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

Seong Hyun Park, Wooseok Ko, Hyun Soo Lee, Injae Shin

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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 S184 via binding to the C-terminal S184 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.

Original languageEnglish
Pages (from-to)4273-4281
Number of pages9
JournalJournal of the American Chemical Society
Volume141
Issue number10
DOIs
Publication statusPublished - 2019 Mar 13

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Genetic Code
Fluorescence Resonance Energy Transfer
Technology
Proteins
Cell death
Apoptosis
Single-Cell Analysis
Phosphorylation
Amino acids
Fusion reactions
Ligands
Amino Acids

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

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abstract = "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 S184 via binding to the C-terminal S184 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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Analysis of Protein-Protein Interaction in a Single Live Cell by Using a FRET System Based on Genetic Code Expansion Technology. / Park, Seong Hyun; Ko, Wooseok; Lee, Hyun Soo; Shin, Injae.

In: Journal of the American Chemical Society, Vol. 141, No. 10, 13.03.2019, p. 4273-4281.

Research output: Contribution to journalArticle

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