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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL072304-02
Application #
6697288
Study Section
Metabolism Study Section (MET)
Program Officer
Applebaum-Bowden, Deborah
Project Start
2003-02-01
Project End
2008-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
2
Fiscal Year
2004
Total Cost
$390,000
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Hobbs, Helen H (2018) Science, serendipity, and the single degree. J Clin Invest 128:4218-4223
Lee, Jyh-Yeuan; Kinch, Lisa N; Borek, Dominika M et al. (2016) Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 533:561-4
Wang, Jin; Mitsche, Matthew A; Lütjohann, Dieter et al. (2015) Relative roles of ABCG5/ABCG8 in liver and intestine. J Lipid Res 56:319-30
Wang, Jin; Grishin, Nick; Kinch, Lisa et al. (2011) Sequences in the nonconsensus nucleotide-binding domain of ABCG5/ABCG8 required for sterol transport. J Biol Chem 286:7308-14
Rios, Jonathan; Stein, Evan; Shendure, Jay et al. (2010) Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia. Hum Mol Genet 19:4313-8
Yi, Ming; Horton, Jay D; Cohen, Jonathan C et al. (2006) WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data. BMC Bioinformatics 7:30
Yang, Chendong; McDonald, Jeffrey G; Patel, Amit et al. (2006) Sterol intermediates from cholesterol biosynthetic pathway as liver X receptor ligands. J Biol Chem 281:27816-26
Zhang, Da-Wei; Graf, Gregory A; Gerard, Robert D et al. (2006) Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8. J Biol Chem 281:4507-16
Wang, Jin; Sun, Fang; Zhang, Da-wei et al. (2006) Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution. J Biol Chem 281:27894-904
Xu, Fang; Rychnovsky, Scott D; Belani, Jitendra D et al. (2005) Dual roles for cholesterol in mammalian cells. Proc Natl Acad Sci U S A 102:14551-6

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