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Analysis of Protein-Protein Interaction in a Single Live Cell by Using a FRET System Based on Genetic Code Expansion Technology

조회수 : 9 등록일 : 2019.04.15 00:00

저자 : Ko, W (Ko, Wooseok); Lee, HS (Lee, Hyun Soo)
출처 : JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
출판일 : 2019.03.13


Analysis of Protein-Protein Interaction in a Single Live Cell by Using a FRET System Based on Genetic Code Expansion Technology


Park, SH (Park, Seong-Hyun)1 ] Ko, W (Ko, Wooseok)2 Lee, HS (Lee, Hyun Soo)2 Shin, I (Shin, Injae)1 ]


[ 1 ] Yonsei Univ, Ctr Biofunct Molecules, Dept Chem, Seoul 03722, South Korea
[ 2 ] Sogang Univ, Dept Chem, Seoul 04107, South Korea


Hsp70 is known to directly bind to Bax for suppression of apoptosis. However, mechanisms on how Bax is dissociated from its complex with Hsp70 during apoptosis remain largely unknown. In the current study, we developed the efficient fluorescence resonance energy transfer (FRET) system which consisted of Hsp70-YFP and fluorescent amino acid (ANAP)-incorporated Bax, which was generated by using genetic code expansion technology, and applied the FRET system to elucidate mechanisms on how apoptosis-inducing substances dissociate Bax from Hsp70. Time-dependent analysis of single live cell images showed that Bax activators binding to Bax trigger sites inhibited the Bax-Hsp70 interaction but a Bax activator, which blocks phosphorylation of 5184 via binding to the C-terminal 5184 site, did not affect this interaction. Additionally, an inhibitor for Hsp70-Hsp40 interaction blocked the Bax-Hsp70 interaction. Furthermore, p53 activators promoted the dissociation of Bax from Hsp70 by reactivating p53 which disrupted the Bax-Hsp70 interaction. We also found that death ligands and a Bcl-2 inhibitor enhanced the dissociation of Bax from Hsp70 by activating activator BH3-only proteins. Results from this effort suggest that FRET systems consisting of the ANAP-incorporated protein and the YFP fusion protein will be valuable tools to gain an understanding of other types of protein-protein interactions.






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