Role of protein-protein interactions in protein import, cholesterol transport and steroid biosynthesis
Rone, Malena Beth.
Thesis (Ph.D.)--Georgetown University, 2010.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Steroid synthesis is initiated by the transfer of cholesterol to the inner membrane of the mitochondria where the conversion of cholesterol to pregnenolone occurs through the C27 cholesterol side chain cleavage cytochrome P450 enzyme (P450scc; CYP11A1). The rate of steroidogenesis is not regulated by the activity of CYP11A1 but by the availability of substrate. As the process of trafficking cholesterol to the mitochondria can occur through a series of cytosolic and mitochondrial proteins and vesicular interactions, we propose that this delivery of cholesterol into the mitochondria occurs through specific protein-protein interactions that drive steroidogenesis.; This hypothesis was explored using MA-10 mouse Leydig cells, which undergo steroidogenesis to produce progesterone. It was identified through cross-linking studies that a protein complex formed at the outer mitochondrial membrane (OMM) upon hormonal stimulation. This complex consisted of mitochondrial protein translocator protein (TSPO, 18 kDa), Golgi protein PBR Associated Protein 7, (PAP7), cytosolic protein PKA-RI, and mitochondrially targeted Steroidogenic Acute Regulatory protein (StAR). TSPO assists with the translocation of cholesterol from the OMM to the IMM and is an integral OMM protein; therefore, we decided to study the mechanisms of its import and integration into the OMM. We identified that the C-terminus and amino acids #103-109 functioned in targeting TSPO to the mitochondria through the assistance of heat shock proteins (HSP). Translocase of Outer Mitochondrial (TOM) Membrane protein Tom70 interacted with the HSP's in an ATP-dependant manner to import TSPO with the aid of theMetaxin1. From these findings, we went on to study the protein-protein interactions formed by TSPO in the mitochondria. Upon hCG treatment TSPO undergoes polymerization, identification of a shift in TSPO molecular weight and proteins that associate with TSPO was observed through Blue Native-PAGE and 2D-SDS PAGE immunoblot (western) analysis after hCG treatment in MA-10 cells. From this we identified a BN-PAGE shift in CYP11A1 from a lower to a higher molecular weight complex, suggesting it interacts specifically with the polymerized TSPO, forming of a steroidogenic protein complex.; In summary, these studies demonstrate that delivery of cholesterol into the mitochondria and thus steroidogenesis are driven by a series of protein-protein interactions.
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