Cholesterol-mediated conformational changes in the steroidogenic acute regulatory protein are essential for steroidogenesis.
Although the mechanism by which the steroidogenic acute regulatory protein (StAR) promotes steroidogenesis has been studied extensively, it remains incompletely characterized. Because structural analysis has revealed a hydrophobic sterol-binding pocket (SBP) within StAR, this study sought to examine the regulatory role of cholesterol concentrations on protein folding and mitochondrial import. Stopped-flow analyses revealed that at low concentrations, cholesterol promotes StAR folding. With increasing cholesterol concentrations, an intermediate state is reached followed by StAR unfolding. With 5 μg/mL cholesterol, the apparent binding was 0.011 s(-1), and the unfolding time (t1/2) was 63 s. The apparent binding increased from 0.036 to 0.049 s(-1) when the cholesterol concentration was increased from 50 μg/mL to 100 μg/mL while t1/2 decreased from 19 to 14 s. These cholesterol-induced conformational changes were not mediated by chemical chaperones. Protein fingerprinting analysis of StAR in the absence and presence of cholesterol by mass spectrometry revealed that the cholesterol binding region, comprising amino acids 132-188, is protected from proteolysis. In the absence of cholesterol, a longer region of amino acids from position 62 to 188 was protected, which is suggestive of organization into smaller, tightly folded regions with cholesterol. In addition, rapid cholesterol metabolism was required for the import of StAR into the mitochondria, suggesting that the mitochondria have a limited capacity for import and processing of steroidogenic proteins, which is dependent on cholesterol storage. Thus, cholesterol regulates StAR conformation, activating it to an intermediate flexible state for mitochondrial import and its enhanced cholesterol transfer capacity.
Department of Biochemistry, Division of Biomedical Sciences, Mercer University School of Medicine , Savannah, Georgia 31404, United States.
Maheshinie
M
Rajapaksha
Jasmeet
J
Kaur
Mahuya
M
Bose
Randy M
RM
Whittal
Himangshu S
HS
Bose
D001665:Binding Sites; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D002784:Cholesterol