分子認識による酸化的フォールディングの1分子観察

• Project Area: Novel measurement techniques for visualizing 'live' protein molecules at work
• Project/Area Number: 15H01626
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: Tohoku University
• Principal Investigator: 奥村 正樹 東北大学, 多元物質科学研究所, 助教 (50635810)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000); Fiscal Year 2016: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000); Fiscal Year 2015: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
• Keywords: 酸化的フォールディング / 高速AFM / 溶液散乱 / PDIファミリー / ジスルフィド結合 / PDI / フォールディング / 分子認識 / folding

Outline of Annual Research Achievements

PDIによる酸化的フォールディング触媒反応の可視化について、タンパク質科学会において若手奨励賞や新生鎖の国際会議でエクセレントポスター賞として高く評価され、国際一流紙に現在投稿準備中である。
またPDI酸化酵素Ero1のCys208-Cys241ジスルフィド結合を開裂させた変異体が、野生型よりも高いPDI酸化活性を示すことを明らかにし、生化学的データとあわせ、Cys208-Cys241ジスルフィド結合が構造安定でなく機能調節に関与するジスルフィド結合であると結論付け、Redox Biology誌とJ Biol Chem誌に論文として報告した。
次に、小胞体内に蓄積するミスフォールド基質のジスルフィド結合を切断するERdj5の1分子動態の観点から作用機序の全容を明らかにするため、これまでの我々が決定しているERdj5の結晶構造(Hagiwara, et al. Mol. Cell 2011)以外に、新たにN-terminal clusterとC-terminal cluster間のチオレドキシンドメインの配向の違う結晶構造を決定するとともに、高速AFMによってERdj5のN-terminal clusterとC-terminal cluster間の可動性の観察に成功した。本成果は、Structure誌に採択された。

Research Progress Status

28年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

28年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] バーゼル大学(スイス)
• [Int'l Joint Research] Max Planck Institute of Biochemistry(ドイツ)
• [Journal Article] Preparation of Selenoinsulin as a Long-Lasting Insulin Analog (2017)
  • Author(s): K. Arai#, T. Takei#, M. Okumura#, S. Watanabe#, Y. Amagai, Y. Asahina, H. Hojo*, K. Inaba*, M. Iwaoka*. (#These authors contributed equally to this work.)
  • Journal Title: Angewandte Chemie., Volume: in press
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] The highly dynamic nature of ERdj5 is key to efficient ERAD of aberrant protein oligomers (2017)
  • Author(s): K. Maegawa, S. Watanabe#, K. Noi#, M. Okumura#, Y. Amagai#, M. Inoue, R. Ushioda, K. Nagata, T. Ogura, and K. Inaba*. (#These authors contributed equally to this work.)
  • Journal Title: Structure Volume: in press
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Human Ero1α undergoes dual regulation through complementary redox interactions with PDI (2016)
  • Author(s): S. Kanemura, M. Okumura, K. Yutani, T. Ramming, T. Hikima, C. Appenzeller-Herzog, S. Akiyama and K. Inaba*.
  • Journal Title: J. Biol. Chem. Volume: 291 Issue: 46 Pages: 23952-23964
  • DOI: 10.1074/jbc.m116.735662
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Redox-assisted regulation of Ca2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5 (2016)
  • Author(s): R. Ushioda, A. Miyamotod, M. Inoue, S. Watanabe, M. Okumurae, K. Maegawa, K. Uegaki, S. Fujii, Y. Fukuda, M. Umitsu, J. Takagi, K. Inaba, K. Mikoshibad, and K. Nagata
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 113(41) Issue: 41 Pages: 6055-6063
  • DOI: 10.1073/pnas.1605818113
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Cysteines 208 and 241 in Ero1 are required for maximal catalytic turnover (2015)
  • Author(s): T. Ramming, S. Kanemura, M. Okumura, K. Inaba and C. Appenzeller-Herzog.
  • Journal Title: Redox Biology Volume: 7 Pages: 14-20
  • DOI: 10.1016/j.redox.2015.11.004
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Structural Stability of Amyloid Fibrils Depends on the Existence of the Peripheral Sequence near the Core Cross-03B2 Region (2015)
  • Author(s): M. Saiki, K. Shiba, and M. Okumura.
  • Journal Title: FEBS Lett. Volume: 589 Issue: 23 Pages: 3541-3547
  • DOI: 10.1016/j.febslet.2015.10.015
  • Peer Reviewed / Open Access
• [Journal Article] A PDI-catalyzed thiol/disulfide switch regulates the production of hydrogen peroxide by human Ero1 (2015)
  • Author(s): T. Ramming, M. Okumura, S. Kanemura, S. Baday, J. Birk, S. Moes, P. Jenö, S. Bernèche, K. Inaba and C. Appenzeller-Herzog.
  • Journal Title: Free Radic Biol Med. Volume: 83 Pages: 361-372
  • DOI: 10.1016/j.freeradbiomed.2015.02.011
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] One-dimensional sliding of p53 along DNA is accelerated in the presence of Ca(2+) or Mg(2+) at millimolar concentrations (2015)
  • Author(s): Murata A, Ito Y, Kashima R, Kanbayashi S, Nanatani K, Igarashi C, Okumura M, Inaba K, Tokino T, Takahashi S, Kamagata K
  • Journal Title: JOURNAL OF MOLECULAR BIOLOGY Volume: 427 Issue: 16 Pages: 2663-2678
  • DOI: 10.1016/j.jmb.2015.06.016
  • Peer Reviewed / Open Access
• [Journal Article] Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum. (2015)
  • Author(s): Okumura M, Kadokura H, Inaba K.
  • Journal Title: Free Radic Biol Med. Volume: 83 Pages: 314-322
  • DOI: 10.1016/j.freeradbiomed.2015.02.010
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Dynamic assembly/disassembly of Protein Disulfide Isomerase during guiding oxidative protein folding (2017)
  • Author(s): M. Okumura, K. Noi, S. Kanemura, S. Masui, T. Hikima, S. Akiyama, T. Ogura, and K. Inaba.
  • Organizer: Grand Challenges in Small-angle Scattering
  • Place of Presentation: 岡崎カンファレンスセンター(愛知県、岡崎市)
  • Year and Date: 2017-03-18
  • Int'l Joint Research
• [Presentation] 高速AFMが明らかにしたPDI酵素による基質の酸化的フォールディング触媒機構の解明 (2017)
  • Author(s): 奥村 正樹
  • Organizer: 熊本大学発生研セミナー
  • Place of Presentation: 熊本大学発生医学研究所(熊本県、熊本市)
  • Year and Date: 2017-01-17
  • Invited
• [Presentation] Dynamic assembly/disassembly of Protein Disulfide Isomerase during the catalysis of oxidative protein folding. (2016)
  • Author(s): M. Okumura, K. Noi, S. Kanemura, S. Masui, T. Hikima, S. Akiyama, T. Ogura, and K. Inaba.
  • Organizer: EMBO Meeting
  • Place of Presentation: Girona, Spain
  • Year and Date: 2016-10-23
  • Int'l Joint Research
• [Presentation] Dual regulation of human Ero1 via redox interplays with PDI. (2016)
  • Author(s): S. Kanemura, M. Okumura, K. Yutani, T. Ramming, C. Appenzeller-Herzog, S. Akiyama, K. Inaba.
  • Organizer: EMBO Meeting
  • Place of Presentation: Girona, Spain
  • Year and Date: 2016-10-23
  • Int'l Joint Research
• [Presentation] Dynamic assembly/disassembly of Protein Disulfide Isomerase during the catalysis of oxidative protein folding. (2016)
  • Author(s): M. Okumura, K. Noi, S. Kanemura, T. Hikima, S. Akiyama, T. Ogura, K. Inaba.
  • Organizer: Nascent Chain Biology 2016
  • Place of Presentation: 富士レークホテル(山梨県、南都留郡)
  • Year and Date: 2016-09-01
  • Int'l Joint Research
• [Presentation] Human Ero1 undergoes dual regulation via redox interplays with PDI. (2016)
  • Author(s): S. Kanemura, M. Okumura, K. Yutani, T. Ramming, C. Appenzeller-Herzog, S. Akiyama, K. Inaba.
  • Organizer: Nascent Chain Biology 2016
  • Place of Presentation: 富士レークホテル(山梨県、南都留郡)
  • Year and Date: 2016-09-01
  • Int'l Joint Research
• [Presentation] 酸化的フォールディングを支える PDI ファミリー酵素の新規触媒機構の解明 (2016)
  • Author(s): 奥村 正樹
  • Organizer: 蛋白質科学会若手奨励賞受賞講演
  • Place of Presentation: 福岡国際会議場(福岡県、福岡市)
  • Year and Date: 2016-06-08
  • Invited
• [Presentation] 高速AFMが明らかにしたPDIによる基質の酸化的フォールディング触媒機構の解明 (2015)
  • Author(s): 奥村 正樹、野井 健太郎、金村 進吾、秋山 修志、小椋 光、稲葉 謙次
  • Organizer: 第38回 分子生物学会年会
  • Place of Presentation: 神戸国際会議場(神戸市)
  • Year and Date: 2015-12-04
• [Presentation] 小胞体におけるタンパク質の品質管理；酵素による基質触媒反応を活写する (2015)
  • Author(s): 奥村 正樹
  • Organizer: 関西学院大学、理工学部講演会
  • Place of Presentation: 関西学院大学(三田市)
  • Year and Date: 2015-11-30
  • Invited
• [Presentation] 小胞体におけるタンパク質の品質管理 (2015)
  • Author(s): 奥村 正樹
  • Organizer: 東海大学
  • Place of Presentation: 東海大学湘南キャンパス(平塚市)
  • Year and Date: 2015-11-17
  • Invited
• [Presentation] Real-time monitoring of PDI-catalyzed oxidative protein folding by high-speed atomic force microscopy (2015)
  • Author(s): 奥村 正樹
  • Organizer: 第53回 生物物理学会年会
  • Place of Presentation: 金沢大学(金沢市)
  • Year and Date: 2015-09-14
  • Invited
• [Presentation] Real-time monitoring of PDI-catalyzed oxidative protein folding by high-speed atomic force microscopy. (2015)
  • Author(s): M. Okumura, K. Noi, S. Kanemura, S. Masui, T. Hikima, S. Akiyama, T. Ogura, and K. Inaba.
  • Organizer: ER&Redox Club Meeting
  • Place of Presentation: Venice, Italy.
  • Year and Date: 2015-04-14
  • Int'l Joint Research
• [Book] ERp46とPDIの異なる構造と機能的役割 (2015)
  • Author(s): 奥村 正樹、金村 進吾、稲葉 謙次
  • Total Pages: 3
  • Publisher: 生物物理