The dysregulation of sterol metabolism contributes to two major human diseases: coronary atherosclerosis, a major cause of death in Western societies, and cholesterol gallstones, a leading indicator 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 molecular mechanism responsible for the secretion of cholesterol by the liver and intestine was revealed by the finding that mutations in either of two ATP binding cassette (ABC) half-transporters, ABCG5 (G5) and ABCG8 (G8) cause sitosterolemia, an autosomal recessive disease characterized by accumulation of cholesterol and plant-derived sterols, and premature coronary atherosclerosis. This discovery was the starting point of this grant. During the last funding period, we elucidated the basic biology and pathophysiology of G5G8: we found that the two proteins form a heterodimer in the endoplasmic reticulum (ER) that is transported to the apical membranes of hepatocytes or enterocytes, and that most mutations causing sitosterolemia interfere with the coupling and transport of G5G8. We developed mice in which G5 and G8 were deleted, or overexpressed, and provided direct evidence that G5G8 prevents the accumulation of cholesterol and non-cholesterol sterols, and are essential for the maintenance of cholesterol homeostasis in the liver and in extra-hepatic tissues such as the adrenal. A major unresolved question arising from these studies is the role of G5G8 in the intestine. It is generally believed that biliary excretion is the major pathway for cholesterol excretion in humans, but we, and others, have found that fecal sterol excretion is normal in mice unable to secrete cholesterol into bile. This finding suggests that the intestine may play a major role in the elimination of cholesterol. Here we have designed a series of studies to address specifically the role of G5G8 in the intestine in protecting against accumulation of dietary sterols and in promoting the reverse cholesterol transport of endogenously-derived sterols. We have also developed the first in vitro assays for sterol transport by G5G8. We have purified and reconstituted the native (and recombinant) proteins, and shown that sterols are direct transport substrates of the heterodimer. We propose now to use these assays to address fundamental questions regarding the mechanism by which G5G8 couples ATP hydrolysis to the translocation of sterols across membranes. How does G5G8 recognize sterols and make them available to acceptors in the bile and intestinal lumen? We have the expertise, molecular tools and infrastructure to address these important physiological and biochemical questions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL072304-07
Application #
7563313
Study Section
Integrative Nutrition and Metabolic Processes Study Section (INMP)
Program Officer
Liu, Lijuan
Project Start
2003-02-01
Project End
2013-01-31
Budget Start
2009-02-01
Budget End
2010-01-31
Support Year
7
Fiscal Year
2009
Total Cost
$421,656
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
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Mitsche, Matthew A; McDonald, Jeffrey G; Hobbs, Helen H et al. (2015) Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways. Elife 4:e07999
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Stiles, Ashlee R; Kozlitina, Julia; Thompson, Bonne M et al. (2014) Genetic, anatomic, and clinical determinants of human serum sterol and vitamin D levels. Proc Natl Acad Sci U S A 111:E4006-14
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
Wang, Jin; Zhang, Da-Wei; Lei, Ying et al. (2008) Purification and reconstitution of sterol transfer by native mouse ABCG5 and ABCG8. Biochemistry 47:5194-204