2022 Fiscal Year Final Research Report
Structural analyses of exosome localizing membrane protein, EAAC1 and CD63
| Project/Area Number |
20K06515
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 43020:Structural biochemistry-related
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| Research Institution | Tohoku University (2022)
Institute of Physical and Chemical Research (2020-2021) |
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Principal Investigator |
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | クライオ電子顕微鏡 / グルタミン酸輸送体 / テトラスパニン / 単粒子構造解析 / エクソソーム |
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| Outline of Final Research Achievements |
Glutamate transporter (EAAC1) and tetraspanin (CD63), membrane proteins that localize to exosomes, were expressed and purified as recombinant proteins using human cells. Each protein was expressed as a GFP fusion protein, and the expression was confirmed under a fluorescence microscope. The proteins were also observed to be secreted as exosomes into the culture medium, suggesting that the expressed proteins were correctly localized to the exosomes.
The obtained proteins were observed by cryo-EM, and particle images of the target protein were obtained for EAAC1.
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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 |
高発現系ヒト細胞(Expi293F)を用いて，EAAC1およびCD63の発現系の構築および組換えタンパク質の調製が可能であることが明らかとなった．EAAC1およびCD63はそれぞれ疾患との関りが強く，神経変性疾患やメラノーマで特異的に発現量が増えることが報告されている．このことから，これらの過剰発現細胞系がそれぞれの疾患のメカニズム解析へと貢献できる可能性を示唆している．
また，重金属を利用したネガティブ染色並びにクライオ電子顕微鏡像からEAAC1については粒子が均一な構造をとっており，単粒子構造解析が可能であることを示唆するデータが得られている．
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