest that these interactions are highly dynamic in solution. Sites of interaction of a client
protein, the p53 DNA-binding domain were then probed both by chemical shift mapping and by NMR saturation
transfer spectroscopy. Specific spectroscopic changes were extremely small and difficult to observe, but were observed
for residues over a wide area of the surface of Hsp90, in the N-terminal, middle and C-terminal domains. These results
are consistent with a highly dynamic and non-specific interaction between Hsp90 and p53 DBD in this simple system,
but an interaction that nevertheless results in changes in the structure of the client protein that are detectable by
spectroscopic and other methods. In addition, we present the result of the interaction study between p23 and p53 DNAbinding domain in the absence of Hsp90 and tried to identify the critical surface features required for this interaction.
We successfully mapped the binding surfaces between p23 and p53 core and found that the interaction was mainly
governed by the electrostatic interactions. These results may provide an insight of the action mechanisms of Hsp90
with client and co-chaperone.
Hsp90 is a good drug target for cancer therapy since it is involved in various cellular signaling. Actually, many
developmental studies are on-going currently and several inhibitors of N-terminal domain of Hsp90 are being clinically
tested. We present here novel compounds that inhibits the C-domain of Hsp90 and discuss the possibility of
development of the C-domain inhibitor as a cancer drug.
References
[1] Park SJ, Borin BN, Martinez-Yamout MA, Dyson HJ. The client protein p53 adopts a molten globule–like state in
the presence of Hsp90. 2011, Nature Structural & Molecular Biology, 18(5):537-41
[2] Park SJ, Kostic M, Dyson HJ. Dynamic Interaction of Hsp90 with Its Client Protein p53. 2011, J Mol Biol.
411(1):158-73.
122
Structural study of TLR8 sensing single stranded RNA in innate
immune system
Toshiyuki Shimizu
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
E-mail: shimizu@mol.f.u-tokyo.ac.jp
The Toll-like receptors (TLRs) are a family of pattern-recognition receptors that recognize microbial components
and initiate subsequent immune responses. Ten members of the human TLR family (TLR1 to TLR10) have been
identified to date. The extracellular domains have leucine rich repeats (LRRs) and are responsible for binding so-called
―pathogen-associated molecular patterns‖.
TLR7 and TLR8 recognize ssRNA and initiate innate immune responses. Moreover, several small molecule
compounds (CL097 etc.) have been identified as TLR7 and TLR8 activators. We determined the crystal structures of
unliganded and ligand-induced activated human TLR8 dimers [1]. Ligand recognition was mediated by a dimerization
interface formed by two protomers. Upon ligand stimulation, the TLR8 dimer was reorganized such that the two Ctermini were brought into proximity. The loop between leucine-rich repeat 14 (LRR14) and LRR15 was cleaved;
however the N- and C-terminal halves remained associated and contributed to ligand recognition and dimerization.
Ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes. A C2-butyl
furo[2,3-c]quinoline was reported with purely TLR8 agonistic activity. This compound (DS877) was successfully cocrystallized with the human TLR8 ectodomain, and the co-crystal structure revealed ligand-induced reorganization of
the binding pocket of TLR8. Focused structure-based ligand design studies led to the identification of 3-pentylquinoline-2-amine as a novel, structurally simple, and highly potent human TLR8-specific agonist [2].
References
[1] Tanji, H, Ohto, U, Shibata, T, Miyake, M, and Shimizu, T, ―Structural reorganization of the Toll-like receptor 8
dimer induced by agonistic ligands‖, Science 339, (2013), pp1426-1429
[2] Kokatla HP, Sil D, Tanji H, Ohto U, Malladi SS, Fox LM, Shimizu T, David SA., ―Structure-Based Design of
Novel Human Toll-like Receptor 8 Agonists‖, ChemMedChem., in press
123
Structural Studies of HIV-1 Co-receptors CXCR4 and CCR5
Qiuxiang Tan1, Ya Zhu1, Gye Won Han2, Xin Xie1, Hualiang Jiang1, Vadim Cherezov2, Hong Liu1, Raymond C.
Stevens2, Qiang Zhao1, Beili Wu1
1
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zuchongzhi Road, Pudong, Shanghai, China 201203
2
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA
E-mail: beiliwu@simm.ac.cn
Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and
development. The G protein-coupled chemokine receptors, CXCR4 and CCR5, are principle co-receptors for HIV-1
infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t
and a cyclic peptide CVX15 at 2.5-3.2 Å resolution, and the 2.7 Å resolution crystal structure of CCR5 bound to the
marked HIV drug maraviroc. Although crystal packing is different, all five CXCR4 structures reveal a consistent
homodimer with an interface involving helices V and VI that may be involved in regulating signaling. CXCR4 and
CCR5 structures provide new clues about the interactions between the receptors and their natural chemokine ligands,
and deepen our understanding of the exact molecular details and mechanism of HIV-1 infection, and address
specificity issues as well as factors that define viral glycoprotein gp120 binding. The location and shape of the ligandbinding sites of CXCR4 and CCR5 differ from other G protein-coupled receptors and are closer to the extracellular
surface. The CCR5 structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for
chemokines and the viral gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling
and viral entry by maraviroc. Structural characterization of ligand binding behavior of CXCR4 and CCR5 lays a
foundation for carrying out next generation drug discovery aimed at inhibiting viral entry of different HIV-1 strains.
124
Finding new tricks of old GPCR modulators —
Drug Repositioning in Autoimmune Diseases
Xin Xie
National Center for Drug Screening,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
We are interested in the roles of G-protein coupled receptors (GPCRs) in the development of Multiple Sclerosis (MS).
We found several GPCRs, which have been targeted to treat other diseased, are involved in the pathogenesis of MS,
such as cysteinyl leukotriene receptor 1 (CysLT1) and adenosine receptor A2B. Several clinically used drugs targeting
these receptors could effectively block the development of EAE, a mouse model of MS. Given our current lack of
effective pharmacological targets for the treatment of MS, the continuous identification and study of GPCRs in MS
pathogenesis may eventually lead to major breakthroughs and new pharmacological strategies.
125
Ultrasensitive immunosensors using redox
cycling combined with enzymatic amplification
Haesik Yang
Department of Chemistry, Pusan National University, Busan 609-735, Korea.
E-mail: hyang@pusan.ac.kr
High signal amplification for the ultrasensitive detection of biomolecules can be obtained by combining enzymatic
reactions of enzyme labels with redox cycling of the products of the enzymatic reactions. Electrochemical detection
corresponds well to redox cycling, because it can trigger and participate in redox cycling [1,2]. In general,
electrochemical signals are limited by the mass transfer of signalling species to electrodes, which becomes worse
when signalling species in a small volume are consumed in a short period [2]. Redox cycling is effective in
regenerating the signalling species consumed and provides high steady-state electrochemical signals [2]. Redox
cycling can be achieved using two working electrodes, such as in an interdigitated array electrode in which an
electroactive species generated at one microband electrode diffuses across a band gap to the second microband
electrode, with the reverse reaction occurring at the second electrode. However, an interdigitated array electrode
requires a microfabricated microband electrode with a short band gap and numerous band pairs to obtain a high redox
cycling efficiency. Redox enzymes can also produce redox cycling via enzymatic reduction or oxidation of
electrooxidized or electroreduced species. The redox-cycling efficiency is highly dependent on the enzyme kinetics
and the amount of a dissolved or immobilized enzyme. We have developed redox cycling schemes that do not need
additional working electrodes and/or redox enzymes. Electrochemical–chemical (EC) redox cycling [3,4] and
electrochemical–chemical–chemical (ECC) redox cycling [5,6,7] allow ultrasensitive detection simply by including
one or two more chemicals in a solution without the use of an additional enzyme and/or electrode. In both cases, the
detection procedures are the same as those in conventional enzyme label-based electrochemical immunosensors. In
this presentation, the signal amplification based on (i) EC redox cycling using a reducing agent and (ii) ECC redox
cycling using a reducing agent and an oxidizing agent will be discussed in detail.
References
[1] Niwa, Electroanalysis 7 (1995) 606.
[2] H. Yang, Curr. Opinion Chem. Biol. 16 (2012) 422.
[3] J. Das, K. Jo, J. W. Lee, H. Yang, Anal. Chem. 79 (2007) 2790.
[4] M. R. H. Akanda, M. A. Aziz, K. Jo, V. Tamilavan, M. H. Hyun, S. Kim, H. Yang, Anal. Chem. 83 (2011)
3926.
[5] M. R. Akanda, Y.-L. Choe, H. Yang, Anal. Chem. 84 (2012) 1049.
[6] M. R. Akanda, T. Vellaiappillai, S. Park, K. Jo, M. Hyun, H. Yang, Anal. Chem. 85 (2013) 1631.
[7] M. R. Akanda, H.-A. Joung, V. Tamilavan, S. Park, S. Kim, M. H. Hyun, M.-G. Kim, H. Yang, H. Analyst 139
(2014) 1420.
126
From High Efficient Protein Micro Chip Toward
Ultra High Sensitive Single Protein Nano Array
Fan-Gang Tseng
Engineering and System Science Dept./NEMS Institute,
National Tsing Hua University, Hsinchu, Taiwan, ROC
Division of Mechanics, Research Center for Applied Sciences,
Academia Sinica, Taiwan, ROC
Protein microarrays have been employed to screen tens to thousands of proteins simultaneously for the observation of
the biochemical activities in the protein-protein, protein-nucleic acid and small molecule interactions. This
presentation provides a novel view from the preparation of high efficient protein micro chip toward ultra high sensitive
single protein molecule array through the technology integration of BioMEMS and Bio-Nanotechnology.
In the high efficient and rapid preparation of protein micro array, micro contact printing system with batch-filling and
parallel-printing capability was employed for rapid generation of protein micro arrays [1], as shown in Fig. 1. This
system provides a passive, gentle, and high throughput way to simultaneously filling and printing tens to thousands of
bio-solutions in seconds into a dense array for disease diagnosis or drug screening [2].
On the other hand, to enhance the signal into single molecule level detection, a novel nano-cone single molecule
detection site was proposed and implemented, as shwon in Fig. 2. This patented binding site can accommodate only
one protein molecule at a time, and allow very low background noise for the detection of single molecule event. The
excitation/detection volume can be reduced into less than sub-aL range (~20-50 nm in diameter), an extremely
localized excitation to greatly reduce background noise [3-6], as shown in Fig. 3. On the other hand, the dynamic range
of the applicable substrate concentrations can be enlarged by localized sample concentration techniques combining the
actions of surface tension gradient and AC electro-osmosis flow[7]. As a result, the detection of substrate
concentration from 1 fM (~1-10 molecules/10000 μm2) to 1 μM is feasible, allowing a 6-9 orders of magnitude of
dynamic range [8].
Fig. 1 3-in-1 protein chip
Fig 2. Single protein
Fig.3 Fabricated device and testing result
nano array
[1] Cheng-En Ho, et al, J. of Microelectromechanical Systems, Vol 17:2, (2008), pp. 309-317.
[2] Shih-Hao Huang, et al, J. Micromech. Microeng. 15, (2005), 2235-2242.
[3] Hui-Wen Cheng, et al, Nanoscale Res. Lett., 8 , (2013), pp 482/1–482/10.
[4] Hui-Wen Cheng, et al, Phys. Chem. C, 117 (25), (2013), pp 13239–13246.
[5] Hsin-Yi Hsieh, et al, Anal. Chem., 81: 19, (2009), pp 7908-7916.
[6] Huang YT, et al, J. of Biological Chemistry, Vol.288(27), (2013), pp 19312-19320.
[7] Ren-Guei Wu, et al, Biosensors and Bioelectronics, Vol.43, (2013), pp 453-460.
[8] Tsung-Yen Lee, et al, Technique Digest of IEEE/ASME MEMS 2010, (2010), pp. 879-882.
127
128
Symposium 5D: Cancer biomarker discovery by proteomics and glycoproteomics
The Advent of High throughput
Glycoproteomics and Application to Biomarker Discovery in Human Plasma
Jong Shin Yoo
1
Department of Mass Spectrometry, Korea Basic Science Institute, Korea
E-mail: jongshin@kbsi.re.kr
Diseases have profound effects on gene expression, including a cell‘s glycosylation machinery. Thus, they produce
glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by
a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate
compared with their protein backbones. Specially N-glycoproteins have the enormously complex heterogeneities in
glycan structures at different attachment sites but few studies characterize their site-specific aberrant glycoforms in
major biological processes.
We developed the method to illustrate the global mapping of major N-glycoproteins in human plasma by high
resolution mass spectrometry with Glycoprotein Analysis (GPA) algorithm. Scoring systems were applied to search the
site-specific N-glycoproteins in UniProt database combined with retrosynthetic glycan library. GlycoProtein Analysis
system with mass spectrometry and automatic scoring algorithm of FDR<1% was first developed for high throughput
analysis of site-specific N-glycoproteins including glycan compositions as well as amino acid sequences.
The GPA platform has been applied for global mapping of total 619 aberrant N-glycopeptides of 123 N-glycoproteins
from Immunoglobulins (~1 mg/ml) to AFP (~20 ng/ml) present in human plasma. We identified distinct glycopeptides
spectra and the aberrant glycoforms in normal and cancer plasma. Evidently the increased levels of fucose, N-acetyl
glucosamine, and highly branched N-glycans in disease specific proteins were found to be associated with cancer
progression, while sialylation decreased. Obviously this method could profoundly facilitate the approach to high
throughput discovery of glycoprotein biomarkers by mass spectrometry.
References
[1] Allison Doerr, ―Glycoproteomics‖, Nature Method 2012, 9, 36
[2] Stavenhagen, K. et. al.,J. Mass Spectrom 2013, 48, 627-639
[3] Pompach and Goldmann et.al., Molecular Cellular Proteomics, 2013, 12, 1281-1293
129
Lung Cancer Proteome Biomarkers:
Discovery, Validation and Clinical Assay Development
Je-Yoel Cho, DVM, PhD
Department of Biochemistry, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
E-mail: jeycho@snu.ac.kr
Without any effective tool for screening and early diagnosis, lung cancer shows the highest mortality in cancer related
death. Molecular biomarkers which can diagnose at earlier stage to predict the disease prognosis or indicate therapeutic
effect of treatment will lead to effective and proper remedy for lung cancer. In this presentation, I will show the
development of our recent lung cancer proteome biomarkers and clinical assay using the identified biomarkers for
simple diagnosis. By using LC-ms/ms–based proteomics approaches combined with glycoprotein or low molecular
weight protein enrichment technologies in the sera of the cancer patients, we discovered several potential lung cancer
protein biomarkers. iTRAQ and label-free quantitative proteomics combined with fucosylated glyprotein enrichment
approaches also revealed that not only the amount of the glycoprotein biomarkers but also their fucosylation levels and
patterns can serve as diagnostic and prognostic serological markers for lung cancers. These biomarkers were validated
by western blot, tissue microarray, lectin-hybrid ELISA, protein chips etc. We also developed multiple reaction
monitoring (MRM) for the quantitative measurement of our lung cancer biomarkers by mass spectrometry in the sera
of the patients. Our MRM data had good agreement with the ELISA data, indicating that the MRM quantification is
reliable and can be a good alternative way for the measurement of serum biomarker levels. Then, we pursued to
develop a pair of target specific monoclonal antibodies which can be used for the pair-test assay development. These
monoclonal antibodies proved for pair-test were produced in high-titer, purified and used for the assay development of
ELISA and Rapid Test Kit. After optimization of the test kits, we finally used them for the test of the sera from lung
cancer patients, healthy controls and other samples in about 500 cases. We are currently applying multiple biomarkers
to the assay kit to improve the specificity and sensitivity for the diagnosis of lung cancers.
References
[1]
Ahn JM, Sung HJ, Yoon YH, Kim BG, Yang WS, Lee C, Park HM, Kim BJ, Kim BG, Lee SY, An HJ, Cho JY.
Mol Cell Proteomics. 2014 Jan; 13(1): 30.
[2]
Ahn JM, Kim MS, Kim YI, Jeong SK, Lee HJ, Lee SH, Paik YK, Pandey A, Cho JY. J Proteome Res.
2014;13(1): 137
[3]
Kang SM, Lee HJ, Cho JY. Cancer Lett. 2013;335(2):487.
[4]
Yoon KA, Cho HS, Shin HI, Cho JY. Stem Cells Dev. 2012;21(18):3391
[5]
Sung HJ, Jeon SA, Ahn JM, Seul KJ, Kim JY, Lee JY, Yoo JS, Lee SY, and Je-Yoel Cho. J Proteomics. 2012;
75(7):2170.
130
The sweet spot of post-translational modifications –
understanding the role of the glycosylation in diseases and health
Hyun Joo An
Graduate School of Analytical Science & Technology, ChungNam National University
Proteins are often modified by the attachment of sugar units called glycans during the normal course of protein
production. It is estimated that over 70% of all human proteins are modified in this way. The development of new
analytical methods, specifically mass spectrometry and separation science, has significantly increased the progress in
understanding the role of the glycome in many biological areas. Research in our group has focused on glycans as
disease markers and their role. For disease markers, a new paradigm for cancer biomarker discovery is proposed. The
detection and analysis of the glycans that decorate the underlying polypeptide in glycoproteins may provide more
specific detection of cancer rather than examining the proteins themselves. There are numerous studies that
demonstrate glycans produced in cancer cells are different from those in normal cells. The aberration in glycosylation
is observed with many types of diseases as well. In the studies in our laboratory, we harvest glycoproteins and extract
the glycans in patient serum to determine whether the glycosylation has changed in cancer patients compared to
healthy patients. This new glycans assay is used to discover biomarkers for the diagnosis of breast, prostate, and
ovarian cancer.
131
Beta-amyloid oligomers inhibit Ca2+-triggered exocytosis by blocking SNARE
assembly
Yoosoo Yang1†, Jaewook Kim1†, YoungSoo Kim2, Hye Yun Kim2, and Yeon-Kyun Shin1,3*
1
Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 136 -791, Republic of
Korea
2
Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 136-791, Republic of Korea
3
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011
It is thought that intracellular beta-amyloid (Aβ), in its soluble oligomeric form, causes cognitive impairment
associated with Alzheimer‘s disease (AD). Essential to cognition is neurotransmitter release at the synapse that
requires soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated vesicle fusion. We
report that in PC12 cells, Aβ oligomers inhibit neurotransmitter release and SNARE assembly. In in vitro settings,
we show that Aβ oligomers bind directly to target membrane SNARE, which results in the inhibition of Ca 2+-triggered
vesicle fusion. Consistently, reduced SNARE-complex assembly is observed in AD transgenic mice. These findings
suggest that intracellular Aβ oligomers impair synaptic transmission by blocking SNARE complex formation.
132
Biomimetic Membranes for
Ion Channel Studies and Engineered Sensor Applications
Tae-Joon Jeon1
1
Department of Biological Engineering, Inha University, Incheon 402-751, Korea
E-mail: tjjeon@inha.ac.kr
Lipid bilayers and their biomimetic analogs are used in a variety of scientific applications and have also been explored
as a platform for highly sensitive and rapid single molecule sensing. Furthermore, ion channels incorporated into such
biomimetic membrane have a number of applications and potential uses including drug screening, drug discovery, and
electrophysiological studies. However, these uses are inhibited by the fragility and short lifetime of the membrane
supporting the protein and lipid bilayer technologies have historically been the purview of experts, precluding its use
as a high-throughput application. In order to address those shortcomings, we are developing an easy-to-use, robust,
automatable, and inexpensive technological platform for membrane formation and channel protein measurement.
Technologies to realize such platform includes membrane stabilization by hydrogel encapsulation/conjugation,
freezing of membrane precursors, and automated high throughput membrane formation. I will present our recent
progress in developing biomimetic membrane platforms for ion channel studies and engineered sensor applications.
References
[1] Kim, Y.R., Jung, S., Ryu, H., Yoo, Y.E., Kim, S.M., Jeon, T.J. ―Review: Synthetic biomimetic membranes and
their sensor applications‖, Sensors, Vol. 12, No. 7, (2012), pp 9530-50.
133
Macromolecular crystallography at SPring-8 and SACLA
Masaki Yamamoto1, Kunio Hirata1, Go Ueno1, Yoshinori Kawano1, Keitaro Yamashita1,
Hideo Ago1 Kazuya Hasegawa1,2, Hideo Okumura1,2 and Takashi Kumasaka1,2
1
RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan.
2
JASRI/SPring-8, Kouto, Sayo, Hyogo 679-5143, Japan.
E-mail: yamamoto@riken.jp
In the past decade, macromolecular crystallography (MX) remarkably developed by generalization of the highbrilliance and the wide energy range of the thirds generation Synchrotron Radiation. We have been developing and
operating seven MX beamlines at SPring-8. An R&D target is the highly precise data collection from small size and/or
weakly-diffracting crystals, for example membrane proteins and large molecule complexes. RIKEN Targeted Proteins
Beamline (BL32XU) [2], has been started user operation since May 2010. In order to analyze micro-crystals smaller
than 10 μm, we have developed the beamline optics and data collection system to acquire high S/N data. The beamline
provide the focused micro-beam with the size and flux of 0.9 × 0.9 μm2 and 6 × 1010 photons/sec, respectively. We
achieved high throughput screening and structure determinations of membrane proteins with the small crystal size.
BL41XU is an undulator beamline with beam size of 10μm to 50μm and the photon flux of 6 ×1011 - 4 × 1013
photons/sec. We are upgrading BL41XU [1] to achieve variable beam size between 5 μm to 50 μm with keeping the
photon flux at the order of 1013 photons/sec for small crystals and high resolution data collection.
In 2012 X-ray lasers SACLA was open for users. We developed a method of femtosecond crystallography using a
goniometer based diffractometer to collect a series of still diffraction images from different parts of a large crystal
cooled at a cryogenic temperature. The combined usage of XFEL and large crystal is expected to provide the
diffraction data free of radiation damage at the high resolution.
We will present the current status and the future prospects of MX at SPring-8 and SACLA. BL32XU project was
supported by Targeted Proteins Research Program and Platform for Drug Discovery, Informatics, and Structural Life
Science from MEXT of Japan.
References
[1]
K. Hirata et.al. ―Achievement of protein micro-crystallography at SPring-8 beamline BL32XU‖, J. Physics: Conf. Series
425, 012002 (2013).
[2]
K.Hasegawa. et al., ―SPring-8 BL41XU, a high-flux macromolecular crystallography beamline.‖, J.
Synchrotron Rad. 20 (6), 910-913 (2013).
134
Crystal Structure of Voltage-gated Proton Channel, Hv1/VSOP
Kohei Takeshita1,2,3, Souhei Sakata2,3, Eiki Yamashita1, Yuichiro Fujiwara2, Akira Kawanabe2, Tatsuki Kurokawa2,4,
Yoshifumi Okochi2, Makoto Matsuda1, Hirotaka Narita1, Yasushi Okamura2,3 and Atsushi Nakagawa1,3
1
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
3
Institute for Academic Initiatives, Osaka University, Suita, Osaka 565-0871, Japan.
4
Present address: Graduate School of Engineering, Kyoto University, Kyoto 615-8530, Japan.
2
E-mail: atsushi@protein.osaka-u.ac.jp
The voltage-gated proton channel, Hv1 (VSOP)1, 2) has a voltage-sensor domain (VSD) but lacks an authentic pore
domain. The VSD of Hv1 plays dual roles of voltage sensing and proton permeation. Hv1 is required for high-level
superoxide production by phagocytes through its tight functional coupling with NADPH oxidase to eliminate
pathogens. Hv1 is also expressed in human sperm and has been suggested to regulate motility through activating
pH-sensitive calcium channels. The activities of Hv1 also have pathological implications, such as exacerbation of
ischemic brain damage and progression of cancer.
We succeeded to solve a crystal structure of mouse Hv1 (mHv1) in the resting-state at 3.45 Å resolution3). The
structure showed a ―closed Wagasa (Japanese umbrella)‖ shape with a long helix consisting of the cytoplasmic
coiled-coil and the voltage-sensing helix, S4, harboring a wide inner-accessible vestibule.
Bijvoet anomalous Fourier map showed that a Zn2+ ion was bound at the extracellular region of mHv1 protomer.
The binding of Zn2+ strongly suggested that the crystal structure of mHv1 represents the resting state, since Zn2+
specifically inhibits activities of voltage-gated proton channels. Actually, two of the sensor residues (R204 (R2) and
R207 (R3)) slide toward the inner membrane side relative to the conserved phenylalanine, F146, on the S2 in a
charge transfer center. On the other hand, many positive residues on the S4 in other VSDs in the activated state
were located above F146. Additionally, the crystal structure of mHv1 showed highlighted two hydrophobic barriers.
Aspartic acid (D108), which is critical for proton selective permeation, would be was located facing intracellular
vestibule below the inner hydrophobic barrier, thereby being accessible to water from the cytoplasm. However,
Another hydrophobic layer of extracellular side would probably ensures interruption of close the proton pathway of
mHv1 in resting state.
These findings provide a novel platform for understanding the general principles of voltage sensing and proton
permeation.
References
[1] Ramsey, I.S., Moran, M.M., Chong, J.A. and Clapham, D.E., ―A voltage-gated proton-selective channel lacking
the pore domain‖, Nature, Vol. 440, (2006), pp 1213-1216.
[2] Sasaki, M., Takagi, M. and Okamura, Y., ―A voltage sensor-domain protein is a voltage-gated proton channel‖,
Science, Vol. 312, (2006), pp 589-592.
[3] Takeshita, K., Sakata, S., Yamashita, E., Fujiwara, Y., Kawanabe, A., Kurokawa, T., Okochi, Y., Matsuda, M.,
Narita, H., Okamura, Y. and Nakagawa, A. ―X-ray Crystal Structure of Voltage-gated Proton Channel‖, Nature
Struct. Mol. Biol., (2014), in press.
135
Protein-protein interaction inhibitors against
drug-resistant cancers
Po-Huang Liang1, Yuan-Feng Lin1, Shih-Hsun Chen1, Tsung-Ching Lai2,
Jinn-Moon Yang3,and Michael Hsiao2
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
2
Genomics Research Center, Academia Sinica, Taipei, Taiwan
3
Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsin-Chu,
Taiwan
E-mail: phliang@gate.sinica.edu.tw
The work in our laboratory has revealed two protein-protein interactions (PPIs) as new anti-cancer targets and
disrupting the PPIs selectively kills drug-resistant cancers. Because PPIs are mediated by amino acids, we previously
generated a library of iodoacetyl amino acids (I-AAs) and found some of these compounds are cytotoxic, by forming
covalent bonds with Cys residues in their targeting proteins. Using the cytotoxic compounds attached with biotin or
fluorescent group, we identified two PPIs, -tubulin:CCT- [1] and XIAP:p19/p12-caspase-7 [3], as the targets of ITrp and I-Lys, respectively. We found I-Trp rendered the cancer cells undergoing cell apoptosis due to covalent
attachment of I-Trp onto Cys354 of CCTat its interface with CCT- , thereby disrupting -tubulin:CCTcomplex, leading to ER-stress to cause cell death [2]. This cell death is more severe in the drug-resistant tumors
because those cells expressed CCT- at higher level. On the other hand, as described in [3], through caspase-3 downregulation (CASP3/DR), which enables cancer cells to survive cancer therapy-induced apoptosis, drug-resistant cancer
cells express structurally and functionally similar caspase-7 for maintaining cellular homeostasis, but caspase-7 forms
complexes with its endogenous inhibitor XIAP (X-linked inhibitor of apoptosis protein). I-Lys is covalently linked
with Cys246 of caspase-7, located in the interface with XIAP, resulting in dissociation of XIAP to trigger caspase-7dependent apoptosis. From clinical samples, CASP3/DR and Caspase-7 accumulation have been found in many
malignancies such as breast, lung and colon cancers, and correlate significantly with poor survival in patients. Because
the XIAP:p19/p12-caspase-7 only exists in the cancer cells, but not in normal cells, and moreover, I-Lys disrupts the
interaction of XIAP with caspase-7, but not caspase-3, the use of I-Lys to induce cancer cell apoptosis is effective and
safe without side-effect, as also revealed by the animal studies. Therefore, this paper provides a basis of developing a
useful cancer targeted therapy. Based on our original observations, we further explored small-molecule reversible
inhibitors by using computer virtual screening for blocking these PPIs to treat drug-resistant cancers (unpublished).
References
[1] Lin Y.F., Tsai W.P., Liu H.G., and Liang P.H.*, ―Intracellular -tubulin/Chaperonin containing TCP1complex serves as a novel chemotherapeutic target against drug-resistant tumors‖, Cancer Research, Vol. 69,
(2009), pp 6879-6888.
[2] Lin Y.F., Lee Y. F., and Liang P.H.*, ―Targeting β-tubulin:CCT-β complexes incurs Hsp90 and VCP-related
protein degradation and induces ER stress-associated apoptosis by triggering capacitative Ca2+ entry,
mitochondrial perturbation and caspase overactivation‖, Cell Death & Disease, Vol. 3, (2012), pp e434.
[3] Lin Y.F., LaiT.C., Chang, C.K., Chen C.L., Huang M.S., Yang C.J., Liu H.G., Dong J.J., Chou Y.A., Teng K.H.,
Chen S.H., Tian W.T., Jan Y.H., Hsiao M., and Liang P.H.* ―Targeting the XIAP/caspase-7 complex selectively
kills caspase-3 deficient malignancies‖, J. Clinical Investigation, Vol. 123, (2013), pp 3861-3875
136
Structural views of glycosylation as potential drug target
Koichi Kato1,2,3,4,5,
1
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
2
Department of Functional Molecular Science, Graduate University for Advanced Studies,
Okazaki 444-8787, Japan.
3
Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural
Sciences, Okazaki 444-8787, Japan.
4
The Glycoscience Institute, Ochanomizu University, Tokyo 112-8610, Japan.
5
GLYENCE Co., Ltd., Nagoya 464-0858, Japan.
E-mail: kkato@phar.nagoya-cu.ac.jp
Many of proteins constituting living systems are modified with sugar chains, which not only affect the physical
properties of proteins such as solubility and thermostability, but also govern their biological properties including serum
half-life and functional protein–protein interactions. Hence, glycosylation is now considered to be one of the most
important factors in the design and development of biopharmaceuticals currently typified by antibody medicines.
Although 3D structural insights into the protein glycosylation are essential for facilitating the biopharmaceutical design,
complexity and heterogeneity of glycan structures, which are not directly encoded in genomes, often discourage
researchers from actively challenging and addressing this important issue. In view of the situation, we have been
developing a systematic method of structural biology for elucidating structures, dynamics, and interactions of
glycoconjugates at atomic level.
In this symposium, I will present atomic views of biopharmaceuticals including therapeutic antibodies provided by Xray crystallography and NMR spectroscopy. The effector functions of immunoglobulin G (IgG) critically depend on Nglycosylation of its Fc region. Our structural data of the complexes formed between IgGreceptor IIIa with N-glycosylation offer structural basis for improved efficacy of therapeutic antibodies on removal of
the core fucose residue from their Fc glycans. Furthermore, our newly developed paramagnetism-assisted NMR
approaches in conjunction with molecular dynamics simulations provide atomic descriptions of dynamic
conformational ensembles of flexible oligosaccharides in solution. Moreover, our NMR approach is also presented for
characterization of glycolipid clusters as unique platforms for interactions of amyloidogenic proteins associated with
neurodegenerative disorders. These approaches will allow new possibilities for structural studies on glycoconjugates of
clinical and pharmaceutical interests, providing insights to design and development of biopharmaceuticals with
glycosylation.
References
[1] Kamiya Y., Satoh T. and Kato K., ―Recent advances in glycoprotein production for structural biology: toward
tailored design of glycoforms‖, Curr. Opin. Struct. Biol. (2014) in press.
[2] Zhang Y., Yamaguchi T. and Kato K., ―New NMR tools for characterizing the dynamic conformations and
interactions of oligosaccharides‖, Chem. Lett., 42, 1455-1462 (2013)
137
Computer-aided drug discovery of Adenosine Receptor modulators using
multiple receptor conformations and network analysis
Sun Choi
National Leading Research Lab of Molecular Modeling & Drug Design, College of Pharmacy, Graduate School of
Pharmaceutical Sciences, and Global Top 5 Research Program, Ewha Womans University, Seoul 120-750, Korea
E-mail: sunchoi@ewha.ac.kr
G-protein coupled receptor (GPCR) plays a role as a major gatekeeper of extracellular signals on plasma membrane,
and it is one of the most important therapeutic targets for various diseases. We recently discovered novel modulators of
A3 adenosine receptor (A3AR), which belongs to GPCR, and found that the minute chemical modifications crossover
the boundary between the agonistic and antagonistic effects. This change was elucidated by our A 3AR homology
models newly constructed using the multiple receptor conformations (MRCs). It turned out that the H-bonding
interaction with Thr94 at the ligand binding site plays a crucial role for the agonistic effect of A 3AR. It is believed that
our new homology models constructed regarding the pharmacological profile of the ligands can facilely predict the
boundary between the agonist and antagonist at the binding site of A3AR.
In order to further investigate the receptor regulation mechanism, we identified the intramolecular signal transduction
of GPCR. Using A2AAR as a model system, we applied the network centrality analysis to the relaxed structures of the
apo and agonist-bound forms of the receptor. Through the analysis of the graph representation of the A2AAR structures,
we calculated the maps of information flow in the receptor and identified the specific residues important for the intramolecular signaling and their pathways. The allosteric hotspot residues identified using our network analyses are in
strong correlation with biochemical data from mutation studies and X-ray crystal structures. Especially, this analysis
precisely identified the location of rotameric micro-switches, which are deemed critical to mediate signal transductions
in GPCRs. Interestingly, Thr88 (Thr94 in A3AR) residue, which plays an important role for the agonistic effect, was
also identified as allosteric hot spot. In addition, the intra-molecular cross-correlation map was calculated based on
equilibrium structural ensemble from molecular dynamics simulation, and the strong signals of long-range
transmembrane communications were observed only in the agonist-bound state. Taken together, these structure-based
modeling studies using MRCs and network analyses provide valuable insights into the agonism and allosteric
modulation of the receptor, and they could be utilized as powerful methods in drug discovery.
138
Developing new anti-malarial agents using
fragment- and structure-based drug discovery
RS Norton1, SS Lim1, SM Devine1, CO Debono1, CA MacRaild1, IR Chandrashekaran1, B Krishnarjuna1, MJ Scanlon1,
PJ Scammells1, SM McGowan2, X Ge3, M Foley3, RF Anders3
1
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville 3052, AUSTRALIA.
2
Department of Biochemistry, Monash University, Clayton 3052, AUSTRALIA.
3
Department of Biochemistry, La Trobe University, Victoria 3086, AUSTRALIA.
E-mail: ray.norton@monash.edu
Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Plasmodium
falciparum to invade human red blood cells. AMA1 has a conserved hydrophobic cleft that is the site of key
interactions with the rhoptry neck protein complex. Our goal is to develop small molecule inhibitors of AMA1 with
broad strain specificity, which we are pursuing using a fragment-based approach [1]. Our screening campaign
identified fragments that bind to the hydrophobic cleft with a high hit rate of 5%, which strongly suggests that a
druggable pocket is present within the cleft. Progress in elaboration of promising fragments will be described [1,2].
Information on the binding sites and binding poses of elaborated fragments is critically important in the efficient
progression of mM fragment hits to µM ligands and eventually nM leads. X-ray crystallography plays a key role, but
rapid solution-state methods are also very valuable. We have employed 19F NMR on AMA1 labelled with fluoro-Trp
residues as a means of confirming binding to the target site on AMA1 and determining whether the bound ligand
triggers conformational changes similar to those induced by peptide ligands for this site [3].
References
[1] Lim SS, Debono CO, MacRaild CA, Chandrashekaran IR, Dolezal O, Anders RF, Simpson JS, Scanlon MJ,
Devine SM, Scammells PJ & Norton RS (2013) Development of inhibitors of Plasmodium falciparum apical
membrane antigen 1 based on fragment screening. Australian Journal of Chemistry 66, 1530–1536.
[2] Devine SM, Lim SS, MacRaild CA, Debono CO, Chandrashekaran IR, Lam R, Anders RF, Scanlon MJ,
Scammells PJ & Norton RS (2014) A critical evaluation of pyrrolo[2,3-d]pyrimidine-4-amines as Plasmodium
falciparum apical membrane antigen 1 (AMA1) inhibitors. Submitted
[3] Ge X, MacRaild CA, Devine SM, Debono CO, Scammells PJ, Anders RF, Foley M & Norton RS (2014)
Ligand-induced conformational changes in Plasmodium falciparum AMA1 detected using 19F NMR. Submitted
139
Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites
for Escherichia Coli D-3-Phosphoglycerate Dehydrogenase
Qian Wang1, Yifei Qi2, Ning Yin2, Luhua Lai1,2*
1
BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Ch
emistry and Molecular Engineering, Peking University, Beijing 100871, China.
2
Center for Quantitative Biology, Peking University, Beijing 100871, China.
E-mail: qwang@mdl.ipc.pku.edu.cn
D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine
biosynthesis [1], and can be allosterically inhibited by serine [2]. In a previous study, we developed a computational
method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation [3]. Two potential
allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and
the other near the regulatory domain (Site II). In the present study [4], we discovered allosteric inhibitors and activators
based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to
eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration
inhibition phenomenon, with IC50 values of 34.8 and 58.0 μM in enzymatic bioassays, respectively. For its activation
activity, compound 2 exhibited an AC50 value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serineand substrate-independent. Enzyme kinetics studies showed that these compounds influenced K m, kcat, and kcat/Km.
Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study
not only identified a novel allosteric site and effectors for PGDH, but also provides a general strategy for designing
new regulators for metabolic enzymes.
References
[1] Al-Rabiee R., Zhang Y., Grant G. A., The mechanism of velocity modulated allosteric regulation in D-3phosphoglycerate dehydrogenase. Site-directed mutagenesis of effector binding site residues, J Biol Chem, 271,
38, (1996), 23235-23238.
[2] Sugimoto E., Pizer L. I., The mechanism of end product inhibition of serine biosynthesis. I. Purification and
kinetics of phosphoglycerate dehydrogenase, J Biol Chem, 243, 9, (1968), 2081-2089.
[3] Qi Y.F., Wang Q., Tang B., Lai L. H., Identifying allosteric binding sites in proteins with a Two-state Gō model
for novel allosteric effector discovery, J Chem Theory Comput, 8, 8, (2012), 2962-2971.
[4] Wang Q., Qi Y. F., Yin N., Lai L. H., Discovery of novel allosteric effectors based on the predicted allosteric
sites for Escherichia coli D-3-phosphoglycerate dehydrogenase, PloS One, in revision.
140
How an intrinsically disordered region functions: a case of CARMIL protein
Motonori Ota1, Syuichi Takeda2, Yuichiro Maéda2 and Ryotaro Koike1
1
Graduate School of Information Science, Nagoya University, Nagoya 464-0848, Japan
2
Structural Biology Research Center, Nagoya University, Nagoya 464-0848, Japan
E-mail: mota@is.nagoya-u.ac.jp
Capping protein (CP) binds to the barbed end of an actin filament and inhibits the further polymerization. V-1 and
CARMIL inhibit CP to bind the barbed end, but their inhibition mechanisms are quite different: V-1 sterically inhibits
the CP binding to actin filament, and allosterically does CARMIL. In addition, CARMIL can uncap CP from actin
filament or V-1. From the X-ray structure of CP/CARMIL complex and the prediction results, it was revealed that an
intrinsically disordered region in CARMIL was used to the inhibition as well as the uncapping.
To elucidate the regulation mechanism of CARMIL, we conducted molecular dynamic simulation for the structures of
free CP, CP/CARMIL and CP/V-1 complexes, and investigate the dynamic properties of CP. For the snapshot
ensemble of CP, Motion Tree was applied and the distribution of rigid bodies was examined. Based on the statistics of
rigid bodies, we found the CARMIL binding suppresses large domain motions of CP, and it would result in the
inhibition and the uncapping.
References
[1] Takeda S et al. ―Two distinct mechanisms for actin capping proteinregulation– steric and allosteric inhibition‖,
PLos Biol. 8 (2010) e1000416.
[2] Takeda S et al. ―Actin capping protein and its inhibitor CARMIL: how intrinsically disordered regions
function‖, Phys. Biol. 8 (2011), 035005
[3] Koike R et al. ―Hierarchical description and extensive classification of protein structural changes by Motion
Tree‖, J. Mol. Biol., 426 (2014), 752-762
141
Systems biology: How to understand the functional interaction
network of proteins?
Kwang-Hyun Cho1
1
Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291
Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
E-mail: ckh@kaist.ac.kr
Proteomics enabled us to observe many protein components in a cell simultaneously, but also led us to encounter
another challenging problem: How to make sense of large-scale proteomics data? Systems biology could provide a
useful answer to this question by integrating such omics data and constructing a 'network' on the basis of the integrated
information. We can then further investigate the dynamics and function of this network through mathematical
modeling and computer simulations. Such systems biological investigation would help us to unravel hidden design
principles of many puzzling biological phenomena and eventually to control critical cell fate decisions. In this talk, I
will show some case studies on biomolecular interaction networks in this regard and use them as a vehicle for
discussing future challenges and opportunities.
References
[1] Kim J., Park S.-M. and Cho K.-H., ―Discovery of a kernel for controlling biomolecular regulatory networks‖,
Scientific Reports (Nature Publishing Group), Vol. 3, No. 2519, (2013), pp. 1-8.
[2] Kim J.-R., Kim J., Kwon Y.-K., Lee H.-Y., Heslop-Harrison P. and Cho K.-H., ―Reduction of complex
signaling networks to a representative kernel‖, Science Signaling, Vol. 4, No. 175, (2011), p
142
Biosynthetic mechanism prediction for a secondary metabolite, ustiloxin B, in
Aspergillus flavus
Nozomi Nagano1, Myco Umemura2, Jin Kawano2, Tomoko Ishii2, Koichi Tamano2, Hideaki Koike3, Miho Izumikawa4,
Kentaro Tomii1, Toshitaka Kumagai5, Goro Terai6, Naoko Nakayama7, Akira Yoshimi8, Keietsu Abe8, Kazuo Shin-ya9,
Kiyoshi Asai1,10 and Masayuki Machida2
1
Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology
(AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
2
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 17-2-1
Higashi-Nijo, Tsukisamu, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan.
3
BioproductionResearchInstitute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1
Higashi, Tsukuba, Ibaraki 305-8566, Japan.
4
Japan Biological Informatics Consortium, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
5
Fermlab Inc., 4-3-1-913 Shirakawa, Koto-ku, Tokyo 135-0021, Japan.
6
INTEC Inc, 1-3-3 Shinsuna Koto-ku, Tokyo 136-8637, Japan.
7
Technology Research Association of Highly Efficient Gene Design, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
8
New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579,
Japan.
9
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi,
Koto-ku, Tokyo 135-0064, Japan.
10
Department of Computational Biology, Graduate School of Frontier Sciences, the University of Tokyo, Chiba 2778568, Japan.
E-mail: n.nagano@aist.go.jp
Ustiloxin B is a toxic cyclic peptide that comprises Tyr-Ala-Ile-Gly (YAIG), a modified tetrapeptide that is
circularized at Tyr and Ile, and norvaline, a non-proteinogenic amino acid.[1] This modified peptide was recently
identified using DNA microarray data and metabolite analyses of the deletion mutants as a secondary metabolite,
coupled with its corresponding biosynthetic gene cluster, in a filamentous fungus, Aspergillus flavus.[2] The gene
cluster contains at least 16 genes, which encode a transcription factor, a transporter, and various enzymes such as
cytochrome P450 and tyrosinase.[2] Initially, this modified peptide was regarded as synthesized by so-called nonribosomal peptide synthetases (NRPS). However, the sequence analyses indicated no NRPS domains were detected in
the gene cluster. [3] Instead, a function-unknown gene, ustA, of which the translated product has a 16-fold repeated
peptide containing the tetrapeptide YAIG, was detected along with two peptidase genes in the ustiloxin B gene cluster.
[3]
These peptidases might be involved in hydrolysis of the repeated peptide of ustA, to produce the ustiloxin
precursors. Consequently, ustiloxin B turned out to be a ribosomal peptide, making it the first case reported for
filamentous fungi. [3] More details of the biosynthetic mechanism of ustiloxin B will be presented in this report.
References
[1] Koiso Y., et al., ―Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by
Ustilaginoidea virens.‖, J Antibiot (Tokyo), Vol. 47, No. 7, (1994), pp 765-773.
[2] Umemura M., Koike H., Nagano N., Ishii T., Kawano J., Yamane N., Kozone I., Horimoto K., Shin-ya K., Asai
K., Yu J., Bennett J.W. and Machida M., ―MIDDAS-M: motif-independent de novo detection of secondary
metabolite gene clusters through the integration of genome sequencing and transcriptome data.‖, PLoS One,
Vol. 8, No. 12, (2013), pp e84028.
[3] Umemura M., Nagano N., Koike H., Kawano J., Ishii T., Miyamura Y., Kikuchi M., Tamano K., Yu J., Shin-ya
K. and Machida M., ―The first identification of a complete biosynthetic gene cluster for a ribosomally
synthesized cyclic peptide in fungi: Ustiloxin B biosynthesis in Aspergillus flavus.‖, submitted to Fungal
Genet Biol.
143
Computer-Aided Drug Design (CADD)
for Human Chymase via Systems Biology
Keun Woo Lee*, Mahreen Arooj, Guang ping Cao, Venkatesh Arulalapperumal
Division of Applied Life Science (BK21 Plus Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant
Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS),
Gyeongsang National University (GNU), 501 Jinju-daero, Jinju, 660-701 Republic of Korea.
E-mail: kwlee@gnu.ac.kr
Chymase (EC 3.4.21.39) is an enzyme of the hydrolase class that catalyzes the hydrolysis of peptide bonds and it is
abundant in secretory granules of mast cells. Chymase is the major extravascular source of vasoactive angiotensin II
(Ang II). The octapeptide hormone, Ang II targets human heart and plays an important role in vascular proliferation,
hypertension and atherosclerosis. Chymase also converts precursors of transforming growth factor-β (TGF-β) and
matrix metalloproteinase (MMP)-9 to their active forms thus contributing to vascular response to injury. The multiple
functions of chymase may play an important role in the development and promotion of various diseases. Therefore,
chymase has become an emerging drug target for many diseases such as cardiovascular diseases, allergic inflammation,
and fibrotic disorders.
The main aim of this research work is to provide a new idea of multi-target drug design thus finding novel uses for
human chymase inhibitors as well as to explore the inhibition mechanism of human chymase. Moreover, various
computational approaches have been applied to find potential chymase inhibitors from various databases. A robust
computational strategy is developed that is applicable to any enzyme system and that allows the prediction of drug
effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural
and functional similarity analyses along with molecular docking studies. Finally, literature survey was carried out to
incorporate these off-targets into various biological pathways and to establish links between pathways and particular
adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin
pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system.
These off-targets and their associated pathways are elucidated for the effect of inflammation, cancer, hemorrhage,
thrombosis and central nervous system diseases (Alzheimer‗s disease). Prospectively, our approach is helpful not only
to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses
for these inhibitors. Moreover, this computational approach is also valuable for applications such as multitarget drug
design and drug combinations. In the next part, to find novel and potent potential chymase inhibitors, various
computational approaches such as structure and ligand-based pharmacophore modeling, molecular docking, and
database screening techniques were applied. Finally, 11 compounds were selected as potential chymase inhibitors
based on their strong binding affinity at the active site of chymase and key interactions with important active site
residues. This study also illustrated that how multiple pharmacophore approach could be more useful in identifying
structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme.
144
Update on the chemokine binding protein from Orf virus
Rafael Couñago1, Karen Knapp1, Stephen Fleming2, Andy Mercer2 and Kurt L. Krause1
1
c
Department of Biochemistry, University of Otago, Dunedin, New Zealand
Department of Microbiology, University of Otago, Dunedin, New Zealand
E-mail: kurt.krause@otago.ac.nz
Poxviruses possess a huge array of non-structural proteins that assist the
virus during. One common poxvirus protein known as CKBP or chemokine
binding protein modulates the host immune response to infection by binding
to chemokines secreted by the host following infection and disrupting the
gradients responsible for leukocyte migration. CKBPs usually bind
chemokines with high affinity and high specificity or with broad specificity
but lower affinity. We have recently described the structure of the
chemokine binding protein from orf virus that displays both high affinity
and broad specificity, an unusual combination in chemokine-protein
interactions. The crystals of this protein were challenging to produce and
the use of small molecule additives resulted in significant optimization.
Although the structure was found to be related to other viral CKBPs it is
also quite distinct in several key areas and displays a broad β sheet on its
surface containing contributions from more than ten β strands. The dimeric
nature of this CKBP appears to be a unique property of its class. Recent
work on complexes of three different chemokines bound to CKBP has
further progressed our understanding of the binding features of this
remarkable protein.
145
Crystal structures of Bax and Bak Reveal Molecular
Events Initiating Apoptosis
Czabotar PE1,2, Brouwer JB1,2, Robin A1,2, Westphal D1,2, Wardak AZ1, Thompson GV1 and Colman PM1,2
1
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia
2
Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
E-mail: presenting_czabotar@wehi.edu.au
The conversion of Bax or Bak from inert monomers into cytotoxic mitochondrial membrane perforating oligomers is a
key event in the pathway to apoptosis. BH3-only relatives can initiate this step through direct interactions, yet the
means by which conformational changes are invoked, the nature of the conformational changes themselves, the
mechanism by which they insert into membranes and the process by which they perforate these barriers has largely
remained a mystery. Our recent structural studies provided the first insights into this process for Bax [1]. We found
that BH3 domains activate Bax by binding to a hydrophobic groove on its surface. Crystal structures of these
complexes revealed an unexpected conformational change involving dissociation of a previously unrecognized ―core‖
domain from a ―latch‘ domain. A further structure of freed Bax ―core‖ domains revealed that these form dimers that
possess a surface of aromatic residues which we hypothesis engages the outer leaflet of the mitochondrial membrane
to induce curvature. We have now extended our structural studies to the related protein Bak and find that it to
undergoes similar conformational transformations and dimerization. These results further our understanding of the
molecular mechanisms by which these highly dynamic proteins control the life/death switch in cells.
References
[1] Czabotar PE, et al., ―Bax Crystal Structures Reveal how BH3 Domains Activate Bax and Nucleate its
Oligomerization to Induce Apoptosis‖, Cell, 152, (2013), pp 519-531
146
A protein allostery that discriminates cyclic nucleotide second messengers
Hyung-Sik Won1, Seung-Hyeon Seok2, Hookang Im2, Min-Duk Seo3, Yoo-Sup Lee2 and Bong-Jin Lee2
1
Department of Biotechnology, Konkuk University GLOCAL Campus, Chungju-si, Chungbuk-do 380-701, Korea.
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742,
Korea.
3
College of Pharmacy, Ajou University, Suwon, Kyeonggi 443-749, Korea.
2
E-mail: wonhs@kku.ac.kr
The prokaryotic global transcription factor CRP has been considered an ideal model for in-depth study of both the
protein allostery and the differential utilization of the homologous cyclic nucleotide second messengers cAMP and
cGMP1-3. Here, atomic details from crystal structures of two inactive CRP species, an apo-form and a cGMP-bound
form, in comparison with a known active conformation, the cAMP-CRP complex4, provide macroscopic and
microscopic insights into the CRP allostery that is coupled with a specific discrimination between the two effectors.
The cAMP-induced conformational transition, including dynamic fluctuations, can be driven by the fundamental
folding forces that cause water-soluble globular proteins to construct an optimized hydrophobic core, including
secondary structure formation. The observed conformational asymmetries underlie a negative cooperativity5 in the
sequential binding of cyclic nucleotides and a stepwise manner of each binding with discrimination between the
effector molecules. Our finding that cGMP, which is specifically recognized in a syn-conformation, induced an
inhibitory conformational change, rather than a null effect, on CRP supports the intriguing possibility that cGMP
signaling could be widely utilized in prokaryotes3,6-9, including in an aggressive inhibition of CRP-like proteins, and
that the cGMP-discriminative mechanics in CRP may have evolved to permit the diversification of its use in other
proteins, including cGMP-regulated protein kinases and ion channels.
References
[1] Seok S.-H. et al., ―Crystal structures of inactive CRP species reveal the atomic details of allosteric transition
that discriminates cyclic nucleotide second messengers‖, Acta Crystallogr. D. (2014) under revision.
[2] Won H.-S., Lee Y.-S., Lee S.-H. and Lee, B.-J., ―Structural overview on the allosteric activation of cyclic AMP
receptor protein‖, Biochim. Biophys. Acta 1794 (2009) 1299-1308.
[3] Won H.-S., Lee T.-W., Park S.-H. and Lee B.-J., ―Stoichiometry and structural effects of the cyclic nucleotide
binding to cyclic AMP receptor protein‖, J. Biol. Chem. 277 (2002) 11450-11455.
[4] Won H.-S. et al., ―Structural understanding of the allosteric conformational change of cyclic AMP receptor
protein by cyclic AMP binding‖ Biochemistry 39 (2000) 13953-13962.
147
Amino Acid Sequence Influences Fibril Formation of the Recombinant
Full-length Prion Proteins
Xu Yan, Jun-Jie Huang, Zheng Zhou, Jie Chen and Yi Liang
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
E-mail: liangyi@whu.edu.cn
It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with
infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases.
Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have
different secondary structures and macromolecular crowding has different effects on fibril formation of the
rabbit/human PrPs, we do not know which amino acids of PrPs cause such differences. Here we construct two PrP
chimeras, in one of which the helical 2 and helix 3 (H2H3) of the human PrP is replaced with that of the rabbit PrP
(rabbit chimera) and in another of which the H2H3 of the rabbit PrP is replaced with that of the human PrP (human
chimera), and use site-directed mutation to analysis how amino acid sequence influences fibrillization of the
recombinant full-length PrPs. We report that the presence of a strong crowding agent dramatically promotes fibril
formation of both PrP chimeras, and amyloid fibrils formed by human chimera have secondary structures and
proteinase K-resistant features similar to those formed by the human PrP. We also find that amyloid fibrils formed by
rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments
different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP.
Furthermore, the amino acids beyond PrP-H2H3 have a remarkable effect on fibrillization of the rabbit PrP but almost
no effect on the human PrP. Our data demonstrate that some PrP chimeras form amyloid fibrils with different
structural features in absence and presence of crowding agents, revealing the importance of macromolecular crowding
on protein misfolding.
References
[1] Zhou Z., Fan J. B., Zhu H. L., Shewmaker F., Yan X., Chen X., Chen J., Xiao G. F., Guo L. and Liang Y.,
―Crowded cell-like environment accelerates the nucleation step of amyloidogenic protein misfolding‖, J. Biol.
Chem., Vol. 284, No. 44, (2009), pp 30148-30158.
[2] Zhou Z., Yan X., Pan K., Chen J., Xie Z. S., Xiao G. F., Yang F. Q. and Liang Y., ―Fibril formation of the
rabbit/human/bovine prion proteins‖, Biophys. J., Vol. 101, No. 6, (2011), pp 1483-1492.
[3] Ma Q., Fan J. B., Zhou Z., Zhou B. R., Meng S. R., Hu J. Y., Chen J. and Liang Y., ―The contrasting effect of
macromolecular crowding on amyloid fibril formation‖, PLoS One, Vol. 7, No. 4, (2012), pp e36288.
148
Structural and functional aspects of viroporins in enveloped viruses
Jaume Torres, Yan Li and Wahyu Surya
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore;
Tel: +65 6316 2857; Fax: +65 6791 3856
E-mail: jtorres@ntu.edu.sg
Coronaviruses and paramyxoviruses are single-strand RNA enveloped-viruses with lethal effects on human populations.
Few effective treatments exist for the infections caused by these viruses, which fuels the search for new molecular
targets. The envelope (E) protein in the severe acute respiratory syndrome coronavirus (SARS-CoV) and the small
hydrophobic (SH) protein in the respiratory syncytial virus (RSV) are short viral-encoded polypeptides, also called
viroporins, that accumulate at the ER-Golgi intermediate (ERGIC) and Golgi compartments, respectively. Deletion of
these viroporins leads to attenuation in animal models and delay of apoptosis in infected cells [1,2]. Both viroporins
have a single α-helical transmembrane (TM) domain, and form pentameric ion channels [3-6] with low or no ion
selectivity [7]. In SARS-CoV E, channel activity appears to increase viral fitness by a poorly understood mechanism
[1]. Structural determination of these viroporins is key to rationalize other effects that depend on interactions with viral
and host binders. We have recently described cytoplasmically exposed membrane-bound α–helices that likely form a
ring around the lumen of the ion channel [5,6]. Good inhibitors for these channels have not been identified, but recent
structural data may offer some solid ground for future drug development.
References
[1] Nieto-Torres, J-L., DeDiego, M.L., Verdiá-Báguena, C., Jimenez-Guardeño, J.M., Regla-Nava, J.A., FernandezDelgado, R., Castaño-Rodriguez, C., Alcaraz, A., Torres, J., Aguilella, V., Enjuanes, L. Severe acute respiratory
syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis (2014) (PLoS
Pathogens, in press).
[2] Fuentes, S., Tran,K.C,. Luthra,P., Teng, M.N., He, B. Function of the Respiratory Syncytial Virus Small
Hydrophobic Protein, J. Virology, 81 (2007) pp 8361–8366
[3] Torres, J., Parthasarathy, K., Lin, X., Saravanan, R., Kukol, A., Liu, D.X. Model of a putative pore: the pentameric
α-helical bundle of SARS coronavirus E protein in lipid bilayers. Biophys. J. 91, (2006) pp 938-947.
[4] Pervushin, K., Tan, E., Parthasarathy, K., Lin, X., Jiang, F.L., Yu, D., Vararattanavech, A., Soong, T.W., Liu D.X.,
and Torres J. Structure and inhibition of the SARS coronavirus envelope protein ion channel. (2009) PLoS Pathogens 5
(7).
[5] Gan, S.W., Tan, E., Lin, X., Yu, D., Wang, J., Tan, G.M., Vararattanavech, A., Yeo, C.Y., Soon, C.H., Soong, T.W.,
Pervushin, K., and Torres, J. The small hydrophobic protein of the human respiratory syncytial virus forms pentameric
ion channels, J. Biol. Chem. 287 (2012), pp 24671-24689.
[6] Li, Y., Surya, W., Claudine, S., and Torres, J. Structure of a Conserved Golgi Complex-targeting Signal in
Coronavirus Envelope Proteins, J. Biol. Chem. (2014, in press).
[7] Verdiá-Báguena, C., Nieto-Torres, J-L., Alcaraz, A., DeDiego, M.L., Torres, J., Aguilella, V.M. and Enjuanes, L.
Coronavirus envelope protein ion channel preference is associated to the polarity of the phospholipid membranes,
Virology 432: (2012) pp 485-494.
149
2D Correlation Analysis of Protein Denaturation
Young Mee Jung1 and Bogusława Czarnik-Matusewicz2
1
Department of Chemistry, Kangwon National University, Chunchon 200-701, Korea.
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland.
2
E-mail: ymjujg@kangwon.ac.kr
Proteins are transformed from the native state into the intermediate states or into the denaturated state under low pH,
high concentrations of denaturant, or heating. The general concept of the intermediate state is currently being
challenged by the results of recent experiments that have enhanced our understanding of the structures of these
proteins. Their structural changes induced by pH and heat have been widely studied elsewhere. In the intermediate
state proteins possess stable regions of localized secondary structure and a largely disordered tertiary structure.
Despite some common features, a coherent picture of the intermediate state cannot be completed. Therefore,
independent investigations are necessary for each case.
The advanced spectral analysis method was utilized to investigate structural changes of protein during transition from
native to the intermediate state. 2D correlation spectroscopy, which is well established analytical technique to interpret
the spectral data obtained under some type of perturbation (e.g., temperature, concentration, pH, etc.), can provide full
scenario of protein unfolding mechanism [1-3]. In this study, the structural changes of protein during transition from
native to intermediate state were investigated in molecular level.
References
Czarnik-Matusewicz B. and Jung Y.M., ―Two-Dimensional Mid-Infrared Correlation Spectroscopy in Protein
Research”, in Optical Spectroscopy and Computational Methods in Biology and Medicine, ed. M. Baranska, Springer,
Germany, 213-250 Vol. 14, (2014).
Czarnik-Matusewicz B., Kim S.B. and Jung Y.M., ―A study of urea-dependent denaturation of -lactoglobulin by
principal component analysis and two-dimensional correlation spectroscopy‖, J. Phys. Chem. B 113 (2009) 559-566.
Litwińczuk A., Ryu S.R., Nafie L.A., Lee J.W., Kim H.I., Jung Y.M. and Czarnik-Matusewicz B., ―The transition
from the native to the acid-state characterized by multi-spectroscopy approach: Study for the holo-form of bovine αlactalbumin‖, Biochim. Biophys. Acta, Proteins Proteomics 1844 (2014) 593–606.
150
Young Scientist Talk I
Temperature-dependent structural changes of Parkinson’s alpha-synuclein
reveal the role of pre-existing oligomers in alpha-synuclein fibrillization
Winny Ariesandi1,2,3, Chi-Fon Chang1, Tseng-Erh Chen1, and Yun-Ru Chen1,2
1
Genomics Research Center, Academia Sinica
Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica
3
Department of Chemistry, National Tsing-Hua University, Hsin-Chu, Taiwan, R.O.C.
2
E-mail: win2y@gate.sinica.edu.tw
Amyloid fibrils of -synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson‘s
disease brains. To enhance the understanding of its structure and functions relationship, we utilized temperature
treatment to study α-synuclein conformational changes. Recombinant human α-synuclein were expressed from E. coli
and purified by osmotic shock procedure. We employed α-synuclein with a short treatment of different temperatures
(20, 40, 60, 80, and 95°C) at the early stage and studied their conformational changes by various biophysical tools. We
found that after 1 hr of high temperature pretreatment, >80°C, -synuclein fibrillization was significantly inhibited.
However, the temperature melting coupled with circular dichroism (CD) spectra showed that -synuclein was fully
reversible and NMR spectroscopy demonstrated no observable structural changes of α-synuclein after 95°C treatment.
By using photo-induced cross-linking of unmodified protein (PICUP) and analytical ultracentrifugation (AUC), rare
amount of pre-existing -synuclein oligomers were found to decrease after high temperature treatment. In addition, a
small portion of C-terminal truncated -synuclein also occurred. The reduction of pre-existing oligomers of αsynuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results
showed that the pre-existing oligomeric species is a key factor contributing to -synuclein fibrillization.
Reference
Ariesandi W., Chang C.F., Chen T.E., Chen Y.R., ―Temperature-dependent structural changes of parkinson‘s alphasynuclein reveal the role of pre-existing oligomers in alpha-synuclein fibrillization‖, PlosONE, Vol. 8, No. 1, (2012),
e53487.
151
In vivo substrate diversity and preference of small heat shock protein IbpB as
revealed by using a genetically incorporated photo-crosslinker
Xinmiao Fu 1, Xiaodong Shi 2 and Zengyi Chang 1
1
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University,
2
Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221002, China
E-mail: fuxinmiao@pku.edu.cn
Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones, are known to be able to protect cells
against stresses and suppress the aggregation of a variety of model substrate proteins under in vitro
conditions. Nevertheless, it is poorly understood what natural substrate proteins sHSPs act upon in living
cells. Here by using a genetically incorporated photo-crosslinker (Bpa), we identified a total of 95 and 54
natural substrate proteins of IbpB (a sHSP from Escherichia coli) in living cells with and without heat
shock, respectively. Functional profiling of these proteins (110 in total) suggests that IbpB, although binding
to a wide range of cellular proteins, has a remarkable substrate preference for translation-related proteins
(e.g., ribosomal proteins and amino-acyl tRNA synthetases) and moderate preference for metabolic
enzymes. Further, these two classes of proteins were found to be more prone to aggregation and/or
inactivation in cells lacking IbpB under stress conditions (e.g., heat shock). Together, our in vivo data offer
novel insights into the chaperone function of IbpB, or sHSPs in general, and suggest that the preferential
protection on protein synthesis machine and metabolic enzymes may dominantly contribute to the wellknown protective effect of sHSPs on cell survival against stresses.
References
Fu X*, Shi X, Yan L, Zhang H, Chang Z.* In vivo substrate diversity and preference of small heat shock protein IbpB
as revealed by using a genetically incorporated photo-crosslinker, J. Biol. Chem., (2013) 288: 31646-54.
152
Conformational dynamics in the regulation of β2-adrenergic receptor signaling
Tae Hun Kim1*, Aashish Manglik2*, Christian Altenbach3, Zhongyu Yang3, Daniel Hilger3, Foon Sun Thian3,
Tong Sun Kobilka3, Wayne L Hubbell2, R Scott Prosser1, and Brian K Kobilka3
1
Departmen of Chemistry, University of Toronto, Mississauga, Ontario L5L 1C6, Canada,
Department of Molecular and Cellular Physiology, Stanford University School of Medicine,
Stanford, California 94305, USA,
3
Jules Stein Eye Institute and Department of Chemistry and Biochemistry,
University of California, Los Angeles, CA 90095, USA
* Both authors contributed equally to this work.
2
E-mail: taehun.kim@mail.utoronto.ca
G protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins 1.
GPCR function relies on allosteric regulation of the intracellular G protein-coupling domain by the extracellular facing
ligand-binding domain1. Despite recently acquired structures of active and inactive conformations of a prototypical
GPCR, the β2 adrenergic receptor (β2AR) 2,3, it remains unclear how ligands regulate conformational changes in
receptors. Here, we utilize 19F-fluorine NMR to directly examine the conformations and dynamics of the G proteincoupling domain of the β2AR4. These studies show that β2AR conformational plasticity is highly regulated by ligand
efficacy and affinity. While NMR reveals a wealth of information regarding the influence of ligand on population and
conformational dynamics, double electron-electron resonance (DEER) spectroscopy provides detail on the distribution
of states5. Taken together, the ligand both influences on-pathway states associated with β2AR activation and lifetimes
of specific spectroscopically detected intermediates. This ―fluid‖ exchange between distinct intermediates stands in
sharp contrast to the tight regulation of protein conformation observed for rhodopsin, and may be responsible for the
complex signaling behavior observed for many GPCRs5.
References
[1]
[2]
[3]
[4]
[5]
Christopoulos, A. & Kenakin, T. G protein-coupled receptor allosterism and complexing. Pharmacol. Rev. 54,
323–374 (2002).
Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–
555 (2011).
Rasmussen, S. G. F. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469,
175–180 (2011).
Kim, T. H. et al. The role of ligands on the equilibria between functional States of a g protein-coupled receptor. J.
Am. Chem. Soc. 135, 9465–9474 (2013).
Altenbach, C., Kusnetzow, A. K., Ernst, O. P., Hofmann, K. P. & Hubbell, W. L. High-resolution distance
mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc Natl Acad Sci USA 105,
7439–7444 (2008).
153
Solubility and supersaturation-dependent protein misfolding revealed by
ultrasonication
Yuxi Lin, Young-Ho Lee, Yuichi Yoshimura, Hisashi Yagi and Yuji Goto
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
E-mail: yxlin@protein.osaka-u.ac.jp
Studies on the phenomenon of amyloid fibrillation by polypeptide molecules have increased dramatically because of
the associations between amyloid fibrillation and various neurodegenerative disorders, e.g., Alzheimer‘s and
Parkinson‘s diseases. In the last decade, a large volume of studies established alcohols can facilitate amyloid
fibrillation under simple and well-controlled environment conditions. So far, however, the underlying mechanism of
alcohol-induced fibrillation is unclear. We studied the alcohol-induced fibrillation of hen egg-white lysozyme at
various concentrations of ethanol, 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Under
the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered
fibrillation. The optimal alcohol concentration depended on the alcohol species. HFIP showed a sharp maximum at 1216%. For TFE, a broad maximum at 40-80% was observed. Ethanol exhibited only an increase of fibrillation above
60%. These profiles were opposite to the equilibrium solubility of lysozyme in water/alcohol mixtures. The results
indicate that, although fibrillation is determined by solubility, supersaturation prevents conformational transition and
that ultrasonication is highly effective in minimizing an effect of supersaturation. We propose an alcohol-dependent
protein misfolding funnel useful for examining the amyloidogenicity (Fig.1). This misfolding funnel will apply to
fibrillation under physiological conditions where biological environments play important roles in decreasing the
solubility [1].
Fig.1 Solubility- and supersaturation-dependent protein misfolding funnels.
References
[1] Lin Y., Lee Y-H., Yoshimura Y., Yagi H. and Goto Y., ―Solubility and Supersaturation-Dependent Protein
Misfolding Revealed by Ultrasonication‖, Langmuir, Vol. 30, No. 7, (2014), pp 1845-1854.
154
MARCH5-mediated quality control on acetylated Mfn1 facilitates
mitochondrial homeostasis and cell survival
Yong-Yea Park1 Oanh T. Kim Nguyen1,2 and Hyeseong Cho1,2
1
Department of Biochemistry, Ajou University School of Medicine, Suwon, Korea
Department of Biomedical Sciences, Graduate School of Ajou University, Suwon, Korea
2
E-mail: yongnye@ajou.ac.kr
Mitochondrial dynamics and quality control play a central role in the maintenance of cellular integrity. Mitochondrial
ubiquitin ligase MARCH5 regulates mitochondrial dynamics. Here, we show that mitochondrial adaptation to stress is
driven by MARCH5-dependent quality control on acetylated Mfn1. Under mitochondria stress conditions, levels of
Mfn1 were elevated two fold and depletion of Mfn1 sensitized these cells to apoptotic death. Interestingly,
overexpression of Mfn1 also promoted cell death in these cells, indicating that a fine-tuning of Mfn1 levels is
necessary for cell survival. MARCH5 binds Mfn1 and the MARCH5-dependent Mfn1 ubiquitylation was significantly
elevated under mitochondria stress conditions along with an increase in acetylated Mfn1. The acetylation deficient
K491R mutant of Mfn1 showed weak interaction with MARCH5 as well as reduced ubiquitylation. Neither was
observed in the acetylation mimetic K491Q mutant. In addition, MARCH5 knockout MEF and MARCH5H43W
expressing HeLa cells lacking ubiquitin ligase activity experienced rapid cell death upon mitochondrial stress. Taken
together, a fine balance of Mfn1 levels is maintained by MARCH5-mediated quality control on acetylated Mfn1 which
is crucial for cell survival under mitochondria stress conditions.
References
[1] Park YY, Lee S, Karbowski M, Neutzner A, Youle RJ, Cho H. Loss of MARCH5 mitochondrial E3 ubiquitin
ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1. J Cell Sci 2010 Feb 15;
123(Pt 4): 619-626.
[2] Yonashiro R, Ishido S, Kyo S, Fukuda T, Goto E, Matsuki Y, et al. A novel mitochondrial ubiquitin ligase plays a
critical role in mitochondrial dynamics. EMBO J 2006 Aug 9; 25(15): 3618-3626.
[3] Tondera D, Grandemange S, Jourdain A, Karbowski M, Mattenberger Y, Herzig S, et al. SLP-2 is required for
stress-induced mitochondrial hyperfusion. The EMBO journal 2009 Jun 3; 28(11): 1589-1600.
[4] Chen H, Vermulst M, Wang YE, Chomyn A, Prolla TA, McCaffery JM, et al. Mitochondrial fusion is required for
mtDNA stability in skeletal muscle and tolerance of mtDNA mutations. Cell 2010 Apr 16; 141(2): 280-289.
155
Membrane Curvature Affects the Fibrillation of Amyloid β
Mayu S. Terakawa1, Hisashi Yagi1,2 and Yuji Goto1
1
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
2
Center for Research on Green Sustainable Chemistry, Tottori University, 4-101 Koyama-naka,
Tottori 680-8552, Japan.
E-mail: ygoto@protein.osaka-u.ac.jp
The deposition of amyloid β peptides (Aβ) is a pathological hallmark of Alzheimer‘s disease. It was reported that Aβ
interacted specifically with lipid rafts. However, several studies suggested that Aβ also interacted with
phosphatidylcholine (PC) by deforming PC membranes with accompanied membrane curvature [1]. Although the
deformation of the membranes has been observed, effect of changing curvature on aggregation kinetics of Aβ remains
unclear.
In this study, we aimed to understand how membrane curvature affects the Aβ fibril formation processes. 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was chosen as a component of the liposomes. This choice enabled us to focus
on the membrane curvature because it eliminated other effects on the fibrillation (e.g. the electrostatic interaction).
Surprisingly, we found that small size liposomes made of DOPC (≤ 50 nm) accelerated the nucleation reaction
significantly. We hypothesized that hydrophobic exposure of liposomes caused the acceleration of the nucleation
reaction. Our analysis suggested that the smaller size liposomes exposed hydrophobic area more and this exposure is
responsible for the shortened lag time. Thus, we need to consider not only the lipid species but also the effects of
membrane curvature on the interactions between Aβ and lipid membranes.
References
[1] Martina P., Danilo M., Antonio R., Mikko K. and Carmelo R., ―Analytical model and multiscale simulations of Aβ
peptide aggregation in lipid membranes: towards a unifying description of conformational transitions,
oligomerization and membrane damage‖, Phys. Chem. Chem. Phys., Vol.15, (2013), pp8940-8951.
156
Young Scientist Talk II
Characteristics of oxidized LDL and technetium 99m for non-invasive
SPECT/CT imaging of atherosclerotic diseases
Chi-Bao Bui1,3, Dieu Hang Bui2 and Tan Son Vo1
1
Department of Medicine, University of medicine and pharmacy Hochiminh, Vietnam.
2
Department of Nuclear Medicine, Cho Ray Hospital , Hochiminh, Vietnam.
5
School of Biotechnology, International Universiry, Vietnam National University, Hochiminh, Vietnam.
E-mail: bcbao@ump.edu.vn
Oxidized low-density lipoproteins (OxLDL) such as malondialdehyde-modified LDL (MDA-LDL) are involved in the
development atherosclerosis lessions. This study was designed an approach to examine and characterize technetium99m (99mTc) labeled MDA-LDL that could serve as standardized protocol to improve detection of OxLDL for the early
diagnosis of asymptomatic atherosclerosis.
Methods: In vitro screening of Biocore kinetics, in which 99mTc is attached to the chip, and peptides interact with
different parts of Malondialdehyde (an OxLDL). Study on twenty-two male Wistar rats (Rattus norvegicus) received a
fat-diet associated with coronary artery injury. After 32 ± 9 days (mean ± SD), and Wistar undergoing coronary
angiography with administration of 403.3 ± 48.1 MBq of 99mTc-OxLDL, and underwent the SPECT-CT. The
immunohistochemistry of aorta sections would be confirmed the labeled 99mTc -OxLDL.
Results: Increase the stable interaction between 99mTc and OxLDL through covalent binding. In rat, atherosclerotic
lesions are mainly level II. Thirteen of 22 showed lesions scan-positive with 99mTc-OxLDL. Percentage of injured
vessels compared to control vessels was 2.38 ± 0.61 and 1.27 ± 0.23 positive to negative (P <0:05).
Conclusion: 99mTc -OxLDL indicates to noninvasive SPECT/CT imaging to localize plaque in the coronary vessels.
A
Figure: 99mTc -OxLDL in assessing atheroclerotic plaque by SPECT/CT imaging.
References
[1] Hansson GK., "Inflammation, atherosclerosis, and coronary artery disease". N Engl J Med, Vol 352 (2005), pp
1685-1695.
[2] Bui CB., Chau CG., Vo TS., Kim HD, Shin J., ―Investigation of the binding oxidized LDL with technetium 99m
in atheroclerosis diseases‖, Clinical Medicine and pharmacy IAEA Vienna, Austria, Vol. 8 (2013), pp 192-197.
[3] Bui CB., Chau CG., Ho NAM, Vo TS., ―Using non-invasive assays to improve detection of oxidized low density
lipoproteins (Ox LDL) in Atherosclerosis‖, International Conference on integrated mdical imaging in
cardiovascular diseases, IAEA Vienna, Austria, Vol. 1, P-263, (2013), pp 168.
[4] Blankenberg FG., Katsikis PD., Tait J., Davis E., Naumovski L., Ohtsuki K.. "Imaging of apoptosis (programmed
cell death) with 99mTc annexin V". J Nucl Med. Vol 40 (1999), pp184–191.
157
The effect of environmental factors on amyloid assembly of insulin
Tae Su Choi1, Hugh I. Kim*,1,2
1
Department of Chemistry, Pohang University of Science and Technology (POSTECH),
Pohang, 790-784, Republic of Korea
2
Division of Advnaced Materials Science, Pohang University of Science and Technology (POSTECH), Pohang, 790784, Republic of Korea
E-mail: hughkim@postech.edu
Amyloidogenic proteins tend to be aggregated into unbranched β-sheet rich amyloid fibril in vivo. This phenomenon is
closely related to amyloidogenic diseases such as Alzheimer‘s disease, Parkinson‘s disease, type 2 diabetes, and
spongiform encephalopathy.[1] Amyloid fibrillation is generally initiated by exposing hydrophobic residues of
amyloidogenic proteins, and self-assembly process is promoted by hydrophobic interaction of the residues. [2] Both
factors are considered as a driving force for amyloid fibrillation, but their correlation is not yet fully understood. In the
present study, we investigate protein-solvent interaction during amyloid assembly process of insulin which causes
injection-localized amyloidosis to understand the correlation between environmental factors and protein fibrillation.
We use formamide derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) as a model system to
systematically control denaturing power and hydrophobicity of solvents. Thioflavin T (ThT) assay and transmission
electron microscopy (TEM) reveal that the binary mixture of water and organic solvents promotes fibrillation rates
and β-sheet abundance of insulin fibril. Solution small-angle X-ray scattering (SAXS) combined with molecular
dynamics (MD) simulation suggests that the structural conversion of B11-B17 core residues from α-helix to random
coil is crucial for the fibrillation kinetics of insulin. Differential scanning calorimetry (DSC) results reveal that
protein-protein interaction for amyloid fibrillation is governed by solvophobicity of the exposed core residues. Our
study suggests that both denaturation and solvophobic interaction of the core residues are prerequisite for the
fibrillation process of insulin.
References
[1] Chiti, F., and Dobson, C. M., ―Protein Misfolding, and Functional Amyloid, and Human Disease‖, Ann. Rev.
Biochem., Vol 75, (2006), pp 333-366.
[2] Cohen, F. E., and Kelly, J. W., ―Therapeutic approaches to protein-misfolding diseases‖, Nature, Vol 426, Issue
6968, (2003), pp 905-909.
158
Toxicoproteomics revealed neuronal projection and developmental
impairment of human neuroblastoma cells upon treatment with fipronil
Khanit Ruangjaroon1, Daranee Chokchaichamnankit2, Chantragan Srisomsap2, Jisnuson Svasti2,3 and
N. Monique Paricharttanakul2
1
Environmental Toxicology, Chulabhorn Graduate Institute, Bangkok, Thailand 10210;
Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand 10210; 3Applied Biological
Sciences, Chulabhorn Graduate Institute, Bangkok, Thailand 10210; Tel:+66-2-5538555; Fax: +66-2-5538560
2
E-mail: d08110304@cgi.ac.th
Fipronil is an N-phenylpyrazole insecticide extensively used in agriculture, domestic households and veterinary fields for pest
control. As the use of fipronil increases to replace older generations of pesticides, the toxicological profiles of fipronil increases
with unclear mechanisms of toxicity, thus posing a human health risk. This study was aimed to investigate the toxicoproteomic
effects and to elucidate the toxic mechanism of fipronil on SH-SY5Y, a human neuroblastoma cell line. Forty-eight hour exposure
of SH-SY5Y to fipronil at the concentrations of 43 µM (75% cell viability) and 78 µM (50% cell viability) led to differential
protein expression of 40 protein spots as compared to vehicle control using two dimensional polyacrylamide gel electrophoresis,
with statistical significance (p-value<0.05). Identification using LC/MS/MS and MASCOT database revealed that six proteins
involved in neuronal projection were up-regulated in expression and six structural proteins down-regulated when treated with
fipronil. From ImageMaster analysis, vimentin, a type III intermediate filament that plays a crucial role in the initiation of
neuronal projection, shows the most dramatic increase in expression and segregation in a dose-response manner to fipronil and
confirmed using Western blot analysis. Further experiments are underway to confirm and validate the toxic mechanism(s) of
fipronil on neuronal cells.
159
Inhibition of α-synuclein oligomer toxicity by epigallocatechin gallate.
Yuichi Yoshimura1
1
Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University,
Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark
E-mail: yuichi@chem.au.dk
Oligomeric species of various proteins are linked to pathogenesis of different neurodegenerative disorders.
Consequently, there is intense focus on the discovery of novel inhibitors to block their toxicity. In Parkinson‘s disease,
α-synuclein forms cytotoxic oligomers, consisting of ~30 monomers on average [1]. Epigallocatechin gallate (EGCG)
has previously been shown to redirect the aggregation of α-synuclein monomers and remodel α-synuclein amyloid
fibrils into disordered aggregates [2,3]. In this study, we investigated the effect of EGCG on the toxicity of the αsynuclein oligomers.
EGCG inhibited the ability of the oligomers to permeabilize membranes and rescued rat neuronal cells from their
toxicity. Liquid-state NMR spectroscopy showed that the N-terminus and the central hydrophobic region of αsynuclein build up the oligomer core whereas the C-terminus remains disordered in the oligomer state, and the
flexibility of the C-terminus decreased upon EGCG binding. EGCG binds to the oligomers without changing either
their secondary structure or size distribution. Thus inhibition of membrane permeabilization and the toxicity is not due
to dissociation or aggregation of the oligomers. Rather, EGCG inhibits the toxicity by reducing their binding affinity
to membranes, highlighted as a viable therapeutic approach against Parkinson‘s disease.
References
[1] Lorenzen, N. et al., J. Am. Chem. Soc., 136, 3859-3868 (2014).
[2] Ehrnhoefer, D. E. et al., Nat. Struct. Mol. Biol., 15, 558-566 (2008).
[3] Bieschke, F. et al., Proc. Natl. Acad. Sci. USA, 107, 7710-7715 (2010)
160
Discovery of Compounds that Can Bind to Intrinsically Disordered Proteins
Chen Yu1, Xiaogang Niu2, Fan Jin3, Zhirong Liu1, 3, Changwen Jin1, 2 and Luhua Lai1, 3
1
BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871, China.
2
Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China
3
Center for Quantitative Biology, AAIS, Peking University, Beijing 100871, China.
Email: cyu@mdl.ipc.pku.edu.cn
Intrinsically disordered proteins (IDPs) show little ordered structures under physiological conditions. Roughly 10%
of proteins in various genomes have been predicted to be fully disordered. IDPs occur more often in disease related
genes and may be used as potential drug design targets. However, due to its dynamic nature, traditional structurebased drug design approaches are ineffective or even infeasible for designing molecules targeting IDPs. Metallo S. J.
et al. discovered several inhibitors targeting a disordered range of cMyc using circular dichroism and NMR
experiments1. These inhibitors induce ordered local structures, suggesting that small molecules can directly target the
IDPs‘ disordered regions and disrupt protein-protein interaction involving IDPs. Inspired by their study, we want to
develop a general approach for discovering compounds that can bind to IDPs. Although IDPs are highly dynamic,
representative lower-energy conformations can be obtained by conformational space sampling and be used in drug
design. We chose the oncoprotein cMyc370-409 as our test system. A low energy conformation was built based on the
NMR data reported2 and used for database screening by molecular docking. CD assay was used to experimentally
screen the purchased compounds. Seven compounds caused large conformational change signals in CD spectra and
their binding constants to cMyc370-409 were estimated to be in the micromolar range. Five out of the seven compounds
and 10074-A41 showed growth inhibition of HL-60 cells at micromolar level and arrested HL-60 cells at S Phase.
Compound DCSD28 and 10074-A41 showed tendency to bind to 375FALRDQIPELE385 in MD simulation results.
Obvious chemical shift changes showed in total correlation spectroscopy (TOCSY) and heteronuclear single quantum
coherence (HSQC) spectroscopy of cMyc370-409 with or without DCSD28. Saturation transfer difference (STD)
spectroscopy also indicated DCSD28 could bind to cMyc370-409. Further SPR studies to confirm the compounds‘
disruption of cMyc and Max heterodimerization are under investigation.
References
[1] Hammoudeh, D. I.; Follis, A. V.; Prochownik, E. V. and Metallo, S. J., Multiple Independent Binding Sites for
Small-Molecule Inhibitors on the Oncoprotein c-Myc, Journal of the American Chemical Society, 2009, Vol. 131,
No. 21, pp 7390-7401.
[2] Jin, F.; Yu, C.; Lai, L.; Liu, Z., Ligand Clouds around Protein Clouds: A Scenario of Ligand Binding with
Intrinsically Disordered Proteins, PLoS Computational Biology, 2013, Vol. 9, No. 10, e1003249.
161
Manipulation of host signal transduction
by secreted effectors from enteropathogenic E. coli
Li Zhang1, 2, Shan Li2, Xiaojun Ding2, Jixin Cui2, Hao Xu2, Qing Yao2, Lin Li2, Wenqing Gao2, Jie Rong3, Xing Chen3,
She Chen2 & Feng Shao2
1
Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College,
2
National Institute of Biological Sciences, Beijing 102206, China.
3
Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology,
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
E-mail: zhangli@nibs.ac.cn
Enteropathogenic E. coli (EPEC) delivers proteins called ―effectors‖ into host cells through type-III secretion system
and manipulates host signal transduction to ensure successful infection. NleE, an effector conserved in many
enterobacteria, is majorly responsible for inhibition of NF-κB pathway. We discover that NleE directly inactivates
human TAB2 and TAB3 to disrupt ubiquitin-chain sensing in NF-κB signaling. NleE harbored an unprecedented Sadenosyl-L-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in
the zinc finger domains in TAB2 and TAB3. Cysteine-methylated TAB2 and TAB3 lost the zinc ion as well as the
ubiquitin-chain binding activity; hence NF-κB activation is blocked subsequently. Another effector called NleB blocks
host death receptor signaling by directly inactivating death domains of several proteins, including TRADD, FADD,
RIPK1 and TNFR1 by transferring an N-acetylglucosamine (GlcNAc) onto a conserved arginine. This unappreciated
arginine GlcNAcylation of death domains blocked homotypic/heterotypic death domain interactions and assembly of
the oligomeric TNFR1 complex and FADD-mediated death-inducing signaling complex. The arginine GlcNAc
transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. Therefore,
EPEC effector repertoires not only provide a unique angle to study host signal transduction but also greatly add to the
diversity of enzyme catalysis.
162
Abstract
(Poster Presentaions)
P1
-
P7
Proteins in emerging fields (1a,4a)
P8
-
P28
Protein folding and dynamics (1c)
P30
-
P37
Protein catabolism & anabolism (2a, 5a)
P38
-
P55
Proteins in diseases (1b, 2b)
P56
-
P59
Proteins analyses techniques (2c, 3c)
P60
-
P71
Protein modification (3a)
P72
-
P80
Protein as therapeutics (3b, 4b)
P81
-
P86
Protein design and engineering, nanobio (4c, 5c)
P87
-
P92
Proteins as drug targets (5b)
P93
- P100
Proteins in membranes (6a)
P101 - P116
Proteins and drug discovery (6b)
P117 - P119
Protein bioinformatics (6c)
P120 - P149
Frontiers in protein sciences (1d,2d,3d,6d, etc)
163
Poster Abstracts
encoding the cytosolic portion of the enzyme was cloned and expressed in a
heterologous expression system and the conditions for its solubility were
optimized. This study will aid in purification of expressed protein and
attempting for their crystallization for structure determination. Till now not
even a single crystal structure from the entire family has been reported.
Proteins in emerging fields
References
[1] Patil C G. 2004. Nuclear DNA amount variation in Cyamopsis D C
(Fabaceae). Cytologia. 69: 59-62
[2] Mishra S. 2008. India guar gum exports up on industry demand. In.
reuters.com. Retrieved 2011-04-18.
[3] Naoumkina, M., Torres-Jerez, I., Allen, S., He, J., Zhao, P. X., Dixon, R. A.
and May, G. D. (2007). Analysis of cDNA libraries from developing seeds of
guar (Cyamopsis tetragonoloba (L.) Taub). BMC plant biology 7(1):62
[4] Dhugga KS, Barreiro R, Whitten B, Stecca K, Hazebroek J, Randhawa
GS, Dolan M, Kinney AJ, Tomes D, Nichols S, Anderson P. 2004. Guar
seed beta-mannan synthase is a member of the cellulose synthase super gene
family. Science. 16;303:363-6.
P1
Structure and function analyses of the vegetative insecticidal protein
Vip3A from Bacillus thuringiensis
Thittaya Kunthic1, Boonhiang Promdonkoy2 and Panadda Boonserm1
1
Institute of Molecular Biosciences, Mahidol University, Nakhonpathom,
Thailand
2
National Center for Genetic Engineering and Biotechnology, National
Science and Technology Development Agency, Pathum Thani, Thailand
E-mail: nailty@hotmail.com
Vip3A insecticidal proteins are produced from Bacillus thuringiensis (Bt)
during vegetative growth and exert a broad spectrum of toxicity against
lepidopteran insect species. The amino acid sequences of Vip proteins have no
homology to those of Bt δ-endotoxins (Cry/Cyt toxins), implying their diverse
mechanisms of toxicity. It is thus highly desirable to integrate Vip proteins
into insect control programs to reduce the development of resistance in insect
populations. So far, no three-dimensional structure of Vip3A proteins has been
available and their mode of action is still unclear. Therefore, this project aims
to study both structure and function of the Vip3Aa. Here, Vip3Aa toxin was
expressed in Escherichia coli as a soluble protoxin. Bioassays using the
protoxin fed to susceptible Spodoptera exigua larvae showed the LC50 value
of 355 ng/cm2. Crystals of Vip3A have been formed under conditions
containing LiSO4 and/or PEG 400-4,000 at pH 6.5-8.5 via hanging drop
vapour-diffusion technique. However, better protein crystals are required for
further X-ray diffraction analysis. Site-directed mutagenesis was used to
investigate the role in the toxicity of selected aromatic and charged amino
acids. Results suggest that Y619 plays an important role in the toxicity of
Vip3Aa toxin.
P3
Crystal structure of the BinB component of Lysinibacillus sphaericus
binary toxin
Kanokporn Srisucharitpanit1,4, Min Yao2, Boonhiang Promdonkoy3, Sarin
Chimnaronk1, Isao Tanaka2, Panadda Boonserm1
1
Institute of Molecular Biosciences, Mahidol University, Salaya,
Phuttamonthon, Nakhon Pathom 73170, Thailand
2
Faculty of Advanced Life Sciences, Hokkaido University, Sapporo 060-0810,
Japan
3
National Center for Genetic Engineering and Biotechnology, National
Science and Technology Development Agency, 113 Pahonyothin Road, Khlong
Nueng, Khlong Luang, Pathum Thani 12120, Thailand
4
Faculty of Allied Health Science, Burapha University, 169 Long-Hard
Bangsaen Road, Saensook, Muang District, Chon Buri , 20131, Thailand
E-mail: panadda.boo@mahidol.ac.th
References
[1] Estruch, J.J. et al. Vip3A, a novel Bacillus thuringiensis vegetative
insecticidal protein with a wide spectrum of activities against lepidopteran
insects. PNAS. 93, (1996), pp 5389-94.
[2] Mesrati, L.A., Tounsi, S. & Jaoua, S. Characterization of a novel vip3-type
gene from Bacillus thuringiensis and evidence of its presence on a large
plasmid. FEMS Microbiol Lett. 244, (2005), pp 353-8.
The binary toxin (Bin) from Lysinibacillus sphaericus is composed of BinA
(42 kDa) and BinB (51 kDa) components, and both of them are required for
full toxicity against Culex and Anopheles mosquito larvae. BinB component is
responsible for the specific binding to a receptor present on the midgut
epithelial membranes, while BinA is proposed to be a toxic component. Here,
we determined the crystal structure of the active form of BinB at a resolution
of 1.75 Å . The overall structure is composed of three domains. The Nterminal domain has a β-trefoil scaffold which is a highly conserved
architecture of some sugar binding proteins or lectins, suggesting a role of this
module in receptor-binding. The Bin -rich C-terminal domains, domains 2
and 3, share similar three-dimensional folding characteristics with aerolysin
type β-pore forming toxins, despite a low sequence identity. The BinB
structure, therefore, is a new member of the aerolysin-like toxin family, which
probably exerts its cytolytic mechanism via pore formation.
P2
Expression of the cytosolic portion of mannan synthase involved in the
biosynthesis of galactomannan in Cyamopsis tetragonoloba
Varun Bansal1, Pallavi Gahlot2, Shilpi Kumari2, Kanwarpal Singh Dhugga3,
and G. S. Randhawa2
1
Department of Molecular Cell Biology, Samsung Biomedical Research
Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 ,
Republic of Korea
2
Department of Biotechnology, Indian Institute of Technology Roorkee,
Roorkee -247667, India
3
Crop Research & Development, 7300 NW 62nd Avenue, Johnston, IA 50131,
U. S. A.
E-mail: vrun.bnsl@gmail.com
References
[1] Srisucharitpanit K., Yao M., Chimnaronk S., Promdonkoy B., Tanaka I.
and Boonserm P., Crystallization and preliminary X-ray crystallographic
analysis of the functional form of BinB binary toxin from Bacillus sphaericus.
Acta Crystallogr.,Vol. F69, (2013), pp 170-173.
[2] Srisucharitpanit K., Inchana P., Rungrod A., Promdonkoy B. and
Boonserm P. Expression and purification of the active soluble form of
Bacillus sphaericus binary toxin for structural analysis. Protein Expr. Purif.,
Vol. 82, (2012), pp 368-372
Cluster bean or Cyamopsis tetragonoloba is an important leguminous crop
grown in north western parts of India. Galactomannan gum from guar
endosperm is useful in various industries as thickener and rheology modifier
which is affected by galactose and mannose ratio.This galactose and mannose
ratio is crucial for the quality of produced gum. Guar gum is exported to an
extent of Rs.1000 crores annually from India. Guar mannan synthase is an
important multi-pass membrane protein, localized in Golgi, responsible for
biosynthesis of galactomannan, a hemicellulosic cell wall storage
polysaccharide belongs to cellulose synthase-like (Csl) family and
constituting more than 90% of the total seed endosperm at maturity. It is a
developmental stage specific gene expressed exclusively in the endosperm of
developing seeds with peak expression at 25-30 DAF. In-silico analysis of the
protein predicted that it possesses five transmembrane helices. The region
P4
Influenza A virus hemagglutinin, a target protein of virus sensing.
Hye-Min Woo, Jin-Moo Lee and Yong-Joo Jeong
Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702,
Korea
The influenza virus envelope carries two major immunogenic surface
glycoproteins: hemagglutinin (HA) and neuraminidase. The viral surface
protein hemagglutinin identifies receptor sialic acid residues present on the
164
host cell surface. It makes hemagglutinin suitable for target to detect the
influenza virus. In this study, we constructed hemagglutinin expression
vectors and purified the GST-tagged HA1 protein, and activity of HA1 was
confirmed by using hemagglutination assay. Using a Counter-SELEX
procedure, we found four ssDNA aptamers, which have specific affinity for
the HA1(H1N1). Selected aptamers were shown the dissociation constant of
about 100nM that confirmed using SPR and ELISA. The interaction of
selected aptamer, purified HA1 and sialic acid on HEK 293T cell were
investigated by using fluorescence-activated cell sorting (FACS). Our selected
aptamer have high affinity to H1 subtype and we hope these aptamers would
have wide applications in diagnosis, therapy and prevention of influenza virus.
glucooligosaccharides [1]. To understand the glucoside binding during the
-mannosidase, Os7BGlu26 and its
acid/base mutant Os7BGlu26 E179Q were crystallized and soaked with 2,4dinitrophenyl-2-deoxy-2-fluoroglucoside (dNPG2F) and the putative
transition state mimic inhibitor glucoimidazole. The structure of the
Os7BGlu26 complex with dNPG2F had one molecule of dNPG2F bound at
the +1 to +2 subsites and another at a position further out in the putative
substrate binding cleft, rather than at the expected subsite -1 to +1 position. In
contrast, the Os7BGlu26 E179Q acid/base mutant soaked with this inhibitor
yielded the structure of a covalent intermediate with 2-fluoroglucoside (G2F),
which was found in a 4C1 chair conformation. The crystal structure of
Os7BGlu26 in complex with glucoimidazole showed the inhibitor bound in
the active site in a 4E envelope conformation, which is close to the 4H3 half
chair transition state conformation reported for glucosides. This study
suggests that D-glucosides transition through the 4H3 half chair or 4E envelope
form near the transition state to a 4C1 chair conformation in the covalent
-mannosidase, in
contrast to the 1S5 skew boat to B2,5 boat to OS2 skew boat pyranoside
conformation trajectory for mannosides [2].
References
Kuntothom T., Luang S., Harvey A. J., Fincher G. B., Opassiri R., Hrmova M.,
and Ketudat Cairns J. R., ―Rice family GH1 glycoside hydrolases with β-Dglucosidase and β-D-mannosidase activity‖, Archives of Biochemistry and
Biophysics. Vol. 491, (2009), pp 85-95.
Tankrathok A., Iglesias-Fernández J., Luang S., Robinson R. C., Kimura A.,
Rovira C., Hrmova M., and Ketudat Cairns J. R., ― Structural analysis and
-DMannosidase‖, Acta Crystallographica Section D. Vol. 69, (2013), pp
P5
Small heat shock protein IbpB acts as a robust chaperone in living cells
by hierarchically activating its multi-type substrate-binding residues
Xinmiao Fu 1, Xiaodong Shi 2 and Zengyi Chang 1
1
State Key Laboratory of Protein and Plant Gene Research, School of Life
Sciences, Peking University, Beijing 100871, China
2
Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical Colleg
e, Xuzhou 221002, China
P7
Structural examination of the in vitro interaction
between the Tid1 J-domain and P53
Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones, are
crucial for protein homeostasis. It is not clear why sHSPs are able to bind a
wide spectrum of non-native substrate proteins and how such binding is
enhanced by heat shock. Here, by utilizing a genetically incorporated photocrosslinker (Bpa), we systematically characterized the substrate-binding
residues in IbpB (a sHSP from Escherichia coli) in living cells over a wide
spectrum of temperatures (from 20oC to 50oC). A total of 20 and 48 residues
were respectively identified at normal and heat shock temperatures. They are
not necessarily hydrophobic and can be classified into three types: type I and
II are respectively activated at low and normal temperatures; type III mediate
oligomerization at low temperature but switch to substrate-binding at heat
shock temperature. In addition, substrate-binding of IbpB in living cells
begins at such a low temperature as 25oC and is further enhanced upon
temperature elevation. Together, these in vivo data provide novel structural
insights into the wide substrate spectrum of sHSP and suggest that sHSP is
able to hierarchically activate its multi-type substrate-binding residues and
thus act as a robust chaperone in cells under fluctuating growth conditions
Department of Biotechnology, Konkuk University, Chungju, Chungbuk 380701, Republic of Korea
E-mail : freedom234@naver.com
Ku-Sung Jo, Dae-Won Sim and Hyung-Sik Won*
The specific pair of a heat shock protein 40 (Hsp40) and a heat shock protein
70 (Hsp70) is an essential chaperone system involved in diverse cellular
processes including apoptosis. Tid1, belonging to the Hsp40-family proteins,
also functions basically as a co-chaperon of cytosolic and mitochondrial
Hsp70. Recently, it has been suggested that the Tid1 interacts with P53,
leading to mitochondrial translocation of the complex and subsequent
triggering of intrinsic apoptosis. In particular, the N-terminal J-domain of
Tid1 (Tid1-JD) was necessary, while either N- or C-terminal domain of P53
was responsible for the interaction. Here, we solved the solution structure of
Tid1-JD by NMR spectroscopy. The structure showed four conserved αheliices and a flexible HPD motif that is critical for the interaction to Hsp70.
Then, the NMR spectra of the Tid1-JD showed no significant perturbation in
the presence of the N-terminal TAD (transactivation domain) or the Cterminal domain of P53. Thus, we suggest that the direct interaction between
the Tid1-JD and P53 would require a certain post-translational modification
or should be mediated by other parts of Tid1 or by another protein, such as
Hsp70.
References
Fu X*, Shi X, Yin L, Liu J, Joo K, Lee J, Chang Z.* “Small heat shock
protein IbpB acts as a robust chaperone in living cells by hierarchically
activating its multi-type substrate-binding residues. ”, J Biol Chem, (2013)
288: 11897-906.
P6
Structural analysis of glucoside binding during the glycosylation step of
hydrolysis by the rice GH1 Os7BGlu26 beta-mannosidase
Protein folding and dynamics
Anupong Tankrathok1, Spencer J. Williams2 and James R. Ketudat Cairns1
P8
Correlated vibrations in ion-pair dynamics in mechanoactivation
identifies functional domains of force-dependent titin kinase
1
School of Biochemistry and Center for Biomolecular Structure, Function and
Application, Institute of Science, Suranaree University of Technology, Nakhon
Ratchasima 30000, Thailand
2
School of Chemistry and Bio21 Molecular Science and Biotechnology
Institute, University of Melbourne, Parkville, Victoria 3010, Australia
E-mail: tankrathok@yahoo.com
Ming-Chya Wu1,2, Jeffrey G. Forbes2 and Kuan Wang2,3,4
1
Research Center for Adaptive Data Analysis, National Central University,
Chungli 32001, Taiwan.
2
Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan.
3
Muscle Proteomics and Nanotechnology Section, Laboratory of Muscle
Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases,
National Institutes of Health, Bethesda, Maryland 20892, USA
-mannosidase is the member of glycoside hydrolase
family 1 (GH1), which prefentially hydrolyzes mannosides and
mannooligosaccharides,but
can
also
hydrolyze
glucosides
and
165
4
Nanomedicine Program, Institute of Biological Chemistry, Academia Sinica,
Nankang, Taipei 11529, Taiwan
E-mail: mcwu@ncu.edu.tw
References
Sipe, J. D. et al. ―Amyloid fibril protein nomenclature: 2012
recommendations from the Nomenclature Committee of the International
Society of Amyloidosis.‖, Amyloid, Vol. 19, No. 4, (2012), pp 167-170.
Yoshimura Y, et al. ―Distinguishing crystal-like amyloid fibrils and glass-like
amorphous aggregates from their kinetics of formation.‖ Proc Natl Acad Sci
USA, Vol. 109, No. 36, (2012), pp 14446-14451.
Hoshino M, et al. ―Mapping the core of the β2-microglobulin amyloid fibril
by H/D exchange.‖, Nat Struct Biol Vol.9, No. 5, (2002), pp 332-336.
Titin kinase is a mechanoenzyme, whose activity is activated by mechanical
stretching and binding of calcium sensors. Stretching causes local and global
conformational changes of secondary structures and complex movements of
ion pairs and transient formations of salt bridges. This paper applies the
adaptive time series analysis approach to study the mechanical responses of
ion-pair movements to stretch unfolding through steered molecular dynamics
(SMD) simulations, focusing on the ion-pair dynamics of mechanoactivation
obtained from the SMD trajectories. Temporal correlation analysis of the ionpair time series shows that the activation process involves changes of
secondary structure. Spectral analysis defined several groups and subgroups
of the ion pairs with vibrational damping/resonance in the scale of ~0.5Å ,
corresponding to vibrational modes of chemical bonds. Examination of these
groups revealed the locations or neighboring structures of the autoinhibitory
loop, ATP binding cleft, catalytic loop, and P+1 loop, all key functional
domains of this kinase. We propose that the correlated vibrations of subgroups
of ion pairs have significant correlations with functional domains, which can
be used to identify, a priori, special functional and structural features of folded
proteins.
P10
Microsecond-dynamics of the unfolded BdpA and ubiquitin by a line
confocal detection of single molecule fluorescence
Hiroyuki Oikawa1, Masataka Saito1,2, Po-Ting Chen3, Hsin-Liang Chen3, Rita
P.-Y. Chen3, Munehito Arai4,5, Kiyoto Kamagata1,2, and Satoshi Takahashi1,2
1
Institute of Multidisciplinary Research for Advanced Materials, Tohoku
University, Sendai 980-8579, Japan.
2
Graduate School of Science, Tohoku University, Sendai 980-8577, Japan.
3
Institute of Biological Chemistry (IBC), Academia Sinica, Taipei 115, Taiwan.
4
PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012,
Japan.
5
Department of Life Sciences, Graduate School of Arts and Sciences, The
University of Tokyo, Tokyo 153-8902, Japan.
E-mail: st@tagen.tohoku.ac.jp
References
Wu M.-C., Forbes J. G. and Wang K., ―Correlated vibrations in ion-pair
dynamics in mechanoactivation identifies functional domains of forcedependent titin kinase‖, Soft Matter Vol. 9, (2013), pp 9897-9906.
High-speed tracking of fluorescence signals from single molecules is
expected to reveal detailed information of protein folding. To improve the
time resolution, we developed a line-confocal microscope combined with fast
sample flow system [1]. The excitation light is focused into a line shape
along the flow path. By imaging the path, we can trace a time evolution of
fluorescence intensities and FRET efficiency from single molecules. The
system achieved the photon count rate of more than 2000 photons/ms, and the
time resolution of less than 20 μs.
As the first demonstration, we investigated the B domain of protein A (BdpA)
labeled with Alexa488 and Alexa633 at residues 22 and 55. In the FRET
efficiency histograms, we assigned peaks appearing at around 0.4 ~ 0.6 at low
denaturant concentrations to the native conformation. The peaks appearing
at ~ 0.2 at the higher denaturant concentrations can be assigned to the
unfolded state.
The time series from the unfolded state suggested
conformational substates.
As the second demonstration, we investigated the unfolded state dynamics of
ubiquitin. We labeled a mutant of ubiquitin, M[C]Q/S65C, by Atto532 and
Alexa647. The single molecule measurements at different denaturant
concentrations suggested the presence of intermediate states.
P9
Heat of supersaturation-limited amyloid burst directly monitored by
isothermal titration calorimetry
Tatsuya Ikenouea, Young-Ho Leea, József Kardosb, Hisashi Yagia, Takahisa
Ikegamia, Hironobu Naikic and Yuji Gotoa
a
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita,
Osaka 565-0871, Japan.
b
Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C,
Budapest, 1117, Hungary.
c
Faculty of Medical Sciences, University of Fukui, Matsuoka, Fukui 910-1193,
Japan.
E-mail: t-ikenoue@protein.osaka-u.ac.jp
Amyloid fibrils have been associated with over 30 degenerative diseases,
including Alzheimer‘s, Parkinson‘s, and prion diseases1. Amyloid fibrils form
in supersaturated solutions via a nucleation and growth mechanism. We
have revisited "supersaturation" and argued its critical role for amyloid
fibrillation2. Although the structural features of amyloid fibrils have become
increasingly clearer3, knowledge on the thermodynamics of fibrillation is
limited. Furthermore, protein aggregation is not a target of calorimetry, one of
the most powerful approaches used to study proteins. Here, with β2microglobulin (β2m), a protein responsible for dialysis-related amyloidosis,
we show direct heat measurements of the formation of amyloid fibrils using
isothermal titration calorimetery (ITC). The spontaneous fibrillation after a
lag phase was accompanied by exothermic heat (Fig. 1A). The
thermodynamic parameters of fibrillation obtained under various protein
concentrations and temperatures were consistent with the main-chain
dominated structural model of fibrils, in which overall packing was less than
that of the native structures. We also characterized the thermodynamics of
amorphous aggregation enabling the comparison of protein folding, amyloid
fibrillation and amorphous aggregation (Fig. 1B, C). These results indicate
that ITC will become a promising approach for clarifying comprehensively
the thermodynamics of protein folding and misfolding.
A
B
3131
°C°C
References
Oikawa H., Suzuki Y., Saito M., Kamagata K., Arai M. and Takahashi S.,
"Microsecond dynamics of an unfolded protein by a line confocal tracking of
single molecule fluorescence" Scientific Reports 3, 2151, (2013)
P11
Multilevel structural characteristics for the natural substrate proteins of
bacterial small heat shock proteins
Xinmiao Fu 1
1
Sciences, Peking University, Beijing 100871, China
Amorpho
3434
°C°C
Small heat shock proteins (sHSPs) play crucial roles for protein quality
control in cells. It is poorly understood what natural substrate proteins, with
respect to structural characteristics, are preferentially bound by sHSPs in cells.
Here I compared the structural characteristics for the natural substrate
proteins of Escherichia coli IbpB and Deinococcus radiodurans Hsp20.2 with
the respective bacterial proteome. Data indicate that both IbpB and Hsp20.2
preferentially bind to substrates of high molecular weight or moderate acidity.
Surprisingly, their substrates contain abundant charged residues but not
abundant hydrophobic residues, thus strongly indicating that ionic interactions
other than hydrophobic interactions also play crucial roles for the substrate
recognition and binding of sHSPs. Further, secondary structure prediction
analysis indicates that the substrates of low percentage of β-sheets or coils but
high percentage of α-helices are un-favored by both IbpB and Hsp20.2. In
-1
Heat flow (µcal sec-1)
Heat flow (µcal sec )
Nativ
3737
°C°C
Amylo
4040
°C°C
fibril
(spontaneous) native native
state
fibril
(spontaneous)
state
amorphous
aggregate
amorphous
aggregate
- 400
- 400
1616
-1
molmol)-1)
G (kJ
G (kJ
1212
- 100
- 10
0
-1
8
- 300
- 200
-1
8
Time
Time
(h)(h)
- 100
- 10
H (kJ mol )
4
- 200
- 20
- 20
-1
4
- 300
- 30
- 30
-1
0
-
40
- 40
-TS (kJ mol )
0
-1
H (kJ mol )
-1
1 µ
1cal
µ cal
sec
sec
-TS (kJ mol )
C
50
-- 50
4343
°C°C
0
0 0
20
40
60
80
0
100
Temperature (°C)
0
20
40
60
80
100
Temperature (°C)
Figure 1. The results obtained enabled the calorimetric
characterization of amyloid fibrils and amorphous
aggregates relative to that of the native globular structures,
which opens a new field for the calorimetric study of protein
aggregates.
166
Yuji Gotoa,1
addition, IbpB preferentially interacts with multi-domain proteins but
unfavorably with α+β proteins as revealed by SCOP analysis. Together, our
data suggest that bacterial sHSPs, though having broad substrate spectrums,
selectively bind to substrates of certain structural features. These structural
characteristic elements may substantially participate in the sHSP-substrate
interaction and/or increase the aggregation tendency of the substrates.
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita,
Osaka 565-0871, Japan
E-mail: h-muta@protein.osaka-u.ac.jp
Amyloid fibrils form in supersaturated solutions via a nucleation and growth
mechanism. We proposed that ultrasonication may be an effective agitation to
trigger nucleation that would otherwise not occur under the persistent
metastability of supersaturation. However, the roles of supersaturation and
effects of ultrasonication have not been elucidated in detail except for limited
cases. Insulin is a typical amyloidogenic protein that is useful for
investigating the mechanisms underlying amyloid fibrillation with biological
relevance. We studied the alcohol-induced amyloid fibrillation of insulin
using various concentrations of 2,2,2-trifluoroethanol, and 1,1,1,3,3,3hexafluoro-2-propanol at pH 2.0 and 4.8. Ultrasonic irradiation effectively
triggered fibrillation under conditions in which insulin retained persistent
supersaturation. Structural analyses by circular dichroism, Fourier transform
infrared spectroscopy, transmission electron microscopy and atomic force
microscopy revealed that the dominant structures of fibrils varied between
parallel and antiparallel -sheets depending on the solvent conditions. pHand alcohol-concentration-dependent phase diagrams showed a marked
difference before and after the ultrasonic treatment, which indicated that the
persistent metastability of supersaturation determined the conformations of
insulin. These results indicate the importance of an alternative view of
amyloid fibrils as supersaturation-limited crystal-like aggregates formed
above the solubility limit[1].
References:
Fu X.*, Chang Z, Shi X, Bu D, Wang C. Multilevel structural characteristics
for the natural substrate proteins of bacterial small heat shock proteins.,
Protein Sci., (2014) 23: 229-237
P12
Allosteric sites can be identified based on the residue-residue interaction
energy difference
Xiaomin Ma1,2, Yifei Qi1,2 and Luhua Lai1,2,3*
1
Center for Quantitative Biology, Peking University, Beijing 100871, China.
BNLMS, State Key Laboratory for Structural Chemistry of Unstable and
Stable Species, College of Chemistry and Molecular Engineering, Peking
University, Beijing 100871, China.
3
Peking-Tsinghua Joint Center for Life Sciences, Peking University, Beijing
100871, China.
E-mail: xmma89@pku.edu.cn
2
Allosteric drugs, taking an action-at-a-distance mechanism to achieve
regulations on the protein functions, have several advantages over
conventional orthosteric drugs, including diverse regulation types and minor
side effects. The rational design of allosteric ligands, however, remains
challenging, especially for the step of allosteric site identification. As
allosteric ligand binding may work by inducing changes in the pattern of
residue-residue interactions, we calculated the residue-residue interaction
energies within the allosteric site based on the MM/GBSA energy
decomposition scheme. For a dataset of 17 allosteric proteins with structural
data for both the apo and the ligand-bound state available, we calculated the
residue-residue interaction energy in both states and compared their
differences. In all the cases, distinct interaction energy differences (>25%)
were observed. We then used 25% energy difference as a cutoff to classify
allosteric site from non-allosteric site. For other 31 ―druggable binding sites‖
identified using a binding site detection program, CAVITY, 21 of them with
residue-residue interaction energy difference >25% were predicted as novel
allosteric sites. Three of them were supported by recent experimental studies.
All the predicted sites may serve as novel allosteric sites for allosteric ligand
design. Our study provides a computational method for identifying novel
allosteric sites.
References
Yoshimura Y, et al. Distinguishing crystal-like amyloid fibrils and glass-like
amorphous aggregates from their kinetics of formation. ―Proc Natl Acad Sci
USA” Volume 109, No.36 (2012), pp 14446-14451.
P15
Structural analysis of regulator of G protein signaling (RGS) by
hydrogen/deuterium exchange mass spectrometry (HDX-MS)
Su Youn Lee*, In Young Ko*, and Ka Young Chung
School of Pharmacy Sungkyunkwan University, Suwon, South Korea
E-mail: youn3887@hanmail.net, koiy@skku.edu
P13
Structural Analysis of Full Length EBP50 by Hydrogen/Deuterium
Exchange Mass
Nguyen Minh Duc*, Dong Kyun Kim* and Ka Young Chung
School of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Korea
E-mail: minhduchup@gmail.com, kurababy@skku.edu
Regulators of G protein signaling (RGS) proteins are key players in regulating
signaling via G protein-coupled receptors. RGS proteins directly bind to the
Gα-subunits of activated heterotrimeric G-proteins, and accelerate the
hydrolysis of GTP, thereby rapidly deactivating G-proteins. The structural
analysis of RGS would provide information on the understanding of its
mechanism. Few structures of RGS including RGS2 and RGS4 are
determined. In this study, we analyzed the conformational dynamics of RGS2
and RGS4 using HDX-MS. The HDX profiles of RGS2 and RGS4 showed
conformational dynamics and provided conformational difference between
RGS2 and RGS4. These results have potential implications for understanding
how the conformational dynamics among RGS proteins may play a key role
in structure and function of RGS.
*equally contributed
P16
Structural study of beta–arrestins in various activation status by
hydrogen/deuterium exchange mass spectrometry
Ezrin-radixin-moesin-binding protein 50 (EBP50) is a scaffolding protein that
contains two post synaptic density-95/disk-large/ZO-1 homology (PDZ)
domains and an ezrin/radixin/moesin-binding (EB) domain. It scaffolds
various proteins including membrane receptors and transporters and signals
them into functional complexes. Several studies defined the high resolution
structures of isolated PDZ domains. However, the structural analysis of fulllength EBP50 is limited. Here, we studied the conformation of full-length
EBP50 using hydrogen/deuterium exchange mass spectrometry (HDX-MS).
The HDX data showed that linker regions and most of the C-terminal region
are disordered whereas PDZ domains are more ordered. The HDX data also
suggested that PDZ1 is more dynamic than PDZ2.
Dong-Kyun Kim*, Young-Joo Yun*, and Ka Young Chung
E-mail: kurababy@skku.edu, dudwn0903@skku.edu
P14
Supersaturation-limited amyloid fibrillation of insulin revealed by
ultrasonication
G protein-coupled receptors (GPCRs) are primary drug targets. Betaarrestins are most important regulators in GPCRs signaling, and mediate G
protein independent signaling. However, in spite of their importance,
structural mechanism of activation and inactivation of beta-arrestins remains
elusive. In the present study, we analyzed the conformation of various active
status beta–arrestins using hydrogen/deuterium exchange mass spectrometry
(HDX-MS). We constructed active mutants and inactive mutants of form of
beta–arrestins. HDX-MS data provided insight for the conformational
changes of beta-arrestins upon their activation and inactivation.
Hiroya Mutaa, Young-Ho Leea, József Kardosb, Yuxi Lina, Hisashi Yagia, and
P17
*Equally contributed
167
Conformational analysis of Beta-arrestins by hydrogen/deuterium exchange
mass spectrometry
compounds in vivo, regulating the formation of amyloid fibrils
YoungJoo Yun, Dong-Kyun Kim and Ka Young Chung
P20
Ultrasonication-dependent
microglobulin
School of Pharmacy Sungkyunkwan University, Suwon 440-746, South Korea
E-mail: dudwn0903@skku.edu, kurababy@skku.edu
inactivation of amyloid fibrils of β2-
Masatyki Adachi1, Masatomo So1, Yuji Goto1
Arrestins are most important regulators in desensitizing G protein-coupled
receptors (GPCRs). Recently, arrestins are immerged as new drug targets
because of their new role in G protein-independent signaling pathway of
GPCR. In this study, we analyzed the conformation of two non-visual
arrestins (e.g. beta-arrestin 1 and beta-arrestin 2) using hydrogen/deuterium
exchange mass spectrometry (HDX-MS). The HDX data showed different
dynamics of the conformation of two closely related beta-arrestins, which may be
involved in the functional differences of beta-arrestin 1 and beta- arrestin 2.
1
Institute for Protein Research, Osaka university, Yamadaoka 3-2, Suita,
E-mail: m-adachi@protein.osaka-u.ac.jp
Amyloid fibrils are misfolded and self-assembled aggregates of proteins
associated with the pathology of more than 20 serious disorders. In the case of
dialysis-related amyloidosis, β2- microglobulin forms amyloid fibrils.
Amyloid fibrils form in supersaturated solutions via a nucleation and growth
mechanism. We proposed that ultrasonication is an effective agitation to
trigger nucleation that would otherwise not occur under the persistent
metastability of supersaturation [1].
When formation of amyloid fibrils of β2-microglobulin was monitored by ThT
fluorescence, the ThT fluorescence increased after a lag time, saturated and
then decreased slowly. As expected, the lag time shortened with an increase in
the strength of ultrasonication. The decrease in the ThT fluorescence was
accelerated with an increase in the strength of ultrasonication, suggesting that
the decrease represents the ultrasonication-dependent degradation or
inactivation of fibrils. Conformational analysis showed that the extensively
ultrasonicated fibrils retain the β structure similar to the mature fibrils. On the
other hand, seeding experiments revealed the loss of seed potentials.
Combined with the effects of ultrasonication on amyloid fibrils of other
proteins, we propose a comprehensive model of the effects of ultrasonication
on the various stages of the formation of fibrils.
P18
Molecular dynamics study on enhancer recognitions by cooperative
binding of Ets1 and partner transcription factors.
Kota Kasahara1, Ikuo Fukuda1 and Haruki Nakamura1
1
Institute for Protein Research, Osaka University, Suita 565-0871, Japan
E-mail: kota.kasahara@protein.osaka-u.ac.jp
Transcription factor (TF) Ets1 recognizes several kinds of enhancer regions
regulating genes related to important biological processes such as cancer
development and autoimmunity. While Ets1–enhancer binding is
accomplished by cooperative binding with partner TFs, e.g., Runx1, CBFbeta,
Pax5, or homo-dimerization of Ets1, molecular mechanisms of how Ets1
communicate with its partner TFs is largely unclear. Here, we performed
molecular dynamics (MD) simulations on Ets1–DNA complexes with partner
TFs and analyzed inter-molecular communications. We applied our GPGPUspecialized simulator ―Psygene-G‖ implementing the original non-Ewald
electrostatic potential estimation method named Zero-dipole summation
method. In addition, a method to quantify correlation of atomic motions
between two atoms based on a pattern recognition technique was newly
developed. As a result of analyses on Ets1 homodimer–DNA complex,
significant correlations at Gly333–Asp380 and Leu337–C110 were detected
and they were consistent with previous experimental studies. The model with
removal of an Ets1 from the homodimer–DNA complex exhibited
significantly weaker correlations among Leu337, Tyr395, and C110 than the
native complex. A simulation in Ets1–Pax5–DNA complex also showed same
results. They imply that this ternary relationship of correlative motions is
essential for communication with partner TFs
References
[1] Yoshimura, Y., So, M., Yagi, H. and Goto, Y., ―Ultrasonication, an efficient
agitation for accelerating the supersaturation-limited amyloid fibrillation of
proteins.‖ Jpn. J. Appl. Phys. 52 (2013) 07HA01
P21
Cold denaturation of alpha-synuclein amyloid fibrils
Young-Ho Lee1, Tatsuya Ikenoue1 and Yuji Goto1
1
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita,
E-mail: mr0505@protein.osaka-u.ac.jp
Although the structural property and the mechanism of formation of amyloid
fibrils have become increasingly clearer, conformational stability of fibrils
remains largely unclear. Here, we showed the systematic study on the
response of amyloid fibrils to temperature changes. Amyloid fibrils of αsynuclein cold-denature to monomers at 0-20 ºC via kinetic intermediates as
well as heat-denature at 60-110 ºC. The fibrils of α-synuclein mutants and
core fragments also showed similar temperature responses. Meanwhile, the
fibrils of β2-microglobulin, Alzheimer‘s Aβ1-40/Aβ1-42 peptides, and insulin
exhibited only heat denaturation. Chemical denaturants revealed the hidden
trends of cold denaturation of amyloid fibrils by increasing the melting
temperature. The heat denaturation of fibrils caused by increasing chain
flexibility resembled that of globular proteins. In contrast, a comparison of
structural parameters with thermodynamic properties which showed opposite
signs to protein folding (i.e., positive enthalpy and heat capacity changes)
indicated that the burial of charged residues in fibril cores was responsible for
cold denaturation. We propose that although the main-chain dominated
structures of fibrils overwhelm the unfavorable burial of charged side-chains,
reinforced electrostatic repulsion combined with increases in hydrophobic
hydration at low temperatures may result in cold denaturation, leading to a
unique thermodynamic property of amyloid fibrils
P19
Effects of various detergents on the amyloid fibrillation of 2microglobulin
Masatomo So1, Akira Ishii1, Hisashi Yagi1, Hironobu Naiki2 and Yuji Goto1
1
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita,
2
Department of Pathological Sciences, Faculty of Medical Sciences,
University of Fukui, Eihei-ji, Fukui 910-1193, Japan.
E-mail: mso@protein.osaka-u.ac.jp
Amyloid fibrils are highly ordered assemblies of misfolded protein and are
associated with various degenerative diseases including Alzheimer‘s disease
and dialysis related amyloidosis. Amyloid fibrillation is dominated by the
environmental factors. Although various additives, such as SDS,
phospholipids and RNA aptamers, are known to accelerate and/or inhibit
fibrillation, little is known about the mechanism how these additives effect
fibrillation. Clarifying the mechanisms of acceleration and inhibition of fibril
formation by additives is useful for treating amyloid-related diseases and
understanding physical properties of proteins. In this study, we investigated
fibril formation in the presence of various concentrations of detergents. At a
concentration bellow the CMC, proteins retain the native structure because
few detergents interact with proteins. As the detergent concentration becomes
close to CMC, 2-microglobulin forms amyloid fibrils probably because
hydrophobic regions partly expose to solvent. At the concentrations well
above the CMC, the protein molecules are solubilized into micelles of
detergents producing large micelles containing proteins. We suggest that such
a competing mechanism of promotion and suppression occurs for various
P22
Structural Identification of a Dynamic Regulatory Region of Hsp33 from
Escherichia coli
Jeong-Hwa Jang1, Yoo-Sup Lee1, Jinhyuk Lee2, Keun Woo Lee3, and HyungSik Won1,*
1
168
Department of Biotechnology, Konkuk University, Chungju, Chungbuk 380-
701, Korea
2
Korean Bioinformation Center, Korea Research Institute of Bioscience and
Biotechnology, Daejeon 305-806, Korea
3
Division of Applied Life Science, Gyeongsang National University, Jinju
660-701, Korea
E-mail: jeonghwa1102@gmail.com
P24
Dynamic Origin of Internal Friction and Its Application in Protein
Dynamics
Jun Wang1, Wei Wang1
Hsp33 is a prokaryotic molecular chaperone of which activity is regulated by
redox potential. The oxidation-induced unfolding of the C-terminal zincbinding domain and concomitant dimerization of the N-terminal core domain
constitutes the activation process of Hsp33. Here, solution structure of the
reduced, inactive Hsp33 from Escherichia coli was characterized by nuclear
magnetic resonance (NMR) spectroscopy in its monomeric state and modeled
by a template-based method using the conformational space annealing (CSA)
calculation with the NMR data. Although its overall conformation was
comparable to that expected from the known dimeric Hsp33 crystal structures,
NMR signals from specific regions were hardly detected, particularly
-helix 1 at the N-termin
-strand
10 at the interdomain linker stretch. NMR spectra of a mutant Hsp33 with the
N-terminal six-residue deletion implied a certain peculiar dynamics
-1 or the
-1 elements resulted in the formation of reduced, but active species, without
unfolding of the zinc domain. Taken all together, it is conclusively suggested
that unchaining the interdomain linker stretch could be the structural
determinant driving the activation process of Hsp33 and it is dynamically
regulated by its contacting counterparts, the N-terminal segments.
1
Department of Physics and National Lab of Solid State Microstructure,
Nanjing University, Nanjing 210093, China.
E-mail: wangj@nju.edu.cn
The internal friction is a topic with long-lasting interests in polymer physics
and protein dynamics. It is believed to be an intrinsic property of the dynamics
of the concerned polymeric systems. In present work, starting from the generic
Langevin equation, the form of the internal friction is derived. This kind of
form is exemplified in some model systems with simulations. As a result, the
features of the internal friction are disclosed. The dependences of the internal
friction on the system size and the temperature are also obtained. Furthermore,
this definition of the internal friction is applied to the protein systems. These
results are compared with the experimental observations, and provide the
elaborate information about the contributions of the internal friction to the
dynamics of protein systems.
P25
An Improved HDX Workflow For Enhanced Separation, Digestion, And
Data Analysis
P23
Pogranulin mutations involved in frontotemporal lobar degradation in
silico modeling.
Kevin Cho, Joomi Ahn,Michael Eggertson,Keith Fadgen, Ying Qing Yu,
Weibin Chen
Sun Oh Bae1, Lingyan Shen1, Seong Soo An1, Sang Yun Kim2
Waters Corp, MA, USA
E-mail : kevin_cho@waters.com
1
Department of Bionano Technology, Gachon university, Seongnam 461-701,
Korea
2
Seoul National University, Bundang Hospital, Seoul 110-744, Korea
E-mail: baesunoh@gmail.com
An integrated hydrogen/deuterium exchange mass spectrometry (HDX MS)
workflow consists of several key components including on-line digestion,
sub-ambient chromatography, MS, and dedicated informaticstool. In recent
years, each component has been significantly developedto improve
efficienciesof the HDX workflow. In this study, we present our effort to
systematically optimize the performance of a HDX platform by using a
prototype microscale-UPLC system that can operate at 15,000 PSI, an
immobilized pepsin column packed with mechanically strong particles, and a
dedicated informatics tool with enhanced functionality.We will demonstrate
that the higheroperatingpressure rendered by the UPLC improves
chromatographic resolution;the digestion efficiency of the pepsin BEH
column is enhanced during higher pressure digestion.
An increased number of overlapping peptic peptides were generated from
pressurized digestion resulting in improved coverage and redundancy score.
Increasing the digestion temperature of the online pepsin column resulted in
higher sequence coverage of cytochrome C from 63% at 0 °C to 100% at
25 °C. The rate of back-exchange from deuterated proteins, which is an
important factor during HDX MS experiments, was carefully measured for
both the high-pressure tolerant BEH pepsin column and commercially
available pepsin columns. Comparable back-exchange rates were achieved.
An enhanced chromatographic resolution was achieved for protein digests
separated in less than 10 min at 0 °C at high pressure. With a prototype
microscale-UPLC system that can operate at 15,000 PSI, the complex peptic
digests of IgG and phosphorlyase b were reproducibly separated at 0 °C. A
set of data interpretation/display tools such as coverage map and heat map
was provided in the informatics tool, facilitating efficient data comparison.
The results demonstrated that enhanced performance of the HDX platform is
achieved when operated high pressure with no compromise in deuterium
recovery.
All proteins folded and had unique 3D structure. This folding of proteins
related to act of proteins. Thus, the normal protein folding is very important in
the function. In fact, the exact structure of protein that needs a large amount of
pure protein required, and expensive experimental instruments required like
NMR, Circular dichroism, X-ray crystallography, etc. However, depending on
amino acid sequence of the protein 3D modeling had an advantageous of
structure prediction.
Progranulin (PGRN), which is granulin precursor, had various biochemical
capabilities (Inflammation modulator, wound healing, etc) and involved in
Familial frontotemporal lobar degeneration(FTLD), the cause of mutations in
PGRN gene. These mutations in PGRN compared to structural differences
using in silico modeling. Therefore, In silico modeling is valuable tool for
protein folding prediction.
P26
Contribution of Different Amino Acids to the Conformational
Thermostabilities of Cytidylate kinases from Thermophilic
Geobacillus bacteria.
References
N. Brouwers, MSc, K. Sleegers, MD, PhD, S. Engelborghs, MD, PhD,
―Genetic variability in progranulin contributes to risk for clinically diagnosed
Alzheimer diseas”, Neurology, Vol. 71, No. 91, 656-664.
Ian R. A. Mackenzie, Matt Baker, Stuart Pickering-Brown.,―The
neuropathology of frontotemporal lobar degeneration caused by mutations in
the progranulin geneThe title of the journal paper‖, Brain, 129, (2006),
3081-3090.
Takanori Satoh1, Momoko Abe2, Shota Takahashi2, Shota Inoue2, Misaki
Nakayama2, Yuto Oe2, and Masahiro Sakata3
1
Institute of Socio-Arts and Sciences, The University of Tokushima, Minami
josanjima-cho 1-1,Tokushima city, Japan, 2 Faculty of Integrated Arts and
Sciences, The University of Tokushima,Minami josanjima-cho 1-1,Tokushima
city, Japan, 3Graduated School of Integrated Arts and Sciences, The
169
University of Tokushima, Minami josanjima-cho 1-1, Tokushima city, Japan.
Email: tsatoh @ ias.tokushima-u.ac.jp
of three decoy sets were chosen for trials. During MD simulations, all nativelike decoys kept each initial complex structure, and some (not all) non-native
decoys dissociated. We calculated an averaged GB/SA score over the last 6-ns
MD trajectories. All of near-native decoys exhibited favorable scores, and
most of non-native decoys did not. We have successfully discriminated
between native and non-native decoys using the averaged GB/SA score over
MD trajectory. Therefore, the method is applicable to evaluate the decoy set
generated by molecular docking between Ab and Ag.
Cytidylate kinase (CMPK) catalyzes the phosphorylation of dCMP to dCDP.
In previous studies, we cloned and expressed CMPK genes from three
thermophilic Geobacillus bacteria (Bst, Gza, and Bcl), and elucidated that the
Bcl CMPK is the most thermostable for the enzyme activity and conformation
among three dimeric CMPKs. Then, we deduced that these differences in
thermostabilities might be caused by different amino acids among them. In
this study, to elucidate the mechanisms for thermostabilization of Bcl CMPK,
we focused three charged amino acids (Arg23, Glu26 and Glu213) only in Bcl
CMPK, followed by constructed and examined characteristics and
thermostabilities of three Bcl CMPK variants (R23L, E26Q and E213A). As
results, it was observed that R23L and E26Q formed trimer in addition to
dimer, and E26Q decreased the enzyme activity and affinity for nucleotides.
Furthermore, we evaluated their thermostabilities of enzymatic activity,
tertiary and quaternary structures. It was revealed that thermostabilities of
R23L and E26Q decreased in all criteria. Especially, it was suggested that the
hydrophobic region in E26Q was exposed above 70ºC, and quaternary
structure of E26Q was eliminated above 75ºC. Therefore, it was elucidated
that Arg23 and Glu26 are crucial for maintaining the thermostable
conformation of Bcl CMPK. The enzyme (0.1mg/ml) was incubated in 20mM
Tris-HCl buffer (pH 8.0) at the indicated temperatures for 1 h. The enzyme
activity was measured after rapid cooling, and the activity after heating at
40ºC was taken as 100%.
References
Mashimo T., Fukunishi Y., Kamiya N., Takano Y., Fukuda I. and Nakamura H.
―Molecular dynamics simulations accelerated by GPU for biological
macromolecules with a non-Ewald scheme for electrostatic interactions‖ J.
Chem. Theory Comput., 9, 5599-5609 (2013).
Fukuda I., Yonezawa Y. and Nakamura H. ―Molecular dynamics scheme for
precise estimation of electrostatic interaction via zero-dipole summation
principle‖ J. Chem. Phys., 134, 164107 (2011).
Fukuda I., Kamiya N., Yonezawa Y. and Nakamura H., ―Simple and accurate
scheme to compute electrostatic interaction: Zero-dipole summation
technique for molecular system and application to bulk water‖ J. Chem. Phys.,
137, 054314 (2012).
Kamiya N., Fukuda I. and Nakamura H. ―Application of zero-dipole
summation method to molecular dynamics simulations of a membrane protein
system‖ Chem. Phys. Lett., 568-569, 26-32 (2013).
Arakawa T., Kamiya N., Nakamura H. and Fukuda I. ―Molecular dynamics
simulations of double-stranded DNA in an explicit solvent model with the
zero-dipole summation method‖ PLoS ONE, 8, e76606 (2013).
P28
Method for the computation of dynamical domains based on graph
theory algorithm
Fig.1 Thermostablities of Bcl CMPK (WT) and its variants for the enzyme activity
Jun Sim,1 Jaehyun Sim,2 Eunsung Park,3 and Julian Lee1
1
Department of Bioinformatics and Life Science, Soongsil University, 369
Sangdo-ro, Dongjak-gu, Seoul 156-743, Korea
2
School of Dentistry, Seoul National University, Seoul 110-749, Korea
3
Apsun Dental Hospital, Seoul 135-590, Korea
E-mail: junsim@ssu.ac.kr
References
Sakata, M., Abe, M., Oe, Y., Kawano, N., Takeuchi, S., Maegawa, A., Kurochi,
J., Endoh, Y. and Satoh, T., ―Thermostabilities of Cytidylate kinases from
three thermophilic Geobacillus bacteria‖, The 13th Annual Meeting of The
Protein Science Society of Japan, Abstracts, 2P-080, (2013), pp.100.
We present an algorithm for computing rigid substructures of a protein, called
the dynamical domains, by comparing two distinct conformations of the
protein. The computation is performed by mapping the problem to that of
finding the maximal cliques in a graph. By utilizing a graph-theory algorithm,
the method can efficiently produce all the rigid domains present, except those
included in other rigid domains. By examining the common residues in the
dynamical domains, the hinge region paying an important role in the
conformational transition can also be identified.
P27
Molecular dynamics study on evaluation of a decoy set generated by
docking between antibody and antigen
Narutoshi Kamiya1, Noriko Shimba2 and Haruki Nakamura1
1
Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita,
Osaka, 565-0871, Japan
2
Device Solutions Center, Panasonic Corporation, 3-4, Hikaridai, Seika-cho,
Soraku-gun, Kyoto, 619-0237, Japan
E-mail: nkamiya@protein.osaka-u.ac.jp
References
[1] Nichols WL, Rose GD, Ten Eyck LF, Zimm BH., ―Rigid domains in
proteins:an algorithmic approach to their identification.‖, Proteins, Vol. 23,
No. 1,(1995), pp 38-48.
[2] Bron C, Kerbosch J., ―Algorithm 457: finding all cliques of an undirected
graph‖, Commun. ACM, Vol. 16, No. 9,(1973),pp 575-577.
A structure prediction method for antibody (Ab) and antigen (Ag) complexes,
which consists of conformer generation of Ab and Ag, molecular docking to
generate a decoy set, and a ranking the set using a scoring function, is one of
the most important techniques to design antibody drugs and biosensors. As
the scoring functions implemented in the conventional docking programs are
not always suitable for Ab and Ag, the prediction result is not accurate. In
addition, there is no evaluation method whether the predicted complex is real
or not. In this work, we present a scoring function and an evaluation method
using molecular dynamics (MD) simulation to improve the accuracy.
We introduced a score using the Generalized-Born/Surface Area method, the
GB/SA score, where interaction energy between Ab and Ag including
solvation free-energy is calculated. To test the GB/SA score, we executed the
ranking of the Ab-Ag decoy sets in the ZDOCK benchmark. The area under
the curve obtained from the GB/SA score (= 0.949) was better than that using
the ZDOCK score (= 0.918), suggesting that our score improved the docking
accuracy.
We used our new MD program, psygene-G [1], which utilizes GPGPU
(general-purpose computing on graphics processing units) for acceleration of
the electrostatic treatment. Electrostatic interaction was treated by our original
zero-dipole summation method [2]. We have attained similar structures and
dynamics properties to the particle mesh Ewald method in a pure TIP3P water
system [3], a membrane protein system [4], and a DNA-water-ion system [5].
We executed short 10-ns molecular dynamics (MD) simulations starting from
the decoy structures in explicit water, where the top 8 to 10 decoys from each
P29
Proteolytic processing of gp37 and the stoichiometry of gp37-gp38
receptor binding complex from T2-like phage PP01
Fumio Arisaka1, Kaname Nishijo1, Yasunori Tanji2 and Shuji Kanamaru1
1
Department of LifeScience and 2Department of Bioengineering, Graduate
School of Bioscience and Biotechnology, Tokyo Institute of Technology,
Yokohama 226-8501, Japan
E-mail: farisaka@bio.titech.ac.jp
T2 and T4 phages are called T-even phages. These phages recognize the
surface of their host E. coli cell with the distal tip of long tail fibers (LTFs). It
is known that T-even phages are very similar with each other, but a few
features are obviously different. One of the differences between T4 and T2 is
the receptor binding protein (RBP) of LTFs. In T4 phage, the C-terminal
domain of gp37 (gp37C) is utilized to recognize the glucose residue of
lipopolysaccharide (LPS) which is located at the surface of the host E. coli.
On the other hand, in phage T2, gp38 is attached to the C-terminus of gp37
and acts as a RBP. In addition, it is known that T2 phage gp37 undergoes
170
proteolytic processing at about 120 residues from the C-terminus and the
resultant C-terminal fragment is removed. It is believed that the processed
gp37 (gp37*) is trimer and one copy of gp38 is bind to trimeric gp37*.
PP01 phage was isolated from swine stool sample in 2002. It is classified as
T2-like phage and specifically infects E. coli O157: H7 strain [1]. It is found
that gp38, RBP of PP01, binds and recognizes the outer membrane protein
OmpC of E. coli O157: H7 [2]. The crystal structure of the C-terminal receptor
binding domain of T4 phage, gp37C, has been already reported [3], but the
structural details of the RBP of T2 type phage is still unknown. We have overexpressed, purified and characterized the C-terminal domain of gp37 (gp37C)
and gp38, the RBP, from PP01 phage. The over-expressed gp38 was insoluble,
but became soluble when co-expressed with gp37C. The co-expressed
proteins were purified as a gp37C-gp38 complex. Based on the ratio of the
band intensity of SDS-PAGE and sedimentation velocity analysis, the
stoichiometry of gp37C and gp38 in the complex has been determined to be
3:1. From the expression profile and bioinformatic analyses, it is suggested
that PP01 gp37C undergoes C-terminal processing as in T2, as expected. The
removed C-terminal 120 residues might function as an intramolecular
chaperone for the folding of gp37 C-terminal domain. The same type of
cleavable intramolecular chaperone was found in endosialidase from phage
K1-F (endoNF) [4] which was assigned as a homologous protein by HHpred,
homology detection & structure prediction server.
References
[1] S. Kim, R.T. Sauer, ―Distinct regulatory mechanisms balance DegP
proteolysis to maintain cellular fitness during heat stress.‖ Genes Dev, (2014)
accepted
[2] S. Kim, R.T. Sauer, ―Cage assembly of DegP protease is not required for
substrate-dependent regulation of proteolytic activity or high-temperature cell
survival.‖ Proc Natl Acad Sci USA,109 (2012), pp 7263-8.
[3] S. Kim, R.A. Grant, R.T. Sauer, ―Covalent linkage of distinct substrate
degrons controls assembly and disassembly of DegP proteolytic cages.‖ Cell,
145 (2011), pp 67-78.
P31
Calcium enhances the fibrinogenolytic activity of a protease from latex of
Euphorbia sp.
Jaruwan Siritapetawee1, Wanwisa Limphirat2 and Sompong Thammasirirak3
1
School of Biochemistry, Institute of Science, Suranaree University of
Technology, Nakhon Ratchasima, Thailand.
2
Synchrotron Light Research Institute (Public Organization), Nakhon
Ratchasima, Thailand.
3
Protein and Proteomics Research Center for Commercial and Industrial
Purposes (ProCCI), Department of Biochemistry, Faculty of Science, Khon
Kaen University, Khon Kaen, Thailand.
E-mail: jaruwan_siritape@yahoo.com
References
Morita, M., Tanji, Y., Mizoguchi, K., Akitsu, T., Kijima, N. and Unno, H.
―Characterization of a virulent bacteriophage specific for Escherichia coli
O157:H7 and analysis of its cellular receptor and two tail fiber genes‖, FEMS
Microbiol Lett 211, (2002), pp 77-83.
Yoichi, M., Abe, M., Miyanaga, K., Unno, H. and Tanji, Y. ―Alteration of tail
fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7‖, J
Biotechnol 115, (2005) pp 101-7.
Bartual, S. G., Otero, J. M., Garcia-Doval, C., Llamas-Saiz, A. L., Kahn, R.,
Fox, G. C. and van Raaij, M. J. ―Structure of the bacteriophage T4 long tail
fiber receptor-binding tip‖ Proc Natl Acad Sci U S A 107, (2010), pp 2028792.
Schwarzer, D., Stummeyer, K., Haselhorst, T., Freiberger, F., Rode, B., Grove,
M., Scheper, T., von Itzstein, M., Muhlenhoff, M. and Gerardy-Schahn, R.
―Proteolytic release of the intramolecular chaperone domain confers
processivity to endosialidase F‖ J Biol Chem 284, (2009), pp 9465-74
A 82 kDa serine protease with human fibrinogenolytic activity, designated as
HFP-82, was purified from the latex of Euphorbia sp. The enzyme efficiently
hydrolyzed Aα followed by Bβ chains of human fibrinogen. HFP-82 had a
broad range of pH stability (3-13). The enzyme had highest fibrinogenolytic
activity at pH 10 and temperature 35 C. In addition, the various metal ions
containing in fresh latex from Euphorbia sp. were also examined using X-ray
fluorescence (XRF) and X-ray absorption near-edge spectroscopy (XANES).
Both techniques indicated that calcium ion (Ca2+) was found high
concentration in this plant latex. Therefore the effect of Ca 2+ on the
fibrinogenolytic activity of the enzyme was investigated. The result showed
that increasing the concentration of Ca2+ (from 1 mM to 10 mM) could
markedly enhance the fibrinogenolytic activity of HFP-82. The
fibrinogenolytic activity of HFP-82 activated by Ca2+ identified in this study,
promoted the potential to develop this enzyme as an antithrombotic agent for
treatment of thromboembolic disorders. The activity of this agent may be
enhanced by plasma Ca2+, which is involved in the hemostasis pathway of
human [1].
Protein catabolism & anabolism
References
Kumar V., Abul K. and Aster J. C., ―Robbins Basic Pathology‖, 9th edition,
W.B. Saunders, (2013).
P30
Harnessing the destructive power: Activity regulation of a bacterial heatshock protease, DegP
Seokhee Kim1 and Robert T. Sauer2
P32
Influence on the stability of melanophilin by interaction with Rab27a
1
Department of Chemistry, College of Natural Sciences, Seoul National
University, Seoul 151-747, Korea
2
Department of Biology, assachusetts Institute of Technology, Cambridge, MA
02139, USA
E-mail: seokheekim@snu.ac.kr
Jong il Park1, Cheol hwan Myung1, Kyung rhim Lee1, Eun ju Oh1, Dae-Hyuk
Kweon2, Choong Hwan Lee3 and Jae Sung Hwang1*
1
Department of Genetic Engineering, College of Natural Sciences, Kyung Hee
University, Yongin, South Korea.
2
School of Life Science and Biotechnology and Center for Human Interface
Nanotechnology, Sungkyunkwan University, Suwon , South Korea
3
Department of Bioscience and Biotechnology, Konkuk University, Seoul,
South Korea
E-mail: jshwang@khu.ac.kr
Proper elimination of toxic misfolded proteins is essential for cellular life.
Proteases in protein quality control (PQC) protect cells by degrading
misfolded proteins, but the uncontrolled proteolysis may be wasteful or
cytotoxic. How the PQC proteases regulate their activity has been largely
unknown. DegP is the major PQC protease in the bacterial periplasm and
belongs to the highly conserved HtrA family, whose members are associated
with bacterial pathogenesis by various species. Here I present that distinct
self-regulatory elements in architecture control DegP proteolysis and maintain
the cellular fitness during misfolded protein stress in vivo. Biochemical,
biophysical, and structural studies revealed mechanisms of activity regulation
in vitro; the bipartite mode of substrate binding, dynamic conversion between
inactive and active states, and assembly of large cages. Mutations that modify
these processes could generate a rogue protease that kills cells by excessive
proteolysis and this lethality could be suppressed by new intragenic mutations
that offset the original mutational effect or a mutant lipoprotein that functions
as a novel inhibitor. These results demonstrate that there is a delicate balance
of quality control proteolysis in bacterial periplasm, and suggest that its
disruption either by inhibition or activation may be a good strategy for
antibiotic development.
Melanosomes are specific melanin-containing intracellular organelles of
epidermal melanocytes. In melanocytes, melanophilin / slac2-a is a Rab27a
effector which forms an interaction of Rab27a on mature melanosome
membrane and actin-based motor protein myosinVa, and the tripartite
complex regulates melanosome transport. In this study, we found that Rab27a
siRNA decreased protein level of melanophilin as well as Rab27a. When
siRab27a was treated with melan-a melanocytes, Rab27a protein was
decreased in 6 h and melanophilin protein was decreased in 24 h. Accordingly,
when we measured mRNA level of Rab27a and melanophilin, Rab27a mRNA
level was decreased but melanophilin mRNA was not changed. Another
siRab27a sequences also decreased the protein level of Rab27a and
melanophilin, but they didn't affect mRNA level of melanophilin. To
determine whether the absence of Rab27a promote degradation of
171
melanophilin, we inhibited the degradation of protein by treatment of
proteasome inhibitor (MG132) and lysosomal enzyme inhibitor (E64D and
pepstatin A) in melan-a melanocytes. After 24h treatment, MG132 inhibited
the degradation of melanophilin, but E64D and pepstatin A didn‘t influenced
on melanophinin. The absence of Rab27a enhanced the ubiquitination of
melanophilin and induced proteasomal degradation. From these results, we
concluded that the interaction with Rab27a is important for the stability of
melanophilin and melanosome transport.
digestion. We identified several endoplasmic reticulum (ER) resident proteins
and mitochondrial proteins among increased proteins. Hsc70 and Hsp90 were
identified to be decreased protein group. We also identified mRNA level
decrease of actin modulating protein, destrin, by comparing differentially
expressed genes of uninfected and infected A549 cells.
References
[1] Wasmeier, C., Hume, A. N., Bolasco, G., and Seabra, M. C. Melanosomes
at a glance. J Cell Sci 121, (2008), 3995-9.
[2] Lee, E. J., Lee, Y. S., Hwang, S., Kim, S., Hwang, J. S., and Kim, T. Y. N(3,5-dimethylphe-nyl)-3-methoxybenzamide (A3B5) targets TRP-2 and inhibits
melanogenesis and melanoma growth. J Invest Dermatol 131, (2011), 1701-9.
[3] Fukuda, M., and Kuroda, T. S. Slac2-c (synaptotagmin-like protein
homologue lacking C2 domains-c), a novel linker protein that interacts with
Rab27, myosin Va / VIIa, and actin. J Biol Chem 277, (2002), 43096-103.
[4] Hume, A. N., Ushakov, D. S., Tarafder, A. K., Ferenczi, M. A., and Seabra,
M. C. Rab27a and MyoVa are the primary Mlph interactors regulating
melanosome transport in melanocytes. J Cell Sci 120, (2007), 3111-22.
References
[1] Paton, J. C., P. W. Andrew., G. J. Boulnois., and T. J, Mitchell. Molecular
analysis of the pathogenicity of Streptococcus pneumoniae: the role of
pneumococcal proteins. Annu Rev Microbiol. Vol. 47, (1993), pp 89-115.
[2] Obaro. S., and Adegbola R., The pneumococcus: carriage, disease and
conjugate vaccines. J. Med. Microbiol. Vol. 51, (2002), pp 98-104.
[3] Breiman RF., Butler JC., Tenover FC., Elliott JA., Facklam RR.
Emergence of drug-resistant pneumococcal infections in the United States.
JAMA. Vol. 271, (1994), pp 1831-5.
P36
Bilberry Anthocyanins Neutralize the Cytotoxicity of Co-Chaperonin
GroES Fibrillation Intermediates
Hiroshi Kameda1, Hisanori Iwasa1, Naoya Fukui1, Sakiho Yoshida1, Kunihiro
Hongo1, Tomohiro Mizobata1, Saori Kobayashi2, and Yasushi Kawata1
P33
Chaperone-mediated protection of the cell membrane integrity enables
Escherichia coli to grow at lethal temperature of 50oC
Anastasia N. Ezemaduka 1, Jiayu Yu 1, Xinmiao Fu 1, and Zengyi Chang 1
1
Department of Chemistry and Biotechnology, Graduate School of
Engineering, Tottori University 680-8552, Japan
2
Wakasa Seikatsu Co., Ltd., Research Park 1st Building, 134 Chudoujiminamicho, Shimogyo-ku, Kyoto 600-8813, Japan
E-mail: d12t3002c@edu.tottori-u.ac.jp
1
Sciences, and Center for Protein Science, Peking University, Beijing 100871,
China
E-mail: anasha@pku.edu.cn
Temperature is a single environmental factor that most profoundly affects
organisms by significantly modulating many biological processes, which are
typically termed as heat shock response and cold shock response. Hence, it is
fundamental for organisms to survive or even grow under stress conditions
(e.g., at high temperature). Escherichia coli, the mostly characterized bacteria,
is known to be unable to grow at above 465 oC, with the mechanism being
poorly understood. Here we report that heterologous expression of a 17kDa
small heat shock protein partially localized in the mitochondria of
Caenorhabditis elegans (CeHSP17) enables E. coli cells to grow at 50oC, the
highest temperature ever reported. Surprisingly, it also rescues the thermal
lethality of E. coli mutant deficient of degP that encodes a protein quality
control factor localized in the periplasm. Mechanistically, CeHSP17 was found
to be secreted into the periplasmic space and associated with the cell
membrane of E. coli. As such, it is able to prevent the misfolding of outer
membrane proteins and maintain the membrane integrity at lethal temperatures.
Together, our study indicate that maintaining the cell envelope integrity plays a
crucial role for the E. coli cells to grow at high temperatures, and also shed
new light on the development of thermophilic bacteria for industrial
application.
P35
Streptococcus pneumonia Infection changed
Chaperones and an Actin Modulating Protein
Level
of
Molecular
Kyung Tae Chung1, Yun Yeong Lee and Jeong Ae Park
1
Department of Clinical Laboratory Science, College of Nursing and
Healthcare Sciences, Dong-Eui University, Busan 614-714, Korea.
E-mail: kchung@deu.ac.kr
Acute respiratory infections are the leading infectious cause of death globally
among both infants and adults [1]. Though colonization with pneumococci is
mostly symptomless, it can progress to respiratory or even systemic diseases
[2]. Streptococcus pneumoniae is one of main bacteria for communityacquired pneumonia throughout the world. The bacteria invade through lung
tissue. if not properly diagnosed and treated, S. pneumoniae infection can
lead to bacteremia, meningitis, pericarditis, empyema, purpura fulminans,
endocarditis and/or arthritis [3].
We, therefore, attempted investigation on bacterial invasion and host cell
response at protein levels using human lung epithelial cells, A549.
Streptococcus pneumoniae D39 strain was infected to A549 cells. Reduced
or increased host cell proteins which were obtained at each time point were
separated by SDS-PAGEs and analyzed using MALDI-TOF after trypsin
172
The co-chaperonin GroES (Hsp10) works with chaperonin GroEL (Hsp60) to
facilitate the folding reactions of various substrate proteins. Upon forming a
specific disordered state in guanidine hydrochloride, GroES is able to selfassemble into amyloid fibrils similar to those observed in various
neurodegenerative diseases. GroES therefore is a suitable model system to
understand the mechanism of amyloid fibril formation. Here, we determined
the cytotoxicity of intermediate GroES species formed during fibrillation. We
found that neuronal cell death was provoked by soluble intermediate
aggregates of GroES, rather than mature fibrils. The data suggest that amyloid
fibril formation and its associated toxicity toward cell might be an inherent
property of proteins irrespective of their correlation with specific diseases.
Furthermore, with the presence of anthocyanins that are abundant in bilberry,
we could inhibit both fibril formation and the toxicity of intermediates.
Addition of bilberry anthocyanins dissolved the toxic intermediates and fibrils,
and the toxicity of the intermediates was thus neutralized. Our results suggest
that anthocyanins may display a general and potent inhibitory effect on the
amyloid fibril formation of various conformational disease-causing proteins.
P37
Functional Analysis of Chaperonin GroEL with Mutants at Gly192 in the
Hinge2 Site
Naoya Fukui, Kiho Araki, Kunihiro Hongo, Tomohiro Mizobata, and Yasushi
Kawata
Department of Chemistry and Biotechnology, Graduate School of Engineering,
Tottori University 680-8552, Japan
E-mail: d13t3002u@edu.tottori-u.ac.jp
The E. coli chaperonin GroEL facilitates the folding of various substrate
proteins in vivo and in vitro. The subunit structure of GroEL is divided into
three domains; the apical domain, the intermediate domain, and the equatorial
domain. These three domains are respectively linked through two ‗Hinge‘
regions, with the ‗Hinge2‘ site connecting the apical and intermediate
domains. Hinge2 contains three glycine residues, Gly192, Gly374, and
Gly375. Our previous experiments have shown that among these three
glycines, Gly192 is essentially important for maintaining chaperonin function.
In this study, in order to understand further the importance of the Gly192 in
Hinge2, we have substituted Gly192 with various amino acid residues with
differing side chain sizes and characteristics; Ala, Asn, Val, Ile, Phe, Tyr, and
Trp. Using the AffinixQNμ QCM system, we determined quantitatively that
some G192X mutants were capable of binding co-chaperonin GroES even in
the absence of ATP. Moreover, we found that larger side chains introduced to
this site resulted in more severe effects on GroEL ATPase activity and
functional refolding ability. These findings showed clearly that the Gly192 at
Hinge2 is critical and alteration of Gly192 severely compromises an essential
movement that allows efficient chaperonin function.
P40
Structural Studies of ToxR, a Transcriptional activator in Vibrio
vulnificus
Proteins in diseases
Saba Imdad1, Na Young Park2, Kun Soo Kim2, Kyeong Kyu Kim1,
P38
Conformational analysis of hIAPP-metal complexation
electrospray ionization ion mobility mass spectrometry
1
Institute, Sungkyunkwan University, Suwon 440-746, Korea.
2
Department of Life Science, Sogang University, Seoul 121-742, Korea.
E-mail: saba.imdad@hotmail.com
using
Shin Jung C. Lee,1 and Hugh I. Kim1,2
Vibrio vulnificus, a lethal human pathogen, is the leading cause of reported
seafood-related deaths in U.S. [1,2]. Case-fatality rates are greater than 50%
for primary septicemia [3]. ToxRS system positively regulates production of
extracellular VvhA, a hemolysin influenced by temperature and/or salinity
change [4]. ToxR, an inner membrane protein with C-terminal periplasmic
domain lack homology to other proteins and N-terminal cytoplasmic domain
with strong homology to the OmpR/PhoB family of winged helix-turn-helix
transcriptional activators [5]. Several environmental factors, such as
temperature, pH, osmolarity and aeration are known to serve as the regulatory
signals of the ToxR regulon. It is not yet known that how the various
environmental signals feed into this signaling cascade via the pivotal ToxR.
To serve this aim, ToxR gene of V. vulnificus was amplified and cleaved into
N- and C-terminal domain and cloned into pet21a vector each. The Nterminal domain of ToxR was purified via Ni-NTA column, Q column and
size exclusion chromatography with high purity. Crystals of protein were
obtained by the microbatch method. Diffraction and modeling experiments
are underway. The structure of ToxR will reveal useful information to block
this upstream regulatory molecule of the virulence cascade of V. vulnificus
and control pathogenesis.
1
Department of Chemistry, Pohang University of Science and Technology
(POSTECH), Pohang 790-784, Korea
2
Division of Advanced Materials Science, Pohang University of Science and
Technology (POSTECH), Pohang 790-784, Korea
E-mail: shinjung@postech.edu
Human islet amyloid polypeptide (hIAPP) is an intrinsically disordered
protein (IDP) which does not have rigid secondary or tertiary structure. Also,
it is categorized as amyloidogenic protein to form insoluble β-structured
fibrils and to role as the pathogen to induce type II diabetes. Due to the
structural heterogeneity, a common characteristic of IDP, it has been
challenging to characterize its‘ conformational properties using conventional
structural biology tools. Normally in structural study of IDP, organic cosolvent or detergent molecule is used to stabilize the protein into a specific
conformation. In this study, however, we identify structural properties of
hIAPP and divalent metal complexes using electrospray ionization ion
mobility mass spectrometry without any stabilizing agent. We also investigate
the metal effect on fibrillation through thioflavin T fluorescence assay and
transmission electron microscopy image and subsequently understand the
influence of metallation on hIAPP fibril formation in relevance with the
portion of β-structured component. Also, the binding site for each metal is
deduced based on electron capture dissociation tandem mass spectrometry.
From the example of hIAPP-metal complexation, we will discuss beneficial
points of ion mobility mass spectrometry in conformational analysis of IDP as
well as the effect of solution-phase structural properties on the protein in gasphase.
References
Gulig P. A., Bourdage K. L and Starks A. M., ―Molecular pathogenesis of
Vibrio vulnificus”, J. Microbiol., Vol. 43, (2005), pp 118–131.
Oliver J. D. and Kaper J. B., ―Vibrio species‖, In Doyle M. P. and Beuchat L.
R. (ed.), “Food microbiology: fundamentals and frontiers”, 3rd edition, ASM
Press, Washington, DC, (2007), pp 343-379.
Bross M. H., Soch K., Morales R. and Mitchell R. B., ―Vibrio vulnificus
infection: diagnosis and treatment‖, Am. Fam. Physician, Vol. 76, (2007), pp
539-44.
Lee S. E., Shin S. H., Kim S.Y., et al., ―Vibrio vulnificus has the
transmembrane transcription activator ToxRS stimulating the expression of
the hemolysin gene vvhA‖. J. Bacteriol., Vol. 182, (2000), pp 3405-15.
Martinez-Hackert E. and Stock A. M., ―Structural relationships in the OmpR
family of winged-helix transcription factors‖, J. Mol. Biol., Vol. 269, (1997),
pp 301–312.
P39
The Co-existence of an Equal Amount of Alzheimer’s Amyloid-β 40 and
42 forms Structurally Stable and Toxic Oligomers through a Distinct
Pathway
Yun-Ru (Ruby) Chen1 and Yu-Jen Chang2
1
Genomics Research Center, Academia Sinica, Taipei, Taiwan. 128, Academia
Rd., Sec. 2, Nankang Dist., Taipei 115, Taiwan.
E-mail: yrchen@gate.sinica.edu.tw
P41
Role of the Disulfide Bond of Cu, Zn-Superoxide Dismutase in the
Amyloid Fibril Formation and Identification of the Amyloid Core
Peptides
Fibrillar amyloid-β (Aβ) is the major constituent of senile plaques in the brain
of patients with Alzheimer‘s disease (AD). Aβ is a short peptide generated
from amyloid precursor protein with two main isoforms, Aβ40 and Aβ42,
with the latter having two additional hydrophobic residues at the C-terminus.
The two isoforms have distinct characteristics, in which Aβ42 plays a more
pathogenic role. Some early-onset familial AD cases possess an elevated
Aβ42/Aβ40 level, and biochemical studies show the two species interact with
each other. Therefore, understanding structural conversion in the aggregation
of mixed Aβ isoforms is essential for elucidating AD pathogenesis. Here, we
systematically examined the differences among Aβ42, Aβ40, and various
A42/Aβ40 mixtures by monitoring the fibrillization kinetics, epitope
changes, assembly, morphology, and induced cytotoxicity. We found the
minor Aβ species in different mixing ratios modulated the major aggregation
pathway. Size-exclusion chromatography, circular dichroism spectroscopy,
and photo-crosslinking assay showed that soluble A42 oligomers were
stabilized after A40 addition, and the equimolar A42/Aβ40 mixture rapidly
formed spherical oligomers. These oligomers were the most toxic among
those examined as evidenced by neurite degeneration and neuronal toxicity.
However, these oligomers were not responsible for intracellular calcium
elevation. Overall, our results demonstrated that differently mixed Aβ species
repartitioned oligomer intermediates on the major aggregation pathway.
Furthermore, the equimolar mixture rapidly formed structurally stable and the
most toxic oligomers. These results provided information on the potential
pathological mechanisms underlying the elevated Aβ42/Aβ40 ratio in familial
AD patients and in the local environment of sporadic AD brains.
Masataka Ida, Mizuho Ando, Asumi Tanaka, Kunihiro Hongo, Tomohiro
Mizobata, and Yasushi Kawata
Department of Chemistry and Biotechnology, Graduate School of
Engineering, Tottori University 680-8552, Japan.
E-mail: ma-ida@nitta-gelatin.co.jp
Cu, Zn-superoxide dismutase (SOD1), one of causative proteins of the
neurodegenerative disease amyotrophic lateral sclerosis (ALS), forms
amyloid fibrils under the conditions in which the structure is perturbed. In this
study, we found that the structural stability of metal-free apo form was
decreased compared to metal-binding holo form of SOD1. Furthermore, the
reduction of disulfide bond between Cys57 and Cys146 of SOD1 destabilized
the tertiary structure and greatly enhanced a typical fibril formation during
incubation at the physiological conditions in vitro. In order to understand the
mechanism of fibril formation of SOD1, we identified fibril core peptides that
are resistant to protease digestion, using a combination of mass-spectroscopy
and Edman degradation analyses. The fibril core peptides were localized at
three regions; N-terminal, central, and C-terminal regions of SOD1.
Specifically synthesized three peptides corresponding to the identified regions
were confirmed to form amyloid fibrils by TEM and fluorescence microscopy
measurements, with either individuals, two, or three of them. These findings
suggest that the disulfide bond formation of SOD1 Cys57/Cys146 is pivotal
for the suppression of amyloid fibril formation and ultimately, of onset of
ALS disease.
173
Collagen triple helix repeat-containing 1 is known to be aberrantly
upregulated in most human solid tumors. In this study, the expression profile
and clinical importance of CTHRC1 were examined by various methods in
normal and tumor patient samples. CTHRC1 was detectable in normal tissues,
but also was highly expressed in tumor specimens. CTHRC1 upregulation
was significantly associated with demethylation of the CTHRC1 promoter in
colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed
that nodal status and expression of CTHRC1 (95% CI 0.999-3.984, p=0.05)
were significant prognostic factors for disease-free survival. Promoter CpG
methylation status were measured by bisulfite sequencing and
pyrosequencing analysis. Furthermore, we showed that overexpression of
CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect
mediated by extracellular signal-regulated kinase (ERK)-dependent
upregulation of matrix metalloproteinase 9. Consistent with this, we found
that knockdown of CTHRC1 attenuated ERK activation and cancer cell
invasivity. These results demonstrate that CTHRC1 expression is elevated in
human colon cancer cell lines and clinical specimens, and promotes cancer
cell invasivity through ERK-dependent induction of MMP9 expression. Our
results further suggest that high levels of CTHRC1 expression are associated
with poor clinical outcomes.
P42
Innate immune sensing of bacterial modification of host Rho
GTPases by the Pyrin inflammasome
Hao Xu1, Jieling Yang1 and Feng Shao1
1
National Institute of Biological Sciences, Beijing, China.
E-mail: xuhao@nibs.ac.cn
The inflammasome is critical for immune defense against pathogen infection
by activating inflammatory caspases like caspase-1 and caspase-11.
Activation of inflammasome is mediated by a pattern recognition receptor
(PRR). Pyrin is a candidate PRR but its physiological function remains
unknown. Mutation in Pyrin-encoding gene MEFV causes familial
Mediterranean fever, an inheritable autoinflammatory disease in human. Here
we show that Pyrin mediates caspase-1 inflammasome activation by Rhoglucosylating cytotoxin TcdB, a major virulence factor of Clostridium difficile
that causes the majority of nosocomial diarrhea. Mefv-/- macrophage did not
respond to TcdB stimulation. Other Rho-inactivating toxins, including FICdomain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus
somni IbpA) and C. botulinum ADP-ribosylating C3 toxin, could also
enzymatically activate the Pyrin inflammasome. Common to these bacterial
toxins/effectors is that they all inactivated the Rho subfamily by covalently
modifying a residue within the GTPase switch I region. Moreover,
Burkholderia cenocepacia infection inactivated Rho GTPase by deamidation
of Asn-41 in the switch I region and thereby triggered robust Pyrin
inflammasome activation. Pyrin-mediated inflammasome activation played an
important role in limiting B. cenocepacia intracellular growth as well as
mediating lung inflammation in the infected mice. Such mode of Pyrin
function represents a new paradigm in mammalian innate immunity.
References
[1] Makrilia N, Kollias A, Manolopoulos L and Syrigos K. Cell adhesion
molecules: role and clinical significance in cancer, Cancer Invest, 27, 10,
(2009):1023-1037.
P45
Crystal structure of the response regulator VraR from Staphylococcus
aureus
Chieh-Shan Lee1,2, Chia-I Liu3,4,5, Wei-Jung Chang3,4, Wen-Yih Jeng1,2,3,4,*
1
Department of Biochemistry and Molecular Biology, National Cheng Kung
University, Tainan 701, Taiwan
2
Center for Bioscience and Biotechnology, National Cheng Kung University,
Tainan 701, Taiwan
3
4
Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei 115,
Taiwan.
5
School of Medical Laboratory Science and Biotechnology, Taipei Medical
University, Taipei 110, Taiwan
E-mail: wyjeng@mail.ncku.edu.tw
P43
Cystatin SN neutralizes the inhibitory effect of cystatin C on cathepsin B
activity
Jong-Tae Kim, Seon-Jin Lee, Byungmoo Oh, Jieun Kang,
Heesoo Lee, and Hee Gu Lee*
Biomedical Genomics Research Center, Korea Research Institute of
Bioscience and Biotechnology, Daejeon, Republic of Korea
Cystatin SN (CST1) is one of the several salivary cystatins that form tight
equimolar complexes with cysteine proteases, such as the cathepsins. High
expression of CST1 is correlated with advanced pTNM stage in gastric cancer.
However, the functional role of CST1 in tumorigenesis has not been
elucidated. In this study, we showed that CST1 was highly expressed in colon
tumor tissues, compared with nontumor regions. Increased cell proliferation
and invasiveness were observed in HCT116 cell lines stably transfected with
CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also
demonstrated that CST1-overexpressing cell lines exhibited increased tumor
growth as well as metastasis in a xenograft nude mouse model. Interestingly,
CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB)
inhibitor, with a higher affinity than the interaction between CST3 and CTSB
in the extracellular space of HCT116 cells. CTSB-mediated cellular
invasiveness and proteolytic activities were strongly inhibited by CST3, but in
the presence of CST1 CTSB activities recovered significantly. Furthermore,
domain mapping of CST1 showed that the disulfide-bonded conformation, or
conserved folding, of CST1 is important for its secretion and for the
neutralization of CST3 activity. These results suggest that CST1 upregulation
might be involved in colorectal tumorigenesis and acts by neutralizing the
inhibition of CTSB proteolytic activity by CST3
Staphylococcus aureus is one of the major causes of nosocomial infections
today. Infections caused by S. aureus are a growing cause of concern owning
to the widespread development of multiple antibiotic-resistant strains,
particularly methicillin and vancomycin-intermediate resistant strains (MRSA
and VISA). The VraSR two component system is reported to be highly related
to the development of vancomycin resistance of S. aureus. VraS is a
membrane-bound sensor histidine kinase that received the signal stimulation
from environment to regulate the phosphorylation status of its cognate
response regulator proteins VraR to control target genes transcription to result
in the development of vancomycin resistance of S. aureus.
Here, we report the crystal structure of unphosphorylated VraR from S. aureus
in a dimeric form at 1.8 Å resolution. The crystals of VraR belong to the
monoclinic space group C2 containing two protein molecules as a dimer in
the asymmetric unit. The first β-strand at N-terminal end of each VraR
subunit was inserted into the other subunit to form an intertwining dimer
structure. Finally, according to previous reports and our crystal structure, we
propose a regulation model for VraR. The feedback control of vraSR operon
would be the unphosphorylated dimeric VraR rather than the
unphosphorylated monomeric VraR.
References:
Belcheva A. and Golemi-Kotra D., ―A close-up view of the VraSR twocomponent system - A mediator of Staphylococcus aureus response to cell
wall damage‖, J Biol Chem, Vol. 283, No. 18, (2008), pp 12354-12364.
Leonard P. G., Golemi-Kotra D. and Stock, A. M., ―Phosphorylationdependent conformational changes and domain rearrangements in
Staphylococcus aureus VraR activation‖, Proc Natl Acad Sci USA, Vol. 110,
No. 21, (2013), pp 8525-8530
P44
Collagen triple helix repeat containing 1 (CTHRC1) acts via ERKdependent induction of MMP9 to promote invasion of colorectal cancer
cells
Tae Woo Kim, Tae Gi Uhm, Suk Ran Yoon, Sang-Yoon Park, Tamina Park,
and Hee Gu Lee
Bioscience and Biotechnology, Daejeon, South Korea, 305-806
E-mail: tae1410 @ naver.com
P46
Structural insights into the catalytic mechanism of human squalene
synthase
174
Chia-I Liu1,2,3,*, Wen-Yih Jeng4, Wei-Jung Chang1,2, Min-Fang Shih1,2,
Tzu-Ping Ko1,2, and Andrew H.-J. Wang1,2,5,*
showed no effect on cell proliferation. Considering the importance of
HASMC migration in the development of atherosclerosis, our study provides
a novel insight into diabetic vascular complications.
1
Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei 115,
Taiwan.
3
School of Medical Laboratory Science and Biotechnology, Taipei Medical
4
Center for Bioscience and Biotechnology, National Cheng Kung University,
Tainan 701, Taiwan
5
College of Medical Science and Technology, Taipei Medical University,
Taipei 110, Taiwan
E-mail: CIL: ponpiqq@gmail.com; AHJW: ahjwang@gate.sinica.edu.tw
2
References
[1] A.K. Srivastava, High glucose-induced activation of protein kinase
signaling pathways in vascular smooth muscle cells: a potential role in the
pathogenesis of vascular dysfunction in diabetes (review), International
journal of molecular medicine, 9 (2002) 85-89.
[2] A. Goldin, J.A. Beckman, A.M. Schmidt, M.A. Creager, Advanced
glycation end products: sparking the development of diabetic vascular injury,
Circulation, 114 (2006) 597-605.
[3] C. Lu, J.C. He, W. Cai, H. Liu, L. Zhu, H. Vlassara, Advanced glycation
endproduct (AGE) receptor 1 is a negative regulator of the inflammatory
response to AGE in mesangial cells, Proceedings of the National Academy of
Sciences of the United States of America, 101 (2004) 11767-11772.
[4] D. Neves, Advanced glycation end-products: a common pathway in
diabetes and age-related erectile dysfunction, Free radical research, 47 Suppl
1 (2013) 49-69.
[5] M. Alikhani, C.M. Maclellan, M. Raptis, S. Vora, P.C. Trackman, D.T.
Graves, Advanced glycation end products induce apoptosis in fibroblasts
through activation of ROS, MAP kinases, and the FOXO1 transcription factor,
American journal of physiology. Cell physiology, 292 (2007) C850-856.
Squalene synthase (SQS) is the first enzyme involved in steroid biosynthesis
and presents an attractive target for development of cholesterol-lowering
drugs without inhibiting biosynthesis of nonsteroid biomolecules. This
enzyme catalyzes a 2-step reductive ―head-to-head‖ condensation of 2
molecules of farnesyl pyrophosphate (FPP) to form squalene by using
presqualene diphosphate (PSPP) as an intermediate. Although structures of
apo- and inhibitor-bound human SQS have been reported, they do not provide
substantial active sites information concerning catalysis. In this paper, we
present several crystals structures of human SQS and its mutants in complex
with various substrate analogs and intermediates coordinated with Mg2+ or
Mn2+, which delineate the stepwise biosynthetic pathway. Extensive study of
the SQS active site has identified several critical residues that are involved in
binding to nicotinamide adenine dinucleotide phosphate (NADPH). Based on
mutagenesis data and a locally closed (JK loop-in) structure observed in the
hSQS-(F288L)/PSPP complex, an NADPH binding model is proposed for
SQS. Our results identified 4 major steps (substrate binding, condensation,
intermediate formation, translocation) of the ordered sequential mechanism
involved in the ―1'-1‖ isoprenoid biosynthetic pathway. These new findings
clarify the previous hypotheses about SQS catalysis.
P48
Dendrite defects caused by nuclear SCA3 proteins involve impaired local
mRNA supply in Drosophila
Min Jee Kwon1*, Keun Hye Jeon1*, Yun Mi Lee1, Yuh-Nung Jan2,
Sung Bae Lee1
1
Department of Brain Science, Daegu Gyeongbuk Institute of Science and
Technology (DGIST), Daegu 711-873, Korea
2
Howard Hughes Medical Institutes, Departments of Physiology,
Biochemistry, and Biophysics, University of California San Francisco, San
Francisco, California 94143-0725, USA
E-mail: minjeek89@dgist.ac.kr
References:
Liu C. I., Jeng W. Y., Chang W. J., Shih M. F., Ko, T. P. and Wang A. H. J.,
―Structural insights into the catalytic mechanism of human squalene
synthase‖, Acta Crystallogr D Biol Crystallogr, Vol. 70, No. 2, (2014), pp
231-241.
Liu C. I., Jeng W. Y., Chang W. J., Ko T. P. and Wang, A. H. J., ―Binding
modes of zaragozic acid A to human squalene synthase and staphylococcal
dehydrosqualene synthase‖, J Biol Chem, Vol. 287, No. 22, (2012), pp 1875018757.
Lin F. Y., Liu C. I, Liu Y. L., Zhang Y., Wang K., Jeng W. Y., Ko T. P., Cao R.,
Wang A. H., Oldfield E., ―Mechanism of action and inhibition of
dehydrosqualene synthase‖, Proc Natl Acad Sci USA, Vol. 107, No. 50, (2010),
pp 21337-21342.
Recently, it has been shown that a subset of polyglutamine (polyQ) proteins
accumulated preferentially within the nucleus can modify the terminal
dendrite structures. These findings raise a crucial question on how pathogenic
SCA3 proteins accumulated within the nucleus can affect the distal dendrite
morphology as well as cytoskeletal structures. Here, we study the molecular
basis of polyQ-induced dendrite defects in Drosophila da neurons. We found
that the overexpression of certain mRNA translational inhibitors induced
dendrite defects similar to those caused by polyQ toxicity. We further showed
that the dendrite defects by this genetic inhibition of mRNA translation
involved F-actin alterations in distal dendrites as well. Moreover, our genetic
studies revealed that nuclear polyQ proteins and mRNA translational
inhibitors synergistically interacted to cause ectopic neuronal cell death, while
loss of these inhibitors failed to suppress polyQ-induced dendrite defects
indicating that these translational inhibitors may be not a direct target of
nuclear polyQ aggregates. Interestingly, it was observed that the genetic
enhancement of mRNA supply or translation could obviously ameliorate the
polyQ-induced dendrite defects. Taken together, our data suggest that specific
cellular processes, mRNA supply and local translation, may account for, at
least partially, the polyQ-induced dendrite defects in distal dendrites.
P47
Lipocalin-2 elicited by advanced glycation end-products promotes the
migration of vascular smooth muscle cells
Hee-Jung Choi1, Tae-Wook Chung1, Cheorl-Ho Kim2, Ki-Tae Ha1,*
1
Division of Applied Medicine, School of Korean Medicine, Pusan National
University, Yangsan, Gyeongsangnam-do 626-870, Republic of Korea.
2
Department of Molecular and Cellular Glycobiology, College of Natural
Science, Sungkyunkwan University, Suwon, Kyungki-do 440-746, Republic
of Korea.
* E-mail: hagis@pusan.ac.kr
P49
Endosomal Defects in Dendrite by PolyQ Toxicity are Associated with Factin Alterations
Advanced glycation end-products (AGEs) play key roles in the development
of diabetic vascular complications by activating the proliferation and
migration of vascular smooth muscle cells. Here, we identified an increase of
the migratory properties of human aortic smooth muscle cells (HASMC)
through AGE-induced expression of lipocalin-2 (LCN2). Because the AGEelicited expression of LCN2 was diminished by an antibody against the AGE
receptor (RAGE), diphenylene iodonium (DPI), N-acetyl cysteine, LY294002,
and SP600125, we suggest that AGEs enhance the expression of LCN2 via a
RAGE-NADPH oxidase-reactive oxygen species pathway, leading to the
phosphorylation of PI3K-Akt and JNK in HASMCs. In addition, a chromatin
immunoprecipitation assay and promoter assay revealed that
CCAAT/enhancer binding protein β is crucial for AGE-induced expression of
LCN2. However, any other AGE-related signaling pathway, including ERK1/2,
p38, NF-κB, and AP-1, did not affect the AGE-induced expression of LCN2.
Knockdown of LCN2 expression by shRNA showed that AGE-elicited LCN2
expression enhanced the invasive and migratory properties of HASMCs, but
Chang Geon Chung1*, Keun Hye Jeon1* , Jeong Hyang Park1 , In Jun Cha1 ,
and Sung Bae Lee1
1
Department of Brain Science, Daegu Institute of Science and Technology,
Daegu 711-873, Korea
E-mail: changgeon@dgist.ac.kr
There are nine known polyglutamine (polyQ) diseases, of which all of them
have a clear genetic cause consisting of expanded glutamine repeats within
the encoded proteins that leads to the formation of intracellular protein
aggregates [1]. While many studies so far have focused on polyQ-induced cell
death, our previous study has recently shown that there are obvious cell-typespecific defects in dendrites involving cytoskeletal alterations affecting
mainly the terminal branches in polyQ-expressing neurons, which occur prior
175
to cell death [2,3]. Here we show a noticeable defect in dendritic distribution
of endosomes and Golgi-outpost, while mitochondria and peroxisomes are
spared in polyQ-expressing Drosophila da neurons. We also found F-actin
alterations to precede endosomal distribution problems. Interestingly,
defective distribution of Golgi-outpost in dendrites appeared to be unrelated
to the alterations in F-actin in polyQ-expressing neurons, suggesting that
polyQ toxicity in neurons could result in multiple and separated cellular
defects. Taken together, we provide evidence that polyQ toxicity causes
selective cytoskeletal alterations and problems with organelle distribution.
activities. In conclusion, the induction of ROS might disturb the proteasome
activity in the cells, suggesting that reduction of ROS may be a therapeutic
target to recover proteasome activity
References
Byung Hoon Lee., Min Jae Lee., Soyeon Park., Dong Chan oh., Suzanne
Elsasser., Ping-Chung Chen., Carlos Gartner., Nevena Dimova., John Hanna.,
Steven P. Gygi., Scott M. Wilson., Randall W. King., and Daniel Finley.,
―Enhancement of proteasome activity by a small-molecule inhibitor of
USP14‖, Nature, Vol. 467, No. 9, (2010), pp 179-184
Charity T. Aiken., Robyn M., Kaake., Xiaorong Wang., and Lan Huang.,
―Oxidative Stress-Mediated Regulation of Proteasome Complexes‖, MCP,
Vol.10, No.5, (2011), pp 1-11.
Xinyu Zhang., Jilin Zhou., Alexandre F. Fernandes., Janet R. Sparrow., Paulo
Pereira., Allen Taylor., and Fu Shang. ―The Proteasome: A Target of Oxidative
Damage in Cultured Human Retina Pigment Epithelial Cells‖, IOVS, Vol. 49,
No. 8, (2008), pp 3622-3630.
References
Zoghbi, H. Y. and H. T. Orr "Glutamine repeats and neurodegeneration," Annu
Rev Neurosci Vol. 23 (2000), pp 217-247.
Lee, S. B., et al. "Pathogenic polyglutamine proteins cause dendrite defects
associated with specific actin cytoskeletal alterations in Drosophila," Proc
Natl Acad Sci U S A Vol. 108 No. 40, (2011), pp 16795-16800.
Chen, L., et al. "Axon injury and stress trigger a microtubule-based
neuroprotective pathway," Proc Natl Acad Sci U S A Vol. 109, No.1, (2012),
pp 11842-11847
P52
Protective role of Bax Inhibitor-1 (BI-1) in High Fat Induced ER Stress
and Insulin Resistance (IR) in mice.
P50
HBxBP degrades mitochondrial targeted HBx viral oncoprotein.
Kashi Raj Bhattarai, Han-Jung Chae
Young-suk Yoo1,2, Yong-Yea Park1, Hyeseong Cho1,2
Department of Pharmacology and Institute of Cardiovascular Research,
School of Medicine, Chonbuk National University, Jeonju, South Korea 560182
Email: meekasik@yahoo.com
Department of Biochemistry, Ajou University School of Medicine, suwon,
443-721
Department of Biomedical sciences, Graduate school of Ajou University,
suwon, 443-721
Republic of Korea
E-mail: goodluck22@daum.net
BI-1, is an evolutionary conserved ER-membrane protein which is identified
as a protective mechanism against apoptosis. The objective of this study was to
show the role of BI-1 in High Fat Diet associated alteration of the Unfolded
Protein Response and systemic IR. BI-1 reduced the expression of ER stress
marker GRP78, CHOP, P-PERK and IRE-1alpha by inhibiting Reactive
Oxygen Species. ER stress produced ROS generation and lipid peroxidation of
the ER membrane, but BI-1 reduced this accumulation. Electron uncoupling
between NADPH-dependent cytochrome P450 reductase (NPR) and
cytochrome P450 2E1 (P450 2E1) is a major source of ROS on the ER
membrane. The expression of CYP2E1 was increased in knock out mice and
partially reduced in adenoviral gene delivered BI-1 overexpressed mice. We
observed a high hepatic lipid accumulation in knock out mice in both acute
and chronic model and down regulation of BI-1 expression in liver. The liver
enzymes are partially reduced in BI-1 overexpressed mice. Importantly, BI-1
overexpression improved glucose metabolism and decreased protein oxidation
in diet induced IR mouse.
These results suggest that BI-1 regulates IRE1 alpha, insulin sensitivity,
hepatic lipid metabolism and protects from ER as well as an oxidative stress.
The hepatitis B virus X protein (HBx), oncogenic protein, plays an important
role in HBV replication and development of hepatocellular carcinoma. HBx
localizes to the mitochondrial outer membrane, which induces mitochondrial
dysfunction, oxidative stress and cell death. HBxBP is a E3 ubiquitin ligase.
We here showed that HBxBP binds HBx and promotes its degradation
whereas the HBxBP mutant lacking ubiquitin ligase activity did not,
indicating that HBxBP serves as a protein quality controller on viral
oncoprotein. Accordingly, overexpression of HBxBP alleviated the HBxinduced ROS generation. In addition, HBx-mediated transactivation activity
on the COX-2 promoter was weakened in the presence of HBxBP. Thus, the
data indicate that HBxBP contributes to protecting the cells from HBxmediated cytotoxic effects.
References
Lee YI., Yu DY and Moon HB., ― Human hepatitis B virus-X protein alters
mitochondrial function and physiology in human liver cells. J Biol Chem , Vol
279:15460-15471 (2004).
Gearhart TL and Bouchard ―The hepatitis B virus X protein modulates
hepatocyte proliferation pathways to st,ulate viral replication.‖ J Virol, Vol
84:2675-2686 (2010).
References
[1] Kim HR., Lee GH., Cho EY., Chae SW., Ahn T., and Chae HJ., ―Bax
inhibitor 1 regulates ER-stress-induced ROS accumulation through the
regulation of cytochrome P450 2E1‖. J Cell Sci. Vol. 122 No. 8 (2009) pp
1126-1133
[2] Bailly-Maitre B., Belgardt BF., Jordan SD., Coornaert B., von Freyend
MJ., Kleinridders A., Mauer J., Cuddy M., Kress CL., Willmes D., Essig M.,
Hampel B., Protzer U., Reed JC.,and Brüning JC., Hepatic Bax inhibitor-1
inhibits IRE1alpha and protects from obesity-associated insulin resistance and
glucose intolerance‖. J Biol Chem. Vol.;285 No.9(2010) pp 6198-207
P51
Negively Regulated Proteasomal Activity by Reactive Oxygen Species in
Acute Retinal Pigment Epithelia
Seung Kyun Shin1, and Min Jae Lee*
1
Department of Applied Chemistry, College of Applied Sciences, Kyung Hee
University, Yongin 446-701, Korea.
E-mail sincoccr@khu.ac.kr
P53
BAX Inhibitor-1 is related with Cyclosporine-induced autophagy in
kidney
Reactive oxygen species (ROS) in cells have been contributed to many
harmful effects such as aging and many human diseases. Ubiquitinproteasome system (UPS) plays an important role in the clearance of not only
protein with damages, mutations, or misfolds, but also oxidatively modified
proteins. Here, we examined the effects of ROS on the UPS dysfunction by
using the proteasomal degradation of N-end rule substrates, that has the Ncrystalin), which is essential to form structures of retina and known to be
sensitive to oxidative stress. We observed DHA- and menadione-induced
ROS in the cells by fluorescence microscopy analysis and ROS affects on
cytotoxicity. We found that total ubiquitin levels were increased by the
presence of ROS, and especially, DHA-induced ROS decreased the activity of
proteasomes, indicating that it might be due to decreased proteasomal
Raj-Kumar Yadav1, Hyung-Ryong Kim2, Han-Jung Chae1
1
Department of Pharmacology, School of medicine, Chonbuk National
University, Jeonju, Chonbuk 561-180, Korea
2
Department of Dental Pharmacology and Wonkwang Biomaterial Implant
Research Institute, School of Dentistry, Wonkwang University, Iksan, Chonbuk
570-749, Korea.
Cyclosporine A (CsA), a calcineurin inhibitor, has improved allograft survival
in solid organ transplantation and was also demonstrated that CsA induces
autophagy, both in vivo in mouse kidneys, vitro in human tubular cells
autophagy serves as a protective mechanism against CsA toxicity, showing
that CsA-induced autophagy is triggered by endoplasmic reticulum (ER)
176
stress. The CsA-induced autophagy is highly stimulated in Bax inhibitor 1
(BI-1)-overexpressing cells. Through in vivo approach, the CsA-induced
autophagosome and Lysosome formation are also increased, relating with the
protective function of BI-1 on CsA-induced nephrotoxicity. The chronic
treatment of cyclosporine causes accumulation of autophagosome and
reduced autophagic clearance. Here we have shown Bi-1 has special activity
for highly maintained lysosome activation. Transcription factor EB (TFEB)
which helps in lysosome biochgenesis is transloacated to nucleus during
autopahgolysosome activation . In CsA treated BI-1 the transcription rate of
tfeb is increased and also the localization of TFEB can be seen inside the
nucleus region during the CsA treatment.
In conclusion the Bax inhibitor 1 has a protective role in human renal tubular
cells under ER stress conditions against CsA toxicity by activation of
lysosome and formation of autophagolysosome activation.
glycosylation of Glut-2 in endoplasmic reticulum.
References
[1] Ohtsubo K., Takamatsu S. and Minowa MT., ―Dietary and Genetic
Control of Glucose Transporter 2 Glycosylation Promotes Insulin
Secretion in Suppressing Diabetes‖, Cell. Vol. 123, No. 7, (2005), pp
1307-1321.
[2] Ohtsubo K., Chen MZ. and Olefsky JM., ―Pathway to diabetes through
attenuation of pancreatic beta cell glycosylation and glucose transport‖,
Nat Med. Vol. 17, No . 9, (2011) pp 1067-1075.
[3] Helenius A., and Aebi M., ―Roles of N-linked glycans in the endoplasmic
reticulum‖, Annu. Rev. Biochem. Vol. 73 (2004) pp 1019–1049
[4] Ohtsubo
K.,
―Targeted
Genetic
Inactivation
of
NAcetylglucosaminyltransferase-IVa Impairs Insulin Secretion from
Pancreatic β Cells and Evokes Type 2 Diabetes‖, Methods Enzymol. Vol.
479(2010) pp 205-222
P54
Bax Inhibitor-1-mediated Inhibition of Mitochondrial Permeability
Transition Pore Opening and Cell Death
Geum-Hwa Lee1, Hwa-Young Lee1, Bo Li1, Hyung-Ryong Kim2, Han-Jung
Proteins analyses techniques
Chae1¶
1
Department of Pharmacology and Cardiovascular Research Institute,
Medical School, Chonbuk National University, Jeonju, 561-181, Republic of
Korea; 2Department of Dental Pharmacology and Wonkwang Dental
Research Institute, School of Dentistry, Wonkwang University, Iksan, 570749, Republic of Korea.
P56
Multi-Parametric Surface Plasmon Resonance(MP-SPR):
new possibilitiesfor characterization of biomolecularlayers.
Willem M. Albers1, Tony Munter2, Inger Vikholm-Lundin3,
Janusz W. Sadowski1, Niko Granqvist1, Annika Jokinen1
An ER stress regulator, Bax inhibitor-1 (BI-1), plays a regulatory role in
mitochondrial Ca2+ levels. In this study, we found both ER- and mitochondriaassociated membrane (MAM)-resident populations of BI-1. In BI-1overexpressing cells (HT1080/BI-1), ER stress relatively less increased
mitochondrial Ca2+ than control cells. We suggest that the regulated
mitochondrial Ca2+ in HT1080/BI-1 is due to their impaired mitochondrial
Ca2+ intake ability, as well as their lower basal levels of intra-ER Ca2+.
Moreover, opening of the Ca2+-induced mitochondrial permeability transition
pore (PTP) and cytochrome c release were regulated in HT1080/BI-1. In
HT1080/BI-1, mitochondrial membrane potential was basally low and also
resistant to Ca2+ compared with control cells. The activity of the
mitochondrial membrane potential-dependent mitochondrial Ca2+ intake pore,
the Ca2+ uniporter, was reduced in the presence of BI-1. This study also
showed that instead of Ca2+, other cations including K+ enter mitochondria
through mitochondrial Ca2+-dependent K+ channels in HT1080/BI-1,
providing a possible mechanism by which mitochondrial Ca2+ intake is
reduced, leading to cell protection. We propose a model in which BI-1mediated sequential regulations of mitochondrial Ca2+ uniporter and Ca2+dependent K+ channel opening inhibits mitochondrial Ca2+ intake, thereby
inhibiting PTP function and leading to cell protection in HT1080/BI-1.
1
BioNavis Ltd, Elopellontie 3.C, 33470 Ylöjärvi, Finland.
VTT Technical Research Centre of Finland, Process Chemistry and
Environmental Engineering, Sinitaival 6, 33720 Tampere, Finland.
3
University of Tampere, Institute of Biomedical Technology (BioMediTech),
Biokatu 6, 33014 University of Tampere, Finland
2
P55
BI-1 regulates insulin secretion induced Chronic hyperglycemia by
enhancing N-glycosylation of glucose transporter 2
Surface Plasmon Resonance is an established method for studies of molecular
interactions now for 24 years. In recent years, the Finnish National Research
Institute (VTT) has developed ―Multi-Parametric Surface Plasmon Resonance‖
(MP-SPR). With MP-SPR not only studies of molecular interactions can be
conducted, but also nanolayers can be further characterized in terms of
thickness, refractive index and adsorption coefficient. MP-SPR provides
absolute calibrated measurements of the whole SPR curve with maximum
angle range and the measurements are possible in various media ranging from
air to ethanol. Therefore, MP-SPR cannot only be used in life sciences, but
also in materials development, gas sensing, analysis of electrodeposition
processes etc.
Recent examples of application of MP-SPR to the characterization of
biofunctional layers with engineered avidins and in self-assembled layers of
hydrophilic polymers and imprinted monomolecular layers are presented.
Also some examples are given of small molecule interactions with proteins.
Hwa- Young Lee1, Hyung-Ryong Kim2, Han-Jung Chae1
1
Department of Pharmacology, School of Medicine, Chonbuk National
University, Jeonju, Chonbuk, South Korea.
2
Department of Dental Pharmacology, School of Dentistry, Wonkwang
University, Iksan, Chonbuk, South Korea.
E-mail: youngat84@gmail.com
Pancreatic beta cell-surface expression of glucose transporter2 (Glut-2) is
essential for glucose-stimulated insulin secretion (GSIS), thereby controlling
blood glucose homeostasis in response to dietary intake. Glut-2 is essential for
the primary GSIS response of mouse beta cells, and reduced Glut-2 expression
abolishes this role of the pancreas coincident with the onset of type 2 diabetes.
N-glycosyltaion of Glut-2 is required for Glut-2 residency. Glut-2 is Nglycosylated in the endoplasmic reticulum and Golgi apparatus prior to being
trafficked to the cell surface by the constitutive pathway. We observed that
Bax inhibitor-1(BI-1) overexpressing human insulinoma cells (INS-1) increase
the glucose uptake and attenuate the decrease of GSIS in the chronic
hyperglycemia condition. BI-1 is an ER-located protein with anti-apoptotic
functions, involved in the suppression of intrinsic cell death mediated by ER
calcium release, ER stress, and ischaemia. In this study, we identify that BI-1
regulates the GSIS in the chronic hyperglycemia condition and enhances N-
177
P57
Application of carbonylcarbonyl correlation NMR experiments with
ultra-high resolution for resonance assignments of disordered proteins
Yuichi Yoshimura1, Natalia V. Kulminskaya1 and Frans A.A. Mulder1
1
Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry,
Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark
E-mail: yuichi@chem.au.dk
Although various types of multidimensional triple resonance NMR
experiments have been developed for resonance assignments of isotopically
labeled proteins, limited chemical shift dispersion for disordered proteins
often hampers reliable assignments. We examined the use of backbone amide
nitrogen (NH) and carbonyl carbon (C‘) for sequential assignments in
uniformly 13C, 15N-labelled proteins. Advantages of the use of those nuclei are
that (1) they have larger chemical shift dispersion than the other backbone
nuclei, and that (2) spin relaxation rates of NH and C‘ in unfolded systems are
slower than those in folded proteins, which enables to record ultra-high
resolution spectra with long acquisition periods for chemical shift evolution.
Furthermore, the exploitation of the three-bond couplings between
sequentially adjoining C‘ nuclei (3JC‘C‘) provides sequential connectivity in
both directions [1,2], which makes the assignments more reliable. Although a
small magnitude of 3JC‘C‘ (about 1 Hz for disordered proteins) requires an
extremely long period for efficient coherence transfer, a modified phase
cycled CarrPurcell multiple pulse sequence with XY16 supercycles
(MOCCA-XY16) allows a significant reduction of relaxation loss [3]. In this
presentation, we will show the usefulness for sequential resonance
assignments of (intrinsically) disordered proteins.
side chain as well as the desired N-terminus. After a long time, more than one
initiator is attached. This phenomenon results in increase the complexity of
the tandem mass spectrometry mass spectra. In order to facilitate the
interpretation of FRIPS mass spectra, a lysine side chain is guanidinated,
using the well-known guanidination protocol. With this method, it is assured
that TEMPO-Bz-C(O)- conjugated group is attached to the desired Nterminus. Peptides prepared in this way were analyzed and it was found that
the FRIPS tandem mass spectra is readily interpreted compared with those of
putative peptides.
References
Minhee Lee, Minhyuck Kang, Bongjin Moon and Han Bin Oh, ―Gas-phase
peptide sequencing by TEMPO-mediated radical generation‖, Analyst, 2009,
134, pp1706–1712
Jihye Lee, Hyeyeon Park, Hyuksu Kwon, Gyemin Kwon, Aeran Jeon, Hugh I.
Kim, Bong June Sung, Bongjin Moon, Han Bin Oh ―One-Step Peptide
Backbone Dissociations in Negative-Ion Free Radical Initiated Peptide
Sequencing Mass Spectrometry‖, Anal. Chem., 2013, 85 (15), 7044–705
Aeran Jeon, Ji Hye Lee, Hyuk Su Kwon, Hyung Soon Park, Bong Jin Moon,
and Han Bin Oh, ―Charge-Directed Peptide Backbone Dissociations of oTEMPO-Bz-C(O)-Peptides‖, Mass Spectrom. Lett. 2013 Vol. 4, No. 4, 71–74
References
Grzesiek, S. and Bax, A., J. Biomol. NMR, 9 (1997) 207-211.
Liu, A., et al., J. Biomol. NMR, 16 (2000) 127-138.
Felli, I. C., et al., J. Biomol. NMR, 43 (2009) 187-196.
P58
Artificially Conjugated Standard, Suitable for Multimer Detection
System (MDS)
P60
Generation of bispecific antibody fragments to inhibit the growth and
migration of tumour cells
Seung Chan Kim1, and Seongmin Kang2, Seong A. An1, SangYun Kim3
1
Department of BioNano, College of BioNano, Gachon University, Gyeonggido, Korea.
2
PeopleBio Inc., Seoul, Korea.
3
Department of Neurology, Seoul National University Bundang Hospital,
Gyeonggi-do, Korea.
E-mail: chanceksc@gmail.com
Lai, A1; Hollier, B1; Shooter, G1; Van Lonkhuyzen, D1
1
Tissue Repair and Regeneration Program, School of Biomedical Science,
Institute of Health & Biomedical Innovation, Queensland University of
Technology, Brisbane, QLD
E-mail: a2.lai@qut.edu.au
In the field of ageing-related disease, protein misfolded proteins are
designated as important factors, such as amyloid beta and tau in Alzheimer‘s
disease, alpha-synuclein in Parkinson‘s disease, prion in CJD, SOD1 in ALS,
Amylin in Type II diabetes, huntingtin in Huntington‘s disease, P53 in cancer,
fibrillin-1 in Marfan syndrome, CFTR protein in cystic fibrosis, and alpha
galactosidase in Fabry disease. Those proteins are susceptible to aggregate
and cause the diseases. Commercial kits for those proteins have been
developed, but few of them were approved for diagnosis by nHTA (New
Health Technology Assessment) in Korea. But those approved commercial
kits cannot be used for oligomeric forms of target proteins.
Among those diseases, Alzheimer‘s disease is the most common disease and
its most suspected target is amyloid beta 42. This peptide is prone to
aggregate and for that reason, it is difficult to construct standard proteins
especially oligomeric forms. To differentiate these proteins more properly,
artificially conjugated standard amyloid beta 42 peptide was constructed with
several types of amyloid beta 42 peptide fragments and immunologically nonrelated carrier proteins. This protein is designed to apply to the Multimer
Detection System (MDS), which was developed to distinguish oligomer from
monomer forms in the body-originated-fluids [1].
Despite advances in the detection and treatment of breast cancer, late stage
metastatic breast cancer remains problematic with a low 5 year survival rate
of 24% [1]. Recent investigations within our laboratory have demonstrated
that the insulin-like growth factor-1 (IGF-I) and vitronectin (VN) systems
cross-interact and play key roles in cancer cell migration and proliferation. To
translate this knowledge into potential therapeutics, we aim to generate bispecific antibody fragments (BsAb) targeting both interacting systems, in
order to disrupt their roles in cancer metastasis.
A commercial single-chain variable fragment (scFv) phage display library
was used to screen independently for displayed scFv that can bind to
immobilised αv integrin or IGF-1R. Once the selection process is completed,
the binding candidiates will be sequenced and bi-specific antibody expresson
constructs will be generated and expressed. In vitro and in vivo studies such as
signaling assays, kinetics studies on the BIAcore system and real time
monitoring of cell migration and proliferation using the xCELLigence system
will be performed on both the mono and bi-specific antibody fragments.
This project aims to apply the knowledge gained by the study of the
interactions between the IGF-I and VN systems and its role in cancer to
design and produce novel anti-cancer therapeutics.
References
S.S.A. An, K.T. Lim, H.J. Oh, B.S. Lee, E. Zukic, Y.R. Ju, T. Yokoyama, S.Y.
Kim, E. Welker, ―Differentiating blood samples from scrapie infected and
non-infected hamsters by detecting disease-associated prion proteins using
Multimer Detection System‖, Biochemical and Biophysical Research
Communications
References
Siegel, R., Ma, J., Zou, Z., & Jemal, A. (2014). Cancer statistics, 2014. CA: A
Cancer Journal for Clinicians, 64(1), 9-29.
P63
The Phosphorylation Site of Lipin1 by Dullard and Activation with
TMEM188
P59
Free radical initiated peptide sequencing (FRIPS) mass spectrometry
facilitates peptide sequencing
Young Jun Kim1 and Hackyoung Kim1
Han Bin Oh
1
Department of Chemistry, Sogang University, Seoul 121-742, Korea
E-mail: hanbinoh@sogang.ac.kr
Department of Applied Biochemistry, Konkuk University, Chung-Ju, 380-701,
Republic of Korea
E-mail: ykim@kku.ac.kr
The TEMPO-based FRIPS method has been previously shown to be a
powerful tandem mass spectrometry tool for peptide sequencing. However, in
TEMPO-based FRIPS, a TEMPO-radical initiator can be attached to a lysine
Dullard is a member of DXDX(T/V) phosphatase family and serine/threonine
protein phosphatase forming an active complex that dephosphorylates and
may activate LIPIN1. LIPIN1 is a phosphatidate phosphatase that catalyzes
178
the conversion of phosphatidic acid to diacylglycerol and controls the
metabolism of fatty acids at different levels. To define the determinants of
substrate recognition and catalysis of dullard, and hence, lipin regulation,
steady-state kinetic analysis was perforned on phosphoserine-bearing
nonapeptides based on the phosphorylation sites of lipin. The metazoan
ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1regulatory subunit 1 (NEP1-R1) was recently identified. Dullard and
TMEM188 together complement a nem1Δspo7Δ strain to block endoplasmic
reticulum proliferation and restore triacylglycerol levels and lipid droplet
number. CTDNEP1 can dephosphorylate lipins in human cells only in the
presence of NEP1-R1. Therefore, NEP1-R1 is functionally conserved from
yeast to humans and functions in the lipin activation pathway. We tried to
identify the phosphorylation site of LIPIN1 by Dullard and activation
mechanism with TMEM188.
phosphorylated at Serine residue through activation of cyclin-dependent
kinase 1 (CDK1) during mitosis. The Plk1-CR-1 interaction requires the polobox domain (PBD) of Plk1 and is enhanced by Cdk-dependent
phosphorylation of CR-1 at Serine residue. Our results suggest that
phosphorylation of CR-1 at Serine residue by Cdk1 creates a docking site for
the PBD of Plk1 and facilitates the kinetochore maintaining Plk1 at
kinetochores during mitosis.
References
[1] Archambault, V., and D.M. Glover. Polo-like kinases: conservation and
divergence in their functions and regulation. Nature reviews. Molecular cell
biology. 10:265-275. (2009).
[2] Goto, H., T. Kiyono, Y. Tomono, A. Kawajiri, T. Urano, K. Furukawa, E.A.
Nigg, and M. Inagaki. Complex formation of Plk1 and INCENP required for
metaphase-anaphase transition. Nature cell biology. 8:180-187. (2006)
[3] Elowe, S., S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. Tensionsensitive Plk1 phosphorylation on BubR1 regulates the stability of
kinetochore microtubule interactions. Genes & development. 21:2205-2219.
(2007).
P64
The Deletion of N-terminal Amino Acid
Mammalian Proteasome Activity through Opening the Gate
Won Hoon Choi1, Min Jae Lee1
P66
Acetylation/deacetylation regulates mitofusin1 stability.
1
Department of Applied Chemistry, College of Applied Science, Kyung Hee
University, Yong In 446-701, Korea.
E-mail: huni@khu.ac.kr
1,2
1
1,2,*
Nguyen Thi Kim Oanh , Yong-Yea Park and Hyesong Cho
1
The proteasome, the ~2.5 MDa holoenzyme complex, plays a major role in
cells that degrades misfolded, damaged, or ubiquitinated protein. It consists of
two distinguishable the 19-subunit regulatory particle (RP, 19S) and the 28subunit core particle (CP, 20S). CP is a cylindrical structure composed of four
stacked rings. The internal two rings are made of seven -subunits (1-7) that
contain catalytic sites, while the outer two rings are made of seven -subunits
(1-7). The -rings function as the ―gate‖ when substrates enter to the barrel.
Previous studies demonstrate that the N-terminus of 3 subunit is a key
component of the ―gate‖. Here we report enhanced proteasome activity
through opening the gate by deletion of N-terminal amino residue of 3
(3N), which mainly addressed by using transient overexpression of 3N
or stable cells expressing the mutant form of 3. Proteasomes with 3N
showed facilitated -synuclein degradation, which is implicated in Parkinson's
disease, suggesting possible beneficial effects of gate opening in treating
various neurodegenerative diseases.
References
Michael G., Monica B., Alwin K., Luis M., David M. R., Robert H., Michael
H. G and Daniel F ―A gated channel into the proteasome core particle”
Nature Structural Biology, Vol. 7, No. 11, (2001), pp 1062-67.
Y. Saeki., E. Isono., and A. Toh-e ―Preparation of ubiquitinated Substrates by
the PY motif-insertion method for monitoring 26S proteasome activity.”
Methods in Enzymology, Vol. 399,(2005), pp 215-27
B.H Lee., M.J Lee., S Park., D.C Oh., P.C Chen., Carlos G., Dimova N., John
H., Steven P.Gygi., Scott M.W., Randall W.K., and Daniel F.―Enhancement of
proteasome activity by a small-molecule inhibitor of usp14” Nature, Vol.
467. ,(2010), pp 179-84
P65
Cdk1 dependent phosphorylation of CR-1 establishes a stable
kinetochore localization of Plk1 in early mitosis
Ho-Soo Lee
1, 2
1
, Sunyoung Chae , Mi-Young Cho
1, 2
and Hyeseong Cho
Department of Biochemistry, School of Medicine, Ajou Univeristy, Suwon,
443-721
2
Department of Biomedical Sciences, Graduate School of Ajou University,
Suwon, 443-721
*
Genomic Instability Research Center, School of Medicine, Ajou University,
Suwon, 443-721
Email: oanh1694@gmail.com
Mitochondria are a highly dynamics organelle population that changes size
and morphology by fusing together or dividing through fission. Several
studies show that mitochondrial fusion protects mitochondrial function, most
likely by enabling exchange of contents and other matrix components
between mitochondria or by preventing loss of mtDNA nucleoids. Mutant
mice lacking mitochondrial fusion activity show serve mitochondrial DNA
mutations and depletions that precedes respiratory defects. Mitofusins,
conserved dynamin-related GTPases in the mitochondrial outer membrane,
mediate the fusion of mitochondria. However, how Mitofusins stability is
controlled still remains unknown. Here, we found that treatment of
Nicotinamide (NAM) – deacetylase inhibitor causes the degradation of Mfn1
in a proteasome-dependent manner indicating that acetylation might control
the degradation of Mfn1. Notably, our results clearly demonstrate that Mfn1
is acetylated by an acetyltransferase (ACT), and this acetylation promotes
Mfn1 ubiquitinylation and degradation. Conversely, the de-acetyltransferase
(DAC) deacetylates and stabilizes Mfn1.Under hypoxic condition, DAC was
upregulated which enhance amount of mitochondrial localization and it
allowed the deacetylation and stabilization of Mfn1. These observations
represent an example that deacetylation targets Mfn1 stability in response to
hypoxic condition changes. Given that the present study may provide insight
into the underlining mechanism of deacetylation-induced stabilization of
Mfn1 in preserving cellular homeostasis.
References
[1] Guedes-Dias P1, Oliveira JM., ―Lysine deacetylases and mitochondrial
dynamics in neurodegeneration‖, Biochim Biophys Acta., 1832(8), (2013),
1345-59.
[2] Lee JS1, Yoon YG, Yoo SH, Jeong NY, Jeong SH, Lee SY, Jung DI, Jeong
SY, Yoo YH., ―Histone deacetylase inhibitors induce mitochondrial
elongation‖, J Cell Physiol. 227(7), (2012), 2856-69.
[3] Kang HT1, Hwang ES.,‖ Nicotinamide enhances mitochondria quality
through autophagy activation in human cells‖ Aging Cell., 8(4), (2009), 42638.
1, 2,*
1
Department of Biochemistry and Molecular Biology, Ajou University, Suwon
443-721, South Korea
2
Genomic Instability Research Center, School of Medicine, Ajou University,
Suwon 443-721, South Korea
E-mail: jpeople77@hotmail.com
CR-1 was shown to be present in the interphase centromere-associated
components but its centromeric function remained obscure. Here, we report
that CR-1 has a unique role at mitotic centromeres. We identified centromeric
localization of CR-1 at the mitotic chromosomes. Depletion of CR-1 resulted
in severe defects in chromosomal congression and kinetochore-microtubule
attachment. In mitosis, CR-1 was co-precipitated with Aurora B and Plk1, but
Plk1 was a direct binding partner of CR-1. Depletion of CR-1 resulted in a
significant loss of Plk1 in mitotic kinetochores. Loss of Plk1 at the mitotic
kinetochore was restored by exogenously introduced CR-1 C-terminal mutant
that retains Plk1 binding. Here, we demonstrate that CR-1 can be
P67
A novel role of a protein tyrosine phosphatase in the regulation of
signaling pathways
Anna Ju and Sayeon Cho
College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of
Korea.
179
E-mail: aisuru_00@hanmail.net
Nguyen Minh Duc*, Dong Kyun Kim* and Ka Young Chung
*Equally contributed
Mitogen-activated protein kinases (MAPKs) regulate a vast array of
physiological processes such as gene expression, cell proliferation, and
programmed cell death. The MAPKs are activated by dual phosphorylation on
threonine and tyrosine residues through sequential activation of protein
kinases. Recent studies have shown that the protein kinases that form MAP
kinase signaling modules may be organized into signaling complexes by a
scaffold protein. These mammalian scaffold proteins are essential regulators
of many key signaling pathways by assembling the relevant molecular
components in mammals. In this study, we report that a dual-specificity
phosphatase (DUSP), a member of the protein tyrosine phosphatase family,
acts as a distinct scaffold protein in particular MAPK signaling. This DUSP
enhanced c-Jun N-terminal kinase (JNK) activation regardless of its
phosphtase activity but had no effects on p38 in HEK 293 cells. Using coimmunoprecipitation analysis, we found that the DUSP enhanced activation
of JNK signaling pathway through the apoptosis signal-regulating kinase 1
(ASK1), MAPK kinase (MAPKK) MKK7, c-Jun N-terminal kinase (JNK)
complexes in vivo and in vitro. In addition, the oligomerization of DUSP was
observed, suggesting that oligomerization of DUSP is involved in the
regulation of JNK signaling pathway. Thus, our data presents that DUSP
regulates JNK activation as a scaffold protein.
School of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Korea
E-mail: minhduchup@gmail.com, kurababy@skku.edu
Ezrin-radixin-moesin-binding protein 50 (EBP50) is a scaffolding protein that
contains two post synaptic density-95/disk-large/ZO-1 homology (PDZ)
domains and an ezrin/radixin/moesin-binding (EB) domain. It scaffolds
various proteins including membrane receptors and transporters and signals
them into functional complexes. Several studies defined the high resolution
structures of isolated PDZ domains. However, the structural analysis of fulllength EBP50 is limited. Here, we studied the conformation of full-length
EBP50 using hydrogen/deuterium exchange mass spectrometry (HDX-MS).
The HDX data showed that linker regions and most of the C-terminal region
are disordered whereas PDZ domains are more ordered. The HDX data also
suggested that PDZ1 is more dynamic than PDZ2.
P70
The effects of ppGpp on cAMP receptor protein (CRP) and histone-like
nucleoid structural protein (H-NS)
References
[1] Good, M. C., Zalatan, J. G., and Lim, W. A., ―Scaffold Proteins: Hubs
for Controlling the Flow of Cellular Information‖, Science, Vol. 332, No.
6030, (2011), pp. 680-686
[2] Haeusgen, W., Herdegen, T., and Waetzig, V., ―The bottleneck of JNK
signaling: Molecular and functional characteristics of MKK4 and MKK‖, Eur
J Cell Biol, Vol 90, No. 6-7, (2011), pp. 536-544.
[3] Shen, Y., Luche, R., Wei, B., Gordon, M. L., Diltz, C. D., and Tonks, N.
K. ―Activation of the Jnk signaling pathway by a dual-specificity phosphatase,
JSP-1‖, P Natl Acad Sci USA, Vol. 98, No.24, (2001), pp. 13613-13618.
Thanh Tuyen Tran1 and Che-Hun Jung1,2
1
Department of Molecular Medicine, 2Department of Chemistry, Chonnam
National University, Gwangju 500-757, Korea
E-mail: nucuoithienthan2812@gmail.com
The global regulation of guanosine 5‘-diphosphate 3‘-diphosphate (ppGpp) in
replication, transcription, and translation has been reported. In this study, we
report the binding of ppGpp on cAMP receptor protein (CRP) and histone-like
nucleoid structural protein (HNS). CRP and H-NS from E. coli were cloned,
over-expressed, and purified by affinity and ion exchange chromatography.
The binding affinities of ppGpp on these proteins were determined by
enzymatic assay and fluorescence spectrometry. The dissociation constants
for cAMP, ppGpp, GTP, and GDP on CRP were determined by fluorescence
spectrometry as 30.7±5.1, 36.1±9.2, 56.3±4.1, and 312±137 µM, respectively.
In the presence of 200 µM cAMP, the KD for ppGpp, GTP, and GDP were
7.1±0.7, 175±88, and 67±16 µM, respectively, which suggested that the
binding affinity for ppGpp to CRP increased 5 fold in the presence of cAMP.
The KD for ppGpp, GTP, and GDP on HNS were also determined as 49±16,
59±13, and 60±17 µM, respectively. This study identifies novel ppGppregulated transcription factors and suggests a regulatory role of ppGpp on
transcription.
P68
The Effect of Cell Cycle-Dependent Phosphatases on Apoptosis signalregulating kinase 1
Jaehee Choun1, Young-Chang Cho1 and Sayeon Cho1
1
College of Pharmacy, Chung-Ang University, Seoul 151-747, Korea.
E-mail: newstart500@hanmail.net
Apoptosis signal-regulating kinase 1 (ASK1) is a member of mitogenactivated protein (MAP) kinase kinase kinase family and plays a critical role
in mediating apoptosis signals initiated by cellular stresses. ASK1 is activated
by phosphorylation at Thr-838. Here, we show that ASK1 is regulated by cell
division cycle 25 homolog C (cdc25C) phosphatase. In cells arrested in G2/M
phase following nocodazole treatment, ASK1 activity increased, which was
concomitant with increased phosphorylation at Thr-838 of ASK1.
Dephosphorylation of phospho-Thr-838 was induced by cdc25C phosphatase
both in vitro and in vivo. In addition, knockdown of cdc25C increased
activities of ASK1 and ASK1 downstream factors in cells and overexpression
of cdc25C inhibited ASK1-mediated apoptosis, suggesting that cdc25C is a
negative regulator of ASK1. Cdc25C was hyperphosphorylated and thus
hyperactivated upon exposure to nocodazole, but hyperphosphorylated
cdc25C had significantly reduced affinity to ASK1 compared to
hypophosphorylated cdc25C with basal phosphatase activity, implying that
enhanced ASK1 activity in G2/M arrested cells is due to reduced binding of
hyperphosphorylated cdc25C to ASK1. Thus, our findings suggest that
cdc25C negatively regulates the proapoptotic ASK1 in a cell cycle-dependent
manner.
References
[1] Takahashi, M., Blazy, B., Baudras, A., ―An equilibrium study of the
cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine
cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate
receptor protein from Escherichia coli‖, Biochemistry, Vol. 19, (1980), pp
5124-5133.
[2] De Crombrugghe, B., Chen, B., Gottesman, M. and Pastan, I., ―Regulation
of lac mRNA synthesis in a soluble cell-free system‖, Nature New Biol. Vol.
230, (1971), pp 137-140.
[3] Dorman, C. J., ―H-NS: a universal regulator for a dynamic genome‖,
Nature Rev. Microbiol. Vol. 2, (2004), pp 391-400.
P71
The regulation of protease DegP with ppGpp
Pilwon Seo1 and Che-Hun Jung1,2
References
Ichijo H, Nishida E, Irie K., ―Induction of apoptosis by ASK1, a mammalian
MAPKKK that activates SAPK/JNK and p38 signaling pathways.‖ Science
Vol. 275, (1997), pp 90-94.
Karlsson-Rosenthal C and Millar JB., ―Cdc25: mechanisms of checkpoint
inhibition and recovery.‖ Trends Cell Biol Vol. 16, (2006), pp 285-292,.
[3] Zou X, Tsutsui T, Ray D., ―The cell cycle-regulatory CDC25A
phosphatase inhibits apoptosis signal-regulating kinase 1.‖ Mol Cell Biol Vol.
21, (2001), pp 4818-4828.
1
Department of Chemistry, 2Department of Molecular Medicine, Chonnam
National University, Gwangju 500-757, Korea
Email: iamdecor@naver.com
DegP (protease do) of Escherichia coli is a heat-shock inducible periplasmic
serine protease which functions as a protease with a chaperone activity. It
degrades abnormal proteins in the periplasm, including mutant proteins,
oxidatively damaged proteins, and aggregated proteins. Guanosine 5‘diphosphate 3‘-diphosphate (ppGpp) acts as an alarmone in the stringent
response under conditions of nutrient and energy starvation or other
environmental stresses in bacteria. In the previous study, we had discovered
that DegP bound to ppGpp column. To elucidate a possible regulation of DegP
by ppGpp, DegP from Escherichia coli was cloned, over-expressed in BL21,
P69
Structural Analysis of Full Length EBP50 by Hydrogen/Deuterium
Exchange Mass
180
and purified by affinity and ion exchange chromatography. The binding
affinities of DegP with ppGpp, GTP, and GDP were determined by
fluorescence spectroscopy. This is the first report showing that a protease is
also regulated by ppGpp, expanding the regulatory roles of ppGpp.
40002, Thailand
E-mail: somkly@kku.ac.th
The anti-inflammatory effect of Crocodylus siamensis hemoglobin (cHb) was
measured by examining its ability to reduce inflammatory damage through
suppressing nitric oxide (NO) production in murine macrophages RAW 264.7
cells stimulated with lipopolysaccharide (LPS). Cells were treated with LPS
(1 µg/ml) and 100 µg/ml of cHb at 37°C for 24 h. Then, 100 µl of culture
medium from each sample was mixed with the same volume of Griess reagent
and incubated at room temperature for 10 min. The absorbance was measured
at 550 nm. As determined by the MTTassay, no cytotoxicity of cHb (100-1000
µg/ml) was observed. LPS showed significant up regulation of nitrite
production in LPS-treated cell when compared to the untreated cell that used
as control. In contrast, cells treated with cHb exhibited a significant
suppressed in the induction of nitrite after stimulation with LPS by
approximately 51% compared to that in LPS-stimulated cells. This result
suggests that cHb has a potent anti-inflammatory activity in RAW264.7 cells.
References
[1] Jiang J. et al., ―Activation of DegP chaperone-protease via formation of
large cage-like oligomers upon binding to substrate proteins‖, PNAS, Vol. 105,
No. 33, (2008), pp 11939–11944
[2] Krojer T. et al, ―Structural basis for the regulated protease and chaperone
function of DegP‖, Nature, Vol. 453, (2008), pp 885-890
Protein as therapeutics
P72
Cloning and production of polyclonal antiserum against sugarcane
sucrose-transporter protein SUT1
P74
Fast Structural Characterization of Antibody Drug Conjugates by
UPLC-MS and Targeted Bioinformatics
Bambang Sugiharto1 , Popy Hartatie Hardjo2, Nurul Holifah1 and Koshun
Ishiki3
1
Department of Biology and Center Development of Advanced Science and
Technology, Jember University, Jl. Kalimantan No. 37, Jember 68121,
Indonesia
2
Department of Biology, Faculty of Techno Biology, University of Surabaya,
Jl. Raya Kalirungkut, Surabaya Indonesia
3
Tropical Agriculture Research Front, Japan International Research Center
for Agricultural Sciences, Maezato-Kawarabaru, Ishigaki, Okinawa 907-0002,
Japan
E-mail: sugiharto.fmipa@unej.ac.id
Asish Chakraborty;Henry Shion; Robert Birdsall; Weibin Chen
1
Waters Corporation, Milford, MA; 2Waters MS Technologies, Manchester,
UK
E-mail: Paul_Park@waters.com
Antibody-drug conjugates (ADCs) are a class of therapeutic agents that are
produced by covalently linking a potent cytotoxic agent to a monoclonal
antibody (mAb) for the treatment of cancers and inflammatory diseases. The
conjugation process, regardless of the site and process used for linkage,
results in heterogeneous ADC molecules with variation in both the
distribution and loading of cytotoxic drug species, and therefore creates great
challenges for structural characterization of these molecules.
In the current study, we performed a series of studies to characterize the
structures of a couple of ADC molecules. We first studies the drug
distribution profile of ADCs by electrospray quadruple time-of-flight mass
spectrometry(ESI-QTOFMS). Two different chromatographic conditions
(SEC and RP) were used to introduce samples to the ESI-MS. The use of MSfriendly SEC with sub-2 micron column packing materials directly coupled to
ESI-MS provided a routine analysis method for profiling of conjugates and
conjugate subunits, degree of conjugations, sequence confirmation (isotope
resolution), and MW measurement. The results showed that both light and
heavy chains contained linked drugs, and the antibody to drug ratio (DARs)
were automatically calculated using a bioinformatics tool (Unifi). The
conjugation sites in both chains were determined by peptide mapping using
trypsin and Asp-N protease digestion. Protease digestion identified the
modified lysine residues through high energy fragmentation data. The
analysis of Asp-N digest revealed the modified peptides by confirming a mass
increase corresponding to the modification during the automatic data
processing, and estimates the percentage of site modification for each lysine
residue. The two digestion methods provided consistent results, leading to the
identification of modified lysine residues in both light and heavy chains. The
UPLC-QTOF MS system combing with a targeted bioinformatics tool
presents a vial approach to thoroughly investigate the structures of ADC
molecules, and facilitates the development of the increasingly important
therapeutic conjugates.
Sucrose is a major photosynthetic product that transported from source to sink
tissues by sucrose transporter proteins (SUT) and among this protein family
SUT1 is a type of sucrose transporter that has a high affinity for sucrose
loading mechanism in plants. To develop immuno diagnostic tools for the
detection of the SUT1 protein, the production of good quality antibodies is a
necessity. The full length cDNA encoding for the SUT1 protein was cloned
by RT-PCR from sugarcane cultivar NiF8 and the cDNA fragment encoded
for a loop domain of the SUT1 protein was sub-cloned into bacterial
expression vector (pET28a). The pET28a plasmid contained the cDNA
fragment of SUT1 was transformed into Escherichia coli BL21 and the
recombinant SUT1 protein was expressed as a fusion protein containing hexa
histidine-tag. The loop domain of SUT1 protein was expressed in the
unexpected insoluble fraction, thus the protein was further purified under
denaturing condition by affinity chromatography and yielding 1.75 mg per
liter bacterial culture. The purified protein was used as an antigen for raising
polyclonal antibody in a rabbit. Specificity of the antibody in the serum was
determined by Ouchterlony and Western blot analysis. The antibody was able
to detect the recombinant SUT1 protein at concentration up to less than 1 ng.
The antibody was also successfully detecting 70 kDa SUT1 protein in the
insoluble fraction of the extractant from sugarcane leafs. This report on
production of the polyclonal antibody is useful tools for studying role of the
SUT1 protein on the sucrose transport and accumulation in sugarcane.
References
Novita H., Sumadi, Restanto DP., Siswoyo TA. and Sugiharto B., ―Isolation
and characterization of the genes encoding for sucrose transporter proteins in
sugarcane (Saccharum officinarum)‖. Jurnal Ilmu Dasar, Vol. 8, No. 2,
(2007), pp 118-127
Rae AL., Perroux JM. and Grof CPL., ―Sucrose partitioning between vascular
bundles and storage parenchyma in the sugarcane stem: potential role for the
ShSUT1 sucrose transporter‖, Planta, Vol. 220 (2005), pp 817–825
P75
Engineering Aglycosylated Antibodies for
Next –Generation Immunotherapeutics
Man Seok Ju, Sang Hwan Ko, Migyeong Jo and Sang Taek Jung
P73
Crocodylus siamensis hemoglobin reduces inflammatory damage to
lipopolysaccharide-stimulated murine macrophages
Department of Bio and Nano Chemistry, Kookmin University, Seoul, Korea,
136-702
E-mail : seok0801@kookmin.ac.kr
Nisachon Jangpromma1,2, Natthiya Poolperm1,2, Santi Phosri1,2,
Sakda Daduang1,2 and Sompong Thammasirirak1,2
Antibody IgG has been evolved in natural immune system against a variety of
pathogens and widely used for therapeutics, diagnostics, and research
reagents due to its high evolvability which adopts new functions at ease with
tolerating destabilizing mutations. In IgG molecules, the glycan appended to
N297 residue of upper CH2 region is indispensable for binding to FcγRs(Fc
gamma receptors) expressed on the surface of various immune effector cells
and eliciting therapeutic effector functions. Therefore, aglycosylated
1
Department of Biochemistry, Faculty of Science, Khon Kaen University,
Khon Kaen, 40002, Thailand
2
Protein and Proteomics Research Center for Commercial and Industrial
Purposes (ProCCI), Faculty of Science, Khon Kaen University, Khon Kaen,
181
antibodies expressed in bacteria have no ADCC (Antibody dependent cell
mediated cytotoxicity) and ADCP (Antibody dependent cell mediated
phagocytosis) activity for the clearance of tumor cells. A set of Fc (fragment
crystallizable) engineered aglycosylated antibodies displaying unique FcγRs
selectivities and therapeutic effector functions have been isolated by high
throughput library screening and will be discussed.
compositions, we generated a series of undecapeptide isomers having the
L5K5W formula. Amino acid sequences were designed to be perfectly
amphipathic when folded into a helical conformation by converging leucines
onto one side and lysines onto the other side of the helical axis. The single
tryptophans, whose positions were varied in the primary structures, were
located commonly at the critical amphipathic interface in the helical wheel
projection. Helical conformations and the tryptophanyl environments of the
11 L5K5W peptides were confirmed and characterized by circular dichroism,
fluorescence and nuclear magnetic resonance spectroscopy. All of the isomers
exhibited a potent, broad-spectrum of antibacterial activity with just a slight
variance in individual potency, whereas their hemolytic activities against
human erythrocytes were significantly diversified. Interestingly, helical
dispositions and fluorescence blue shifts of the peptides in aqueous
trifluoroethanol solutions, rather than in detergent micelles, showed a marked
linear correlation with their hemolytic potency. These results demonstrate that
our de novo design strategy for amphipathic helical model peptides is
effective for developing novel antimicrobial peptides and their hemolytic
activities can be estimated in correlation with structural parameters.
References
Jung ST, et al., ―Aglycosylated IgG variants expressed in bacteria that
selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic
cells‖, Proc Natl Acad Sci USA, 107(2), (2010), 604-609.
Jung ST, Kang TH, & Georgiou G, ―Efficient expression and purification of
human aglycosylated Fcγ receptors in Escherichia coli‖, Biotechnol Bioeng
107(1), (2010), 21-30.
Jung ST, Kang TH, Kelton W, & Georgiou G, ―Bypassing glycosylation:
engineering aglycosylated full-length IgG antibodies for human therapy‖,
Curr Opin Biotechnol 22(6), (2011) 858-867.
Jung ST, Kelton W, Kang TH, Ng DT, Andersen JT, Sandlie I, Sarkar CA,
Georgiou G, ―Effective phagocytosis of low Her2 tumor cell lines with
engineered, aglycosylated IgG displaying high FcγRIIa affinity and
selectivity‖, ACS Chem Biol 15(8), (2013), 368-375.
Ju MS & Jung ST, ―Aglycosylated antibodies: steps toward next-generation
immunotherapeutics‖, Curr Opin Biotechnol, (2014), article in review.
P78
Size-controlled doxorubicin-loaded fibrinogen microspheres for the
application in tumor targeting drug delivery
Jae Yeon Joo1, Do Hyun Kim1 and Seong Soo A. An1
P76
Faciliateted Degradation of Proteotoxic Proteins by
Proteasome-Mesoporous Silica Nanoparticle Complexes
1
Department of Bionano, Gachon University, Seongnam 461-701, Korea.
E-mail: joojy21@gmail.com
Delivering
Dong Hoon Han, and Min Jae Lee
Doxorubicin (DOX) is one of the widely used anti-tumor agents for various
cancer chemotherapies, which can slow down the growth of cancer cells by
intercalating DNA. Despite its efficient medicinal effect, many side effects of
DOX such as myelosuppression and cardiotoxicity have been reported. Our
group has fabricated fibrinogen (Fbg) microspheres as a DOX carrier to
reduce unintended toxicity by free-DOX administration and to increase the
drug efficiency through site specific and targeted delivery. Size-controlled
Fbg microspheres were manufactured in microchannel including T-junction.
In addition, DOX was conjugated with Fbg by using proper linker and the
drug loading and releasing test was conducted. In vitro drug efficacy test of
DOX-loaded Fbg microspheres was conducted with RFP or GFP-expressing
U2-OS, and time-lapse live cell images were captured. As a result, the U2-OS
cell death by DOX-loaded Fbg microspheres was confirmed through gradual
cell shrinkage and decreasing confluences by cell detachment from the culture
plate. In conclusion, the Fbg microspheres might be applied in various drug
delivery fields.
Department of Applied Chemistry, College of Applied Sciences,
Kyung Hee university, Yongin 446-701, Korea.
E-mail: ehd37@khu.ac.kr
Oligomerization of tau proteins is closely implicated in the prevalence of
Alzheimer‘s disease (AD), which may link to decreased or impaired
proteasome activities. We attempted here the delivery of active human
proteasomes using mesoporous silica nanoparticles (MSNs) into the cells for
enhancement of proteolytic activities against tau aggregates related with AD.
The binding of His-tagged proteasomes that were affinity-purified from
HEK293-pre1-HTBH cells to Nickel-charged MSNs (MSNPNs) was noncovalent interaction, and confirmed with the increase of surface charge and
transmission electron microscopy (TEM) analysis. The complexes, megadalton sized-proteasomes and MSNPNs, can be efficiently internalized into
the cells through clathrin-mediated endocytosis without cytotoxicity, and still
retained proteolytic activities recovered from the acidic endosomal condition.
The delivered human proteasomes degraded tau proteins which are
overexpressed and aggregation-prone in cultured cell models. In addition to
tau degradation, ROS-induced cytotoxicity might be reversed by proteasome
delivery. Accordingly, we suggest that proteasome delivery using MSNPNs
may be a beneficial strategy to facilitate degradation of proteotoxic proteins
which are implicated in cellular stress or neurodegenerative disorders.
References
Grossman, J. H. & McNeil, S. E. Nanotechnology in Cancer Medicine.
Physics Today 65, 38, (2012).
Rajangam, T., Paik, H.-j. & An, S. Development of fibrinogen microspheres
as a biodegradable carrier for tissue engineering. BioChip J 5, 175-183,
(2011).
Miyazaki, S., Hashiguchi, N., Yokouchi, C., Takada, M. & Hou, W. M.
Antitumour effect of fibrinogen microspheres containing doxorubicin on
Ehrlich ascites carcinoma. The Journal of pharmacy and pharmacology 38,
618-620 (1986).
References
Min Jae Lee, Jung Hoon Lee and David C. Rubinztein., ―Tau degradation:
The ubiquitin-proteasome system versus the autophagy-lysosome system‖,
Prog Neurobiol, Vol.105, (2013), pp 49-59
Hee-Kyung Na, Mi-Hee Kim, Kihyun Park, Soo-Ryoon Ryoo, Kyung Eun
Lee, Hyesung Jeon, Ryong Ryoo, Changbong Hyeon, and Dal-Hee Min.,
―Efficient functional delivery of siRNA using mesoporous silica nanoparticles
with ultralarge pores‖, Small, Vol.8, No. 15, (2012),
pp 1752-61.
Bhaswati Bandyopadhyay, Guibin Li, Haishan Yin and Jeff Kuret., ―Tau
aggregation and toxicity in a cell culture model of tauopathy‖, Journal of
Biology and Chemistry, Vol 282, No. 22, (2007), pp 16454-16464.
P79
De novo designed nonapeptide isomers with antimicrobial activities and
anti-inflammatory potentials
Na-Hyun Oh, Xiao Sun, Jae-Wan Choi, Do-Wan Sim, Hyung-Sik Won and
Kwang-Ho Lee*
Department of Biotechnology, Research Institute of Inflammatory
Diseases(RID), Konkuk University, Chungju, Chungbuk 380-701, Republic of
Korea
E-mail: annie90222@naver.com
P77
De novo designed L5K5W model peptide isomers with potent
antimicrobial and varied hemolytic activities
Antimicrobial peptides (AMPs) are appreciated as multifunctional host
defense peptides, including their immunomodulatory properties. Here, a series
of nonapeptide isomers with the L4K4W formula were generated. Amino acid
sequences were designed to be perfectly amphipathic when folded into a
helical conformation by converging leucines onto one side and lysines onto
the other side of the helical axis. The single tryptophans, whose positions
were varied in the primary structures, were located commonly at the critical
amphipathic interface in the helical wheel projection. Helical conformations
Jae-Wan Choi, Sung-Hee Lee and Hyung-Sik Won*
Department of Biotechnology, Konkuk University, Chungju, Chungbuk 380701, Republic of Korea
E-mail: cjw2650@gmail.com
In an effort to develop short antimicrobial peptides with simple amino acid
182
in a membrane-mimetic environment were confirmed and characterized by
circular dichroism spectroscopy. Most of the isomers exhibited a potent,
broad-spectrum of antibacterial activity with neither cytotoxicity nor
hemolytic activity against human erythrocytes. Additionally, some of them
showed potent anti-inflammatory potentials, which were verified in the
lipopolysaccharide (LPS)-stimulated RAW264.7 cells. These results
demonstrate that our de novo design strategy for amphipathic helical model
peptides is effective for developing novel peptide agents with both
antimicrobial and anti-inflammatory activities and we expect that some our
L4K4W peptides can be suggested as useful molecules for such therapeutic
development.
industrial productions. To investigate the mechanism of enzyme stability,
establish efficient enzyme stabilization strategies, is not only a challenging
hotspot in biology and protein engineering, but also the urgent problem to be
solved in industrial production.
Comparative studies of the relationship between enzyme conformation and
activity during denaturation suggest that the active site is more fragile than
the enzyme as a whole Because the active site plays a key role in enzymatic
catalysis, maintaining its correct conformation is the key to engineering its
kinetic stability, understanding how rigidity in the active site affects protein
kinetic stability will provide new insight into enzyme stabilization. In this
study, we demonstrated enhanced kinetic stability of Candida antarctica
lipase B (CalB) by mutating the structurally flexible residues within the active
site. Six residues within 10 Å of catalytic Ser105 residue with a high B factor
were selected for iterative saturation mutagenesis. After screening 2200
colons, we obtained the D223G/L278M mutant, which exhibited a 13-fold
increase in half-life at 48°C and a 12°C higher T5015. Further characterization
showed that global unfolding resistance against both thermal and chemical
denaturation also improved. Analysis of the crystal structures of wild-type
CalB and the D223G/L278M mutant revealed that the latter formed an extra
main chain hydrogen bond network with seven structurally coupled residues
within the flexible α10 helix, which are primarily involved in forming the
active site. Further investigation of the relative B factor profile and molecular
dynamic simulation confirmed that the enhanced rigidity decreased
fluctuation of the active site residues at high temperature. These results
indicate that enhancing the rigidity of the flexible segment within the active
site may provide an efficient method for improving enzyme kinetic stability.
P80
The role of Dual-specificity phosphatase 5 in the restraint of proinflammatory cytokines production in lipopolysaccharide-stimulated
RAW 264.7 macrophages
Huiyun Seo and Sayeon Cho
College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea
E-mail: huiyunseo@snu.ac.kr
Mitogen-activated protein kinases (MAPKs) are key players of physiological
responses by signal cascade. For immune cells, MAPK pathway is regulated
delicately during inflammation through the unique substrates. Dual-specificity
phosphatases (DUSPs) are a subfamily of protein tyrosine phosphatases
(PTPs), many of which specifically dephosphorylate MAPKs on both tyrosine
and serine/threonine residues. In present report, we investigated that DUSP5
regulates pro-inflammatory cytokines in RAW 264.7 mouse macrophage cells
stimulated with lipopolysaccharide (LPS). DUSP5 mRNA and protein
expression levels were rapidly increased and its overexpression suppressed
secretion of TNF-α and IL-6 in LPS-stimulated RAW 264.7 cells. Using small
interfering RNA (siRNA) analysis, we confirmed that knock-down of DUSP5
in RAW 264.7 cells increased TNF-α and IL-6 production. DUSP5
phosphatase activity negatively influences MAPK activation, especially
extracellular signal regulated kinase 1/2 (ERK1/2). This regulation also may
be involved in NF-κB activation. In addition, promoter-reporter assay showed
that DUSP5 reduces AP-1 and NF- κB transcriptional activity. Based on these
findings, we propose that DUSP5 is a negative regulator of TNF-α and IL-6
production and might play a role in prevention of inflammation.
References
Ostman, A., Yang, Q. and Tonks, N.K., Expression of DEP-1, a receptor-like
protein-tyrosine-phosphatase, is enhanced with increasing cell density. Proc
Natl Acad Sci US, 91, (1994), 9680-4
Camps, M., Nichols, A. and Arkinstall, S., Dual specificity phosphatases: a
gene family for control of MAP kinase function. FASEB, where it took place,
20, (2000), 6-16
Dong, C., Davis, R.J. and Flavell, R.A., MAP kinases in the immune
response., Annu Rev Immunol, 20, (2002), 55-72
P82
Engineering of peptide assembly as a template of nanomaterial array:
newly approach to improve the oxygen reduction reaction activity in
energy materials
Ji Hun Kim1, Sun Hwa Park2, Yong Tae Kim1, Yong Ho Kim1,2,*
1
Department of Sungkyunkwan University Advanced Institute of
Nanotechnology(SAINT), Sungkyunkwan University, Suwon, 440-746, Korea.
2
Department of Chemistry, College of Natural Sciences, Sungkyunkwan
University, Suwon, 440-746, Korea.
E-mail: kprhan@skku.ac.kr
Protein design and engineering, nanobio
Protein design aims to engineer geometrical structure and unique properties of
protein. Because of the characteristics of protein assemblies [1], it is paid
attention to the chemical, biological, and medical scientists and also regards
possible way to break through the traditional limitation of other research
area[2-3]. Despite the fact, until now there have been few examples of
application of protein design in the energy science. Here we suggest a novel
approach of protein assembly for energy material, engineering the peptide to
be a template of metal nanoparticle array on single wall carbon nanotube
(SWNT) for high oxygen reduction reaction (ORR) activity. Previously, we
were developed the self-assembly peptide-SWNT superstructure[4], by
mutagenesis of the peptide assembly, HexCoil-Ala-2C were built for the
metal template. Considerable optimization of the location of cysteine residues
decides the metal binding. Consequently, the gold-platinum alloy
nanoparticles synthesized on the SWNT by reduction method without any
surfactant, and we were confirmed the structure by TEM, XRD, and Raman.
ORR activity of the material showed as high as the commercial Pt/C. In
addition, we controlled the size and composition of the nanoparticle by
changing reduction time and concentration of the precursor and confirmed the
factors directly influenced the ORR activity.
P81
Enhanced enzyme kinetic stability by increasing rigidity within
the active site
Yuan Xie1, 2, Jiao An1, 2, Guangyu Yang1, Geng Wu1,Yong Zhang1, Li Cui1, and
Yan Feng1
1
The State Key Laboratory of Microbial Metabolism, School of Life Sciences
and Biotechnology,
Shanghai Jiao Tong University, Shanghai 200240, China
2
Key Laboratory for Molecular Enzymology and Engineering of Ministry of
Education,
Jilin University, Changchun 130023, China
E-mail: ytmito1986@163.com
Enzymatic catalysis has the characteristic features of high efficiency, high
selectivity and environmental friendliness, which endow enzymes great
potential for the bio-industry applications. However，low stability of enzyme
limited the widely application in harsh conditions，such as high temperature,
extreme pH and organic solvents, which were required by some specific
183
References
Neil. P.K, and Yen-Ting L, ―Practical approaches to design novel protein
assemblies‖, Current Opinion in Structural Biology, Vol. 23, (2013), pp 632638.
Zhang S., ―Fabrication of novel biomaterial through molecular self-assembly‖,
Nature Biotechnology, Vol. 21, (2003), pp 1171-1178.
Nurxat. N, et al., ―Biotemplated Synthesis of Perovskite Nanomaterial for
Solar energy Conversion‖, Advanced Materials, Vol. 24, (2012), pp 28852889.
Gevorg. G., et al., ―Computational Design of Virus-Like Protein Assemblies
on Carbon Nanotube Surfaces‖, Science, Vol. 332, (2011), pp 1071-1076
Email: rnzaliha@upm.edu.my
Nature has designed lipase to be one of the great biocatalysts. The ability of
lipase to catalyze variety of reactions in aqueous and non-aqueous media
offers various advantages for biotechnology and industrial applications [1].
The search of lipases with new and exciting properties therefore, remains a
continuous pursuit. An organic solvent tolerant lipase gene from
Staphylococcus epidermidis AT2 was successfully cloned and expressed with
pTrcHis2 in E. coli TOP10 [2]. Sequence analysis revealed an open reading
frame (ORF) of 1,933 bp in length which coded for a polypeptide of 643
amino acid residues. The polypeptide comprised of a signal peptide (37 amino
acids), pro-peptide and a mature protein of 390 amino acids. Expression of
AT2 lipase resulted in an 18-fold increase in activity, upon the induction of
0.6 mM IPTG after a 10 h incubation period. Interestingly, this lipase was
stable in various organic solvents (25% (v/v), mainly toluene, octanol, pxylene and n-hexane). Literature shows that most of the organic solvent stable
bacterial lipases were produced by Pseudomonas sp. and Bacillus sp., but
very few from Staphylococcus sp. This lipase demonstrates great potential to
be employed in various industrial applications.
P83
Rational Design of Amyloid-like supramolecular Protein Assemblies on a
primitive Graphene surface
Young Hyun No1, Yong Tae Kim2 , Yoo Young Ahn2 , Seung Min Shin2 and
Yong Ho Kim1,2
1
SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan
University, Suwon 440-746, Republic of Korea.
E-mail: shxl114@gmail.com
Self-assembly of proteins has
attracted vast interests for
diverse applications like biosensitized solar cells, biobatteries and drug delivery
because proteins have unique
properties like photovoltaic
property, catalytic activity and
biological specific binding
capability[1-3]. So self-assembly
of proteins shows the potential to replace existing materials with performance
that overcomes the limits of structures and chemical properties. Here we
designed short peptides as nano-building blocks that recognize surface lattice,
POSIs (Peptides On Solid Interfaces). The repeating backbones of POSIs are
selected to satisfy two restraints : Presentation of surface groups in geometries
to match the lattice repeat and inter-subunit highly-ordered packing similar to
that found in natural protein structures. POSIs were designed to self-assemble
two-dimensionally on the primitive chemical vapor deposition (CVD)
graphene. POSIs render self-assembly of amyloid-like peptides controllable
along graphene carbon trace and demonstrate conserved hydrogen-bonding
providing geometric specificity for large area 2D assembly. We observed the
self-assembly of peptides with AFM and confirmed the intended assembling
geometry, however peptides are partially or not assembled in the absence of
graphene. In addition, molecular dynamics (MD) simulation suggests that
POSIs have strong π-π interaction with graphene and hydrogen-bonding
interaction makes POSIs ordered β-stranded structure and self-assembly in
structural array of graphene.
References
[1] Jaeger K-E., Dijkstra BW and Reetz MT., Bacterial Biocatalysts:
Molecular Biology, Three-Dimensional Structures and Biotechnological
Applications of Lipases. Annual Review of Microbiology, 53 (1999), pp 315351.
[2] Rahman RNZRA., Kamarudin NHA., Yunus J.,Salleh AB and Basri M.,
Expression of an Organic Solvent Stable Lipase from Staphylococcus
epidermidis AT2. International Journal of Molecular Sciences, 11 (2010), pp
3195-3208.
P85
Conformational preferences of the 2-methylproline residue and its role in
stabilizing -turn and polyproline II structures of peptides
Hae Sook Park1, Young Kee Kang2
1
Department of Nursing, Cheju Halla University, Cheju 690-708, Korea.
Department of Chemistry and BK21 PLUS Research Team, Chungbuk
National University, Chungbuk 361-763, Korea.
E-mail: ykkang@chungbuk.ac.kr
2
Conformational preferences of the 2-methylproline (2-MePro) residue and its
role in stabilizing -turn and polyproline II (PII) structures of peptides have
been studied using density functional methods in the gas phase and in water.
The population of the C7 H-bonded structures for Ac-2-MePro-NHMe is
calculated to be dominant in the gas phase, whereas they become significantly
depopulated and the populations of polyproline and -helical structures
increase in water. Due to the increased stability of the up-puckered PII
structure, the populations of its PII and PI structures are more increased by
10.9% and decreased by 12.2% than Ac-Pro-NHMe in water, respectively.
There is a large decrease in the cis population by 20.6% for the prolyl peptide
bond by replacing Pro with 2-MePro in water, which is consistent with 13C
NMR experiments. For Ac-Ala-2-MePro-NHMe, the conformer with a Iturn is found to be most preferred with the population of 25.2% in water,
whereas the open conformers are dominantly populated for Ac-Ala-ProNHMe. This is consistent with experimental results that the Ala-Pro sequence
of the antigen mimotope associated with autoimmune recurrent thrombosis
have been known to adopt a stable I-turn structure with the replacement of
Pro by 2-MePro in water. With the replacement of a single Pro by 2-MePro
in the middle of the sequences of the polyproline model peptide Ac-(Pro)5NMe2 and the single-strand model peptide of collagen Ac-(Hyp-Gly-Pro)2NMe2, the relative stabilities of the PII structures are enhanced by 8.11 and
3.91 kcal/mol, respectively, although there are only small changes in
backbone torsion angles for both PII and PI structures. It can be deduced that
the single strands of (Hyp-Gly-2-MePro)n with the stable PII structure might
provide more stability to their triple helix.
References
A. M. Kannan, V. Renugopalakrishnan, S. Filipek, P. Li, G. F. Audette, and L.
Munukutla, "Bio-Batteries and Bio-Fuel Cells: Leveraging on Electronic
Charge Transfer Proteins", Journal of Nanoscience and Nanotechnology, vol.
8, (2008), pp 1-13.
Chih-Wei Chang, Chin-Hao Chang, Hsueh-Pei Lu, Tung-Kung Wu, and Eric
Wei-Guang Diau, ―Fabrication and Photovoltaic Characterization of BioSensitized Solar Cells Using Myoglobin-Based Sensitizers‖, Journal of
Nanoscience and Nanotechnology , vol. 9, (2009), pp 1688-1695.
Masaki Uchida, Michael T. Klem, Mark Allen, Peter Suci, Michelle Flenniken,
Eric Gillitzer, Zachary Varpness, Lars O. Liepold, Mark Young, and Trevor
Douglas, ―Biological Containers: Protein Cages as Multifunctional
Nanoplatforms‖, Advanced Materials, vol. 19, (2007), pp 1025-1042.
P84
Expression of an Organic Solvent Stable Lipase from Staphylococcus
epidermidis AT2
Raja Noor Zaliha Raja Abd. Rahman1,2,3, Nor Hafizah Ahmad Kamarudin1,2,
Jalimah Yunus1,2, Abu Bakar Salleh1,2,3, Mahiran Basri1,3,4
1
Enzyme and Microbial Technology Research Centre.
2
Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia
3
Institute of Biosciences, Universiti Putra Malaysia, Serdang, Selangor,
Malaysia
4
Faculty of Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
184
P86
Rational Design of hierarchical self-assembly of β-peptide
toward electrically and mechanically enhanced hydrogel
Jae Hyun Jun1, Hye Jin Kwon1,Se Young Ann1,Yong Ho Kim1
1
SKKU Advanced Institute of Nanotechnology (SAINT) & Department of
Chemistry Sungkyunkwan University ,Republic of Korea Protein Materials &
Protein Design Lab
Email: jhskku02@gmail.com
A potent strategy to obtain electrically and mechanically enhanced hydrogel is
the bottom-up hierarchical assembly of β peptide. In this study, hierarchical
structure of β peptide is proposed as new tool to solubilize single-walled
cabon nanotubes (SWNTs) and modify property of hydrogel. Due to
remarkable properties in electrical and mechanical devices, SWNTs have
potential application in biomaterials.
Our first work is to focus on manipulating SWNTs' solubility without
decrease electric and mechanical property. To make dispersion of SWNTs in
aqueous milieu , we overcome the noncovalent force between SWNTs. We
have designed β‑Peptide which takes form of hierarchical nano-fiber structure.
Peptide fibers should wrapped SWNTs around with hydrophobic interaction
and consequentially modified surfaces of SWNTs play diverse important role
in improvement of nanotube's solubility. Those peptide fibers not easily
dissociated from the SWNTs, thereby providing extremely stable SWNTs
dispersion. The other our focus is the generation of peptide/SWNTs hydrogels.
The concentration of dispersed SWNTs in solution have contribute to
hydrogel formation. The intensity of hydrogel is decided by concentration of
peptide-SWNTs.
Fig. PLP dihedral angles in crystallographic intermediate structures (a)
Superimposition between the active-sites. (b) Comparison of the average
dihedral angles. (c) Tetrahedral geometry of PLP and attacking and leaving
amino groups in PGD. (d) Schematic representations of chemical structures of
Schiff-base-linked PLP.
References
Ngo, H. P., Cerqueira, N. M., Kim, J. K., Hong, M. K., Fernandes, P. A.,
Ramos, M. J. & Kang, L. W. (2014). Acta crystallographica. Section D,
Biological crystallography 70, 596-606.
P88
Examining molecular characterizations of breast cancer in drug
resistance with proteomic tools
Minh Nguyen Anh Ho1, Vinh The Nguyen2, Bao Chi Bui2.
1
Department of Molecular Biology, School of Medicine, Sungkyunkwan
University, Suwon, Korea.
2
Centre for Molecular Biomedicine, University of Medicine and Pharmacy
HCMC, Vietnam.
E-mail: minh_ho74@yahoo.com
References
[1] R. H. Baughman, A. A. Zakhidov and W. A. de
Heer,Science,( 2002),297,787
[2] W. Zhao, C. H. Song and P. E. Pehrsson,J. Am. Chem. Soc., 2002,124,
12418
P87
PLP undergoes conformational changes during the course of an enzymatic
reaction
Thanh Thi Ngoc Doan1, Jin-Kwang Kim1, Myoung-Ki Hong1, Pedro
Alexandrino Fernandes2, Maria João Ramos2, Lin-Woo Kang1
1
Department of Biological Sciences, Konkuk University, 1 Hwayang dong,
Gwangjin-gu, Seoul 143-701, Korea
2
Requimte/Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
E-mail: lkang@konkuk.ac.kr
Numerous enzymes, such as the pyridoxal 5‘-phosphate (PLP)-dependent
enzymes, require cofactors for their activities. By x-ray crystallography, we
determined the structural snapshots of L-serine dehydratase catalytic reactions
of a bacterial PLP-dependent enzyme. In each structure, the dihedral angle
between the pyridine ring and the Schiff base linkage of PLP varied from 18°
to 52°. We proposed the organic cofactor PLP directly catalyzes reactions with
the active conformational changes, and the novel catalytic mechanism
involving the PLP cofactor was confirmed by high-level quantum mechanical
calculations. The conformational change was essential for the nucleophilic
attack of the substrate on PLP, for the concerted proton transfer from the
substrate to the protein, and for the directing of the carbanion formation of the
substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a
series of reactions like the enzyme. The conformational change of the PLP
cofactor in catalysis serves as a starting point for identifying the previously
unknown catalytic roles of organic cofactors.
185
Molecular proteomic cancer research based on the structural and functional
features have led to discover biomarkers that supplement to genomic tools.
The current study focuses on two basic characteristics of breast tumor in
paclitaxel resistance which are epithelial cell‘s morphology and protein
expression. The morphology of epithelial cells of breast cancer can be
detected by atomic force microscopy (IT-AFM) that measures the stiffness
over mass of elasticity; however, there were no differences between sensitive
and resistant breast tissues to paclitaxel (Figure 3). The expression of proteins
can be analyzed by two-dimensional electrophoresis. Worth mentioning, the
presence of over expressed Nrf2 protein is a necessary precondition for
sensitivity to paclitaxel therapies that many patients develop resistance
(Figure 1). Similar results were reported in the over expression of Nrf2 in
certain types of lung and liver cancer. Overall, this study has emphasized the
role of the transcription factor genes against oxidative free radicals lead to
drug resistance of cancer cells. Identification of the downstream effectors of
this pathway will help understanding mechanism(s) of chemo-resistance and
may have the potential to be used as therapeutic targets.
References:
[1] Kaspar JW, Niture SK, Jaiswal AK, ―NRF2:INRF2 (Keap1) signaling in
oxidative stress‖, Free Radic Biol Med, 47(9), (2009), 1304-1309.
[2]Taguchi K, Motohashi H, Yamamoto M, ―Molecular mechanisms of the
Keap1-NRF2 pathway in stress response and cancer evolution‖, Genes to
cells: devoted to molecular & cellular mechanisms, 16(2), (2011), 123-140.
[3]Mizutani H, ―Mechanism of DNA damage and apoptosis induced by
anticancer drugs through generation of reactive oxygen species‖, Yakugaku
Zasshi, 127(11), (2007), 1837-1842.
[4]Alexandre J, Hu Y, Lu W, Pelicano H, Huang P, ―Novel action of paclitaxel
against cancer cells: bystander effect mediated by reactive oxygen species‖,
Cancer Res, 67(8), (2007), 3512-3517.
[5] Plodinec M, Loparic M, Monnier CA, Obermann EC, Zanetti-Dallenbach
R, Oertle P, Hyotyla JT, Aebi U, Bentires-Alj M, Lim RY et al, ―The
nanomechanical signature of breast cancer‖, Nature nanotechnology, 7(11),
(2012), 757-765.
[6] Kannan S, Sujitha MV, Sundarraj S, Thirumurugan R, ―Two Dimensional
Gel Electrophoresis in Cancer proteomics‖, Gel Electrophoresis–Advanced
Techniques, In Tech, Rijeka, (2012),359-390
P91
An inhibitor of Hsc70 that promotes membrane trafficking through
restoration of mutant CFTR chloride channel activity
P89
Apoptozole : a small molecule that induces caspase-dependent apoptosis
through binding to HSP70
CheolWan Park1, Kyung-Hwa Baek1 and Injae Shin1
Sanghyeob Lee1, Kyung-Hwa Baek1, Sung-Kyun Ko1,2 and Injae Shin1
1
Department of Chemistry, College of Sciences, Yonsei University, Seoul 120749, Korea
E-mail: pcw1000@yonsei.ac.kr
1
Department of Chemistry, College of Sciences, Yonsei University, Seoul 120749, Korea.
2
Chemical Biology Research Center, Korea Research Institute of Bioscience
and Biotechnology, Ochang, Korea.
E-mail: lsh788@yonsei.ac.kr
The most frequent mutation of cystic fibrosis transmembrane conductance
regulator(CFTR) that cause cystic fibrosis is deletion of phenylalanine at
position 508(∆F508). This mutation leads to defects in protein folding and
cellular trafficking to the plasma membrane. The lack of the cell surface
CFTR result in a reduction in the lifespan owing to chronic lung infection
with progressive deterioration of lung function. Hcs70 plays a crucial role in
degradation of misfolded CFTR by the ubiquitin-proteasome system. Unlike
other known Hsc70 regulators, a small molecule apoptozole(Az) that inhibits
the Hsc70 activity promotes membrane trafficking of mutant CFTR and
restoration of its chloride channel activity in cells treated with nanomolar
concentrations of Az at 37℃. This susbstance was found to inhibit Hsc70
activity by binding to its ATP-binding site, a phenomenon which leads to
suppression of ubiquitination of ∆F508-CFTR by blocking interaction of the
mutant with Hsc70 and CHIP, promotion of membrane trafficking of the
mutant CFTR and restoration of its chloride channel activity in cells.
Small molecules related to cell death have significant potential as therapeutic
agents to treat apoptosis or autophagy-related diseases. To select molecules
with cell death-inducing activity, cell-based screening with a small molecule
library composed by imidazole scaffolds is performed to identify bioactive
compounds that induce cell death. We have identified from the cell-based
assay a small molecule, apoptozole that interacts with Hsc70 and Hsp70. In
biochemical studies, apoptozole binds ATP-binding domain of Hsc70 and
inhibits the ATPase activity of Hsp70/Hsc70 with dissociation constant of
submicromolar concentration. It is likely that this compound induces cell
death by inhibiting the function of Hsp70 and/or Hsc70, which antagonize
apoptosis by interfering with multiple checkpoints in the cell death pathways.
Apoptozole induces caspase-dependent apoptotic cell death of cancer cells
and accumulates LC3-II in concentration-dependent manner. In animal study,
apoptozole reduces tumor size of cancer cell-transplanted nude mice. As an
Hsp70/Hsc70 inhibitor, apoptozole holds considerable potential as a cancer
therapeutic and can also be used to further understand the molecular basis of
Hsp70-related apoptotic and autophagic process.
References
Darren R.Williams, sung-Kyun Ko, Sungjin Park, Myung-Ryul Lee, Injae
Shin, Angew. Chem. Int. Ed. 2008, 47, 7466-7469.
Hyungseoph J. Cho, Heon Yung Gee, Kyung-Hwa Baek, Sung-Kyun Ko,
Jong-Moon Park, Hookeun Lee, Nam-Doo Kim, Min Goo Lee, Injae Shin, J.
Am, Chem. Soc. 2011, 133, 20267-20276
References
Darren R. Williams, Sung-Kyun Ko, Sungjin Park, Myung-Ryul Lee and
Injae Shin, ―An Apoptosis-Inducing Small Molecule That Binds to Heat
Shock Protein 70‖, Angew. Chem. Int. Ed., Vol. 47, No. 39, (2008), pp 74667469.
P92
UBA linker is important for the activation of MPK38.
Yong-Soon Cho, YingJin Kang, Kuglae Kim, Young Je Cha,
Jeong Seok Cha and Hyun-Soo Cho
P90
NMR Structural Study of modified LPcin with Enhanced Antimicrobial
Activities
Department of Biology, College of Life Science and Biotechnology,
Yonsei University, Seoul, 120-749, Korea
E-mail: pickcha1121@yonsei.ac.kr
Murine protein serine/threonine kinase 38 (MPK38) is the murine ortholog of
human maternal embryonic leucine zipper kinase (MELK), which belongs to
the SNF1/AMPK family. MELK is considered as a promising drug target for
anticancer therapy, because MELK overexpression and hyper-activation
correlate with several human cancers. Activation of MPK38 requires the
extended sequence (ExS) containing the ubiquitin-associated (UBA) linker and
UBA domain, and phosphorylation of the activation loop. However, the
activation mechanism of MPK38 is unknown. This paper reports the crystal
structure of MPK38 (T167E), which mimics a phosphorylation state of the
activation loop in complex with AMP-PNP. In the MPK38 structure, the UBA
linker forces the inward movement of the αC helix into an intermediate
conformation, in which the activation loop might not be stabilized. Then,
phosphorylation of the activation loop induces movement of the activation
loop toward the C-lobe and results in closing of interlobar cleft. These
processes generate a fully active state of MPK38. This structure suggests that
MPK38 has a similar molecular activation mechanism as that of other kinases
of the SNF1/AMPK family. Recently, we also reported the structure of MPK38
in complex with OTSSP167, which is an orally administrative MELK selective
inhibitor, conferring an IC50 of 0.41 nM. OTSSP167 effectively fits into the
active site of MPK38, thus offering an opportunity for structure-based
development and optimization of MELK inhibitors
Ji-Ho Jeong, Ji-Sun Kim, and Yongae Kim
Department of Chemistr, Hankuk University of Foreign Studies, Yong-In, 449791, Korea
E-mail: yakim@hufs.ac.kr
Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer
peptide, corresponds to the carboxy terminal 113–135 region of Component-3
of proteose-peptone. LPcin has been designed and modified using mutation,
sequence shuffling, and better amphipathic characteristics to improve
antimicrobial activities. Three different analogs of LPcin, LPcin-yk1 that has
shorter amino acids in C-terminus, LPcin-yk2 which has mutant amino acids,
and LPcin-yk3 that has shorter and mutant amino acids, were recently
developed by using peptide engineering techniques in our lab. In order to
comprehend the correlation between the structure of LPcin analogs and
antimicrobial activity interacting with bacterial membrane surface, we
optimized the process of overexpress in the form of fusion protein in
Escherichia coli and purified. In order to identify their characteristic structure
of the purified peptides in membrane environment, we performed 1D and 2D
solution NMR experiments and solid-state NMR experiments in membrane
environments like micelle and bicelle samples as well as MS spectrometry
and CD spectroscopy. A home-built solid-state NMR probe for 800MHz NB
magnet was used for bicelle samples. In here, we will present the optimizing
processes for high-yield expression, purification and introduce various
solution and solid-state NMR experimental techniques for investigating antibacterial mechanisms in membrane environments.
References
Park T. J., Kim J. S., Choi S. S and Kim Y. A., ―Cloning, expression, isotope
labeling, purification, and characterization of bovine antimicrobial peptide,
lactophoricin in Escherichia coli‖, Protein Expr Puri., 65, 1, (2009), 23-9.
Park T. J., Kim J. S., Ahn H. C and Kim Y. A., ―Solution and Solid-State
NMR Structural Studies of Antimicrobial Peptides LPcin-I and LPcin-II‖,
Biophysical Journal 101, (2011), 1193–1201.
P93
Conformational variation of the peptide permeable channel chapero
ne revealed by electron microscopy
Kazuhiro Mio1, Masaaki Kawata1, Toshio Moriya1, Yoshikazu Sasaki2 and
Chikara Sato1
186
hematophagous nature avoiding decomposing food sources.
1
National Institute of Advanced Industrial Science and Technology ,
Tokyo 135-0064, Japan
2
Jeol Ltd. Akishima Tokyo 196-8558, Japan
E-mail: kazu.mio@aist.go.jp
The Sec translocon facilitates transportation of newly synthesized
polypeptides from the cytoplasm to the lumen/periplasm across the
phospholipid membrane. Although the polypeptide-conducting machinery is
formed by the SecYEG-SecA complex in bacteria, its transportation
efficiency is markedly enhanced by SecDF. A previous study suggested that
SecDF assumes at least two conformations differing by a 120° rotation in the
spatial orientation of the P1 head subdomain to the rigid base, and that the
conformational dynamics plays a critical role in polypeptide translocation. In
this study we addressed this hypothesis by analyzing the 3D structure of
SecDF using electron tomography and single particle reconstruction.
Reconstruction of wild type SecDF showed two major conformations; one
resembles the crystal structure of full-length SecDF (F-form structure), while
the other is similar to the hypothetical structural variant based on the crystal
structure of the isolated P1 domain (I-form structure). The transmembrane
domain of the I-form structure has a scissor like cleft open to the periplasmic
side. We also report the structure of a double cysteine mutant designed to
constrain SecDF to the I-form. This reconstruction has a protrusion at the
periplasmic end that nicely fits the orientation of P1 in the I-from. These
results provide firm evidence for the occurrence of the I-form in solution and
support the proposed F- to I-transition of wild type SecDF during polypeptide
translocation.
References
[1] Kang, K. et al. Analysis of Drosophila TRPA1 reveals an ancient origin
for human chemical nociception. Nature 464, 597–600 (2010).
[2] Ogawa, H. et al. H2S functions as a nociceptive messenger through
transient receptor potential ankyrin 1 (TRPA1) activation. Neuroscience 218,
335–43 (2012).
[3] Kang, K. et al. Modulation of TRPA1 thermal sensitivity enables sensory
discrimination in Drosophila. Nature 481, 76–80 (2012).
P95
Uracil-dependent innate immune response facilitates pathogen clearance
via a specific TRPA1 isoform
Ji Hye Lee1, Jeong-Ho Park2, Eun Jo Du1, Tae Jung Ahn1, Jae Young Kwon2,
KyeongJin Kang1.
1 Samsung Biomedical Research Institute and Department of Anatomy and
Cell Biology, School of Medicine, 2 Department of Biological Sciences,
Sungkyunkwan University, Suwon
E-mail: ggang0826@naver.com,
Host-microbe interaction in the gut has to be well balanced for animals‘
health and survival. In Drosophila melanogaster, dual oxidase (Duox)
produces microbicidal reactive oxygen species (ROS)1 as upregulated by G
protein signaling pathways2, which uracil released by certain bacteria was
recently discovered to actuate3. This innate immune response is critical for
elimination of uracil-producing bacterial pathogens, thus ensuring survival of
the host. Here, we show that uracil feeding increases defecation frequency
via the conserved electrophile receptor TRPA1 expressed in the gut. The
effect of uracil was absent in TrpA1 mutant flies, and suppressed by coingestion of uracil with ROS-counteracting dithiothreitol (DTT).
Interestingly, a TRPA1(A) variant containing alternative exon 10 (TRPA1
A10b) restored the uracil-dependent increase of defecation frequency in
TrpA1 mutant animals, while the other isoform, TRPA1 A10a, did not.
Immunohistochemical study revealed that TRPA1 is co-expressed with
Prospero, an entero-endocrine cell marker in the midgut. These results
suggest that not only pathogen inactivation by ROS toxicity, but also TRPA1dependent pathogen clearance out of the gut result from uracil-dependent
ROS production. It will be intriguing to determine in the future how
important the TRPA1-dependent facilitation of defecation is for host survival,
when virulent microbes are introduced in the gut.
References
Mio K et al.,‖ Conformational variation of the translocon enhancing
chaperone SecDF.‖ J Struct Funct Genomics. (2013)
Tsukazaki T et al.,‖Structure and function of a membrane component SecDF
that enhances protein export.‖ Nature.(2011) 474, 235-238.
Mio K et al.,‖Single particle reconstruction of membrane proteins: a tool for
understanding the 3D structure of disease-related macromolecules.‖ Prog
Biophys Mol Biol. (2010) 103, 122-130.
P94
TRPA1 of malaria-transmitting mosquito, Anopheles gambiae, is a
protein sensor for both electrophiles and nucleophiles
Eun Jo Du1, Young Seon Shin1, Ji Hye Lee1 and KyeongJin Kang1
1
Samsung Biomedical Research Institute, and Department of Anatomy and
Cell Biology, School of Medicine, Sungkyunkwan University, Suwon 440-746,
Korea.
E-mail: love-pradice@hanmail.net, bluekid326@naver.com
References
Ha, E., Oh, C., Bae, Y. & Lee, W. A direct role for dual oxidase in Drosophila
gut immunity. Science (80-. ). 847, (2005).
Ha, E.-M. et al. Regulation of DUOX by the Galphaq-phospholipase CbetaCa2+ pathway in Drosophila gut immunity. Dev. Cell 16, 386–97 (2009).
Lee, K.-A. et al. Bacterial-derived uracil as a modulator of mucosal immunity
and gut-microbe homeostasis in Drosophila. Cell 153, 797–811 (2013)
Ability of TRPA1 to sense tissuedamaging electrophiles is well
conserved in bilaterians ranging from
fruit flies to humans and enables them
to avoid harmful chemical stimuli from
the hostile environment1. TRPA1dependent detection of oxidizing
electrophiles is, however, somewhat
insufficient to secure animals‘ safety in
this regard, as strong nucleophiles can
be equally dangerous due to their
reactivity and inflammability. While
mammalian TRPA1 was shown to
respond to the well-known reductant hydrogen sulfide, the action of the
chemical must have resulted from its electrophilic capability to oxidize
sulfhydryl residues as the activation was reversed by application of reductant
dithiothreitol (DTT)2. Here, we show that Anopheles gambiae TRPA1
(agTRPA1) exhibits high sensitivity to DTT (EC50: 7.3+/isoform-specific manner that led us to speculate the N-terminus of the
agTRPA1(A) isoform3 is responsible for DTT sensitivity. Moreover, TRPA1
from Drosophila melanogaster little responded to high concentrations of DTT.
Unexpectedly, the only cysteine residue Cys25 unique in the N-terminal
domain of agTRPA1 could not be attributed to DTT reception, as substitution
of Cys25 to Ser, did not affect DTT-dependent activation of agTRPA1 (EC50
of TRPA1 C25S: 5.7+/activated by metal-chelating chemicals, which raises the possibility that DTT
triggers activation of agTRPA1 by removing metal ions from the agTRPA1 Nterminus. Taken together, agTRPA1 serves as a nucleophile sensor not relying
on sulfhydryl residues, and might be evolved in the mosquito for their
P96
Structure of a Conserved Golgi Complex-targeting Signal in Coronavirus
Envelope Proteins
Wahyu Surya1, Yan Li1, Stephanie Claudine1 and Jaume Torres1
1
School of Biological Sciences, Nanyang Technological University, 60
Nanyang Drive, Singapore 637551
E-mail: wsurya1@e.ntu.edu.sg
Coronavirus envelope proteins (CoV E) are minor components of the virions,
but are present abundantly in ER and Golgi complex of infected cells. CoV E
proteins form pentameric oligomers through their transmembrane (TM)
domains, and show channel activity with mild selectivity for cations. While
some structural data of the TM domain are available for the E protein from
severe acute respiratory syndrome (SARS) CoV, the structure of its predicted
extramembrane domains is lacking. The C-terminal domain of E proteins in βand γ- coronaviruses has a predicted β-coil-β motif with a completely
conserved central proline residue. In SARS-CoV, this motif has been reported
to constitute a Golgi targeting signal, and and based on its hydrophobicity it
has been proposed to form a second TM domain. The C-terminal tail of E
protein is important for its interaction with the C-terminal domain of the
membrane (M) protein at the cytoplasmic side of the ER-Golgi intermediate
187
compartment (ERGIC). The C-terminal tail of SARS-CoV E protein interacts
with PALS1, which is thus depleted from the tight junctions in epithelial cells.
Finally, SARS-CoV E interacts with the 7-domain SARS non-structural
protein 3 (nsp3). We show that the C-terminal extramembrane domain of
SARS CoV E forms a membrane-associated α-helix instead of a β-coil-β
motif. However, complementary data suggest that this domain is
conformationally flexible and may exist in dynamic equilibrium with a lessabundant β-structure form.
Human serotonin receptor 3A (5-HT3A) is a ligand-gated ion channel
regulated by serotonin. In a previous study1, we highly expressed P9-5-HT3A,
a fusion protein which is composed of 5-HT3A and an envelope protein P9
found in bacteriophage Phi6, in E.coli membrane fraction. And the expressed
protein was purified to homogeneity using an affinity chromatography. But
only 7% of P9-5-HT3A were observed as pentameric oligomers in sarkosyl. In
this study, pentameric condition was optimized to reassemble the receptor in
complex with detergent in a functional pentameric state. This material would
provide biophysical and structural analysis of 5-HT3A.
References
Surya W., Samso M. and Torres J., “Structural and Functional Aspects of
Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and
Coronaviruses, Respiratory Disease and Infection - A New Insight”, InTech,
(2013), pp 47-76.
Li Y., Surya W., Claudine S., and Torres J., "Structure of a Conserved Golgi
Complex-targeting Signal in Coronavirus Envelope Proteins", Journal of
Biological Chemistry, in press, (2014).
References
Na. J-.H. et al. Bacterially expressed human serotonin receptor 3A is
functionally reconstituted in proteoliposomes, Protein Expr. Purif. 88, 190195 (2013)
Hassaine. G. et al. Large scale expression and purification of the mouse 5TH3 receptor, Biochim. Biophys. Acta. 1828, 2544-2552 (2013)
P97
Recombinant expression, purification and NMR based structure
determination of syndecan4-TM
P99
Development of a novel polymer detergent derived from poly-γ-glutamic
acid to study membrane proteins
Ji-Sun Kim, Ji-Ho Jeong and Yongae Kim
Seong-Gu, Han1, Jung-Hyun, Na1,2, Won-Kyu, Lee1, Dongkook, Park1, Jihye,
Oh1, Sung-Ho, Yoon1, Cheng-Kang, Lee3, Moon-Hee, Sung4, Yeon-Kyun,
Shin2,5 and Yeon Gyu, Yu1
Department of Chemistry, Hankuk University of Foreign Studies, Yong-In,
449-791, Korea
E-mail: yakim@hufs.ac.kr
1
Department of Chemistry, Kookmin University, 861-1 Jeongneung-dong,
Seongbuk-gu, Seoul, 136-702, Republic of Korea.
2
Biomedical Research Institute, Korea Institute of Science and Technology,
Seoul 136-791, Republic of Korea.
3
Department of Chemical Engineering, National Taiwan University of Science
and Technology, 43, Keelung Rd, Section 4, Taipei 10607, Taiwan.
4
Department of Advanced Fermentation Fusion Science and Technology,
Kookmin University, 861-1 Jeongneung-dong, Seongbuk-gu, Seoul, 136-702,
Republic of Korea.
5
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State
University, Ames, IA 50011, USA.
E-mail: seongguhan@kookmin.ac.kr
Syndecan-4 of heparan sulfate proteoglycan (HSPG) is participate in
biologically important cell-to-cell interactions, cell adhesion, tissue
development and repair as well as the pathogenesis of numerous diseases(e.g.
tumor progression and metastasis). All types of the syndecan have a major
sections: modified extracellular domains, conserved transmembrane and
cytoplasmic domains. The structural studies of each sections are necessary to
understanding their relationship between structure and function but it have
been limited because the syndecan4-TM(transmembrane domain) had been
difficult to achieve proper amounts and purity owing to its low solubility. 1-3
We previously reported the successful expression and purification of several
transmembrane proteins. Here, we demonstrated optimized method for
recombinant expression and purification of three kinds of syndecan4-TM:
wild type Syd4-TM (wt-Syd4), mutant Syd4-TM (mSyd4), and Syd4-eTC.
All peptides were released from the fusion protein, and then purified by
biophysical techniques like affinity chromatography, dialysis, chemical
cleavage, and reversed-phase semiprep HPLC. Enough amounts of purified
Syndecan4-TM for the analysis was obtained under optimized conditions.
Their biophysical properties were studied by circular dichroism (CD), mass
spectrometry, and solution & solid nuclear magnetic resonance (NMR)
spectroscopy.
Molecular studies about membrane proteins have hindered due to some
hurdles containing extraction of membrane proteins from cell membrane with
their native conformations. We synthesized novel amphipathic polymer (APG)
by conjugation of octyl and glucosyl groups to poly-γ-glutamic acid (PGA).
The APG made mono-disperse oligomers by self-assembly. And a critical
micelle concentration (CMC) of the APG was 100-1000 folds lower than βoctylglucoside (βOG) and sodium dodecylsulfate (SDS) determined by using
environment sensitive fluorescent dye, pyrene[1,2]. Bacteriorhodopsin (BR)
and human endothelin receptor type A (ETA) were effectively stabilized in
their active conformation with the APG. Furthermore, the complex composed
of ETA and APG was incorporated into liposomes with simple add-and-wash
step without disruption. The APG elevated expression level of G-protein
coupled receptors (GPCRs) in a cell-free transcription/translation system.
These results suggest that the APG provides solutions for the obstacles to
study membrane proteins.
References
Park T. J., Lee M. H., Kim J. S., and Kim Y. A., "Recombinant expression,
purification, and characterization of transmembrane domain of syndecan-4",
Process Biochemistry, Vol. 46, No. 5 (2011), pp 1166-1171.
Chio S. S., Kim J. S., Song J. Y., and Kim Y. A., "High-yield Expression and
Characterization of Syndecan-4 Extracellular, Transmembrane and
Cytoplasmic Domains", Bull. Korean Chem. Soc., Vol. 34, No. 4, (2013), pp
1120-1126.
Song J. Y., Kim J. S., Chio S. S., andKim Y. A., "Structural Effects of the
GXXXG Motif on the Oligomer Formation of Transmembrane Domain of
Syndecan-4", Bull. Korean Chem. Soc., Vol. 34, No. 12, (2013), pp 3577-3585.
References
Dominguez A., Fernandez A., Gonzalez N., Iglesias E., and Montenegro L. ,
―Determination of critical micelle concentration of some surfactants by three
techniques.‖, J. Chem. Educ., Vol. 74, (1997), pp 1227-1231.
Goddard E.D., Turro N.J., Kuo P.L., and Ananthapadmanabhan K.P.,
―Fluorescence probes for critical micelle concentration determination.‖,
Langmuir : the ACS journal of surfaces and colloids, Vol. 1, (1985), pp 352355.
P98
Bacterially expressed human serotonin receptor 3A in complex with
detergent in a functional pentameric state
P100
Expression, purification and reconstitution of human lysophosphatidic
acid receptor 1 (LPA1) in the active conformation.
Jung-Hyun Na1,2, Saet-Byeol Choi1, Won-Kyu Lee1, Yeon-Kyun Shin2,3*,
Yeon Gyu Yu1*
Seong-Gu, Han1, Seung-Il, Baek1, Won-Kyu, Lee1, Saet-Byeol, Choi1,
Dongbin, Lim2, and Yeon Gyu, Yu1
1
Department of Chemistry, Kookmin University, Seoul 136-702, Republic of
Korea
2
Biomedical Research Institute, Korea Institute of Science and Technology,
Seoul 136-791, Republic of Korea
3
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State
University, Ames, Iowa 50011, USA
E-mail: protein@kookmin.ac.kr
1
Department of Chemistry, Kookmin University, 861-1 Jeongneung-dong,
Seongbuk-gu, Seoul, 136-702, Republic of Korea.
2
School of Systems Biomedical Science, Soongsil University, Seoul 156-743,
Republic of Korea.
E-mail: seongguhan@kookmin.ac.kr
188
University, Korea
Email: jyxialei@gmail.com, mjlee@khu.ac.kr
Lysophosphatidic acid receptor 1 (LPA1) is one of lysophspholipid receptors
(LPL-Rs), a member of the G-protein coupled receptor (GPCR) family. LPA1
is considered as a potential target for anti-cancer therapeutics since it
enhances metastasis of cancer by stimulation of cell motility[1,2]. High level
expression (up to 1 mg/2 g wet cell) and purification (up to 90% purity) were
achieved by using P9 expression system in E.coli and IMAC (immobilized
metal affinity chromatography), respectively. The purified LPA1 was
reconstituted with amphipathic poly-γ-glutamic acid (APG) in an active form.
The LPA1 complex with APG showed mono-disperse oligomer, whereas the
LPA1 solubilized with n-dodecyl-D-maltopyranoside (DDM) was polydisperse oligomers. Furthermore, The LPA1 complex with APG bound
specifically to the α-subunit of Gi3. Preparation of a highly purified active
LPA1 using P9 expression system and APG could accelerate not only the
molecular analysis of LPA1, but also the development of LPA1-target drugs.
The N-end rule pathway, which states that the N-terminal amino acid of a
protein determines its half-life, has been implicated in various essential
cellular processes and human diseases. However, the physiological functions
of the N-end rule pathway have not been fully understood, and the discovery
of N-end rule inhibitors has become more crucial, considering its clinical
applications. In eukaryotes, the N-end rule pathway consists of two branches,
the Arg/N-end rule pathway and the Ac/N-end rule pathway. In the Arg/N-end
rule branch, unacetylated N-terminal destabilizing residues function as
essential determinants of protein degradation signals (N-degrons). Our
research identified a specific Arg/N-end rule pathway inhibitor, which showed
significant inhibitory effects on the degradation of its physiological substrates,
including regulators of G protein signaling 4(RGS4), not only in vitro and in
vivo but also in the mice brains. Taking advantage of in silico docking
computational assay, we found that this small-molecule has strong binding
affinity with UBR proteins. Additionally, pathological C-terminal fragments
of TDP43 bearing N-degrons (Arg208-TDP25), which are implicated in the
sporadic and familial pathogenesis of frontotemporal lobar degeneration with
Ub-positive, tau-negative inclusions (FTLD-U) and amyotrophic lateral
sclerosis (ALS), formed a significantly increased amount of cytosolic
aggregation in the presence of this inhibitor. We anticipate this blood brain
barrier-permeable inhibitor can be used to understand the pathophysiological
processes regulated by the Arg/N-end rule pathway.
References
Dai S., Joji K., Hironori Y., Yurai O., Nelson H.T., Toshiaki W., Yoh T., and
Hirokazu N., ―Lysophosphatidic acid(LPA) enhances the metastatic potential
of human colon carcinoma DLD1 cells through LPA1‖, Cancer Res., Vol. 63,
(2003), pp 1706-1711.
Kotaro H., Junken A., Masahiro F., Yasuhiro K., Teruyuki S., Rika S., Hideo
O., Takao Y., Masahiko W., Jerold C., and Hiroyuki A., ―Lysophosphatidic
acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic
cells through LPA1.‖, J. Biol. Chem., Vol. 279, (2004), pp 17634-17639.
References
[1] Sriram SM, Kim BY, & Kwon YT., The N-end rule pathway: emerging
functions and molecular principles of substrate recognition. Nat Rev Mol Cell
Biol,Vol.12, (2011), pp735-747.
[2] Varshavsky A, The N-end rule pathway and regulation by proteolysis.
Protein Sci. (2011).
[3] Lee MJ, et al., RGS4 and RGS5 are in vivo substrates of the N-end rule
pathway. ProcNatl Acad Sci U S A, Vol.102, (2005), pp15030-15035.
[4] Igaz LM, et al., Expression of TDP-43 C-terminal Fragments in Vitro
Recapitulates Pathological Features of TDP-43 Proteinopathies. J Biol Chem,
Vol.284, (2009), pp8516-8524.
Proteins and drug discovery
P101
Scavenging Hydroxyl Radical Activity and DNA Damage Protective
Effect of Hydrolyzed Protein Isolate from Melinjo Seeds ( Gnetum
gnemon)
Tri Agus Siswoyo1
1
P103
A rational designed inhibitor for Alzheimer’s Amyloid-Beta
Center for Development of Science and Technology and Faculty of
Agriculture, University of Jember, Jember 68121, Indonesia
E-mail: triagus.faperta@unej.ac.id
Gnemon tree, Gnetum gnemon, L (Gnataceae) is cultivated in Indonesia,
Malaysia and other south-east Asian islands for its seeds and is used as food
in Indonesia. Gnetum gnemon (melinjo) seeds contain a high concentration of
protein by 9 - 11% of the seed. These seeds may be considered as a suitable
source of functional protein. In this study, Gnetum gnemon protein isolate
(Gg-PI) hydrolysates were prepared by three enzymes (alcalase, trypsin and
pepsin). The antioxidant activities and protective effect against oxidative
DNA damage of Gg-PI hydrolysates were investigated. Alcalase hydrolysates
exhibited the highest hydroxyl radical-scavenging activity (IC50 1.74 mg mL−1)
(P < 0.05). Compared with other two hydrolysates, the hydrolysates obtained
by alcalase had the most abundant <3-kDa fractions. In addition, below 3-kDa
fractions of alcalase hydrolysates showed the highest antioxidant activities
and protective effects against DNA damage through both scavenging
hydroxyl radicals, which was probably because of the increase in several
antioxidant amino acids, such as His, Met, Cys, Tyr and Phe, as well as the
hydrophobic amino acids. The results suggested that enzymatic hydrolysis
could be used as an effective technique to produce high value-added peptides
products from Gnetum gnemon seed.
References
Siswoyo T. A., Eka M., Lee K.O. and Hosokawa K., Isolation and
characterization of Antioxidant Protein Fractions from Melinjo (Gnetum
gnemon) Seed, Journal of Agricultural and Food Chemistry. 59 (2011) pp.
5648-5656.
Siswoyo T.A., Aldino M. and Hosokawa K., Free Radical Scavenging
Activity and DNA damage protective effect of Mlinjo (Gnetum gnemon).
Journal of Medical Plants Research. 7 (2013) pp. 1399-2406.
P102
A specific Arg/N-end rule inhibitor delays the degradation of its
physiological substrates in the mouse brain
Ning-Hsuan Tseng, Ya-Ru Tsai, Rong-Jie Chen, Jien-Lin Charng, Jim-Min
Fang* and Yun-Ru Chen*
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
E-mail: nhtseng@sinica.edu.tw
Alzheimer‘s disease (AD) is a progressive neurodegenerative disorder,
which is characterized by the accumulation of insoluble extracellular plaques
comprising amyloid-β (Aβ) fibrils and intracellular neurofibrillary tangles
comprising hyperphosphorylated tau. Blocking Aβ self-assembly by bloodbrain-barrier (BBB) penetrable small molecules is a potential therapeutic
strategy to ameliorate Aβ aggregation-induced neuronal loss in AD. In this
study, we rationally designed and chemically synthesized a series of dimeric
compounds with different linker regions based on the Aβ fibril structure
model to examine their potency as Aβ binder and inhibitor. We calculated the
logP value using software Marvin Sketch to evaluate the druglikeness of these
molecules and their ability to penetrate BBB. The calculated logP values of
these candidate compounds are around 1.60, ranging from 1.48 to 1.78,
showing a potential of penetrating BBB. Fluorescence titration was used to
examine the binding affinity of these compounds to Aβ42 monomer. We
found the bivalent compound with a shorter linker region processes better
affinity. The bivalent compound has higher affinity in comparison to the
monovalent one. To investigate the inhibitory effect of the compounds on Aβ
aggregation, Thioflavin T (ThT) assay was performed and the end-point
products were examined by dot-blot and western blot to examine the oligomer
appearance, transmission electron microscopy to image Aβ morphology, and
MTT assay to examine Aβ-induced neuronal toxicity. Finally, the compounds
were fed to AD C. elegans model for paralysis assay. Overall, our data
demonstrated that the newly synthesized bivalent compound is a better
inhibitor against Aβ aggregation and has beneficial potential against AD.
P104
Korean mistletoe (Viscum album coloratum) extract regulates the lifespan
through CHICO, SIR2 and FOXO protein in Drosophila melanogaster.
Yanxialei Jiang , Min Jae Lee
Department of Applied Chemistry, College of Applied Sciences, Kyung Hee
Hye-Yeon Lee, Shin-Hae Lee, Kyung-Jin Min
189
Chuankhayan P., Tao T.-T., Lin C.-C., Guan H.-H., Nakagawa A., Fu T.-F. and
Chen C.-J., ―Structural insights into the hydrolysis and polymorphism of
methotrexate polyglutamate by zebrafish -glutamyl hydrolase‖, J. Med.
Chem. Vol. 56 (2013), pp 7625-7635.
Kao T.-T., Chang W.-N., Wu H.-L., Shi G.-Y. and Fu T.-F., ―Recombinant
zebrafish -glutamyl hydrolase exhibits properties and catalytic activities
comparable with those of mammalian enzyme‖, Drug Metab. Dispos. Vol. 37,
No. 2, (2009), pp 302-309.
Department of Biological Sciences, Inha University, Incheon 402-751, Korea.
E-mail: lyou5276@naver.com
Korean mistletoe (Viscum album coloratum) is a hemi-parasite plant living on
the tree and has been used for treating tumor, hypertension, and diabetes.
Recently, our group found that Korean mistletoe extract (KME) extends the
lifespan of worms and fruit flies and regulates the lifespan through
mechanisms similar with dietary restriction. In this study, we investigated the
target proteins of KME in Drosophila melanogaster. The effects of KME on
lifespan extension were abolished in CHICO, SIR2 or FOXO null mutant flies.
CHICO and FOXO are proteins in insulin/IGF-1 signaling pathway and SIR2
is a sensory protein that detects NAD:NADH ratio. These results suggest that
the KME regulates the lifespan by modulating insulin/IGF-1 signaling
pathway and activity of SIR2 protein.
Keywords: Korean mistletoe extract, Lifespan, Dietary restriction, Drosophila
melanogaster
P106
Molecular modeling study of dipeptidyl amides as Plasmodium
falciparum calpain inhibitors for the anti-malarial drug discovery
Minghua Cui1, Pankaz K. Sharma1, Hyun Park2 and Sun Choi1,*
1
National Leading Research Laboratory of Molecular Modeling & Drug
Design, College of Pharmacy, Graduate School of Pharmaceutical Sciences,
and Global Top 5 Research Program, Ewha Womans University, Seoul 120750, Korea
2
Zoonosis Research Center, Department of Infection Biology, School of
Medicine, Wonkwang University, Iksan, Jeonbuk 570-749, Korea
Email: babana1987@ewhain.net
References
[1] Lee SH, An HS, Jung YW, Lee EJ, Lee HY, Choi ES, An SW, Son HH,
Lee SJ, et al., ―Korean mistletoe (Viscum album coloratum) extract extends
the lifespan of nematodes and fruit flies‖, Biogerontology, Vol. 15, (2014), pp
153-164
[2] Lee SH and Min KJ, ―Caloric restriction and its mimetics‖, BMB reports,
Vol. 46, (2013), pp 181-187
[3] David J. Clancy et al., ―Extension of Life-Span by Loss of CHICO, a
Drosophila Insulin Receptor Substrate Protein‖, Science, Vol. 292, (2001), pp
104-106
Malaria causes estimated 300-500 million clinical cases as well as 2-3 million
deaths annually. Most deaths by malaria are caused by Plasmodium
falciparum, which is one of the five species of malaria parasites. Although
there are several agents, such as Chloroquine, available for the treatment of
malaria, increasing resistance to available drugs urges us to develop new antimalarial agents. Since the Plasmodium falciparum calpain (Pf-calpain) is
considered to be an essential mediator of merozoite invasion, it could be a
promising target for the discovery of anti-malarial drugs.
Pf-calpain is a cysteine protease which has an alternative Ca2+-independent
regulatory mechanism. Recently, it was reported that Pf-calpain can be active
only with the catalytic subdomain IIa. Our multiple sequence alignment
(MSA) study indicated that the catalytic subdomain IIa contains all the three
amino acid residues, which are collectively known as the catalytic triad (CysHis-Asn). Moreover, homology modeling study suggested that the subdomain
IIa of Pf-calpain constitutes the active site, holding the catalytic triad residues
in their appropriate orientation for the catalysis. The mutational study further
supported that those amino acid residues are play a functional role to have the
enzymatic activity of Pf-calpain.
A series of dipeptidyl amides, based on the known calpain inhibitor, were
designed and synthesized to discover potent inhibitors of Pf-calpain. Some
compounds among them showed strong anti-malaria activities. Using our Pfcalpain homology model, we carried out docking studies of these compounds
and found that they fit the binding site very well. These results could provide
the opportunity for structure-based anti-malarial drug design and discovery.
P105
Structural insights into hydrolysis of methotrexate polyglutamate by
zebrafish -glutamyl hydrolase
Phimonphan Chuankhayan1, Tseng-Ting Kao2, Chien-Chih Lin1, HongHsiang Guan1, Atsushi Nakagawa5, Tzu-Fun Fu2, and Chun-Jung Chen1,3,4
1
Life Science Group, Scientific Research Division, National Synchrotron
Radiation Research Center, Hsinchu 30076, Taiwan.
2
Department of Medical Laboratory Science and Biotechnology, National
Cheng Kung University, Tainan 701, Taiwan.
3
Institute of Biotechnology and University Center for Bioscience and
Biotechnology, National Cheng Kung University, Tainan 701, Taiwan.
4
Department of Physics, National Tsing Hua University, Hsinchu 30043,
Taiwan.
5
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871,
Japan.
E-mail: phimonphan_c@yahoo.com
 Glutamyl hydrolases (GH) catalyze the hydrolysis of -linked glutamate
residues from the polyglutamyl of folates and antifolates, such as
methotrexate (MTX), a widely used anti-cancer drug. We describe the first
crystal structures of the endopeptidase-type GH (zGH) from zebrafish and
the mutant complexes with MTX(Glu)5 and hydrolyzed MTX(Glu)1, revealing
the complete key residues involved in the substrate recognition and hydrolysis.
The structure of zGH folds into sandwiched-like domains with the central 7Structures of mutant zGH-substrate complexes reveal the complete three
substrate-binding subsites (–1 to +2) inside the catalytic pocket. Cys108,
His218 and Ser168 govern the binding of the glutamic moiety at the –1
subsite and the catalytic reaction. Lys221 and His218 define the +1 subsite for
binding the glutamic moiety. Arg228, Ser283 and Ala284 stabilize the
glutamic moiety at the +2 subsite. The positions of the +3 and +4 subsites are
located near the molecular surface for substrates with higher structural
flexibilities. The pteroyl group of MTX(Glu)5 is stabilized through a watermediated hydrogen bond between Glu112 and the oxygen atom of the paminobenzoic ring. The side chain of Phe20 and the 6-methylpterin ring of
MTX(Glu)5
conformational alterations involving ~90° rotations in the complexes with the
zGH-C108A and zGH-H218N mutant proteins. Structural geometries of the
MTX(Glu)5 and hydrolyzed MTX(Glu)1 in the mutant complexes differ
significantly from those of the previously known MTX(Glu)1, providing
polymorphic information. Together with the structural comparison and the
activity analysis, these results shed light on the catalytic mechanism and
substrate recognition of zGH and other 
P107
Activation and efficiency enhancement of cardiomyocyte-specific
commitment pathways in P19 embryonic stem cells
by small molecules.
Jin A Lee1 and Kyeong Kyu Kim1*
1
Institute, Sungkyunkwan University School of Medicine, Suwon, 440-746,
Korea
E-mail: lja0217a@gmail.com
Cardiovascular diseases are among the leading causes of death worldwide.
The loss of cardiomyocytes poses a major difficulty for normal functioning of
heart, since terminally differentiated cardiomyocytes have limited
regeneration potential. Recently, stem cells have been used to treat a variety
of cardiovascular diseases [1, 2]. However, through knowledge about
signaling pathways involved in stem cell differentiation is required for a
better understanding and enhancing the efficiency of their differentiation. For
this reason, we are aiming to develop new differentiation strategies and in
turn focus on signaling pathways which can be modulated to enhance the
overall efficiency of commitment. For this purpose we used mouse embryonal
carcinoma cells and engineered them to harbor a reporter construct expressing
mCherry fluorescent protein driven by α-MHC (myosin heavy chain)
promoter. This system apart from being qualitative can be used quantitatively
by measuring the mCherry fluorescence intensity. Using this system, we
screened small molecule libraries to identify chemicals which can trigger
cardiac-specific transcriptional activity in P19 cells and thus forms a platform
References
190
to investigate the mechanism of enhancing cardiac muscle commitment.
conjugated protein induces the change from tolerogenic to immune
stimulatory microenvironment in Peyer‘s patch, we monitored the expression
of chemokines and cytokines after LL-37 treatment. We found that it
constructs stimulating microenvironment by enhancing IL-6 expression and
maturation of innate cells. Moreover, we also found that Peyer‘s patch
follicular dendritic cells not only express formyl peptide receptor-2, one of
LL-37 receptors, but also express some chemokines and cytokines by
stimulation with LL-37. Therefore, chemotactic attraction and modulation of
dendritic cells by LL-37 suggested the adjuvant activity of LL-37 as being
sensitization of innate immunity and programming of adaptive immune
environment toward immune-stimulating conditions. Collectively, we
conclude that LL-37 has potential as a mucosal modulator and then oral
mucosal adjuvant by triggering and connecting innate and adaptive
immunity.(This study was supported by the Basic Science Research Program
(2013R1A2A2A01014459) through the NRF).
References
1.Tuch, B.E., Stem cells--a clinical update. Aust Fam Physician, 2006. 35(9):
p. 719-21.
2.Sykova, E. and S. Forostyak, Stem cells in regenerative medicine. Laser
Ther, 2013. 22(2): p. 87-92.
P108
Cathelicidin LL-37 can play a role as a mucosal immune modulator
Sae-Hae Kim1,2, Ha-Yan Lee2, Xi Yin2, Kwang-Yeop Jahng3,
Yong-Suk Jang1,2
1
Department of Molecular Biology and the Institute for Molecular Biology
and Genetics, Chonbuk National University, Jeonju 561-756, Korea
2
BK21 PLUS Program in the department of Bioactive Material Sciences and
Research Center of Bioactive Materials, Chonbuk National University, Jeonju
561-756, Korea
3
Department of Life Sciences, Chonbuk National University, Jeonju 561-756,
Korea.
E-mail: yongsuk@jbnu.ac.kr
References
[1] Davidson, D.J., Currie, A.J., Reid, G.S., Bowdish, D.M., MacDonald, K.L.,
Ma, R.C., Hancock, R.E., and Speert, D.P. The cationic antimicrobial peptide
LL-37 modulates dendritic cell differentiation and dendritic cell-induced T
cell polarization. J Immunol, 172, (2004), pp 1146-1156.
[2] Kahlenberg, J.M., and Kaplan, M.J.. Little peptide, big effects: the role of
LL-37 in inflammation and autoimmune disease. J Immunol, 191, (2013), pp
4895-4901.
Human cathelicidin LL-37 is expressed in neutrophils and epithelial cells. LL37 functions as a host-derived antimicrobial peptide against various infectious
agents, ranging from bacteria to fungi. In addition, LL-37 is known to have
chemotactic and modulating activity on various cells including monocytes, T
cells, and epithelial cells. In this study, we suggest that LL-37 functions as a
mucosal immune modulator. To understand whether LL-37 peptide or LL-37conjugated protein induces the change from tolerogenic to immune
stimulatory microenvironment in Peyer‘s patch, we monitored the expression
of chemokines and cytokines after LL-37 treatment. We found that it
constructs stimulating microenvironment by enhancing IL-6 expression and
maturation of innate cells. Moreover, we also found that Peyer‘s patch
follicular dendritic cells not only express formyl peptide receptor-2, one of
LL-37 receptors, but also express some chemokines and cytokines by
stimulation with LL-37. Therefore, chemotactic attraction and modulation of
dendritic cells by LL-37 suggested the adjuvant activity of LL-37 as being
sensitization of innate immunity and programming of adaptive immune
environment toward immune-stimulating conditions. Collectively, we
conclude that LL-37 has potential as a mucosal modulator and then oral
mucosal adjuvant by triggering and connecting innate and adaptive
immunity.(This study was supported by the Basic Science Research Program
(2013R1A2A2A01014459) through the NRF).
P110
Design, Synthesis, and Biological Evaluation of Phenyl-PiperazineTriazine-Based α-Helix Mimetics Targeting Protein-Protein Interactions
Heejo Moon, Woo-Sirl Lee, Misook Oh, and Hyun-Suk Lim*
Department of Chemistry, Pohang University of Science and Technology,
Pohang 790-784, Korea
E-mail: mhj0804@postech.ac.kr
Many cellular events occur via protein-protein interactions (PPIs) where
protein secondary structures, such as α-helix, often play an important role as a
recognition motif. From the fact that α-helix accounts more than 30% of
protein secondary structure in nature [1], designing α-helical mimetics is of
great importance to modulate PPIs.
Strategies to mimic α-helical structure have been tried over past decades [2-6].
One of the most remarkable compounds is terphenyl structure in which three
substituted phenyl rings exist in array. The low solubility in water and
difficulty in synthesis, however, were pointed out as the fatal problems that
prevent this scaffold from the further extensive application [6]. Therefore, it
has been a big issue among chemical biologists to develop a new class of
scaffold with better water solubility and synthetic accessibility.
Here we describe the design and efficient solid-phase synthesis of phenylpiperazine-triazine scaffold as a novel class of α-helix mimetic small
molecules. The scaffold showed good aqueous solubility. Subsequent
screening of a focused library of the designed molecules identified a potent
and selective inhibitor of MCL-1/BH3 interaction, demonstrating their ability
to act as inhibitors of α-helix-mediated PPIs. Consequently, our scaffold,
along with simple synthetic route, will provide an excellent source of PPI
modulators.
References
[1] Davidson, D.J., Currie, A.J., Reid, G.S., Bowdish, D.M., MacDonald, K.L.,
Ma, R.C., Hancock, R.E., and Speert, D.P. The cationic antimicrobial peptide
LL-37 modulates dendritic cell differentiation and dendritic cell-induced T
cell polarization. J Immunol, 172, (2004), pp 1146-1156.
[2] Kahlenberg, J.M., and Kaplan, M.J.. Little peptide, big effects: the role of
LL-37 in inflammation and autoimmune disease. J Immunol, 191, (2013), pp
4895-4901.
References
[1] Valeria. A., Long. K., Murphy. N. S., and Wilson. A. J., ―Inhibition of αhelix-mediated protein-protein interactions using designed molecules‖, Nature
Chem., Vol. 5, (2013), pp 161-173.
[2] Maity. P., and Konig. B., ―Synthesis and structure of 1,4-dipiperazino
benzenes: chiral terphenyl-type peptide helix mimetics‖, Org. Lett., Vol. 10,
(2008), 1473-1476.
[3] Lee J. H., Zhang Q., Jo S., Chai S. C., Oh M., Im W., Lu H., and Lim H.
S., ―Novel pyrrolopyrimidine-based α-helix mimetics: cell permeable
inhibitors of protein-protein interactions‖, J. Am Chem. Soc., Vol. 133, (2011),
pp 676-679.
[4] Lee. J. H., and Lim H. S., ―Solid-phase synthesis of tetra-substituted
pyrrolo[2,3-d]pyrimidines‖, Org. Biomol. Chem, Vol. 10, (2012), pp 42294235.
[5] Orner. B. P., Ernst. J. T., and Hamilton. A. D., ―Toward proteomimetics:
terphenyl derivatives as structural and functional mimics of extended regions
of an α-helix‖, J. Am. Chem. Soc., Vol. 123, (2001), pp 5382-5383.
[6] Cummings. C. G., and Hamilton. A. D., ―Disrupting protein-protein
interactions with non-peptidic, small molecule α-helix mimetics‖, Curr. Opin.
Chem. Biol., Vol. 14, (2010), 341-346.
P109
Cathelicidin LL-37 can play a role as a mucosal immune modulator
Sae-Hae Kim1,2, Ha-Yan Lee2, Xi Yin2, Kwang-Yeop Jahng3,
Yong-Suk Jang1,2
1
Department of Molecular Biology and the Institute for Molecular Biology
and Genetics, Chonbuk National University, Jeonju 561-756, Korea
2
BK21 PLUS Program in the department of Bioactive Material Sciences and
Research Center of Bioactive Materials, Chonbuk National University, Jeonju
561-756, Korea
3
Department of Life Sciences, Chonbuk National University, Jeonju 561-756,
Korea.
E-mail: yongsuk@jbnu.ac.kr
Human cathelicidin LL-37 is expressed in neutrophils and epithelial cells. LL37 functions as a host-derived antimicrobial peptide against various infectious
agents, ranging from bacteria to fungi. In addition, LL-37 is known to have
chemotactic and modulating activity on various cells including monocytes, T
cells, and epithelial cells. In this study, we suggest that LL-37 functions as a
mucosal immune modulator. To understand whether LL-37 peptide or LL-37-
191
P111
Aquaporin water channels as drug targets
Janet To, Chiew Ying Yeo, Cin Huang Soon, Yin Hoe Yau, Susana GeifmanShochat and Jaume Torres
Division of Structural Biology and Biochemistry, School of Biological
Sciences,
Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
E-mail: yyto1@ntu.edu.sg
Aquaporins (AQPs) are membrane water channels whose major cellular
function is to facilitate the highly efficient yet strictly selective osmotic
transport of water across biological membranes. In humans, thirteen
aquaporin types control water (and glycerol for the aquaglyceroporins)
homeostasis [1]. Evidences from AQP-knockout studies suggest that AQP
modulators may have broad clinical indications, including in nephrology,
neurology, oncology, ophthalmology and in the treatment of obesity and
dermatological indications [2]. However, progression in this field has been
slow partly due to challenges in developing small-molecule AQP inhibitors,
including conceptual and technical issues associated with screening assays. To
this end, we have recently developed a generic high throughput screen [3] to
discover aquaporin water channel inhibitors and have identified a number of
candidates from a small library of 10,000 compounds. Hits were tested for
binding to human aquaporin 1 (hAQP1) in detergent using surface plasmon
resonance to give affinity constants in the low micromolar range. Inhibitory
effect on hAQP1 was also tested using human erythrocytes and hAQP1proteoliposomes, in stopped-flow water permeability functional assays. One
of these compounds reversibly inhibited hAQP1 in erythrocytes but has no
effect on Escherichia coli aquaporin Z (AqpZ), and also significantly
increased the thermostability of hAQP1 in a thermal shift assay.
References
Driggers, E. M., Hale, S. P., Lee, J., Terrett, N. K., ―The exploration of
macrocycles for drug discovery - an underexploited structural class‖, Nat. Rev.
Drug Discov., 7, (2008), pp 608-624.
Redman, J. E., Wilcoxen, K. M., Ghadiri, M. R., ―Automated mass
spectrometric sequence determination of cyclic peptide library members‖, J.
Comb. Chem., 5, (2003), pp 33-40.
P113
Chemical substances that inhibit the activities of Hepatitis C virus NS3
helicase
Da-Hee Jung, Jin-Moo Lee and Yong-Joo Jeong
Department of Bio and Nanochemistry, Kookmin University,
Seoul 136-702, Korea
Hepatitis C is an infectious disease that affects the liver. The primary infection
route is blood-to-blood contact. Hepatitis C is caused by Hepatitis C virus
(HCV) that belongs to Flaviviridae family. The virus was proven in 1989. It
has a positive sense single strand RNA as genetic material. The Hepatitis C
NS3 helicase is an essential protein for viral replication. The purpose of this
study is preventing the replication of HCV as inhibit the activity of
Hepatitis C NS3 helicase. We expressed the helicase in E.coli BL21 (DE3)
cells and purified it. We did ATP hydrolysis and unwinding experiments to
check the activity of the helicase. We performed screening assay to find
chemical compounds inhibit the activity of the helicase as drug candidates.
We did dsDNA unwinding activity inhibition assay and ATPase activity
inhibition assay. Through this screening assay, we found several chemical
compounds inhibiting the activity of HCV NS3 helicase. We measured IC50
value to find the concentration of the discovered inhibitors which is required
to inhibit the activity of HCV NS3 helicase by half.
References
Agre P., King L.S., Yasui M., Guggino W.B., Ottersen O.P., Fujyoshi Y.,
Engel A. and Nielsen S. Aquaporin water channels- From atomic structure to
clinical medicine. Journal of Physiology, Vol. 542, No. 1, (2002), pp 3-16.
Verkman A.S., Anderson M.O. and Papadopoulos M.C. Aquaporins:
important but elusive drug targets. Nature Reviews Drug Discovery, (2014),
article in press.
To J., Yeo C.Y., Soon C.H., Yau Y.H., Geifman-Shochat S. and Torres J. A
generic high throughput screen to discover aquaporin water channel inhibitors.
Journal of Medicinal Chemistry, submitted.
P112
A Simple Ring-Opening Strategy for Cyclic Peptide and Cyclic Peptoid
Libraries
P114
Peptides for Inhibiting MDM2 Function as New Anticancer Therapeutics
Kang Ju Lee, Hyun-Suk Lim*
Si-Hyung Lee1, Do-Hyoung Kim1, In-Hoo Kim2, Ji-Eun Lim1, Ye-Jin Cho1,
Eun-Ji Cha1, Joan J. Han1, Seung-Hui Hong2, Chewook Lee1, Kyung-Tae
Kim2, and Kyou-Hoon Han1,3*
Pohang 790-784, Korea.
E-mail: kjulee0130@gmail.com
1
Division of Biomedical Research, Korea Research Institute of Bioscience and
2
Research Institute and Hospital, National Cancer Center, Goyang 410-769,
Korea
3
Department of Bioinformatics, University of Science and Technology,
Daejeon 305-333, Korea
E-mail : shlee@kribb.re.kr
Cyclic peptides and cyclic peptidomimetics have shown promising properties
as protein-binding molecules relative to their linear counterparts [1]. However,
they cannot be easily sequenced using general methods [2]. The limitation is
critical in sequencing the ―hit‖ compounds from high-throughput screening
(HTS) based on one-bead one-compound (OBOC) libraries.
Here, we describe a simple ring-opening strategy for cyclic peptide and cyclic
peptoid OBOC libraries. Macrocyclic compounds, containing a homocysteine
as a key residue, were re-opened and even cleaved from beads by one-pot
reaction when CNBr was treated. The reaction occurred under general
methionine cleavage condition, thus a lot of reactive amino acids or amines
can be applied in our system. Several cyclic peptoids and cyclic peptides were
synthesized, and then sequenced clearly by tandem MS/MS spectroscopy
from one bead. This easy and simple strategy can be utilized as a powerful
tool in screening with any kind of cyclic peptoid or cyclic peptide library.
The p53 tumor suppressor is a hub protein in the control of cell growth and
differentiation. MDM2 regulates p53 level through a negative-feedback loop.
MDM2 is the E3 ubiquitin ligase that promotes ubiquitin-dependent
proteolysis of p53 reducing the level of p53. Activation of p53 by the use of
antagonists acting on MDM2 have been considered as an attractive strategy
for anticancer therapeutic, and an increasing number of p53-MDM2 binding
inhibitors have been discovered in the past decade [1-2]. MDM2 protein is
known to be stabilized by binding to SUMO-1 protein. A SUMO-1 specific
protease 4 (SUSP4) produced in response to DNA damage has been reported
to remove SUMO-1 from MDM2. This removal of SUMO-1 lead to
promotion of MDM2 self-ubiquitination and digestion, resulting in p53
stabilization [3]. In order to understand the mechanism of SUSP4 antagonistic
activity we have investigated the interaction between various fragment of
SUSP4 and MDM2 using NMR and SPR techniques. Our results show that
SUSP4 peptides bind to the N-terminal region of MDM2. Elucidation of the
interaction between SUSP4 and MDM2 give us insight into the antagonistic
mechanism of SUSP4 involving p53, MDM2 and SUMO-1 suggesting a
192
valuable approach for the treatment of cancers.
References
Chene, P., ―Inhibiting the p53-MDM2 interaction: an important target for
cancer therapy‖, Nat. Rev. Cancer, Vol 3, (2003), pp 102-109
Fotouhi, N. and Graves, B., ―Small molecule inhibitors of p53/MDM2
interaction‖, Curr. Top. Med. Chem., Vol. 5, (2005), pp 159-165
Lee, MH., et al., ―SUMO-specific protease SUSP4 positively regulates p53 by
promoting Mdm2 self-ubiquitination‖, Nat. Cell Biol., Vol. 8, No. 12, (2006),
pp 1424-1431
P117
Protein Data Bank Japan (PDBj): maintaining structural data archive
and integration of structure data with other life sciences data resources
by semantic web technologies
P115
Discovering Candidate Substances of MDM2 Inhibitors using SAR-byNMR
Akira R. Kinjo1, Haruki Nakamura1, Hirofumi Suzuki1, Reiko Yamashita1,
Yasuyo Ikegawa1, Takahiro Kudou1, Gert-Jan Bekker1, Reiko Igarashi1,
Yumiko Kengaku1, Hasumi Cho1, Junko Sato1, Nahoko Haruki1, Daron M.
Standley2, Atsushi Nakagawa1
Do-Hyoung Kim1, Si-Hyung Lee1, and Kyou-Hoon Han1,2*
1
Japan.
2
Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871, Japan
E-mail: harukin@protein.osaka-u.ac.jp
1
Division of Biomedical Research, Korea Research Institute of Bioscience and
2
Daejeon 305-333, Korea
E-mail : organic2@kribb.re.kr
The Protein Data Bank Japan (PDBj, http://pdbj.org/) is a member of the
worldwide Protein Data Bank (wwPDB: http://wwpdb.org/) [1, 2]. PDBj
accepts and processes experimental macromolecular structure data determined
by X-ray crystallography, SAXS/SANS, NMR, and Electron Microscopy as a
Data-in service. While maintaining the archive in collaboration with other
wwPDB partners, PDBj also provides a wide range of Data-out services and
tools for analyzing structures and functions of proteins, as shown in Table 1.
To facilitate the integration of structure data with other life sciences data
resources, we have developed data in formats compatible with semantic web
technologies: PDB/RDF (http://rdf.wwpdb.org/pdb/) and Chem_Comp/RDF
(http://rdf.wwpdb.org/cc/), PDB and chemical compounds data in the
Resource Description Framework (RDF) format, along with its ontology in
Web Ontology Language (OWL) based on the PDB mmCIF Exchange
Dictionary [1, 2]. NMR experimental data in BMRB database are also being
described in RDF as BMRB/RDF, and those data are integrated through the
linked-data technology.
The mouse double minute 2 (MDM2) oncogenic protein is the primary
cellular inhibitor of the p53 tumor suppressor. In addition, the cellular levels
of p53 and MDM2 are mutually regulated through an autoregulatory feedback
loop. Preventing the interaction of MDM2-p53 has been proposed as a
potential cancer therapeutic strategy. In previous NMR studies, we obtain the
structural information about p53-mdm2 interactions [1]. From these results,
we investigated the interactions between MDM2 and extracted mixture using
NMR methods for drug design and screening such as SAR by NMR. The
extracted mixture from rare plants is purchased from the gene bank of
International Biological Material Research Center. In this study, 1H NMR
experiments were performed. Here we will describe the discovery of MDM2
inhibitors based on the change of NMR spectra such as peak position and
peak broadening.
References
Chi, SW., et al., ―Structural details on mdm2-p53 interaction‖ J. Biol. Chem.,
Vol. 280, No. 46, (2005), pp 38795-38802
P116
Regulator screening of protein tyrosine phosphatasome against natural
products
Hyo Jin Kang2, Sunyoung Park2, Kyungmin Roh1, Kisun Park1, Kwang-Hee
Bae3 and Sang. J. Chung1,2
1
Department of Chemistry, College of Natural Sciences, Dongguk University,
Seoul 100-715, Korea.
2
Research Institute for Biomolecular Chemistry, Dongguk University, Seoul
100-715, Korea
3
Systemic Proteomics Research Center, KRIBB, Yuseong, Daejeon 305-806,
Korea
E-mail: jin0305@dongguk.edu
References
[1] Kinjo A. R. et al., Nucl. Acids Res., Vol. 40, (2012) D453-D460. [2]
Berman H. M. et al., Structure, Vol. 21, (2013) pp 1485-1491. [3] Kinjo A. R.
et al., Database, Vol. 2010, (2010) baq021. [4] Standley D. M. et al.,
Bioinformatics, Vol. 26, (2010) pp 1258-1259. [5] Kinjo A. R. and Nakamura,
H., Biophysics, Vol. 3, (2007) pp 75-84. [6] Kinjo A. R. and Nakamura H.,
Structure, Vol. 17, (2009) pp 234-246. [7] Kinjo A. R. and Nakamura H., J.
Mol. Biol., Vol. 399, (2010) pp 526-540. [8] Kinjo A. R. and Nakamura H.,
PLoS One, Vol. 7, (2012) e31437. [9] Kinjo A. R. and Nakamura H.,
Biophysics, Vol. 8, (2012) pp 79-84. [10] Kinoshita K. and Nakamura H.,
Bioinformatics, Vol. 20, (2004) pp 1329-1330. [11] Lis M. et al., Immunome
Res., Vol. 7 (2011) pp 1-8. [12] Kinoshita K. and Nakamura H., Protein
Science, Vol. 12, (2003) pp 1589-1595. [13] Kinoshita K. et al., Nucl. Acids
Res., Vol. 35, (2007) W398-W402. [14] Murakami Y. et al., Protein Science,
(2013) DOI: 10.1002/pro.2329. [15] Wako H. and Endo S., Comput. Biology.
Chem., VOL. 44, (2013) pp 22-30.
Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes that
remove phosphate group from phosphorylated tyrosine residue on protein.
PTKs (protein tyrosine kinases) and PTPs (protein tyrosine phsophsatases)
control phosphorylation-dephosphorylation on protein tyrosines each other.
PTPs together with PTKs are important modulators in regulating important
cellular signal transduction controlling the shape and motility of cells, cell
proliferation, organ development, and immune system. Disfunctions in PTP
activity is implicated in several human diseases, such as cancer, diabetes, and
autoimmune disorders. Therefore, activity regulators of PTPs have been very
important to design the biological lead compound. Natural products are
widely used in drug discovery and structures in drug discovery efforts. Herein,
we report the preparation of protein tyrosine phosphatasome library and the
screening results against 650 natural compounds.
References
Nicholas K. T., ―Protein tyrosine phosphatases: from genes, to function, to
disease‖, Nature Reviews, Vol. 7, (2006), 833-846
Gavin C., Fabrice B., Saranyoo K., Isabelle P., Melissa L. and Russell G. K.,
―Natural products with protein tyrosine phosphatase inhibitory activity‖
Methods, Vol. 65, (2014), 229-238
P118
Identification of an Ideal-like Fingerprint for a Protein Fold using
Overlapped Conserved Residues based Approach
193
Amit Goyal, Sriram Sokalingam, Kyu-Suk Hwang, Sun-Gu Lee
Labeling of Amino Acid in Cell Culture (SILAC)
School of Chemical and Biomolecular Engineering, Pusan National
University, Busan 609-735, Korea.
E-mail: amitgsir@pusan.ac.kr
Jeong Hwan Hur1, Hannah Lee Foon Swa2, Doyoun Kim1, Jayantha
Gunaratne2, Kyeong Kyu Kim1
1
Department of Molecular Cell Biology Samsung Biomedical Research
Institute, Sungkyunkwan University School of Medicine, Suwon 440-746,
Korea.
2
Mass Spectrometry & Systems Biology Laboratory, Institute of Molecular
and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis
Drive, 138673 Singapore.
E-mail: jhhur88@gmail.com
Identification of an ideal fingerprint that represents a structural fold at distant
sequence identity has been a great challenge. This paper proposes a strategy
to extract such ideal-like fingerprints that should be composed of the critical
residues that can detect all of the homologous proteins with perfect specificity
and without the non-essential residues that can decrease the sensitivity.
Identification of such critical residues that may be conserved in three aspects,
i.e. sequence, structure, and intramolecular interaction, can be an efficient
way to generate such ideal like fingerprint. The fingerprint was generated by
selecting the overlapped conserved residues (OCR) from the conserved
residues obtained using three independent alignment methods. The OCR
fingerprints showed more than 90% detection efficiency and identified to be
almost the minimal fingerprints composed of only critical residues. This study
is expected to provide an important conceptual improvement in the
identification of ideal fingerprints for a protein fold.
Left handed helical Z-DNA is a comparably higher energy form of double
stranded DNA structure. It can be stabilized through negative supercoiling
generated by unwrapping of the nucleosomes [1]. Z-DNA can also be
stabilized through protein interaction, and its stability may result significant
biological consequences. The formation of Z-DNA nearby the transcriptional
start site often regulates the transcriptional activities [2]. Furthermore, the
formation of Z-DNA might lead to double strand breaks (DSBs) which can
cause large-scale mutations and genetic instability [3]. In this study, we would
like to explore the novel Z-DNA binding proteins using Stable Isotope
Labeling of Amino Acid in Cell Culture (SILAC) to elucidate the role of ZDNA in transcriptional activity and in genetic instability. SILAC is a novel
approach for relative quantification of the protein in vivo cells by mass
spectrometry (MS), and it is powerful method to investigate protein-DNA
interaction [4]. The relative abundance of novel Z-DNA binding proteins have
been identified and analyzed by using SILAC method in combination with
parallel pull-down of in vivo proteins with B- and Z-DNA oligonucleotides.
Through various validation procedures of these proteins, this study will
provide significant insights for the role of Z-DNA in the various biological
conditions and the genetic instability in more detail.
References
Stevens, F. J., ―Efficient recognition of protein fold at low sequence identity
by conservative application of Psi-BLAST: validation‖, J. Mol. Recognit, 18,
(2005), pp 139-149.
Kister, A.E. & Gelfand, I., ―Finding of residues crucial for supersecondary
structure formation‖, Proc. Natl. Acad. Sci. USA, 106, (2009), pp 1899619000.
P119
Characterization of PDZ domain-ligand interaction using position of
residues in PDZ domain and ligand
References
[1] Rich A. & Zhang, S. ―Timeline: Z-DNA: the long road to biological
function‖. Nature Rev. Genet. 4, 566-572 (2003).
[2] Oh, D. B., Kim, Y. G. & Rich, A., ―Z-DNA binding proteins can act as
potent effectors of gene expression in vivo”. Proc. Natl Acad. Sci. USA 99,
16666-16671 (2002)
[3] Kha, D. T., et al., ―Pathways for Double-strand Break Repair in
Genetically Unstable Z-DNA-forming Sequences‖. Journal of Molecular
Biology. 398, 471-480 (2010)
[4] Mittler, G., Falk, B., Mann, M., ―A SILAC-based DNA protein interaction
screen that identifies candidate binding proteins to functional DNA elements,”
Genome Research, 19, 284–293 (2009).
Changsuk Oh1 and Kyeong Kyu Kim1
1
Department of Molecular and Cellular Biology, Sungkyunkwan Untiversity
School of Medicine, Suwon 440-746, Korea
E-mail: changs.oh@gmail.com
PDZ (PSD95/DLG/ZO-1) domain, a structural domain involved in proteinprotein interaction, is well conserved among species including virus, bacteria,
plant and mammals. The proteins containing this domain play key role in
signaling by interacting with their putative ligands[1]. Generally, the Cterminal residues of ligands are recognized by the central cleft of PDZ.
Specially, residue at (-2) position [P(-2)] is critical for determining specificity
of PDZ recognition. However, it is also proposed that the ligand specificity is
dependent on all seven C-terminal residues [P(-6)-P(0)][2]. To investigate the
importance of these additional residues in PDZ domains, we modified the
position pairing method described elsewhere [3]. We superimposed all the
crystal structures of PDZ available in PDB to NHERF1 1st PDZ domain.
Structures which aligned with RMSD below 1.5Å were selected.
Subsequently, we identified 58 exclusive pairs involved in the interaction. We
extended this study by subsequently aligning the sequences pertaining to PDZ
and ligand from protein microarray and phage display data [2, 4]. Results thus
obtained helped us to determine all possible interaction types and define
critical residues involved. This generalization can be well utilized to make a
platform to tailor synthetic PDZ domains to target important ligands with
several important physiological implications.
P121
In vivo protein biotinylation for identification of cell-specific multiprotein
complexes
Min Jung Lee1,2, Tae Jeong Ahn1, KyeongJin Kang1
1
Department of Anatomy and Cell Biology, School of Medicine,
Sungkyunkwan University, Suwon 440-746, Korea.
2
Research Institute, Dong-A ST Co., Ltd., Yongin 446-905, Korea.
E-mail: liferadar@gmail.com
Assembly of multiprotein complexes is an essential and initial process for
many biological functions in living organisms. BioID is a recently developed
approach to identify protein-protein interaction partners and near-neighbors,
taking advantage of the fact that a promiscuous biotin protein ligase (BirA*)
fusion protein promotes biotinylation of proximal and interacting proteins in
mammalian cells [1]. Here we aim to develop a proteomic strategy based on
in vivo biotinylation to isolate components of protein complexes from specific
cells of growing or fully developed Drosophila melanogaster. For Drosophila
BioID, transgenic flies were generated to express BirA* fusion protein in
specific cells via GAL4/UAS system. Our first applied fusion protein was
Drosophila transient receptor potential ankyrin 1 (dTRPA1), a critical sensory
receptor responsible for avoidance behaviors of tissue-damaging chemicals
[2]. In vivo biotinylation will be conducted by feeding biotin-containing meal.
Fusion protein and its stably or transiently interacting proteins will be
enriched by capturing biotin-conjugated proteins, separated by one
dimensional SDS-PAGE and cataloged by mass spectroscopy. One of the
most exciting points of our method is the combination of BioID proteomics
and Drosophila genetics, allowing the identification of multiprotein
complexes in specific cell types and specific developmental stages in vivo.
Successful application will provide the basis to establish cell-specific
References
1.Kim, J., et al., Rewiring of PDZ domain-ligand interaction network
contributed to eukaryotic evolution. PLoS Genet. 8(2): p. e1002510.
2.Tonikian, R., et al., A specificity map for the PDZ domain family. PLoS Biol,
2008. 6(9): p. e239.
3.Chen, J.R., et al., Predicting PDZ domain-peptide interactions from primary
sequences. Nat Biotechnol, 2008. 26(9): p. 1041-5.
4.Stiffler, M.A., et al., PDZ domain binding selectivity is optimized across the
mouse proteome. Science, 2007. 317(5836): p. 364-9.
Frontiers in protein sciences
P120
Investigation of Novel Z-DNA Binding Protein by Using Stable Isotope
194
proteome database that may help understand the cell-specific function of
proteins and protein-protein interaction.
important role in transcriptional regulation. The former research has revealed
an interaction between IE2 and SUMO, and the SUMO binding by IE2 is
necessary for its efficient transactivation function and for viral growth.
Structurally, IE2 is one of intrinsically unfolded proteins (IUPs), peculiar
proteins that carry out functions without forming a three-dimensional
structure. A structural prediction based on IUPred program shows that
SUMO-interaction region of IE2 has high disorder tendency, which indicates
structural importance of this region. In this study, the predicted disordered
region of IE2, which includes SUMO-interaction region, was labeled with 15N
and expressed. After purification, the structure of IE2 was studied by using
2D 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC).
References
Kyle J. Roux, Dae In Kim, Manfred Raida, and Brian Burke, ―A promiscuous
biotin ligase fusion protein identifies proximal and interacting proteins in
mammalian cells‖, J. Cell Biol., Vol. 196, No.6, (2012), pp 801-810.
Kang K., Pulver S.R., Panzano V.C., Chang E.C., Griffith L.C., Theobald
D.L., Garrity P.A., ―Analysis of Drosophila TRPA1 reveals an ancient origin
for human chemical nociception‖, Nature, Vol. 464, No. 7288, (2010), pp
597-600.
References
Kim E.T., Kim K.K., Matunis M.J. and Ahn J.H., ―Enhanced SUMOylation of
proteins
containing a SUMO-interacting motif by SUMO-Ubc9 fusion‖,
Biochemical and Biophysical Research Communications, Vol. 388, Issue 1,
(2009), pp 41-45.
Kim E.T., Kim Y.E., Huh Y.H. and Ahn J.H., ―Role of noncovalent SUMO
binding by the human cytomegalovirus IE2 transactivator in lytic growth‖,
Journal of Virology, vol. 84, no. 16, (2010), pp 8111-8123.
Caswell R., Hagemeier C., Chiou C.J., Hayward G. and Kouzarides T.,
―Sinclair J The human cytomegalovirus 86K immediate early (IE) 2 protein
requires the basic region of the TATA-box binding protein (TBP) for binding,
and interacts with TBP and transcription factor TFIIB via regions of IE2
required for transcriptional regulation‖, The Journal of General Virology, vol.
74, no. 12, (1993), pp 2691-2698.
Lee J.M., Kang H.J., Lee H.R., Choi C.Y., Jang W.J. and Ahn J.H., ―PIAS1
enhances SUMO-1 modification and the transactivation activity of the major
immediate-early IE2 protein of human cytomegalovirus‖, FEBS Letters, Vol.
555, Issue 2, (2003), pp 322-328.
Wüthrich, K., ―Protein structure determination in solution by NMR
spectroscopy‖, The Journal of Biological Chemistry, Vol. 265, (1990), pp
22059-22062.
P122
Contribution of proline to the pre-structuring tendency of transient
helical secondary structure elements in intrinsically disordered proteins
Chewook Lee a, Lajos Kalmar b, Bin Xue c, Peter Tompa b,
GaryW. Daughdrill d,e, Vladimir N. Uversky c,f,g, Kyou-Hoon Han a,h*
a
Division of Convergent Biomedical Research, Biomedical Translational
Research Center, Korea Research Institute of Bioscience and Biotechnology,
125 Gwahak-ro, Yuseong-gu,
Daejeon 305-806, Republic of Korea
b
Institute of Enzymology, Biological Research Center, Hungarian Academy of
Sciences, 1518 Budapest, P.O. Box 7, Hungary
c
Department of Molecular Medicine, University of South Florida, Tampa, FL
33612, USA
d
Department of Cell Biology, Microbiology, and Molecular Biology,
University of South Florida, Tampa, FL 33612, USA
e
Center for Drug Discovery and Innovation, University of South Florida,
Tampa, FL 33612, USA
f
USF Health Byrd Alzheimer's Research Institute, Department of Molecular
Medicine, Morsani College of Medicine, University of South Florida, Tampa,
FL 33612, USA
g
Institute for Biological Instrumentation, Russian Academy of Sciences,
142292 Pushchino, Moscow Region, Russia
h
Department of Bioinformatics, University of Science and Technology, 113
Gwahak-ro, Yuseong-gu, Daejeon 305-333, Republic of Korea
E-mail : austin89@kribb.re.kr
P124
Structure of the CCR5 chemokine receptor – HIV entry inhibitor
Maraviroc complex
Qiuxiang Tan1†, Ya Zhu1†, Jian Li1, Zhuxi Chen2, Gye Won Han3, Irina
Kufareva4, Tingting Li1, Limin Ma1, Gustavo Fenalti3, Jing Li1, Wenru Zhang1,
Xin Xie1, Huaiyu Yang2, Hualiang Jiang2, Vadim Cherezov3, Hong Liu1,
Raymond C. Stevens1,3,5, Qiang Zhao1, Beili Wu1*
IDPs function without relying on three-dimensional structures. No clear
rationale for such a behavior is available yet. PreSMos are transient secondary
structures observed in the target-free IDPs and serve as the target-binding
―active‖ motifs in IDPs. Prolines are frequently found in the flanking regions
of PreSMos. Contribution of prolines to the conformational stability of the
helical PreSMos in IDPs is investigated. MD simulations are performed for
several IDP segments containing a helical PreSMo and the flanking prolines.
To measure the influence of flanking-prolines on the structural content of a
helical PreSMo calculations were done for wild type as well as for mutant
segments with Pro → Asp, His, Lys, or Ala. The change in the helicity due to
removal of a proline was measured both for the PreSMo region and for the
flanking regions. The α-helical content in ~70% of the helical PreSMos at the
early stage of simulation decreases due to replacement of an N-terminal
flanking proline by other residues whereas the helix content in nearly all
PreSMos increases when the same replacements occur at the C-terminal
flanking region. The helix destabilizing/terminating role of the C-terminal
flanking prolines is more pronounced than the helix promoting effect of the
N-terminal flanking prolines. This work represents a novel example
demonstrating that a proline is encoded in an IDP with a defined purpose. The
helical PreSMos presage their target-bound conformations. As they most
likely mediate IDP-target binding via conformational selection their helical
content can be an important feature for IDP function.
1
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia
Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China
201203
2
Drug Discovery and Design Center, Shanghai Institute of Materia Medica,
Chinese Academy of
Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203
3
Department of Integrative Structural and Computational Biology, The
Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA
92037, USA
4
University of California, San Diego, Skaggs School of Pharmacy and
Pharmaceutical Sciences, La Jolla, CA 92093,USA
5
iHuman Institute, ShanghaiTech University, Shanghai, China 201203
†These authors contributed equally to this work. *Corresponding author.
E-mail: beiliwu@simm.ac.cn
The chemokine receptors CXCR4 and CCR5 serve as HIV-1 co-receptors.
Through the process of HIV entry to the host cells, the HIV-1 envelope
glycoprotein gp120 first binds to the host cell receptor CD4, and is followed
by the exposure of the third variable loop of gp120, V3 loop, which then
interacts with the co-receptor. Then, another HIV-1 glycoprotein gp41 refolds
and triggers the membrane fusion [1]. Several high resolution crystal
structures have revealed detailed pictures of HIV-1 gp120 in complex with
soluble CD4 and gp41, but the structural information of CCR5 was not known,
because GPCRs crystallization is extremely challenging. Following the
determination of CXCR4 crystal structure [2], here we report the 2.7 Å
resolution crystal structure of human CCR5 bound to the marketed HIV drug
maraviroc (Figure). The N-terminal and ECL2 regions of CCR5 have been
identified as major recognition sites for its chemokine ligands, and play an
important role in gp120 binding and HIV-1 infection [3]. The inverse agonist
maraviroc is buried in a cavity within the 7TM domain, and makes no
contacts with N-terminal or ECL2 of CCR5. The distinct ligand binding site
provides insights into the mechanism of allosteric inhibition of chemokine
signaling and viral entry by maraviroc. Individual HIV strains exhibit specific
P123
A Heteronuclear NMR Study on the Intrinsically Unfolded IE2
Jiulong Su 1, 2, Si-Hyung Lee 1, Do-Hyoung Kim 1, and Kyou-Hoon Han 1, 2
1
Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806,
Korea.
2
Daejeon 305-350, Korea.
E-mail: ericsue@kribb.re.kr
Immediate-early protein 2 (IE2) is an 86 kDa protein expressed by immediate
early genes in human cytomegalovirus (HCMV) infection, which plays an
195
cellular tropisms for the co-receptors CXCR4 and CCR5. And the co-receptor
selectivity is mainly determined by the V3 loop of HIV-1 gp120 [4]. A
comparison between CCR5 and CXCR4 crystal structures, along with models
of co-receptor/gp120-V3 complexes, suggests that different charge
distributions and steric hindrances caused by residue substitutions may be
major determinants of HIV-1 co-receptor selectivity. These high-resolution
insights into CCR5 can enable structure-based drug discovery for the
treatment of HIV-1 infection.
In this report, we successfully optimized high yield procedure of hMC4R TM2
and stable nmr condition. Finally, we determined energy minimized structure
ensemble of hMC4R TM2 in SDS micelle condition at 303K. hMC4R TM2
consists of two α-helices (Residues from Met 79 to Ser 96 and Glu 101 to Thr
105) and Asn 97 to Ser 100 region is represented structural dynamics. The
dynamic movement between two helices of hMC4R TM2 should be related to
its biological function. In summary, NMR structure and bio activity assay
strongly supports the fact that hMC4R TM2 D90N mutant is strong
heterozygous mutant with loss of function.
Fig. The crystal structure of CCR5 bound to maraviroc
References
Craig B. Wilen
John C. Tilton
and Robert W. Doms, ―Viral
Molecular Machines”, Rossmann, Springer, (2012), 223-242.
B. Wu et al., Structures of the CXCR4 chemokine GPCR with small-molecule
and cyclic peptide antagonists. Science 330, 1066 (2010), 1066-1071.
L. Duma, D. Haussinger, M. Rogowski, P. Lusso, S. Grzesiek, Recognition of
RANTES by extracellular parts of the CCR5 receptor. J Mol Biol 365, 1063
(2007), 1063-1075.
Hwang, S.S., Boyle, T.J., Lyerly, H.K., Cullen, B.R., 1991. Identification of
the envelope V3 loop as the primary determinant of cell tropism in HIV-1.
Science.253 (5015), 71–74.
References
[1] Huszar, D., Lynch, C.A., Fairchild-Huntress, V., Dunmore, J.H., Fang,
Q., Berkemeier, L.R., Gu, W., Kesterson, R.A., Boston, B.A., Cone,
R.D., ― Targeted disruption of the melanocortin-4 recepto results in
obesity in mice‖, Cell, 88, (1997), pp 131-141.
[2] Chai, B.X., Pogozheva, I.D., Lai, Y.M., Li, J.Y., Neubig, R.R., Mosberg,
H.I., Gantz, I., “Receptor antagonist interactions in the complexes of
agouti and agouti-related protein with human melanocortin 1 and 4
receptors‖, Biochemistry, 44, (2005), pp 3418-3431.
[3] Wuthrich, K., ―NMR structures of biological macromolecules. In
Encyclopedia of Nuclear Magnetic Resonance‖ D.M. Grant and R.K.
Harris, eds. (New York: Wiley, (1996), pp 710–719.
P125
Structure analysis of muscarinic acetylcholine receptor intracellular loop
3 by hydrogen/deuterium exchange mass spectrometry
In Young Ko*, Dong-Kyun Kim* and Ka Young Chung
P127
NMR structural studies on extracellular domain of hMC4R
E-mail: koiy@skku.edu, kurababy@skku.edu
Dongju Lee1, Ji-Hye Yun1, Daeseok Oh1, Eunhee Kim2, Chaejoon Cheong2 and
Weontae Lee1*
The muscarinic acetylcholine receptor (mAchR) belongs to the G protein-coupled
receptors (GPCRs) and plays important roles in various organs. There are five
subtypes of mAchRs; M1, M2, M3, M4, and M5. mAchRs have a long
intracellular loop 3 (ICL3). However, the only crystal structure of mAchR (e.g. the
crystal structure of M3) lacks ICL3, a region that is reported to be important for the
interaction with downstream signaling molecules. The structure analysis of ICL3
would provide information on the understanding of its mechanism. In this study,
we analyzed the conformations of M1-ICL3 and M3-ICL3 using
hydrogen/deuterium exchange mass spectrometry (HDX-MS). The HDX profiles
of M1-ICL3 and M3-ICL3 showed conformational dynamics and provided
conformational difference between M1-ICL3 and M3-ICL3.
1
Structural Biochemistry and Molecular Biophysics Laboratory, Department
of Biochemistry, College
of Life Science and Biotechnology, Yonsei University, Seoul 120-749,
Republic of Korea.
2
Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang,
Chungbuk 363-883, Korea
E-mail: dongju@spin.yonsei.ac.kr
Melanocortin 4 receptor (MC4R) is a member of G-protein coupled receptor
(GPCR) A families. Previous studies report that extracellular (ecto) domain is
related to obesity disease. Ecto domain of human MC4R (hMC4R) has critical
region for interacting with hMC4R ligand such as SHU9119. Cyclic peptide
SHU9119 (Ac-Nle-c[Asp-His-D-Nal-Arg-Trp-Lys]-NH2) is well known potent
antagonist of hMC4R. In order to determine molecular interaction between
antagonist peptide and ecto domain of hMC4R, we performed NMR
experiment. We observed specific interaction sites between hMC4R ecto
domain and SHU9119 from NMR chemical shift perturbations. SHU9119
binds to Val2, Arg7, Trp16, Asn17, Leu23 and Lys33 residues of hMC4R ecto
domain in 200mM DPC micelle. In addition, data from 1H-15N 2D-HSQC
suggests that ecto domain of hMC4R is stable at 200mM salt. Therefore, Our
result will be applicable to understanding functional role of hMC4R ecto
domain related to human disease.
*equally contributed
P126
Structural and functional studies on transmembrane II domain of human
melanocortin-4 receptor
Ji-Hye Yun1, Dongju Lee1, Daeseok Oh1 and Weontae Lee1
1
Department of Biochemistry, College of Life Science and Biotechnology,
Yonsei University, Seoul, 120-749, Korea
E-mail: jihye@spin.yonsei.ac.kr
The melanocortin receptors are members of the G protein-coupled receptor
(GPCR) 1 superfamily that have seven trans-membrane (TM) domains.
Among them, the melanocortin-4 receptor (MC4R) subtype has been
highlighted recently by genetic studies in obese humans. Especially, in patients
with severe early-onset obesity, novel heterozygous mutation in the MC4R
gene was detected resulting in an exchange of aspartic acid to asparagine in
90th amino acid residue located in the second transmembrane domain (TM2).
Our research aim is to figure out the structural and functional characterization
of hMC4R TM2 and its mutant form (D90N) using Nuclear magnetic
resonance (NMR) and biochemical experiments. In order to characterize
hMC4R TM2 structure using NMR, it is essential to optimize efficient
procedure for high yield purification of recombinant transmembrane peptide.
196
References
Chul-Jin Lee., et al., ―Solution structures and molecular interactions of
selective melanocortin receptor antagonists.‖ Mol. Cells. 2010. 30: p. 551-556
Juan Ji An., et al., ―Peripheral effect of α-Melanocyte-stimulating Hormone on
fatty acid oxidation in skeletal muscle.‖ Journal of biological chemistry. 2007.
282(5): p. 2862-2870
Giles S.H. Yeo, et al., ―Mutations in the human melanocortin-4 receptor gene
associated with severe familial obesity disrupts receptor function through
multiple molecular mechanisms.‖ Mol. Genet. 2003. 12(5): p. 561-574
P128
Cystatin SN neutralizes the inhibitory effect of cystatin C on cathepsin B
activity
Jong-Tae Kim, Seon-Jin Lee, Byungmoo Oh, Jieun Kang, Heesoo Lee, and
Hee Gu Lee*
Bioscience and Biotechnology, Daejeon, Republic of Korea
E-mail: jongtae@kribb.re.kr
Fig.1 Comparison of interaction energies of the -helices (AH) and parallel
-sheets (PB and APB) calculated by quantum chemical (QC)
methods (B97D/6-31+G(d)) and molecular mechanics (MM) (AMBER99SB) .
Cystatin SN is one of the several salivary cystatins that form tight equimolar
complexes with cysteine proteases, such as the cathepsins. High expression of
CST1 is correlated with advanced pTNM stage in gastric cancer. In this study,
we showed that CST1 was highly expressed in colon tumor tissues, compared
with nontumor regions. Increased cell proliferation and invasiveness were
observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116CST1) but not in CST3-transfected cells. We also demonstrated that CST1overexpressing cell lines exhibited increased tumor growth as well as
metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted
with cystatin C, a potent cathepsin B inhibitor, with a higher affinity than the
interaction between CST3 and CTSB in the extracellular space of HCT116
cells. CTSB-mediated cellular invasiveness and proteolytic activities were
strongly inhibited by CST3, but in the presence of CST1 CTSB activities
recovered significantly. Furthermore, domain mapping of CST1 showed that
the disulfide-bonded conformation, or conserved folding, of CST1 is
important for its secretion and for the neutralization of CST3 activity. These
results suggest that CST1 upregulation might be involved in colorectal
tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic
activity by CST3.
References
Grimme S., "Semiempirical GGA-type density functional constructed with a
long-range dispersion correction", J. Comput. Chem. Vol. 27, No. 15 (2006),
pp 1787-1799.
Wang J., Cieplak P., Kollman P. A., "How well does a restrained electrostatic
potential (RESP) model perform in calculating conformational energies of
organic and biological molecules?", J. Comput. Chem. Vol. 21, No. 12 (2000),
pp 1049-1074.
P130
Structural characterization of Mycobacterium tuberculosis heparinbinding hemagglutinin and its interaction with synthetic heparan sulfate
analogs
Deli Irene1,2, Tzu-Jui Dai1, Shih-Han Lian1, Chiao-Chu Ku2, Shang-Cheng
Hung2*, and Chia-Lin Chyan1*
P129
Density functional study of molecular interactions in secondary
structures of proteins
1
Departement of Chemistry, National Dong Hwa University, No. 1, Sec. 2, Da
Hsueh Road, Shou-feng, Hualien 974, Taiwan.
2
Genomics Research Center, Academia Sinica, No. 128, Sec. 2, Academia
Road, Taipei 115, Taiwan.
E-mail: deli24@gate.sinica.edu.tw
Yu Takano1 and Haruki Nakamura1
1
Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
E-mail: ytakano@protein.osaka-u.ac.jp
Mycobacterium tuberculosis is one of the most devastating human
microbaterial pathogens. The heparin binding hemagglutinin (HBHA) of
Mycobacterium tuberculosis mediates microbacterial adherence to epithelial
cells and therefore responsible for extrapulmonary dissemination of
tuberculosis. Through the C-terminal lysine-rich domain, HBHA binds to
heparin sulfate proteoglycans on the surface of epithelial cells [1]. Here we
report a thorough thermodynamic characterization of the interaction of HBHA
with synthetic heparan-sulfate analogs in different lengths. HBHA was found
to bind at least six sugar units with the interaction notably being entropically
driven. Circular dichroism data demonstrated that the interaction of the
HBHA and the synthetic heparan-sulfate analogs increases the secondary
structure contents of HBHA. To derive a high resolution structural picture of
this molecular recognition process, the interactions of a truncated and soluble
form of HBHA (residue 110-199; HBHA110-199) with a synthetic heparansulfate analog with eight sugar units were analyzed by NMR spectroscopy.
These data assisted with docking simulations demonstrated that the synthetic
heparan-sulfate analog binds to the C-terminal domain of HBHA and then
induced the stable dimeric complex structure. The study of the interactions
of HBHA with heparan-sulfate analogs not only for better understands the
molecular mechanism of mycobacterial adherence but also facilitates the
development of the novel heparan sulfate-based drugs.
The three-dimensional structure of a protein determines its functions and
-sheets
are important components for the protein architecture. The local and nonlocal
molecular interactions, in particular hydrogen bonding, play significant roles
in the formation of the secondary structures. Quantitative estimate of these
interactions is required to understand the principle of the formation of the
three-dimensional protein structure. In the present study, to improve the force
field for accurate description of protein behavior, we have investigated the
-helices (AH) and parallel
-sheets (PB and APB) composed of alanine residues, using
quantum chemical (QC) methods (B97D/6-31+G(d) [1]) and molecular
mechanics (MM) (AMBER99-SB [2]) . The characteristic interactions
essential for forming the secondary structures are discussed quantitatively. As
shown in Fig. 1, the MM force field overestimates the interaction energies of
-sheet, implying that the
improvement of the electrostatic and hydrogen bonding interactions in the
force field are required.
References
Zulueata MM., Chyan CL., Wong CH., and Hung SC., ―α-Glycosylation by
D-glucosamine-derived donors: synthesis of heparosan and heparin analogues
that interact with mycobacterial heparin-binding hemagglutinin‖, J. Am.
Chem. Soc.,Vol. 134, No. 21, (2012), pp 8988–8995.
197
by congo red, J Biomol Struct Dyn, 30, (2012), 160-169.
[4] Chen, Y. R., et al. Effect of alanine replacement of L17 and F19 on the
aggregation and neurotoxicity of arctic-type Aβ40, PLoS One, 8, (2013),
e61874.
P131
Structural basis for the replication of genomic RNA of porcine enteric
sapovirus by VPg
Hye Jung Min, Hyo-Jeong Hwang, Jeong-Sun Kim, and Chul Won Lee
P133
Structural characterization of DARPP-32 by NMR spectroscopy
Department of Chemistry, Chonnam National University, Gwangju 500-757,
Korea.
Kai-Cyuan He1, Chu-Ting Liang2, Hsien-Bin Huang3 and Ta-Hsien Lin1,2,4
Viral protein genome-linked (VPg) plays a critical role in the life cycle of
vertebrate and plant positive-sense (+) RNA viruses by acting as a protein
primer for RNA synthesis. The replication of the viruses is primed by the VPg
protein by attaching to the 5‘-end of the viral RNA genome. Here we show
the solution structure of VPg of porcine enteric sapovirus (PSV) determined
by multi-dimensional NMR spectroscopy. The structure of VPg comprises
three helices (denoted 1, 2, and 3) which were tightly packed together
through hydrophobic interactions formed by several hydrophobic residues to
form a three-helix bundle structure. The orientation and length of three helices
of PSV VPg are similar to those of feline calicivirus (FCV) VPg with similar
core structure formed by conserved hydrophobic residues. The tyrosine
residue (Tyr956) within the conserved motif for nucleotide acceptor is
exposed to the solvent surface to facilitate nucleotidylation by viral RNA
polymerases. These results may provide the molecular basis for the
replication of positive-RNA viruses.
1Institute of Biochemistry and Molecular biology, National Yang-Ming
2Department of Life Sciences and Institute of Genome Sciences, National
Yang-Ming University,
Taipei 112, Taiwan
3Institute of Molecular Biology, National Chung Cheng University, Chia-Yi
621, Taiwan
4Department of Medical Research & Education, Taipei Veterans General
Hospital, Taipei 112, Taiwan
E-mail: kay5402@hotmail.com
Protein phosphatase 1 (PP1) is one of the major serine/threonine eukaryotic
protein phosphatases. It plays a critical role in the regulation of various
cellular functions, including carbohydrate metabolism, protein synthesis, cell
cycle, muscle contraction and neuronal signaling. The catalytic subunit of PP1
in cells is associated with different binding proteins to form a variety of
holoenzymes. These binding proteins may target the enzyme to specific
subcellular compartments in which PP1 regulates the functions of its
substrates. The catalytic subunit of PP1 is specifically regulated by three
protein inhibitors, inhibitor-1, DARPP-32 (dopamine and cAMP-regulated
phosphoprotein of apparent Mr 32,000) and inhibitor-2. Both inhibitor-1 and
DARPP-32 under the phosphorylated state can suppress the function of PP1,
while inhibitor-2 can only block the activity of PP1 under the nonphosphorylated state. The inhibition of PP1 is regulated through
phosphorylation of these protein inhibitors, however, the underlying
molecular mechanism is not fully understood. To investigate this issue, we
have performed structure-functional analyses of these protein inhibitors. So
far we have characterized the structures of inhibitor-1[1, 2], inhibitor-2 [3, 4]
and N-terminal domain of DARPP-32 [5] under the phosphorylated and nonphosphorylated states. In the present study, we performed structural
characterization of full-length DARPP-32 and phospho-DARPP-32 by using
molecular and structural biology techniques, such as stable isotope labeling
(13C and/or 15N) and nuclear magnetic resonance spectroscopy. The
structural information of these protein inhibitors may provide us with a
structural basis for further studies of their interactions with PP1 from the
structural point of view.
P132
Structural characterization of L17A/F19A mutated β-amyloid peptides
by NMR spectroscopy
Chu-Ting Liang1, Yu-Wen Shen2, Kai-Cyuan He 2 and Ta-Hsien Lin1,2,3
1
Department of Life Sciences and Institute of Genome Sciences, National
Yang-Ming University,Taipei 112, Taiwan
2
Institute of Biochemistry and Molecular biology, National Yang-Ming
3
Department of Medical Research & Education, Taipei Veterans General
Hospital,Taipei 112, Taiwan
E-mail:d49808015@ym.edu.tw
β-amyloid peptide (Aβ), which consists of 39-42 residues, is a proteolytic
product of β-amyloid precursor protein (APP). Alzheimer‘s disease (AD) is
thought to be associated with aggregation of Aβ. The molecular mechanism
underlying Aβ aggregation is not yet clear. The process of Aβ aggregation into
amyloid fibril involves conformational change, suggesting that the
conformational stability of Aβ play a key role in the aggregation process. In
silico study proposed that Aβ contains a discordant sequence, spanning from
residue 14 to 23, which is predicted to form a β-strand but adopts an α-helix
in some environments [1]. The discordant α-helix is thought to be predisposed
to undergo α-helix to β-strand conversion. It also predicted that inhibition of
Aβ aggregation can be achieved by introducing amino acid with higher αhelix propensity, such as alanine, into the discordant sequence. However,
there is a lack of experimental evidences to support these inferences. By using
nuclear magnetic resonance (NMR) spectroscopy in combination with
equilibrium denaturation and site-directed mutation approaches [2-4], we
previously showed that binding of small molecule to the region of Aβ
discordant α-helix and mutation of amino acid residues in this region by
alanine (L17A/F19A) can inhibit conformational change and fibril formation
of wild-type Aβ40 and Arctic Aβ40 variant (Aβ40(E22G)), a pathogenic Aβ40
mutant which has a higher aggregative ability than wild-type Aβ40 and causes
Familial Alzheimer‘s disease (FAD). From the structural point of view, it is
likely that alanine mutation increase the α-helix propensity of Aβ discordant
region, resulting in an increase of the conformational stability of Aβ. Owing
to the lack of structural information, this hypothetic mechanism remains
elusive. To demonstrate this hypothesis, the structures of L17A/F19A mutated
wild-type Aβ40 and Aβ40(E22G) were characterized by using NMR
spectroscopy, and compared to those of their native forms. An increase of αhelix propensity of the discordant region was observed for both L17A/F19A
mutated peptides. This result may provide a structural basis toward
understanding the molecular mechanism of Aβ aggregation.
References
[1] Chyan C. L., Tang T. C., Chen Y. C., Liu H. T., Lin F. M., Liu C. K., Hsieh
M. J., Shiao M. S., H. B. and Lin T. H., ―Backbone 1H, 15N, and 13C
Resonance Assignments of Inhibitor-1 – a Protein Inhibitor of Protein
Phosphatase-1‖, J. Biomol. NMR, Vol. 21, (2001), pp 287-288.
[2] Huang Y. C., Chen Y. C., Tsay H. J., Chyan C. L., Chen C. Y., Hsien-bin
Huang H. B. and Lin T. H., ―The effect of PKA-Phosphorylation on the
structure of inhibitor-1 studied by NMR spectroscopy‖, , J. Biochem., Vol.
147, (2010), pp 273-278.
[3] Huang H. B., Chen Y. C., Tsai L. H., Wang H. C., Lin F. M., Horiuchi A.,
Greengard P., Nairn A. C., Shiao M. S. and Lin T. H., ―Backbone 1H, 15N,
and 13C resonance assignments of inhibitor-2 - a protein inhibitor of protein
phosphatase-1‖, J. Biomol. NMR, Vol. 17, (2000), pp 359-360.
[4] Lin T. H., Chen Y. C., Chyan C. L., Tsay L .H., Tang T. C., Jeng H. H., Lin
F. M. and Huang H. B., ―Phosphorylation by glycogen synthase kinase of
inhibitor-2 does not change its structure in free state‖, FEBS Lett., Vol. 554,
(2003), pp 253-256.
[5] Lin T. H., Huang Y. C., Chin M. L., Chen Y. C., Jeng H. H., Lin F. M.,
Shiao M. S., Horiuchi A., Greengard P., Nairn A. C., and Huang H. B., ―1H,
15N, and 13C resonance Assignments of DARPP-32 (dopamine and cAMPregulated phosphoprotein, Mr. 32,000) – a Protein Inhibitor of Protein
Phosphatase-1‖, J. Biomol. NMR, Vol. 28, (2004), pp 413-414.
P134
Structural characterization of ApoE(222-271) by NMR spectroscopy
References
[1] Kallberg, Y., et al. Prediction of amyloid fibril-forming proteins, J Biol
Chem, 276, (2001), 12945-12950.
[2] Chen, Y. R., et al. Aβ40(L17A/F19A) mutant diminishes the aggregation
and neurotoxicity of Aβ40, BBRC, 405, (2011), 91-95.
[3] Wang, C. C. et al. Characterization of Aβ aggregation mechanism probed
Yu-Wen Shen1, Chi-Jen Lo1, Chu-Ting Liang2, Hsien-Bin Huang3 and TaHsien Lin1,2,4
198
1
Institute of Biochemistry and Molecular biology, National Yang-Ming
2
Department of Life Sciences and Institute of Genome Sciences, National
Yang-Ming University, Taipei 112, Taiwan
3
Institute of Molecular Biology, National Chung Cheng University, Chia-Yi
621, Taiwan
4
Department of Medical Research & Education, Taipei Veterans General
Hospital, Taipei 112, Taiwan
E-mail: fioma800210@hotmail.com
action with endo-xylanase, promising a great potential application for
lignocelluloses saccharification in industry.
References
[1] Shallom, D., Shoham, Y. 2003. Microbial hemicellulases. Curr. Opin.
Microbiol., 6(3), 219-228.
[2] Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V.,
Henrissat, B. 2009. The Carbohydrate-Active EnZymes database (CAZy): an
expert resource for Glycogenomics. Nucleic. Acids. Research., 37, 233-238.
Human apolipoprotein E (ApoE) is a polymorphous protein with 299 residues
(M.W. ~34 kDa). It contains two independently folded structural domains.
One is a 22 kDa N-terminal domain (residues 1-191), which is responsible for
LDL receptor binding activity. The other one, a 10 kDa C-terminal domain
(residues 216-299), is responsible for lipid binding and might be responsible
for tetramerization of ApoE. These two domains are separated by a flexible
hinge region. ApoE has three isoforms, ApoE2, ApoE3, and ApoE4, and these
isoforms differ from one another only by a single residue, but have marked
differences in their biological functions. ApoE4 is a major risk factor for
Alzheimer‘s disease and atherosclerosis. It is thought that the isoform-specific
domain interactions play a key role in their biological function. The molecular
mechanism of isoform-specific domain interactions is still unclear. Our
ultimate goal is to elucidate the molecular mechanism of domain interactions
from the structural point of view. The best approach is to solve the 3D
structures of intact ApoE isoforms. The 3D structures of N-terminal domain
of ApoE isoforms have been solved by X-ray crystallography. Recently, 3D
structure of intact human apoE3 with a mutated C-terminal domain
(F257A/W264R/V269A/L279Q/V287E) has also been solved [1]. However,
the structural information of the C-terminal domain of wild-type human apoE
in a lipid-free and lipid-bound state is still limited.
In the absence of lipid, the C-terminal domain tends to form oligomer. This
might hinder structural determination of ApoE isoforms by X-ray
crystallography or NMR spectroscopy. In this study, we perform structural
characterization on a C-terminal fragment of ApoE (ApoE(222-271)) which
has been thought to be involved in isoform-specific domain interactions.
Structural characterization of ApoE(222-271) in the absence and presence of
lipid was done by using circular dichroism (CD), analytic ultracentrifugation
(AUC) and NMR techniques. The structural information of ApoE(222-271)
may provide us with a structural basis for further studies of the isoformspecific domain interactions from the structural point of view.
P136
Structure-Activity Relationship Study of α-Helix Mimetics built on
Triazine-Piperazine-Triazine
Scaffold
Targeting
Protein-Protein
Interactions
Woo Sirl Lee1, Ji Hoon Lee1, Misook Oh1 and Hyun-Suk Lim1*
1
Pohang 790-784, Korea.
E-mail: wsirl@postech.ac.kr (Correspondence: hslim@postech.ac.kr)
The α-helix-mediated protein-protein interactions (PPIs) are considered one
of the most important focus in chemical biology and medicinal chemistry. [1-3]
Recently, we identified a α-helix mimetic hit compound 9c based on triazinepiperazine-triazine scaffold showing moderate affinity to MCL-1 via highthroughput screening (HTS) based on on-bead screen. In this research, we
investigated the structure-activity relationships of the hit compound using two
different approaches. Fisrt, we introduced hydrophilic functionality such as
carboxylic acid or amino group to the hit compound to give amphiphilicity.
Second, we introduced hydrocarbon-bridge by macrocyclization with a series
of different length of linkers to reduce rotational movement of α-helix
scaffold. As the result of in vitro binding assay using fluorescence
polarization (FP) method, the derivatives bearing negatively charged residue
showed much higher binding affinity to MCL-1 than the original hit
compound. In summary, the inhibitory effect of compound 9c-19 which was
structurally modified from the original α-helix mimetic hit compound out of a
library for MCL-1 was substantially increased by introducing carboxylic acid
because hydrophobic interactions at the binding interface are reinforced by a
complementary polar interaction network.
References
[1] Chen J., Li Q. and Wang J. Topology of human apolipoprotein E3
uniquely regulates its diverse biological functions. Proceedings of the
National Academy of Sciences of the United States of America, Vol. 108,
(2011), pp 14813-14818.
References
A. J. Wilson, ―Inhibition of protein–protein interactions using designed
molecules‖, Chem. Soc. Rev. Vol. 38, (2009), pp 3289-3300.
J. A. Wells and C. L. McClendon, ―Reaching for high-hanging fruit in drug
discovery at protein-protein interfaces‖, Nature, Vol. 450, (2007), pp 10011009.
H. Yin and A. D. Hamilton, ―Strategies for targeting protein-protein
interactions with synthetic agents‖, Angew. Chem. Int. Ed. Vol. 44, No. 27,
(2005), pp 4130-4163.
P135
Characterization a novel β-xylosidase which belongs in glycoside
hydrolase family 5 (GH5) from Phanerochaete chrysosporium
Keum-Kang So1, Dae-Hyuk Kim1, Seung-Moon Park2
P137
Glycan microarray for analysis of carbohydrate-density-dependent
binding of lectins
1
Department of Molecular Biology, College of Natural Sciences, Chonbuk
National University, Jeonju 561-756, Korea
2
Division of Biotechnology, College of Environmental and Bioresource
Sciences, Chonbuk National University, Jeonju 561-756, Korea
E-mail: smpark@jbnu.ac.kr
Jiyoung Hyun1, Jaeyoung Pai1, Xizhe Tian1 and Injae Shin1
1
Department of Chemistry, College of Natural Sciences, Yonsei University,
Seoul 120-749, Korea
E-mail: hyunjy@yonsei.ac.kr
β-xylosidases (E.C 3.2.1.37), exo-type of xylan degrading enzymes, which
catalyze the hydrolysis of β-1,4 linkage of short xylo-oligomer, releasing
xylose single units [1]. According to Carbohydrate Active Enzyme (CAZy)
database (www.cazy.org), β-xylosidases are classified into ten glycoside
hydrolase (GH) families 1, 3, 30, 39, 43, 51, 52, 54, 116, 120 (Cantarel et al.,
2009).
-Xylosidase, PcXyl5, which belongs in glycoside
hydrolase family 5 (GH5) from Phanerochaete chysosporium was cloned and
expressed in Pichia pastoris. Gene sequence analysis indicated that PcXyl5
did not show any homology with known β-xylosidases coding gene.
Recombinant PcXyl5 (rPcXyl5) displayed a protein band of 50 kDa on SDSPAGE, which is considered larger than polypeptide predicted size. EndoH
treatment did not decrease protein size. Optimum pH of rPcXyl5was found to
be 5.5, whereas optimum temperature was 50°C. rPcXyl5 not only catalyzed
hydrolysis of xylo-oligomers to xylose but also displayed transglycosylation
activity using alcohol as receptor. rPcXyl5 increased reducing sugar release of
birchwood xylan, beechwood xylan, arabinoxylan, and NaOH-pretreated
barley straw by 6.4 %, 13 %, 15.8 %, and 9.5 %, respectively in synergistic
To examine density-dependent binding of carbohydrates by lectins, we have
constructed glycan microarrays. In the microarray format, glycans are densly
immobilized on the solid surface and thus inter and intramolecular binding to
lectins are both taking place unlike glycan binding to lectins in solutions,
where this does not occur so readily. For this study, a number of C-terminal
hydrazide-conjugated glycopeptides with various valences and different
spatial arrangements of the sugar ligands were prepared on a solid support.
Glycan microarrays were prepared by attaching the glycopeptides to the
epoxide-modified glass slide and then incubated with various lectins to
analyze density-dependent binding of glycans. The results of binding property
determinations indicate that lectin binding is highly dependent on the surface
glycan density. The number of glycans on glycopeptides, the linker lengths
between the individual sugars, and the distance between glycopeptides on the
surface are all important factors to governing lectin binding properties. This
work could present valuable information on designing and preparing glycan
microarrays that are useful for studies of glycan-mediated recognition
199
events.[1]
resorption on Tmem173 overexpressed RAW264.7 cells. The overexpression
of Tmem173 reduced RANKL-mediated induction of TRAP mRNA level and
protein activity from osteoclast precursor cells by down-regulation c-Fos and
NFATc1. In addition, we observed down-regulation of ERK phosphorylation
in Tmem173 overexpressed RAW264.7 cells by RANKL-stimulation.
Interestingly, we discovered up-regulation of the Tmem173 by
osteoprotegerin (OPG) on RAW264.7 cells. OPG prevents interaction of
RANKL to RANK by competitive binding to RANKL. These results provide
new additional information that Tmem173 played a regulatory role in
RANKL-mediated osteoclast differentiation.
(This research was supported by the Regional Specialized Technology
Convergence R&D Program funded by the Ministry of Trade, Industry and
Energy.)
Figure 1. Illustration of binding of lectins on carbohydrate microarrays
Fucose
Mannobiose
Mannose
LacNAc
GlcNAc
P140
High expression of dengue virus antigen protein using viral expression
system
Byeong-Young Kim, and Moon-Sik Yang
Figure 2. Fluorescence images of glycan microarrays after probing with
Cy3-labeled ConA.
Department of Bioactive Material Sciences, Chonbuk National University,
Jeonju 561-756, Republic of Korea
E-Mail: mskyang@bjnu.ac.kr
References
Xizhe Tian, Jaeyoung Pai and Injae Shin, Chem. Asian J., 2012, 7, 2052–2060
Although plant-based vaccines have many advantages, their use has a
limitation because of low expression level of antigen genes in transgenic
plants, which elicits low immune response and immune tolerance. To
overcome this problem, antigen protein of dengue virus was produced via
agroinfiltration with plant virus based expression systems. Dengue virus
enters into host cells by binding envelope glycoprotein (E) to a receptor. A
DNA fragment encoding antigen protein and M cell-targeting ligand (Co1)
fusion antigen protein were constructed into viral vector and introduced into
tobacco plant cells (Nicotiana benthamiana) by Agrobacterium-mediated
infiltration. The antigen and Co1 fusion antigen proteins were detected in
protein extracts from agoinfiltrated leaf by western blot analysis. The
expression level of plant-produced antigen and Co1-fusion antigen proteins
were 0.98% and 2.03% of the total soluble protein, respectively. These results
suggest that the high expression of dengue virus antigen protein in plants
using plant virus based expression system can improve the feasibility of plantbased vaccines to overcome low expression level and low immune response
for preventing dengue virus infection.
Acknowledgement: Byeong-Young Kim was supported by BK21 Plus
Program in the Department of Bioactive Material Sciences
P138
Rapid profiling of RNA-peptide interactions using peptide microarray
Jaeyoung Pai1, Taejin, Yoon1 and Injae Shin1*
1
Department of Chemistry, Center for biofunctioal Molecules, Yonsei
University, Seoul, 120-749, Korea
E-mail: rookery81@yonsei.ac.kr
A rapid and quantitative method to evaluate binding properties of hairpin
RNAs to peptides using peptide microarrays has been developed. The
microarray technology was shown to be a powerful tool for high-throughput
analysis of RNA−peptide interactions by its application to profiling
interactions between 111 peptides and six hairpin RNAs. The peptide
microarrays were also employed to measure hundreds of dissociation
constants (Kd) of RNA−peptide complexes. Our results reveal that both
hydrophobic and hydrophilic faces of amphiphilic peptides are likely involved
in interactions with RNAs. Furthermore, these results also show that most of
the tested peptides bind hairpin RNAs with submicromolar Kd values. One of
the peptides identified by using this method was found to have good
inhibitory activity against TAR−Tat interactions in cells. Because of their
great applicability to evaluation of nearly all types of RNA−peptide
interactions, peptide microarrays are expected to serve as robust tools for
rapid assessment of peptide−RNA interactions and development of peptide
ligands against RNA targets.
P141
Production and Characterization of Human Pepsin II in Transgenic Plant
Cell Suspension Culture
Moon-Sik Yang
Department of Molecular Biology, Chonbuk National University, Jeonju 561756, Republic of Korea
E-Mail: mskyang@jbnu.ac.kr
References
Jaeyoung Pai, Taejin Yoon, Nam Doo Kim, Im-Soon Lee, Jaehoon Yu, Injae
Shin ―High-Throughput Profiling of Peptide-RNA Interactions Using Peptide
Microarrays‖, J. Am. Chem. Soc. Vol. 134, (2012), 19287–19296
Yeongran Lee, Soonsil Hyun, Hyun Jin Kim and Jaehoon Yu ―Amphiphilic
Helical Peptides Containing Two Acridine Moieties Display Picomolar
Affinity toward HIV-1 RRE and TAR" Angew. Chem., Int. Ed. Vol. 47,
(2008),134−137.
Human progastriscin (hPGC), which acts as a proteolytic enzyme of gastric
secretion, was produced and secreted from transgenic plant suspension cell
culture. The synthetic hPGC gene was synthesized on the basis of riceoptimized codon usage and was cloned into binary vector under the control of
the sucrose starvation-inducible rice α-amylase 3D (Ramy 3D) promoter and
3‘UTR as a terminator. In addition, a Ramy 3D leader sequence was fused to
the N-terminal end and 6-His tag was added to the 3‘ end of the hPGC gene
for effective secretion and easy purification. The plant expression vector was
introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by particle
bombardment. The integration of the hPGC gene into the chromosome of the
transgenic rice callus was verified via genomic DNA PCR amplification and
hPGA expression in transgenic rice suspension cells was confirmed via
Northern blot analysis. Western blot analysis detected both pro and mature
proteins with masses from 40~35 kDa in the culture medium following
induction with sugar starvation. The plant-derived recombinant hPGC was
activated by autocatalysis and low pH condition and the resulting mature
pepsin. It could be bound to nickel affinity matrix, indicating the 6-His tag
was showed hemoglobin degradation activity.
Acknowledgment: This work was supported by National Research Foundation
of Korea Grant funded by the Korean Government (2012R1A1A3015724)
and supported by Advanced Production Technology Development Program,
Ministry for Food, Agriculture, Forestry and Fisheries (312037-05), Republic
P139
Overexpression of Tmem173 inhibits osteoclast differentiation of
RAW264.7 cells by modulating RANKL signaling
CHOE Chung Hyeon1, PARK In Sun1, YU Kang-Yeol1,
Jang Yong-Suk2 and KIM Ju1
1
Jeonju Biomaterials Institute, Jeonju 561-360, South Korea.
Chonbuk National University, Jeonju 561-756, South Korea.
E-mail : dkzudk@jbmi.re.kr
2
The c-Fos and nuclear factor of activated T cells (NFAT) c1 play a crucial role
in osteoclast differentiation by osteoclast precursor cells. In this research, we
investigated the effect of Tmem173 on RANKL-mediated osteoclastogenesis
via the study of osteoclast-specific gene expression, TRAP activity, bone
200
of Korea
reactive hydroxyl radicals.
ZIF-268 is a classical zinc finger protein that uses a Cys2His2 ligand set to
bind zinc and fold. This protein‘s DNA binding properties are well understood
and it is possible to modify the protein sequence to tune DNA recognition. We
hypothesize that iron substituted ZIF-268 may be capable of cleaving target
DNA possibly with sequence selectivity. Work to address this hypothesis, we
have been over expressed and purified ZIF-268 and characterized its iron
(ferric and ferrous) and zinc binding properties. We have learned that ZIF-268
binds DNA with the same affinity when zinc, ferric and ferrous iron is bound.
Future studies will examine DNA cleavage initiated by ferrous-ZIF268
P142
Preparation of fluorophore-labeled glycoclusters and their use for
detection of cell-surface lectins
Sookil Park1, Xizhe Tian1,2, Jaeyoung Pai1, Kyung-Hwa Baek1,
Sung-Kyun Ko1 and Injae Shin1
1
Department of Chemistry, College of Sciences, Yonsei University, Seoul 120749, Korea.
2
Department of Chemistry, College of Sciences, Yanbian University, Yanji
133000, China.
E-mail: chemskpark@yonsei.ac.kr
P144
Design, synthesis and evaluation of cancer cell-specific delivery system
that dual targets Somatostatin receptors and Cathepsin B.
Hui Li1, Xizhe Tian2, Kyung-Hwa Baek1 and Injae Shin1
To detect cell-surface lectins, we prepared peptide-based glycoclusters with
various valences and different spatial arrangements of the sugar ligands. The
synthetic strategy includes 1) solid-phase synthesis of fluorophore-labeled,
alkyne-containing peptides, 2) coupling of azide-linked, unprotected mono-,
di-, and trisaccharides to the alkyne-conjugated peptides on a solid support by
click chemistry, and 3) release of the fluorophore-labeled glycoclusters from
the solid support. By using this methodology, thirty two fluorescent glycolclusters with a valence ranging from 1 to 4 and different spatial arrangements
of the sugar ligands were synthesized. Lectin binding properties of the
glycoclusters were initially examined by using microarrays immobilized by
various lectins.
These glycoclusters were then employed to detect the cell-surface
carbohydrate- binding proteins in bacteria. We also evaluated the uptake of
glycoclusters by mammalian cells through receptor-mediated endocytosis.
The results indicate that the binding affinities toward immobilized and cellsurface proteins are highly dependent on the valence and spatial arrangements
of the sugar ligands in glycoclusters.[1]
1
Department of Chemistry, College of Science, Yonsei University, Seoul 120749, Korea.
2
Department of Chemistry, College of Science, Yanbian University, Yanji
133000, China.
E-mail: lee921@yonsei.ac.kr
The currently available anticancer agents frequently cause unwanted normal
cell death mainly owing to their lack of selectivity for cancer cells. In addition,
molecular imaging agents for tumors exhibit low target-to-background ratios.
As a consequence, elegant methods that more specifically target cancer cells
need to be developed for the improvement of chemotherapeutic efficacy and
diagnosis. In a recent effort aimed at improving the tumor selectivity of
therapeutic and imaging agents, we designed, synthesized and explored the
effectiveness of a dual-targeting delivery system that targets cancer cells more
selectively. The new delivery system is composed of a synthetic ligand
(octreotide) of somatostatin receptors, a dipeptide substrate for cathepsin B,
and a fluorophore or an anticancer agent. The fluorophore-conjugated
delivery system was found to be applicable for specificfluorescence imaging
of cancer cells that express both somatostatin receptors and cathepsin B. In
addition, the anticancer agentcontaining delivery system leads to the death of
cancer cells specifically. In contrast to cancer cells, normal cells that do not
produce both somatostatin receptors and cathepsin B at high levels are
unaffected by the delivery system. The new dual-targeting approach has the
capability of overcoming obstacles associated with current chemotherapeutic
and imaging methods.
References
Xizhe Tian, Jaeyoung Pai, Kyung-Hwa Baek, Sung-Kyun Ko and Injae Shin,
―Fluorophore-labeled, Peptide-based Glycoclusters: Synthesis, Binding
Properties for Lectins, and Detection of Carbohydrate-Binding Proteins in
Cells‖, Chem. Asian J., Vol. 6, No. 8, (2011), pp 2107-2113.
P143
Characterization and Oxidation study of Iron Substituted Classical and
Non-Classical Zinc Finger Proteins
References
Kamb A., Wee S. and C. Lengauer., Nat. Rev. Drug Discovery, 6, (2007), pp
115–120.
Baek K., Park J., and Shin I., Chem. Soc. Rev, 41, (2012), pp 3245–3263.
Williams D R., Lee M R., Song Y A., Ko S K., Kim G H and Shin I., J. Am.
Chem. Soc, 129, (2007), pp 9258–9259.
Beom-Tae Kim2,3,4, Seung Jae Lee1.4 , Jun Young Kim2,4
1
Department of Chemistry, Chonbuk National University,
Jeonju 561-756, Rep. of Korea
2
Department of Bioactive Material Sciences, Chonbuk National University,
3
Institute of Basic Liberal Arts Education, Chonbuk National University,
4
Research Center for Bioactive Materials, Chonbuk Natioanl University,
E-mail: slee026@jbnu.ac.kr
P145
Structure of the entire stalk region of dynein motor domain
Yosuke Nishikawa1,2, Takuji Oyama1, Narutoshi Kamiya1, Tkahide Kon1,
Yoko Y. Toyoshima3, Haruki Nakamura1 and Genji Kurisu1,2
Zinc finger proteins are one of the most common families of proteins found in
eukaryotes. These proteins utilize zinc to fold into the correct three
dimensional structure and mediate a variety of biological functions including
transcriptional regulation, post-transcriptional control and protein-protein
recognition. The research project of my lab has focused understanding the
consequences of iron substitution in zinc finger proteins (ZFP). Although zinc
is typically thought to be the correct metal ion to bind to zinc finger proteins,
there is evidence that iron might substitute for zinc in certain types of zinc
finger proteins. This presentation focused on two zinc finger proteins:
Tristetraprolin (Non Classical ZFP) and ZIF-268 (Classical ZFP).
Tristetraprolin (TTP) uses three cysteine and one histidine residue to bind
zinc. TTP regulates the expression of cytokines such as tumor necrosis factor
α (TNF- α) by binding to AU-rich sequence elements (AREs) located at the 3‘
untranslated region (3‘UTR) of the cytokines‘ mRNA. Upon binding, the
TTP/mRNA complex is degraded by exosomes. I have discovered that both
ferric and ferrous iron will coordinate to TTP and still bind to RNA. In
addition, ferrous (Fe(II)) coordinated TTP may be detrimental to the cell
because it is redox active and may generate damaging reactive oxygen species
(ROS). I have developed a novel spectroscopic assay to measure oxidation
rates of zinc finger proteins as a function of metal ion coordination and I have
discovered that iron substitution promotes oxidation via the formation of
1
Japan
2
Department of Macromolecular Science, Graduate School of Science,
Toyonaka, Osaka 560-0043, Japan
3
Department of Life Sciences, Graduate School of Arts and Sciences,
University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
E-mail: gkurisu@protein.osaka-u.ac.jp
Dyneins are large microtubule-based motor complexes that power a range of
cellular processes. The motor domain is located in the dynein heavy chain and
consists of an N-terminal linker of mechanical element, a central ring of six
AAA+ modules four of which bind or hydrolyze ATP, and a long stalk
extending from the AAA+ ring with a microtubule-binding domain (MTBD)
at its tip. A crucial mechanism underlying the motile activity of cytoskeletal
motor proteins is precise coupling between the ATPase and track binding
activities. In dynein, a stalk region, made up of a long (~15 nm) antiparallel
coiled-coil, separates these two activities, which must facilitate
communication between them. Previous functional studies have provided
evidence that this communication is mediated by a small degree of helix
sliding in the coiled-coil [1]. However, no high-resolution structure is
201
available of the entire stalk region including the MTBD. We have solved the
structure of the entire stalk region of mouse cytoplasmic dynein in a weak
microtubule-binding state by X-ray crystallography and compared it with
related structures i.e., dynein motor domain from Dictyostelium discoideum in
a strong microtubule-binding state [2] and mouse MTBD whose distal portion
of the coiled-coil is fused with seryl tRNA synthetase from Thermus
thermophiles [3]. Our results strongly support the helix-sliding model based
on the complete structure of the dynein stalk with a different form of coiledcoil packing. We also propose a plausible mechanism of helix sliding,
together with further analysis by molecular dynamic simulations.
the physiological effects of MSG using Drosophila melanogaster. The
eclosion and pupation rate was not changed when less than 200mM MGS was
treated, but flies were not eclosed when 500mM MSG was treated. MSG
treatment on adult flies decreased life span by almost tested concentrations
except 500mM MSG. In addition, MSG decreased motility of male flies.
When the flies fed MSG from the larval stage were switched to feed normal
food from adult stage, the lifespan is reduced. Interestingly, the flies fed MSG
from the larval to adult stage increased the lifespan compared to flies fed
MSG in larval or adult stage only. In conclusion, MSG may affect the
physiology of Drosophila in high concentrations.
References
Kon, T., Imamula, K., Roberts, A. J., Ohkura, R., Knight, P. J., Gibbons, I. R.,
Burgess, S. A. and Sutoh, K., ―Helix sliding in the stalk coiled coil of dynein
couples ATPase and microtubule binding‖, Nature Struct. Mol. Biol., Vol. 16,
No. 3, (2009), pp 325-333.
Kon, T., Oyama, T., Shimo-Kon, R., Imamula, K., Shima, T., Sutoh, K. and
Kurisu, G., ―The 2.8 Å crystal structure of the dynein motor domain‖, Nature,
Vol. 484, (2012), pp 345-350.
Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C.,
Milligan, R. A., Vale, R. D. and Gibbons, I. R., ―Structure and functional role
of dynein‘s microtubule-binding domain‖, Science, Vol. 322, (2008), pp 16911695.
References
[1] Olney, John W. "Brain lesions, obesity, and other disturbances in mice
treated with monosodium glutamate." Science 164.3880 (1969): 719-721.
[2] Zanda, G., et al. "A double blind study on the effects of monosodium
glutamate in man." Biomedicine/[publiée pour l'AAICIG] 19.5 (1973): 202.
[3] Chung, Y. J., and H. J. Hong. ―A study of the effect of monosodium
glutamate on the deveolpment of Drosophila melanogaster.‖ Kor. J. Zool.,
16(1973): 127-137
P146
Parental effects of protein restriction on offspring fitness in Drosophila
melanogaster
Moon-Sik Yang
P148
Production and Characterization of Human Pepsin II in Transgenic Plant
Cell Suspension Culture
E-mail: mskyang@jbnu.ac.kr
Bora Lee, Eun-Ji Lee and Kyung-Jin Min
E-mail: violetoy26@gmail.com
Human progastriscin (hPGC), which acts as a proteolytic enzyme of gastric
secretion, was produced and secreted from transgenic plant suspension cell
culture. The synthetic hPGC gene was synthesized on the basis of riceoptimized codon usage and was cloned into binary vector under the control of
the sucrose starvation-inducible rice α-amylase 3D (Ramy 3D) promoter and
3‘UTR as a terminator. In addition, a Ramy 3D leader sequence was fused to
the N-terminal end and 6-His tag was added to the 3‘ end of the hPGC gene
for effective secretion and easy purification. The plant expression vector was
introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by
Agrobacterium tumefacience. The integration of the hPGC gene into the
chromosome of the transgenic rice callus was verified via genomic DNA PCR
amplification and hPGA expression in transgenic rice suspension cells was
confirmed via Northern blot analysis. Western blot analysis detected both pro
and mature proteins with masses from 40~35 kDa in the culture medium
following induction with sugar starvation. The plant-derived recombinant
hPGC was activated by autocatalysis and low pH condition and the resulting
mature pepsin. It could be bound to nickel affinity matrix, indicating the 6-His
tag was showed hemoglobin degradation activity.
Dietary restriction has been found to increase the longevity and stress
resistance in a wide array of eukaryotic species and it has been known that
protein, rather than carbohydrate restriction is important to increase the
lifespan in fruit flies. However, it has never been tested whether the effects of
parental protein restriction on offspring fitness. Adult flies were exposed to a
regular diet or protein restricted diet. We then measured the pupation/eclosion
frequency, longevity, and stress resistance in the offspring of these flies.
Compared to control flies, flies held on a protein-restricted diet were more
likely to develop from eggs to pupae, and from pupae into adults. Female
offspring of flies fed a restricted diet showed a consistent increase in
resistance to starvation and oxidative stress. The effect of parental diet on
male offspring was less consistent. Parental diet also seemed to affect the
longevity of offspring. Offspring of flies fed a protein-restricted diet tended to
outlive offspring of flies held on a regular diet, although results varied
somewhat among trials. This study suggests that the impact of diet restriction
can extend beyond exposed individuals to their offspring, and should be
considered in both theoretical and empirical studies of senescence.
P149
Production of Functional Human Matrix metalloproteinase-1 (hMMP1)
in Transgenic Plant Cell Suspension Culture
References
Martin B, Golden E, Carlson OD, Egan JM, Mattson MP, Maudsley S,
―Caloric restriction: impact upon pituitary function and reproduction‖, Ageing
research reviews, Vol. 7, (2008), pp 209-224.
Mousseau TA, Fox CW, ―The adaptive significance of maternal effects‖,
Trends in Ecology & Evolution, Vol. 13, (1998), pp 403-407.
Moon-Sik Yang and Nan-Sun Kim
E-mail: mskyang@jbnu.ac.kr
P147
Study on the physiological effects of L-glutamic acid (monosodium
glutamate, MSG) in Drosophila melanogaster.
Human matrix metalloproteinase-1 (MMP1), which is also termed as
collagenase I, is involved in the degradation of interstitial collagen and thus
thought to play a role in tumor development and metastasis and rheumatoid
arthritis. Here, we report that hMMP1 was expressed and secreted into culture
medium in a biologically active form under the control of the sucrose
starvation-inducible rice α-amylase 3D (Ramy 3D) promoter and 3‘UTR as a
terminator. The integration and expression of mRNA transcript of hMMP1
gene were verified via genomic DNA PCR and Northern blot analysis. Under
reducing conditions, the hMMP1 from Western blotting in the rice suspended
cell extracts were detected glycoprotein form at 55 kDa that comigrated with
the major band of the murine myeloma cell standard, also, contained hMMP1
fragments of lower molecular weight at 22~27kDa. However, medium from
the suspended cell cultures contained the 22- and 27 kDa fragments only. In
this study, we suggested that during fermentative production of a secreted
recombinant hMMP1 in transgenic rice suspension culture, the protein was
degraded by proteolytic activity. The secreted form, 22~25 kDa of hMMP1
was biologically active and can act as an activator for collagenase via
Woong Seo, Shin-Hae Lee, Bora Lee, Dea-Sub Song, Seok-Woo Go, TaeHyun Kim, Jun-Hyuk Moon, Kuk-Young Jang, Ah-Young Ham, Kyung-Jin
Min
E-mail: asvdo12@naver.com
L-glutamic acid (monosodium glutamate, MSG) is one of the famous artificial
seasonings and commonly exists in nature as sodium salt of glutamic acid.
There are many debates regarding the effects of MSG on human health. MSG
feeding in mice caused hypothalamus damage and vision impairment in some
trials, but no detectable detrimental changes in other trials. Moreover, there
has been not much information regarding the long term effects of MSG like
the effects of MSG on life span and development. In this study, we examined
202
resembling a type II' turn of -peptides, which can be used as a -turn motif
in -hairpins of Ala-based -peptides. The Ac6a dipeptide with homochiral
(1R,2S)-(1R,2S) configurations but different cyclohexyl puckerings shows the
capability to be incorporated into one of two -turn motifs of gramicidin S.
The overall structure of this gramicidin S analogue is quite similar to the
native gramicidin S with the same patterns and geometries of hydrogen bonds.
Our calculated results and the recently observed results may imply the wider
applicability of chirospecific -peptides with a cyclohexyl constraint on the
backbone to form various peptide foldamers.
collagenase activity.
P150
Computationally Designed -Turn Foldamers of -Peptides Based on 2Aminocyclohexylacetic Acid
In Kee Yoo and Young Kee Kang
Department of Chemistry and BK21 PLUS Research Team, Chungbuk
National University, Cheongju 361-763, Korea.
E-mail: ykkang@chungbuk.ac.kr
The -peptide -turn structures have been designed computationally by the
combination of chirospecific -residues of 2-aminocyclohexylacetic acid
(Ac6a) with a cyclohexyl constraint on the CC bond using density
functional methods in water. The chirospecific Ac6a dipeptide with the
(1S,2R)-(1R,2S) configurations forms a stable turn structure in water,
203
Guide to Local Activity
204
205
206
207
208
209
210
211
212
213
Author/Invited Speaker Index
(Nobel, KN: Key Note, PL: Plenary Lecture, S: Symposia, P: Poster)
A, B
Choi Jae-Wan
P77
Choi Sun
S6B
Adachi Masayuki
P20
Choi Tae Su
RSII
Albers Willem
P56
Choi Won Hoon
P64
Albers Willem M.
S3C
Chou Shan-Ho
S1A
Amit Goyal
P118
Choun Jaehee
P68
An Hyun Joo
S5D
Chuankhayan Phimonphan
P105
Ariesandi Winny
RSI
Chung Chang Geon
P49
Arisaka Fumio
P29
Chung Junho
S3B
Bae Sun Oh
P23
Chung Ka Young
S4D
Bang Jeong-Kyu
S1D
Chung Kyung Tae
P35
Bansal Varun
P2
Chung Taijoon
S2A
Bharat Madan
S4C
Churchill David
S1B
Bhattarai Kashi Raj
P52
Cong Yao
S1B
Boonserm Panadda
P3
Cui Minghua
P106
Buck Matthias
S1C
Czabotar Peter
S6D
Bui Chi Bao
RSII
D, F, G, H
C
Daughdrill Gary
S3D
Caaverio Jose M.M.
S2C
Du Eun Jo
P94
Cairns James Ketudat
S4A
Fu Xinmiao
RS I, P5, P11
Cha Jeong Seok
P92
Fukui Naoya
P37
Chae Han-Jung
S5A
Gao George Fu
PL
Chan Nei-Li
S1A
Han Dong Hoon
P76
Chang Tse Wen
S3B
Han Kyou-Hoon
S3D
Chen Chinpan
S2B
Han Seong-Gu
P99, P100
Chen Yong
S3A
Han Weiping
S5A
Chen Yun-Ru
P39
He Kai-Cyuan
P133
Cho Je Yeol
S5D
Heo Won Do
S3C
Cho Kevin
P25
Ho Minh Nguyen Anh
P88
Cho Kwang-Hyun
S6C
Hong Wanjin
PL
Cho Yunje
S1D
Hu Hong-Yu
S3D
Choe Chung Hyeon
P139
Hui li
P144
Choe Han Suk
S4D
Hur Jeong Hwan
P120
Choe Senyon
S2B
Hwang Daehee
S2D
Choi Hee-Jung
P47
Hwang Kwang Yeon
S1D
214
Hyun Jiyoung
P137
I, J,
Kim Ji Hun
P82
Kim Ji-Sun
P97
Kim Jong-Tae
P43, P128
Kim Joon
S2B
Ida Masataka
P41
Kim Sae-Hae
P108, P109
Ikenoue Tatsuya
P9
Kim SangYun
S1B
Imdad Saba
P40
Kim Seokhee
P30
Irene Deli
P130
Kim Seung Chan
P58
Jang Jeong-Hwa
P22
Kim Sunghoon
KL
Jangpromma Nisachon
P73
Kim Tae Hun
RS I
Jeng Wen-Yih
P45
Kim Tae Woo
P44
Jeon Tae-Joon
S6A
Kim Young Jun
P63
Jeon Young Ho
S1D
Kim Youngsoo
S2D
Jeong Ji-ho
P90
Kinjo Akira R.
P117
Jiang Yanxialei
P102
Ko In Young
P125
Jo Ku-Sung
P7
Kornberg Roger D.
Nobel
Jon Sangyong
S4B
Krause Kurt L.
S6D
Joo JaeYeon
P78
Kunthic Thittaya
P1
Ju Anna
P67
Kurisu Genji
P145
Ju Man Seok
P75
Kuwajima Kunihiro
S1C
Jun Jae Hyun
P86
Kwon Min Jee
P48
Jung Da-Hee
P113
Jung Sang Taek
S3B
L
Jung Young Mee
S6D
Lai Andrew
P60
Lee Bora
P146
Lee Chewook
P122
K
Kaibuchi Kozo
S3C
Lee Chul Won
P131
Kameda Hiroshi
P36
Lee Dongju
P127
Kamiya Narutoshi
P27
Lee Geum-Hwa
P54
Kang Ho Chul
S3A
Lee Ho-Soo
P65
Kang Hyo Jin
P116
Lee Hwa-Young
P55
Kang Linwoo
S1A
Lee Hye-Yeon
P104
Kang Lin-Woo
P87
Lee Ji Hye
P95
Kasahara Kota
P18
Lee Jin A
P107
Kato Koichi
S6B
Lee Jooyoung
S1C
Kim Beom-Tae
P143
Lee Kang Ju
P112
Kim Do-Hyoung
P115
Lee Keun Woo
S6C
Kim Dong-Kyun
P16
Lee Min Jung
P121
Kim Hajin
S2C
Lee Sanghyeob
P89
Kim Hugh
S3D
Lee Seung-Goo
S4C
Kim Jeong-Sun
S1A
Lee SeungJae
S1B
215
Lee Shin Jung C.
P38
Ota Motonori
S6C
Lee Si-Hyung
P114
Paek Eunok
S2D
Lee Su Youn
P15
Pai Jaeyoung
P138
Lee Woo Sirl
P136
Park Hae Sook
P85
Lee Young-Ho
P21
Park CheolWan
P91
Lei Lu
S5A
Park Chiyoul
P74
Liang Chu-Ting
P132
Park Jong il
P32, P34
Liang Po-Huang
S6B
Park Sookil
P142
Liang Yi
S6D
Park Sung-Jean
S5B
Lim Carmay
S3D
Park Yong-Yea
RS I
Lim Hyun-Suk
S4A
Rahman Raja Noor
P84
Lim Ji-Hong
S3A
Ren Ruibao
S4B
Lin Yuxi
RS I
Roy Rahul
S3C
Liu Chia-I
P46
Ruangjaroon Khanit
RSII
Liu Cong
S1B
Liu Jinsong
S5A
S
Liu Wei
S4D
Sakoh-Nakatogawa Machiko
S2A
Salleh Abu Bakar
S4C
Sato Mamoru
S3D
M, N
Ma Xiaomin
P12
Satoh Tadashi
S3A
Maenaka Katsumi
S4C
Satoh Takanori
P26
Mio Kazuhiro
P93
Seo Huiyun
P80
Moon Heejo
P110
Seo Pilwon
P71
Muta Hiroya
P14
Seo Woong
P147
Na Jung-Hyun
P98
Seong Jae Young
S4D
Nagano Nozomi
S6C
Shao Feng
S2B
Nakagawa Atsushi
S6A
Shen Yu-Wen
P134
Nakamura Haruki
PL
Shimizu Toshiyuki
S5B
Ngozi Ezemaduka Anastasia
P33
Shin Seung Kyun
P51
No Young Hyun
P83
Shin Injae
S4A
Noda Nobuo N.
S2A
Shin Yeon-Kyun
S6A
Norton Raymond S.
S6B
Sim Jun
P28
Nguyen Duc Minh
P13
Siritapetawee Jaruwan
P31
Nguyen Duc Tien
P69
Siswoyo Tri Agus
P101
Nguyen Thi Kim Oanh
P66
Siyu Guan
S4C
So Keum-Kang
P135
So Masatomo
P19
O, P, R
Oh Changsuk
P119
Song Byeong Doo
S4B
Oh Han Bin
P59
Song Hyun Kyu
S2A
Oh Na-Hyun
P79
Songyang Zhou
S4B
Okamoto Koji
S2A
Su Jiulong
P123
216
Sugase Kenji
S2C
Wu Ming-Chya
P8
Sugiharto Bambang
P72
Xie Xin
S5B
Surya Wahyu
P96
Xie Yuan
P81
Xu Hao
P42
T, W
Takahashi Satoshi
P10
Y, Z
Takano Yu
P129
Yadav Raj-Kumar
P53
Tankrathok Anupong
P6
Yamamoto Masaki
S6A
Terakawa Mayu S.
RS I
Yang Haesik
S5C
To Janet
P111
Yang Moonsik
P140, P141
Torres James
S6D
Yang Moonsik
P148, P149
Tran Thanh Tuyen
P70
Yokokawa Ryuji
S5C
Tsai Ming-Daw
PL
Yoo In Kee
P150
Tseng Fan-Gang
S5C
Yoo Jong Shin
S5D
Tseng Ning-Hsuan
P103
Yoo Young Suk
P50
Tsumoto Kouhei
S3B
Yoon Tae-Young
S2C
Udgaonkar Jayant B.
S1C
Yoshimura Yuichi
RSII
Wang Andrew H.-J.
S4A
Yoshimura Yuichi
P57
Wang Hong-Wei
S2C
Yu Chen
RSII
Wang Jun
P24
Yu Myeong-Hee
S2D
Wang Qian
S6B
Yun Ji-Hye
P126
Wang Rui
S1A
Yun YoungJoo
P17
Wang Wei
S1C
Zhang Li
RSII
Won Hyung-Sik
S6D
Zhu Ya
P124
Woo Hye-Min
P4
Wu Beili
S5B
217
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APPA2014 Jeju

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