2. Academic Validation
  3. Bak inserts into Mcl-1-puma and Mcl-1-BimL dimers to form hetero-trimers analyzed using quantitative live-cell FRET imaging

Bak inserts into Mcl-1-puma and Mcl-1-BimL dimers to form hetero-trimers analyzed using quantitative live-cell FRET imaging

  • J Biotechnol. 2025 Oct:406:168-178. doi: 10.1016/j.jbiotec.2025.07.013.
Hongce Chen 1,  Wenhua Su 1,  Lingyu Wang 1,  Zhirui Wu 1,  Beini Sun 1,  Xiaoping Wang 2,  Tongsheng Chen 3
Affiliations

Affiliations

  • 1 MOE Key Laboratory & Guangdong provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China.
  • 2 Department of Pain Management, the First Affiliated Hospital of Jinan University, Guangzhou 510632, China. Electronic address: txp2938@jnu.edu.cn.
  • 3 MOE Key Laboratory & Guangdong provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China. Electronic address: chentsh@scnu.edu.cn.
PMID: 40680860 DOI: 10.1016/j.jbiotec.2025.07.013
Abstract

This report uses fluorescence resonance energy transfer (FRET) to explore protein-protein interactions and the relative priorities of Bcl-2 Family proteins in living cells, focusing on four proteins: Mcl-1, Bak, BimL and Puma. FRET analysis of cells coexpressing CFP-Mcl-1 and YFP-Bak/Puma/BimL demonstrates the direct binding of Mcl-1 to Bak/Puma/BimL. We constructed Bak-P2A-CFP-Mcl-1, BimL-P2A-CFP-Mcl-1, and Puma-P2A-CFP-Mcl-1 plasmids to achieve the co-expression of Bak/Puma/BimL and Mcl-1. FRET analysis for the cells coexpressing Bak-P2A-CFP-Mcl-1 and YFP-Puma/BimL shows that co-expression of Bak and Mcl-1 significantly increased the EDmax values between CFP-Mcl-1 and YFP-Puma/BimL, indicating that Bak makes the sites of Mcl-1 and Puma/BimL close by inserting into the Mcl-1-Puma/BimL dimers to form Bak-Mcl-1-Puma/BimL trimers, which has also been further verified by Co-IP analysis. For the cells coexpressing Puma/BimL-P2A-CFP-Mcl-1 and YFP-Bak, the ED values between Mcl-1 and Bak were very small, demonstrating that Mcl-1 binds preferentially to Puma or BimL in the presence of both Puma or BimL and Bak. Collectively, Puma/BimL preferentially binds to Mcl-1 to form Mcl-1-Puma and Mcl-1-BimL dimers, and Bak can insert into the Mcl-1-Puma and Mcl-1-BimL hetero-dimers to form hetero-trimers (Bak-Mcl-1-Puma and Bak-Mcl-1-BimL).

Keywords

Bak; BimL; FRET; Mcl-1; Puma.

Figures
Products
Inhibitors & Agonists
Other Products