2018 Fiscal Year Final Research Report
Real-time observation of the biological events within living cells using in-cell NMR
Project Area | Novel measurement techniques for visualizing 'live' protein molecules at work |
Project/Area Number |
26119005
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Research Category |
Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
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Allocation Type | Single-year Grants |
Review Section |
Complex systems
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Research Institution | The University of Tokyo |
Principal Investigator |
Nishida Noritaka 東京大学, 大学院薬学系研究科(薬学部), 准教授 (50456183)
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Co-Investigator(Kenkyū-buntansha) |
甲斐荘 正恒 首都大学東京, 理学研究科, 客員教授 (20137029)
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Research Collaborator |
Shimada Ichio
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Project Period (FY) |
2014-07-10 – 2019-03-31
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Keywords | 核磁気共鳴法 / in-cell NMR / 酸化ストレス / シグナル伝達 |
Outline of Final Research Achievements |
We have established the in-cell NMR methodology that enables the observation of the intracellular biological events in real-time manner. To observe the intracellular molecular response against oxidative stresses, we prepared the HeLa cells, in which both thioredoxin and glutathione were isotopically labeled, and monitored the redox status of Trx and glutathione by alternate in-cell NMR measurements. This study demonstrated the redox status of Trx is regulated not only by the intracellular redox potential but also by various endogenous regulatory molecules, such as Trx reductases. We also performed the in-cell NMR observation of small GTPase Ras and its oncogenic variants. We demonstrated that the GTP-bound ratio of Ras in the steady state is decreased in the cell for all Ras constructs, due to the increase of khy and the decrease of kex. We also succeeded in quantifying the contribution of the specific regulatory proteins by in-cell NMR experiments using the knockout cells.
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Free Research Field |
構造生物学
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Academic Significance and Societal Importance of the Research Achievements |
細胞内は多種多様なタンパク質が複雑なネットワークを形成する分子混雑環境であり、細胞内のタンパク質はin vitroとは異なる構造や活性を示す。本研究ではin-cell NMR法を高度化し、細胞内タンパク質の構造や活性変化をリアルタイム観測する手法を確立して、細胞内酸化ストレスやシグナル伝達タンパク質の活性評価に適用した。本研究成果は、細胞内生命現象の可視化のみならず、細胞内で創薬標的タンパク質に対する候補化合物の薬効を直接評価するツールとしての応用が期待できる。
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