Progesterone is a critical hormone in the maintenance of pregnancy. Although the general pathway of biosynthesis of this steroid has been known for some time, a major deficiency in our knowledge of the regulation of steroidogenesis relates to the way in which the precursor, cholesterol, is transported to the mitochondria and subsequently within the mitochondria to reach the cholesterol side chain cleavage system, which catalyzes the first and rate-determining reaction. Cholesterol transport is known to be under tropic regulation, but the specific factors involved in cholesterol movement, their mode of action and control remain to be elucidated. We have based the planned experiments on a working model which envisions key roles for two proteins: sterol carrier protein2 (SCP2), and endozepine, also called acyl CoA binding protein. SCP2 is postulated to move cholesterol to the outer mitochondrial membrane. Movement of cholesterol from the outer to inner mitochondrial membranes is hypothesized to be mediated by endozepine, through interaction with outer mitochondrial membrane proteins which may include a specific receptor (peripheral benzodiazepine receptor). Endozepine may also deliver acyl CoA to mitochondria, providing substrate for generation of reducing equivalents required to support steroidogenesis. We intend to explore the function of the lipid transport proteins in intact cells by altering their levels using molecular biology techniques including hyperexpression of their cDNAs and suppression of translation of endogenous mRNAs with constructs producing antisense RNA. The effects of these alterations on steroid synthesis and aspects of cholesterol metabolism including de novo sterol synthesis, movement of newly synthesized sterol and LDL-delivered sterol to mitochondria and plasma membranes, sterol esterification and cellular and organelle lipid profiles will be assessed. Critical structural features of SCP2 and endozepine required for their steroidogenic action will also be defined through use of site-directed mutagenesis and subsequent expression of the mutated cDNAs. The regulation of expression of the genes encoding these proteins will be examined in human ovarian and trophoblast cells. The ability of tropic hormones to alter protein levels will be determined by Western blotting mRNA levels will be quantitated by Northern blotting and gene transcription by nuclear run-off assays. These studies should provide a test of the working hypothesis using intact cells and yield important insight into key steps in the process of hormone synthesis. They will also establish an experimental paradigm with which the roles of other proteins involved in cellular cholesterol movement can be explored.
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