2. Academic Validation
  3. Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue

Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue

  • J Biochem. 2021 Oct 11;170(2):239-243. doi: 10.1093/jb/mvab031.
Elias Georges 1 2,  Chantal Sottas 2,  Yuchang Li 2,  Vassilios Papadopoulos 2
Affiliations

Affiliations

  • 1 Institute of Parasitology, McGill University, Montreal, Quebec H9X1C0, Canada.
  • 2 Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
PMID: 33846725 DOI: 10.1093/jb/mvab031
Abstract

The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive Cholesterol analogue. PhotoClick Cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess Cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick Cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).

Keywords

cholesterol; click chemistry; photoaffinity labelling; translocator protein (TSPO).

Figures
Products