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がん細胞転移を可視化するシステインプロテアーゼ蛍光プローブ

Research Project

Project/Area Number 18F18116
Research Category

Grant-in-Aid for JSPS Fellows

Allocation TypeSingle-year Grants
Section外国
Research Field Chemical biology
Research InstitutionOsaka University

Principal Investigator

菊地 和也  大阪大学, 工学研究科, 教授 (70292951)

Co-Investigator(Kenkyū-buntansha) REJA SHAHI  大阪大学, 工学(系)研究科(研究院), 外国人特別研究員
Project Period (FY) 2018-04-25 – 2020-03-31
Project Status Completed (Fiscal Year 2019)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2019: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2018: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywords蛍光プローブ / 蛋白質ラベル化 / 超解像イメージング / 1分子イメージング / 破骨細胞
Outline of Annual Research Achievements

本年度はタンパク質を超解像で解析するため、蛍光の点滅を1分子単位で取得するイメージング法を利用したタンパク質ラベル化プローブの開発に取り組んだ。具体的には、標的タンパク質にラベル化する時は蛍光を発し、解離すると蛍光を発しない可逆性のラベル化プローブを設計した。このプローブには、標的タンパク質に対して特異的かつ可逆に結合が起こること、ならびに結合時のみに蛍光を発することが求められる。 融合発現可能なタグとして機能する標的タンパク質として、疎水性ポケットを有し、疎水性の匂い物質をリガンドとして可逆的に結合するOdorant Binding Protein (OBP)を選択した。リガンドとしては中程度のアルキル鎖長を有する脂肪酸を選択した。蛍光色素として、環境応答性を示す7-ジメチルアミノクマリン及びその誘導体を選択し、脂肪酸と連結しプローブを設計、合成した。OBPの組み換えタンパク質に対し、結合特性、蛍光特性を評価したところ、プローブを加えた際に蛍光強度の上昇が見られ、結合に伴い発蛍光性を有することが明らかとなった。またその結合定数はμM以上のオーダーであり、可逆的な結合に適した範囲の強さであることが分かった。また、リガンドおよびOBP配列の検討を行い、脂肪酸のアルキル鎖の最適化とOBPの結合ポケットにおけるアミノ酸変異によって結合時に20倍以上の蛍光上昇が観察された。このプローブを用いて生細胞におけるイメージングを行ったところ、細胞内局所に発現させたOBPをプローブにより迅速にラベル化できることが確認できた。さらに、洗浄操作によりプローブが解離し、蛍光シグナルが観察されなくなったことから、プローブが可逆的にタンパク質と結合し蛍光をスイッチさせることを示した。

Research Progress Status

令和元年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和元年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2019 Annual Research Report
  • 2018 Annual Research Report
  • Research Products

    (19 results)

All 2020 2019 2018

All Journal Article (10 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 10 results,  Open Access: 3 results) Presentation (9 results) (of which Int'l Joint Research: 7 results,  Invited: 8 results)

  • [Journal Article] Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin 32020

    • Author(s)
      Gao J, Hori Y, Shimomura T, Bordy M, Hasserodt J, Kikuchi K
    • Journal Title

      Chembiochem

      Volume: 21 Issue: 5 Pages: 656-662

    • DOI

      10.1002/cbic.201900568

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch2020

    • Author(s)
      Gao J, Hori Y, Takeuchi O, Kikuchi K
    • Journal Title

      Bioconjug Chem

      Volume: 31 Issue: 3 Pages: 577-583

    • DOI

      10.1021/acs.bioconjchem.9b00696

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] In Vivo Multicolor Imaging with Fluorescent Probes Revealed the Dynamics and Function of Osteoclast Proton Pumps2019

    • Author(s)
      Minoshima Masafumi、Kikuta Junichi、Omori Yuta、Seno Shigeto、Suehara Riko、Maeda Hiroki、Matsuda Hideo、Ishii Masaru、Kikuchi Kazuya
    • Journal Title

      ACS Central Science

      Volume: 5 Issue: 6 Pages: 1059-1066

    • DOI

      10.1021/acscentsci.9b00220

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Photolytic Release of a Caged Inhibitor of an Endogenous Transcription Factor Enables Optochemical Control of CREB-Mediated Gene Expression2019

    • Author(s)
      Imoto Takuma、Kawase Akihiro、Minoshima Masafumi、Yokoyama Tatsushi、Bito Haruhiko、Kikuchi Kazuya
    • Journal Title

      Organic Letters

      Volume: 22 Issue: 1 Pages: 22-25

    • DOI

      10.1021/acs.orglett.9b03568

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Development of an effective protein-labeling system based on smart fluorogenic probes2019

    • Author(s)
      Reja SI, Minoshima M, Hori Y, Kikuchi K
    • Journal Title

      J Biol Inorg Chem

      Volume: 24 Issue: 4 Pages: 443-455

    • DOI

      10.1007/s00775-019-01669-y

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes2018

    • Author(s)
      Akazawa Kazuki、Sugihara Fuminori、Nakamura Tatsuya、Mizukami Shin、Kikuchi Kazuya
    • Journal Title

      Bioconjugate Chemistry

      Volume: 29 Issue: 5 Pages: 1720-1728

    • DOI

      10.1021/acs.bioconjchem.8b00167

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Sensing caspase-1 activity using activatable 19F MRI nanoprobes with improved turn-on kinetics2018

    • Author(s)
      Akazawa Kazuki、Sugihara Fuminori、Minoshima Masafumi、Mizukami Shin、Kikuchi Kazuya
    • Journal Title

      Chemical Communications

      Volume: 54 Issue: 83 Pages: 11785-11788

    • DOI

      10.1039/c8cc05381b

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Perfluorocarbon-based 19F MRI Nanoprobes for In Vivo Multicolor Imaging2018

    • Author(s)
      Akazawa Kazuki、Sugihara Fuminori、Nakamura Tatsuya、Matsushita Hisashi, Mukai Hiroaki, Akimoto Rena, Minoshima Masafumi, Mizukami Shin、Kikuchi Kazuya
    • Journal Title

      Angewante Chemie International Edition

      Volume: 57 Pages: 16742-16747

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] In vivo visualisation of different modes of action of biologic DMARDs inhibiting osteoclastic bone resorption2018

    • Author(s)
      Matsuura Y, Kikuta J, Kishi Y, Hasegawa T, Okuzaki D, Hirano T, Minoshima M, Kikuchi K, Kumanogoh A, Ishii M
    • Journal Title

      Annals of the Rheumatic Diseases

      Volume: 印刷中 Issue: 8 Pages: 1219-1225

    • DOI

      10.1136/annrheumdis-2017-212880

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Dynamic Analyses of the Short-Term Effects of Different Bisphosphonates Using Intravital Two-Photon Microscopy2018

    • Author(s)
      Kikuta Junichi、Shirazaki Mai、Sudo Takao、Mizuno Hiroki、Morimoto Akito、Suehara Riko、Minoshima Masafumi、Kikuchi Kazuya、Ishii Masaru
    • Journal Title

      JBMR Plus

      Volume: 2 Issue: 6 Pages: 362-366

    • DOI

      10.1002/jbm4.10057

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Presentation] New Biological Findings which were Revealed by Designed Fluorescent Probes2019

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      Chemical Biology & Physiology Conference 2019
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] In vivo Multicolor Imaging with Fluorescent Probes Revealed Dynamics and Function of Osteoclast Proton Pumps2019

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      16th International, Methods & Applications of Fluorescence (Roger Y Tsien Memorial Symposium)
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] In vivo Chemical Probes for MRI and Fluorescence Imaging2019

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      ICBIC-19, International Conference on Biological Inorganic Chemistry
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Development of Multifunctional 19F MRI Contrast Agents Based on Fluorine-encapsulated Silica Nanoparticle2019

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      The Future of Molecular MR: A Cellular and Molecular Imaging Workshop
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] イメージングプローブのデザイン・合成によるケミカルバイオロジー研究2019

    • Author(s)
      菊地 和也
    • 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 合同年次学会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] Development of PYP-tag far-red fluorogenic probes for intracellular protein imaging2019

    • Author(s)
      Reja Imam Shahi、Hori Yuichiro、Kikuchi Kazuya
    • Organizer
      The 99th Annual Meeting of Chemical Society of Japan
    • Related Report
      2018 Annual Research Report
  • [Presentation] In vivo Chemical Probes for MRI and Fluorescence Imaging2018

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      The 9th Asian Biological Inorganic Chemistry Conference (AsBIC IX)
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Development of Multifunctional 19F MRI Contrast Agents Based on Fluorine-encapsulated Silica Nanoparticle2018

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      Imaging in 2020
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] New Biological Findings Which Were Clarified by Designed Chemical Probes2018

    • Author(s)
      Kikuchi Kazuya
    • Organizer
      MSMLG 2018, 6th International Conference on Molecular Sensors and Molecular Logic Gates
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited

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Published: 2018-05-01   Modified: 2024-03-26  

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