| dc.description.abstract | The rate-limiting step in steroidogenesis is the delivery of cholesterol to the mitochondrial inner membrane by the steroidogenic acute regulatory protein (StAR). A number of developmental and reproductive abnormalities in humans are related to the misregulation of StAR expression and/or to a nonfunctioning StAR gene. For example, lipoid congenital adrenal hyperplasia, a potentially lethal autosomal recessive disease that manifests as cholesterol accumulation in steroidogenic cells of newborn, is a consequence of StAR deficiency. The exact mechanism of cholesterol transfer by StAR is still under debate. On one hand, a molten globular state of StAR observed under acidic conditions, namely pH 3.5, has been suggested to bind and transfer cholesterol. On the other hand, we have proposed that the state of StAR that binds cholesterol has a well-defined tertiary structure. Therefore, the purpose of this project was to investigate the biological and structural features involved in binding of StAR to cholesterol and its steroidogenic activity. There were three specific aims to achieve this goal: the first was to determine the steroidogenic activity of StAR and also to find the environment in which it could bind to cholesterol to promote steroidogenesis, using a quantitative in vitro activity and binding assays. The second was to inquire the various conformational changes of the StAR in the presence of cholesterol through circular dichroism (CD) and the third, to measure the binding affinity of StAR/cholesterol complex. To construct biologically active recombinant StAR and study its activity on isolated mitochondria we elected to produce human StAR proteins lacking the mitochondrial import sequence in bacteria, and to incorporate a [subscript 6]His-N-62 StAR to facilitate their purification. Using these constructs, we could examine the action of StAR independent of the protein import process. Here we show that the [subscript 6]His-tag StAR is biologically active and binding of protein to free cholesterol is necessary to trigger steroidogenesis. As a prerequisite for clarifying the mechanism of StAR action, it was first decided to determine the mechanism by which StAR bound to cholesterol. The mechanism of binding was evaluated under acidic and physiological pH to compare molten globule theory with our proposed model. Thermal denaturation monitored by CD at physiological pH revealed that StAR had a well-defined tertiary structure as depicted by an increase in cooperative denaturation transition. However, the denaturation profile of StAR at pH 3.5 was non-cooperative, as expected for a molten globule. The cooperativity, thermal unfolding transition and an increase in secondary structure content of StAR were observed upon addition of cholesterol. This was a clear indication of the formation of a stable StAR/choleterol complex at physiological pH. In addition, a CD-monitored time course study showed that binding of StAR to cholesterol requires secondary structural changes (unfolding-refolding) that eventually resulted in a more folded protein. Titration of StAR with cholesterol monitored by CD allowed us to determine the stoichiometry of 1:1 and a Kd ~1 x 10[superscript -6] M. Binding assays at lower pHs demonstrated that the interaction of StAR with cholesterol was pH sensitive and diminished as the pH was lowered. Furthermore, a decrease of pH resulted in the dissociation of the protein-ligand complex. Taken together, the results presented herein suggest that a native partially unfolded StAR binds to cholesterol at physiological pH while release of cholesterol may coincide with a transition of StAR to a molten globule state, which is yet to be proven. | fr |
