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Creation of a protease with a catalytic site that is distant from the substrate binding site

Research Project

Project/Area Number 17K07359
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Biophysics
Research InstitutionTokyo Medical and Dental University

Principal Investigator

Ikura Teikichi  東京医科歯科大学, 難治疾患研究所, 准教授 (50251393)

Project Period (FY) 2017-04-01 – 2020-03-31
Project Status Completed (Fiscal Year 2019)
Budget Amount *help
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2019: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2017: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
KeywordsPin1 / プロリン異性化酵素 / タンパク質分解酵素 / 分子動力学シミュレーション / 蛋白質 / バイオテクノロジー / 酵素
Outline of Final Research Achievements

In this research, I aimed to create a protease with a catalytic site that is distant from the substrate binding site. I selected a mutant of peptidyl-prolyl isomerase Pin1 as a prototype for this purpose because the mutant happened to cleave the peptide bond between the 4th and 5th residues before a particular proline residue. First, I determined the sequence specificity of the substrate of the mutant Pin1 by the screening system I developed. Next, I optimized the catalytic site of the mutant Pin1 by substituting amino acid residues. Finally, I micronized the protease derived from the mutant Pin1. To this end, I deleted the N-terminal domain distal to the catalytic site and introduced several mutations to improve stability.

Academic Significance and Societal Importance of the Research Achievements

タンパク質分解は生命活動にとって重要な役割を担っている。一方、生命科学分野の研究において、特異性の高いプロテアーゼは、in vivoとin vitroの両面で利用されてきた。また、その中には将来の医療現場での活用の可能性も期待されているものもある。ところが、自然界に存在するプロテアーゼは、基質認識部位の中に触媒部位を含むため、その利用には限界がある。本研究では、基質認識部位と触媒部位とが完全に分離したプロテアーゼの創製を目的として研究を行い、一定の成果を得た。さらなる特異性と触媒活性の向上が得られれば、将来の商品化の対象として展開が可能である。

Report

(4 results)
  • 2019 Annual Research Report   Final Research Report ( PDF )
  • 2018 Research-status Report
  • 2017 Research-status Report
  • Research Products

    (12 results)

All 2019 2018 2017

All Journal Article (2 results) (of which Peer Reviewed: 2 results) Presentation (9 results) (of which Int'l Joint Research: 1 results) Book (1 results)

  • [Journal Article] Mutational effects of Cys113 on structural dynamics of Pin12019

    • Author(s)
      Ikura Teikichi、Yonezawa Yasushige、Ito Nobutoshi
    • Journal Title

      Biophysics and Physicobiology

      Volume: 16 Issue: 0 Pages: 452-465

    • DOI

      10.2142/biophysico.16.0_452

    • NAID

      130007752754

    • ISSN
      2189-4779
    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation2018

    • Author(s)
      Teikichi Ikura, Naoya Tochio, Ryosuke Kawasaki, Mizuki Matsuzaki, Akihiro Narita, Mahito Kikumoto, Naoko Utsunomiya-Tate, Shin-ichi Tate, Nobutoshi Ito
    • Journal Title

      FEBS Letters

      Volume: 592 Issue: 18 Pages: 3082

    • DOI

      10.1002/1873-3468.13218

    • Related Report
      2018 Research-status Report
    • Peer Reviewed
  • [Presentation] プロリン異性化酵素Pin1の1残基変異C113Aに伴うダイナミクスの劇的変化2019

    • Author(s)
      伊倉貞吉, 米澤康滋, 伊藤暢聡
    • Organizer
      第19回日本蛋白質科学会年会・第71回日本細胞生物学会大会 合同年次大会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Micronization of a protease derived from Pin12019

    • Author(s)
      Teikichi Ikura, Nobutoshi Ito
    • Organizer
      The 57th annual meeting of the Biophysical Society of Japan
    • Related Report
      2019 Annual Research Report
  • [Presentation] タンパク質機能における階層的様相2019

    • Author(s)
      伊倉貞吉
    • Organizer
      理論生物物理学の現在と未来
    • Related Report
      2018 Research-status Report
  • [Presentation] Pin1由来のタンパク質分解酵素の触媒部位の構造ダイナミクス2018

    • Author(s)
      伊倉貞吉, 米澤康滋, 伊藤暢聡
    • Organizer
      第18回日本蛋白質科学会年
    • Related Report
      2018 Research-status Report
  • [Presentation] Mutational analysis on the catalytic site of a protease derived from Pin12018

    • Author(s)
      Teikichi Ikura and Nobutoshi Ito
    • Organizer
      The 56th Annual Meeting of the Biophysical Society of Japan
    • Related Report
      2018 Research-status Report
  • [Presentation] Pin1のWWドメインは、タウペプチドのトランス異性体の凝集化を阻害する2018

    • Author(s)
      伊倉貞吉,栃尾尚哉,川嵜亮祐,松崎瑞季,成田哲博,菊本真人,楯直子,楯真一,伊藤暢聡
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Research-status Report
  • [Presentation] Functional conversion from peptidyl-prolyl isomerase to protease by a single amino acid substitution.2017

    • Author(s)
      Ikura, T., Ito, N.
    • Organizer
      2017 International Biophysics Congress (IUPAB)
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] Pin1由来のタンパク質分解酵素の触媒機構2017

    • Author(s)
      伊倉貞吉, 伊藤暢聡
    • Organizer
      第17回日本蛋白質科学会年会
    • Related Report
      2017 Research-status Report
  • [Presentation] タウ蛋白質に対するPin1由来のプロテアーゼの活性の定量的評価2017

    • Author(s)
      伊倉貞吉, 伊藤暢聡
    • Organizer
      第55回日本生物物理学会年会
    • Related Report
      2017 Research-status Report
  • [Book] タンパク質のアモルファス凝集と溶解性2019

    • Author(s)
      赤澤陽子,浅野竜太郎,荒川力,有坂文雄,安藤昭一朗,伊倉貞吉,池口雅道,石井明子,石原智彦,伊豆津健一,今村比呂志,岩下和輝,千賀由佳子,内山進,江島大輔,太田里子,小澤大作,小野寺理,加藤昌人,河村義史,城所俊一,黒田裕,黒谷篤之,五島直樹,後藤祐児,柴田寛子,白木賢太郎,杉山正明,千賀由佳子,田口英樹,武内敏秀,津本浩平,デミエン ホール 他
    • Total Pages
      283
    • Publisher
      シーエムシー出版
    • ISBN
      9784781313979
    • Related Report
      2018 Research-status Report

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Published: 2017-04-28   Modified: 2021-02-19  

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