The dysregulation of sterol metabolism is a critical factor in the development of two major human diseases: coronary atherosclerosis, the leading cause of death in the Western societies, and cholesterol gallstones, one of the leading indicators for surgery in the United States. The basic pathways of sterol transport in the body have been well defined: sterols enter the body via endogenous synthesis and intestinal absorption and are excreted via the liver into the bile, either as the free sterol or after conversion to bile acids. The two major organ systems involved in these processes, the liver and intestine, provide critical barriers to the accumulation of sterols in the body, but the molecular constituents and mechanisms of action of these barriers are poorly understood. A major unresolved issue that remains to be addressed is whether specific cell membrane transporters facilitate the translocation of cholesterol across the intestinal and bile canalicular membranes. An important clue to the molecular mechanisms that defend against sterol accumulation has recently emerged from studies of sitosterolemia, a genetic disorder associated with increased sterol absorption and decreased sterol excretion. Recently, we showed that sitosterolemia is due to mutations in two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8. The profound alteration in sterol homeostasis observed in sitosterolemic patients indicates that ABCG5 and ABCG8 serve to limit sterol absorption in the intestine and promote sterol excretion in the bile, but almost nothing is known about the basic function and characteristics of these two proteins. The overall goal of this grant proposal is to elucidate the basic mechanisms by which ABCG5 and ABCG8 limit sterol absorption in the intestine and facilitate sterol excretion in hepatocytes. The studies proposed are designed to address three critical questions: First, where are ABCG5 and ABCG8 located in the cell? Second, what is the functional form of ABCG5 and ABCG8? Third, what substrate(s) do ABCG5 and ABCG8 transport? To address these questions, we propose a comprehensive series of biochemical studies that will allow a direct and quantitative investigation of the ABCG5/8 transport system. The subcellular location of ABCG5 and ABCG8 will be determined by microscopy and cell fractionation. The functional form of ABCG5 and ABCG8 will be determined by co-immunoprecipitation studies to identify the primary dimerization partners, and then by purification of the native proteins to identify other components of the transport complex. Finally, a functional transport complex will be reconstituted in proteoliposomes to define the substrate and basic enzymatic properties of the transporter. These studies will provide a comprehensive understanding of the molecular mechanisms of this important transport system that protects against hypercholesterolemia and coronary atherosclerosis.
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