Journal
JOURNAL OF BIOCHEMISTRY
Volume 170, Issue 2, Pages 239-243Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvab031
Keywords
cholesterol; click chemistry; photoaffinity labelling; translocator protein (TSPO)
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Funding
- School of Pharmacy
- John Stauffer Dean's Chair in Pharmaceutical Sciences (University of Southern California)
- Natural Sciences and Engineering Council (NSERC) of Canada [RGPIN 2017-05009]
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The study demonstrated the direct binding of PhotoClick cholesterol to TSPO, with this interaction occurring at physiologically relevant sites. Excess cholesterol was found to reduce the photolabelling of TSPO.
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 immuno-precipitated 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). [GRAPHICS] .
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