Elucidation of mechanisms underlying disulfide bond formation in nascent chains

2018 Fiscal Year Annual Research Report

• Project Area: Nascent-chain Biology
• Project/Area Number: 26116005
• Research Institution: Tohoku University
• Principal Investigator: 稲葉 謙次 東北大学, 多元物質科学研究所, 教授 (10423039)
• Co-Investigator(Kenkyū-buntansha): 門倉 広 東北大学, 多元物質科学研究所, 准教授 (70224558)
• Project Period (FY): 2014-07-10 – 2019-03-31
• Keywords: タンパク質品質管理 / ジスルフィド結合 / X線結晶構造解析 / 高速原子間力顕微鏡

Outline of Annual Research Achievements

LDL受容体のN末端に存在するR1ドメインに対する立体構造特異的抗体と、ジスルフィド結合形成モニタリング系を使った解析を進めた。その結果、R1の下流のβプロペラ領域の約半分が翻訳された時に、R1中の非天然型ジスルフィド結合が天然型に組換えられることを見出した。ジスルフィドの組換え反応は、多くの細胞表層タンパク質の立体構造形成において鍵となるステップであるが、従来の考えに反して翻訳と共役し精緻な制御のもと進行することが初めて判明した(Proc. Natl. Acad. Sci. USA誌,印刷中)。更に、PDIファミリー酵素PDIpは膵臓の外分泌細胞で特異的に発現し、様々な消化酵素の前駆体と相互作用すること、および、PDIpには新合成された消化酵素の前駆体が凝集体を形成するのを抑制し、その正しい折り畳み過程を促進する作用があることを突き止めてJ. Biol. Chem.誌に報告した。
ヒト由来PDIがアンフォールド基質BPTIの酸化的フォールディングを触媒する機構を解明するため、高速原子間力顕微鏡を用いて一分子観察を行った。その結果、PDIが基質依存的に二量体を形成し、その結果生じる二量体の中央キャビティに基質が取り込まれる様子が観察された。BPTIより酸化的フォールディング速度が遅いRNaseAを基質として用いた場合、PDI二量体形成の寿命が顕著に長くなり、この二量体形成が基質の酸化的フォールディング触媒と密接に関係することが示唆された。本成果は、Nat. Chem. Biol.誌に受理された。

Research Progress Status

平成30年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

平成30年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] San Raffaele Scientific Institute(イタリア)
  • Country Name: ITALY
  • Counterpart Institution: San Raffaele Scientific Institute
• [Int'l Joint Research] University of Utah(米国)
  • Country Name: U.S.A.
  • Counterpart Institution: University of Utah
• [Int'l Joint Research] Uniiversity of Copenhagen(デンマーク)
  • Country Name: DENMARK
  • Counterpart Institution: Uniiversity of Copenhagen
• [Journal Article] Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells (2020)
  • Author(s): Kadokura, H.*, Dazai, Y., Fukuda, Y., Hirai, N., Nakamura, O., Inaba, K.
  • Journal Title: Proc. Natl. Acad. Sci. USA（印刷中） Volume: － Pages: －
  • Peer Reviewed
• [Journal Article] Methods to identify the substrates of thiol‐disulfide oxidoreductases (2019)
  • Author(s): Fujimoto, T., Inaba K., Kadokura H.*
  • Journal Title: Protein Science Volume: 28 Pages: 30-40
  • DOI: 10.1002/pro.3530
  • Peer Reviewed / Open Access
• [Journal Article] Coupling effects of thiol and urea-type groups for promotion of oxidative protein folding (2019)
  • Author(s): Okada, S., Matsusaki, M., Araki, K., Hidaka, Y., Inaba, K., Okumura, M.*, and Muraoka, T.
  • Journal Title: Chem. Commun. Volume: 55 Pages: 759-762
  • DOI: 10.1039/c8cc08657e
  • Peer Reviewed
• [Journal Article] Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding (2019)
  • Author(s): Okumura, M., Noi, K., Kanemura, M., Kinoshita, M., Saio, T., Inoue, Y., Hikima, T., Akiyama, S., Ogura, T. and Inaba, K.
  • Journal Title: Nat. Chem. Biol. Volume: 15 Pages: 499-509
  • DOI: 10.1038/s41589-019-0268-8
  • Peer Reviewed
• [Journal Article] Zinc regulates ERp44-dependent protein quality control in the early secretory pathway (2019)
  • Author(s): Watanabe S., Amagai Y., Sannino S., Tempio T., Anelli T., Harayama M., Masui S., Sorrentino I., Yamada M., Sitia R, and Inaba K
  • Journal Title: Nat. Commun. Volume: 10 Pages: 603
  • DOI: 10.1038/s41467-019-08429-1
  • Peer Reviewed / Open Access
• [Journal Article] Identification of the physiological substrates of PDIp, a pancreas-specific protein-disulfide isomerase family member (2018)
  • Author(s): Fujimoto, T., Nakamura, O., Saito, M., Tsuru, A., Matsumoto, M., Kohno, K, Inaba K., Kadokura, H.*
  • Journal Title: Journal of Biological Chemistry Volume: 293 Pages: 18421～18433
  • DOI: 10.1074/jbc.RA118.003694
  • Peer Reviewed / Open Access
• [Journal Article] Ero1-mediated reoxidation of PDI accelerates the folding of cone-snail toxins. (2018)
  • Author(s): O'Brien, H., Kanemura, S., Okumura, M., Baskin, P., Bandyopadhyay, P., Ellgaard, L., Inaba, K., and Safavi-Hemami, H.
  • Journal Title: Int. J. Mol. Sci. Volume: 19 Pages: 3418
  • DOI: 10.3390/ijms19113418
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] ヒト細胞小胞体機能の低下を鋭敏に検出するレポーターの開発と応用 (2019)
  • Author(s): 八巻聡、河野憲二、稲葉謙次、門倉広
  • Organizer: 第42回 日本分子生物学会年会
• [Presentation] Mechanistic basis and physiological functions of GPx7 and GPx8, newly identified PDI oxidases, in the mammalian endoplasmic reticulum (2019)
  • Author(s): Elza Firdiani Sofia, Shingo Kanemura, Hiroshi Kadokura, Masaki Okumura, Kenji Inaba
  • Organizer: 第19回日本蛋白質科学会年会
• [Presentation] Observing co-translational oxidative folding of a cell surface protein in the ER of mammalian cell (2018)
  • Author(s): Hiroshi Kadokura
  • Organizer: International Symposium on“Proteins; from the Cradle to the Grave”, Shiga, Japan
  • Int'l Joint Research / Invited
• [Presentation] Physiological substrates of PDIp, a pancreas-specific protein disulfide isomerase family member protein (2018)
  • Author(s): Takushi Fujimoto, Orie Nakamura, Michiko Saito, Akio Tsuru, Masaki Matsumoto, Kenji Kohno, Kenji Inaba, Hiroshi Kadokura
  • Organizer: FASEB conference, Protein Folding in the Cell
  • Int'l Joint Research
• [Presentation] Physiological function of PDIp, a pancreas-specific protein disulfide isomerase family member protein (2018)
  • Author(s): Takushi Fujimoto, Orie Nakamura, Michiko Saito, Akio Tsuru, Masaki Matsumoto, Kenji Kohno, Kenji Inaba, Hiroshi Kadokura
  • Organizer: 5th CWRU-Tohoku Joint Workshop
  • Int'l Joint Research
• [Presentation] Observing co-translational oxidative folding of a cell surface protein in the ER of mammalian cells (2018)
  • Author(s): Yo Fukuda, Yui Dazai, Naoya Hirai, Orie Nakamura, Kenji Inaba, and Hiroshi Kadokura
  • Organizer: International Symposium on“Proteins; from the Cradle to the Grave”
  • Int'l Joint Research
• [Presentation] Elucidation of physiological function of PDIp, a pancreas-specific protein disulfide isomerase family member protein (2018)
  • Author(s): Takushi Fujimoto, Orie Nakamura, Michiko Saito, Akio Tsuru, Masaki Matsumoto, Kenji Kohno, Kenji Inaba, and Hiroshi Kadokura
  • Organizer: International Symposium on“Proteins; from the Cradle to the Grave”
  • Int'l Joint Research
• [Presentation] Mechanistic basis of hydrogen peroxide-dependent PDI family oxidation catalyzed by GPx7 and GPx8 (2018)
  • Author(s): Elza Firdiani Sofia, Shingo Kanemura, Hiroshi Kadokura, Masaki Okumura, Kenji Inaba
  • Organizer: International Symposium on New Horizon in Advanced Materials Tohoku University-Taipei Tech Joint Symposium
  • Int'l Joint Research
• [Presentation] Zinc ions regulate ERp44-mediated protein quality control in the early secretory pathway (2018)
  • Author(s): Kenji Inaba
  • Organizer: International Meeting “Proteins: from the cradle to the grave
  • Int'l Joint Research / Invited
• [Presentation] Zinc ions regulate ERp44-dependent protein quality control in the early secretory pathway (2018)
  • Author(s): Kenji Inaba
  • Organizer: International Symposium on New Horizon in Advanced Materials Tohoku University
  • Int'l Joint Research / Invited
• [Presentation] Zinc ions have a new physiological role in protein quality control in the early secretory pathway (2018)
  • Author(s): Kenji Inaba
  • Organizer: CWRU-Tohoku joint symposium
  • Int'l Joint Research / Invited
• [Presentation] Structural basis of zinc-dependent ERp44 regulation for protein quality control in the early secretory pathway (2018)
  • Author(s): Satoshi Watanabe, Yuta Amagai, Sara Sannino, Manami Harayama, Shoji Masui, Momo Yamada, Roberto Sitia, and Kenji Inaba
  • Organizer: International Symposium on “Proteins; from the Cradle to the Grave
  • Int'l Joint Research
• [Presentation] Zn2+ controls the function of ERp44 in the early secretory pathway (2018)
  • Author(s): Yuta Amagai, Satoshi Watanabe, Sara Sannino, Momo Yamada, Tiziana Tempio, Manami Harayama, Roberto Sitia and Kenji Inaba
  • Organizer: International Symposium on “Proteins; from the Cradle to the Grave
  • Int'l Joint Research
• [Presentation] Structural and mechanistic insights into substrate recognition by P5, a member of PDI family (2018)
  • Author(s): M. Okumura, S. Kanemura, M. Matsusaki, M. Kinoshita, K. Arai, S. Akiyama, and K. Inaba
  • Organizer: International Symposium on “Proteins ; from the cradle to the grave
  • Int'l Joint Research
• [Presentation] Elucidation of molecular mechanisms of PDI family enzymes acting on nascent chains in early-stage of translation (2018)
  • Author(s): Chihiro Hirayama, Masaki Okumura, Kodai Machida, Kentro Noi, Teru Ogura, Hiroaki Imataka, Kenji Inaba
  • Organizer: International Meeting “Proteins: from the cradle to the grave”
  • Int'l Joint Research
• [Presentation] Identification of the Zinc Transporter critical for regulation of ERp44, a key protein quality controller in the early secretory pathway (2018)
  • Author(s): Momo Yamada, Yuta Amagai, Satoshi Watanabe, Roberto Sitia, Kenji Inaba
  • Organizer: International Symposium on New Horizon in Advanced Materials Tohoku University
  • Int'l Joint Research
• [Presentation] Mechanistic basis of hydrogen peroxide-dependent PDI family oxidation catalyzed by GPx7 and GPx8 (2018)
  • Author(s): Elza Firdiani Sofia, Shingo Kanemura, Hiroshi Kadokura, Masaki Okumura, Kenji Inaba
  • Organizer: International Symposium on Proteins: From the Cradle to the Grave
  • Int'l Joint Research
• [Presentation] Molecular basis of the ERp44-Zn2+ cooperation for protein quality control in the early secretory pathway (2018)
  • Author(s): Kenji Inaba
  • Organizer: 第91回日本生化学会年会
• [Presentation] ERp44の機能制御に関わる亜鉛トランスポーターの同定 (2018)
  • Author(s): 山田 桃、天貝佑太、渡部 聡、Roberto Sitia、稲葉 謙次
  • Organizer: 第41回分子日本分子生物学会
• [Presentation] Structural basis of pH- and zinc-dependent multiple client recognition by ERp44 (2018)
  • Author(s): Watanabe S., Amagai Y. Sitia R. Inaba K
  • Organizer: 第56回日本生物物理学会年会
  • Invited
• [Presentation] 筋小胞体カルシウムATPアーゼSERCA2bの膜貫通ヘリックス相互作用による制御機構の構造基盤 (2018)
  • Author(s): 井上 道雄、作田 菜奈美、渡部 聡、田中 良樹、潮田 亮、加藤 幸成、高木 淳一、塚崎 智也、永田 和宏、稲葉 謙次
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] 初期分泌経路内のタンパク質品質管理におけるERp44の新たな生理的機能の探索 (2018)
  • Author(s): 天貝佑太、稲葉謙次
  • Organizer: 第４１回日本分子生物学会年会
• [Presentation] 小胞体内腔における新生のジスルフィド結合形成反応の解析 (2018)
  • Author(s): 金村進吾、奥村正樹、Neil Bulleid、稲葉謙次
  • Organizer: 第18回日本蛋白質科学会年会
• [Presentation] P5による基質認識の分子構造基盤 (2018)
  • Author(s): 奥村正樹、金村進吾、松崎元紀、荒井堅太、秋山修志、稲葉謙次
  • Organizer: 第18回日本蛋白質科学会年会
• [Presentation] Newly identified insight into mechanism by which PDI/ERp46 catalyze oxidative protein folding of nascent chain (2018)
  • Author(s): 平山千尋、奥村正樹、町田幸大、野井健太郎、小椋光、今高寛晃、稲葉謙次
  • Organizer: 第18回日本蛋白質科学会年会
• [Presentation] Structure and mechanisms of substrate recognition of P5, a member of the PDI family (2018)
  • Author(s): 奥村正樹、金村進吾、松崎元紀、木下岬、荒井堅太、秋山修志、稲葉謙次
  • Organizer: 第41回日本分子生物学学会
• [Book] Oxidative folding of proteins 2nd Edition (2018)
  • Author(s): Okumura, M., Watanabe, S. and Inaba, K.
  • Total Pages: 25
  • Publisher: Royal Chemical Society
  • ISBN: 978-1-78262-990-0
• [Remarks] 東北大学多元物質科学研究所生体分子構造研究分野 稲葉研究室
  • URL: http://www2.tagen.tohoku.ac.jp/lab/inaba/html/
• [Remarks] 東北大学大学院生命科学研究科｜稲葉謙次
  • URL: https://www.lifesci.tohoku.ac.jp/research/teacher/detail---id-16689.html
• [Remarks] 東北大学大学院生命科学研究科｜門倉 広
  • URL: https://www.lifesci.tohoku.ac.jp/research/teacher/detail---id-19734.html
• [Patent(Industrial Property Rights)] タンパク質のリフォールディング剤、タンパク質のリフォールディング方法及びタンパク質の再生方法 (2018)
  • Inventor(s): 村岡 貴博、岡田 隼輔、奥村 正樹、稲葉 謙次、松﨑 元紀
  • Industrial Property Rights Holder: 村岡 貴博、岡田 隼輔、奥村 正樹、稲葉 謙次、松﨑 元紀
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: P180015088