The precise mechanism by which LH accelerates the transport of the substrate cholesterol from the cytosol to the inner mitochondrial membrane, the rate-determining step in steroidogenesis, is unknown. The mitochondrial high affinity cholesterol binding protein peripheral-type benzodiazepine receptor (PBR) and the hormone-induced cytosolic steroidogenic acute regulatory protein (StAR) were found to function in a coordinated manner to transfer cholesterol into mitochondria. We recently demonstrated that in Leydig cells the StAR-induced cholesterol import into mitochondria is mediated by PBR. Based on these findings we hypothesize that changes in the structure and levels of PBR determine the amount of StAR-mobilized cholesterol available for testosterone synthesis. We will test this hypothesis with 3 Specific Aims.
In Aim 1, we will determine how changes in PBR structure affect cholesterol transport. Using biophysical techniques coupled with studies of in vitro reconstituted wild-type and mutant PBR, we will investigate the sequence of molecular events involved in the transfer of cholesterol mobilized by StAR to the cytochrome P450scc via PBR.
In Aim 2, we will investigate the impact of PBR and StAR inactivation on cholesterol transport and steroidogenesis. PBR and StAR structure-based drug design identified lead compounds that target the cholesterol-binding domains of these proteins and inhibit steroidogenesis. The specificity of these in vitro effects and the in vivo effect of these compounds on steroid synthesis will be investigated.
In Aim 3, we will test the hypothesis that the steroidogenic potential of Leydig cells is dictated by PBR protein levels, which in turn, are primarily regulated by PBR gene transcription. In search of molecules inhibiting PBR gene expression and steroidogenesis, we identified peroxisome proliferators (PPs), environmental agents known to exert testis-specific toxic effects. In vitro gene transcription and in vivo studies using transgenic mice showing restricted Leydig cell expression of the PBR gene promoter will be used to understand PBR expression and steroidogenesis in normal testes and testes exposed to the toxic effects of PPs. These studies should provide detailed understanding of the role of PBR and PBR-StAR interaction in cholesterol transport and steroidogenesis, and lead to better understanding of how environmental toxicants might function to elicit male reproductive pathologies as well as to therapies for such pathologies.
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