Dynamics and protein folding mechanism of prefoldin and group II chaperonin system

• Project Area: Dynamical ordering of biomolecular systems for creation of integrated functions
• Project/Area Number: 16H00753
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Tokyo University of Agriculture and Technology
• Principal Investigator: 養王田 正文 東京農工大学, 工学(系)研究科(研究院), 教授 (50250105)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2017: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2016: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
• Keywords: シャペロン / シャペロニン / フォールディング / 結晶化 / 相互作用 / プレフォルディン / ダイナミクス

Outline of Annual Research Achievements

好熱性真菌Chaetomium thermophilum由来プレフォルディン(CtPFD)の機能・構造解析、及びC. thermophilum由来グループ２型シャペロニン(CtCCT)との協調作用を解析した。CtPFDが豚脳由来チューブリンと結合することをPull Down Assayで解析した。CtPFDの結晶化スクリーニングを行い、結晶を得ることに成功した。得られた結晶にX線を照射したが、分解能が低く詳細な構造の解明には至らなかった。古細菌由来PFDの結晶構造において末端領域に存在するDisorder領域の構造が明らかとなっていないことから、Disorder領域が分解能低下の原因である可能性が予想された。そこで、長い末端を有するアルファ型サブユニットである PFD3の両末端を28残基、PFD5のN末端20残基及びC末端21残基削った変異体を構築した。この変異体の機能及び結晶構造解析を継続して進めている。CtPFDとCtCCTの相互作用をBiacoreを用いた表面プラスモン共鳴により解析した。獲得したセンサーグラムに対しカーブフィッティング行い、結合・解離速度定数及び解離定数を決定した。解離定数は先行研究より報告されている超好熱性古細菌Pyrococcus horikoshii OT3由来PFDとCPNのものとおよそ一致していた。また、高速AFM及び電子顕微鏡によりCtCCTとCtPFDの相互作用を解析し、CtCCTにCtPFDが結合している様子が観察された。また、アクチンがCtPFDからCtCCTに受け渡されることを実験的に証明した。再構成型大腸菌無細胞タンパク質合成系（Pure System）を用い、CtPFDとCtCCTの種々の真核生物由来細胞質タンパク質のフォールディングへの効果を解析した。

Research Progress Status

29年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

29年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Prefoldin, a jellyfish-like molecular chaperone: functional cooperation with a group II chaperonin and beyond (2018)
  • Author(s): Muhamad Sahlan, Tamotsu Zako, Masafumi Yohda
  • Journal Title: Biophysical Reviews Volume: 10 Issue: 2 Pages: 339-345
  • DOI: 10.1007/s12551-018-0400-0
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Time-Resolved Measurement of the ATP-Dependent Motion of the Group II Chaperonin by Diffracted Electron Tracking (2018)
  • Author(s): Naoki Ogawa, Yohei Y. Yamamoto, Keisuke Abe, Hiroshi Sekiguchi, Yuji Y. Sasaki, Akira Ishikawa, Judith Frydman, Masafumi Yohda
  • Journal Title: International Journal of Molecular Sciences Volume: 19 Issue: 4 Pages: 950-950
  • DOI: 10.3390/ijms19040950
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Modification of the response of olfactory receptors to acetophenone by CYP1a2 (2017)
  • Author(s): Masashi Asakawa, Yosuke Fukutani, Aulaphan Savangsuksa, Keiich Noguchi, Hiroaki Matsunami, Masafumi Yohda
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 10167-10167
  • DOI: 10.1038/s41598-017-10862-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC (2017)
  • Author(s): Yohei Y. YAMAMOTO, Yuko UNO, Eiryo SHA, Kentaro IKEGAMI, Noriyuki ISHII, Naoshi DOHMAE, Hiroshi SEKIGUCHI, Yuji C. SASAKI, Masafumi YOHDA
  • Journal Title: PLOS ONE Volume: 12(5) Issue: 5 Pages: 0176054-0176054
  • DOI: 10.1371/journal.pone.0176054
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Successful PEGylation of hollow encapsulin nanoparticles from Rhodococcus erythropolis N771 without affecting their disassembly and reassembly properties (2017)
  • Author(s): Seiichi Sonotaki, Taku Takami, Keiichi Noguchi, Masafumi Odaka, Masafumi Yohda. Yoshihiko Murakami
  • Journal Title: Biomaterials Science Volume: 5 Issue: 6 Pages: 1082-1089
  • DOI: 10.1039/c7bm00207f
  • Peer Reviewed
• [Journal Article] Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy (2017)
  • Author(s): M. M. Islam, M. Yohda, S. I. Kidokoro, Y. Kuroda
  • Journal Title: Sci. Rep. Volume: ７ Issue: 1 Pages: 41205-41205
  • DOI: 10.1038/srep41205
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Olfactory receptor accessory proteins play crucial roles in receptor function and gene choice (2017)
  • Author(s): R. Sharma, Y. Ishimaru, I. Davison, K. Ikegami, M. S. Chien, H. You, Q. Chi, M. Kubota, M. Yohda, M. Ehlers, H. Matsunami
  • Journal Title: Elife Volume: 印刷中
  • DOI: 10.7554/elife.21895
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Split luciferase complementation assay for the analysis of G protein-coupled receptor ligand response in Saccharomyces cerevisiae (2017)
  • Author(s): Y. Fukutani, J. Ishii, A. Kondo, T. Ozawa, H. Matsunami, M. Yohda
  • Journal Title: Biotechnol. Bioeng. Volume: 114 Issue: 6 Pages: 1354-1361
  • DOI: 10.1002/bit.26255
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A method of expression for an oxygen-tolerant group III alcohol dehydrogenase from Pyrococcus horikoshii OT3 (2017)
  • Author(s): C. Sugimoto, K. Takeda, Y. Kariya, H. Matsumura, M. Yohda, H. Ohno, N. Nakamura
  • Journal Title: J. Biol. Inorg. Chem. Volume: 482 Issue: 4 Pages: 700-705
  • DOI: 10.1007/s00775-017-1439-2
  • Peer Reviewed
• [Journal Article] Expression and characterization of the Plasmodium translocon of the exported proteins component EXP2 (2016)
  • Author(s): K. Hakamada, H. Watanabe, R. Kawano, K. Noguchi, M. Yohda
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 482 Issue: 4 Pages: 751-764
  • DOI: 10.1016/j.bbrc.2016.11.097
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Characterization of group II chaperonins from an acidothermophilic archaeon Picrophilus torridus (2016)
  • Author(s): Y. Y. Yamamoto, K. Tsuchida, K. Noguchi, N. Ogawa, H. Sekiguchi, Y. C. Sasaki, M. Yohda
  • Journal Title: FEBS Open Bio. Volume: 6 Issue: 7 Pages: 751-764
  • DOI: 10.1002/2211-5463.12090
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074 (2016)
  • Author(s): F. Watanabe, F. Yu, A. Ohtaki, Y. Yamanaka, K. Noguchi, M. Odaka, M. Yohda
  • Journal Title: J. Biosci. Bioeng. Volume: 122 Issue: 3 Pages: 270-275
  • DOI: 10.1016/j.jbiosc.2016.02.003
  • NAID: 40020949768
  • Peer Reviewed
• [Journal Article] Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus, (2016)
  • Author(s): T. Aihara, T. Ito, Y. Yamanaka, K. Noguchi, M. Odaka, M. Sekine, H. Homma, M. Yohda
  • Journal Title: Extremophiles Volume: 20 Issue: 4 Pages: 385-393
  • DOI: 10.1007/s00792-016-0829-7
• [Journal Article] Contribution of the C-Terminal Region of a Group II Chaperonin to its Interaction with Prefoldin and Substrate Transfer. (2016)
  • Author(s): Zako T, Sahlan M, Fujii S, Yamamoto YY, Tai PT, Sakai K, Maeda M, Yohda M.
  • Journal Title: J Mol Biol. Volume: 印刷中 Issue: 11 Pages: 2405-2417
  • DOI: 10.1016/j.jmb.2016.04.006
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study (2016)
  • Author(s): M. Kayanuma, M. Shoji, M. Yohda, M. Odaka, Y. Shigeta
  • Journal Title: The Journal of Physical Chemistry B Volume: 123(13) Issue: 13 Pages: 3259-3266
  • DOI: 10.1021/acs.jpcb.5b11363
  • Peer Reviewed / Open Access
• [Presentation] Molecular Characterization and protein folding mechanism of the prefoldin-CCT system (2018)
  • Author(s): Yohei Y.Yamamoto、Yuko Uno、Kento Morita、Tomohito Obata、Ayaka Fukui、Masafumi Yohda
  • Organizer: The 6th International Symposium on Dynamical Ordering of Biomolecular Systems for Creation of Integrated Functions
  • Int'l Joint Research
• [Presentation] Protein folding mechanism of group II chaperonin (2017)
  • Author(s): Masafumi Yohda
  • Organizer: 第44回生体分子科学討論会
  • Invited
• [Presentation] Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC (2017)
  • Author(s): Masafumi Yohda、Yohei Y.Yamamoto、Yuko Uno
  • Organizer: The 5th Asia Pacific Protein Association Conference
  • Int'l Joint Research
• [Presentation] Molecular characterization and protein folding mechanism of the prefoldin-CCT system (2017)
  • Author(s): Masafumi Yohda、Yohei Y.Yamamoto、Yuko Uno、Kento Morita
  • Organizer: Thermophiles 2017 ( 14th International Meeting on Thermophile Biology)
  • Int'l Joint Research
• [Presentation] 好熱性真菌Chaetomium thermophilum由来プレフォルディンの機能構造解析 (2017)
  • Author(s): 森田健斗、堀綾香、山本陽平、宇野裕子、野井健太郎、小椋光、Jose M. Valpuesta、野口恵一、養王田正文
  • Organizer: 第12回臨床ストレス応答学会大会
• [Presentation] 真核生物プレフォルディン-CCTシステムの基質特異性解析 (2017)
  • Author(s): 福井彩花、小畑智弘、森田健斗、宇野裕子、山本陽平、丹羽達也、田口英樹、養王田正文
  • Organizer: 第12回臨床ストレス応答学会大会
• [Presentation] 真核生物プレフォルディン-CCTシステムの基質特異性の解析 (2017)
  • Author(s): 小畑智弘、福井彩花、森田健斗、宇野裕子、山本陽平、丹羽達也、田口英樹、養王田正文
  • Organizer: 2017年度 生命化学系学会合同年次大会
• [Presentation] 好熱性真菌由来の脱凝集因子 Hsp104の機能構造解析 (2017)
  • Author(s): 篠原恭介、井上耀介、花園祐矢、野井健太郎、川本晃大、竹田一旗、岩政菜津記、野口恵一、難波啓一、小椋光、三木邦夫、養王田正文
  • Organizer: 2017年度 生命化学系学会合同年次大会
• [Presentation] 好熱性真菌 Chaetomium thermophilum 由来プレフォルディンの機能構造解析 (2017)
  • Author(s): 森田健斗、堀綾香、山本陽平、宇野裕子、野井健太郎、小椋光、Jose M. Valpuesta、野口恵一、養王田正文
  • Organizer: 2017年度 生命化学系学会合同年次大会
• [Presentation] 好熱性真菌Chaetomium thermophilum由来プレフォルディンの機能構造解析 (2016)
  • Author(s): 養王田 正文、森田 健斗、山本 陽平、宇野 祐子、野口 恵一
  • Organizer: 第11回臨床ストレス応答学会
  • Place of Presentation: 山口大学医学部霜仁会館、宇部
  • Year and Date: 2016-11-12
• [Presentation] 真核生物型プレフォルデイン－グループII型シャペロニンシステムのタンパク質フォールディング機能 (2016)
  • Author(s): 養王田正文、山本陽平、宇野裕子、森田健斗、野口恵一
  • Organizer: 第89回 日本生化学会大会
  • Place of Presentation: 仙台国際センター、仙台
  • Year and Date: 2016-09-26
• [Presentation] Protein Folding Mechanism of Prefoldin - Group II Chaperonin System Revealed by the Study on That from Hyperthermophilic Archaea (2016)
  • Author(s): Masafumi Yohda, Kohei Y.Yamamoto, Tamotsu Zako
  • Organizer: Extremophiles2016 －11th International Congress in Extremophiles－
  • Place of Presentation: Kyoto University Clock Tower Centennial Hall, Kyoto
  • Year and Date: 2016-09-16
  • Int'l Joint Research
• [Presentation] Functional Expression of Olfacｔory Receptors in Yeast for The Development of Odorant Sensor (2016)
  • Author(s): Masafumi Yohda, Yosuke Fukutani
  • Organizer: INTERNATIONAL SYMPOSIUM ON BIOMEDICAL ENGINEERING
  • Place of Presentation: The Margo Hotel, Depok Indonesia
  • Year and Date: 2016-05-30
  • Int'l Joint Research / Invited
• [Presentation] EXPRESSION, PURIFICATION, CHARACTERIZATION AND CRYSTALLIZATION OF THE MOLECULAR CHAPERONE PREFOLDIN FROM CHAETOMIUM THEMOPHILUM (2016)
  • Author(s): Masafumi Yohda, Kento Morita, Yohei Y Yamamoto, Ayaka Hori, Yuko Uno, Keiichi Noguchi
  • Organizer: PROTEIN HOMEOSTASIS IN HEALTH & DISEASE
  • Place of Presentation: Cold Spring Harbor Laboratory, USA
  • Year and Date: 2016-04-20
  • Int'l Joint Research
• [Presentation] EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE GROUP II CHAPERONIN CCT FROM CHAETOMIUM THEMOPHILUM (2016)
  • Author(s): Yohei Y Yamamoto、Eiryo Sha、Yuko Uno、Kentaro Ikegami、Keiichi Noguchi, Naoshi Dohmae, Hiroshi Sekiguchi, Yuji C Sasaki, Masafumi Yohda
  • Organizer: PROTEIN HOMEOSTASIS IN HEALTH & DISEASE
  • Place of Presentation: Cold Spring Harbor Laboratory, USA
  • Year and Date: 2016-04-19
  • Int'l Joint Research