Cholesterol-Mediated Conformational Changes in the Steroidogenic Acute Regulatory Protein Are Essential for Steroidogenesis

Abstract

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.

The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis

“…StAR phosphorylation by PKA is neither necessary nor dependent on the presence of cholesterol in the reaction media (Baker et al, 2007), in agreement with our observations (Poderoso et al, 2008). This result indicates that StAR phosphorylation by kinases other than PKA is modulated by the endogenous ligand of the protein, in agreement with recent work showing that cholesterol is a potent activator of StAR metabolism (Rajapaksha et al, 2013).…”

Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Regulates Steroidogenic Activity via Steroidogenic Acute Regulatory Protein (StAR)-Voltage-dependent Anion Channel 2 (VDAC2) Interaction

“…Mitochondria-StAR import is slower than that observed for other mitochondrial targeting sequences (18), and it competes only with its own N-terminal sequence (38); however, the impact of the N-terminal sequence is unknown. Therefore, we constructed a recombinant StAR in which 30 amino acids of the N-terminal StAR sequence were substituted with the P450scc mitochondrial sequence (SCC/StAR) and determined whether it still transits through the MAM fraction.…”

“…Under stress, the flux of cholesterol changes dramatically due to enhanced hydrolysis of stored cholesterol esters, increased uptake of plasma cholesterol, and transport of free cholesterol into the mitochondria for the synthesis of steroid hormones in response to ACTH. Free cholesterol induces tighter StAR folding; however, excess cholesterol results in the unfolding of StAR (38). Thus, in the presence of excess cholesterol, StAR may possibly remain associated with the other MAM proteins, and after most of the cholesterol pool is imported into the mitochondria, the remaining cholesterol may promote StAR refolding.…”

“…StAR is expressed in acute conditions, and thus a master regulator chaperone of the uncoupling protein response (46,47) would be essential for its appropriate folding, which is required for its targeting to the mitochondria. StAR has a mitochondrial targeting sequence, and its import is slower than that of other mitochondrial targeting proteins (18,38). In the absence of proper folding, StAR would be proteolyzed by the proteasomal protease (15).…”

Regulation of Human 3β-Hydroxysteroid Dehydrogenase Type 2 by Adrenal Corticosteroids and Product-Feedback by Androstenedione in Human Adrenarche

“…As previously described (Rajapaksha et al, 2011(Rajapaksha et al, , 2013a, hydrophilicity analysis has shown that the N-terminal 3bHSD2 sequence is more hydrophobic up to amino acid 120, and the amino acid sequence of the rest of the protein is weakly hydrophobic and does not form an amphiphilic helix, resulting in a favored association with the membrane. The association may be the result of an electrostatic interaction between the positively charged residues of the C terminus of the protein and the less polar cortisone and androstenedione.…”