The long-term goal of our research program is to understand the molecular basis whereby exchangeable apolipoproteins regulate plasma lipid homeostasis. Recent comparative studies of human and mouse genome sequences led to the discovery of a new member of the exchangeable apolipoprotein family, apolipoprotein A-V (apoA-V). Transgenic and gene disruption experiments in mice have revealed a correlation between apoA-V and plasma triacylglycerol (TG) levels. Given the strong relationship between elevated plasma TG and risk for cardiovascular disease, apoA-V is a potential target for therapeutic intervention. The hypothesis that apoA-V modulates plasma TG levels by influencing hepatic lipoprotein assembly or secretion will be tested in primary hepatocytes from apoA-V knockout and transgenic mice as well as cultured human hepatoma cells. Studies will evaluate the extent to which apoA-V expression in liver cells affects the assembly or secretion of TG-rich lipoproteins. Conditioned medium from cultured cells will be analyzed for lipoprotein content and composition, including apoA-V levels. Fluorescence studies will examine the intracellular localization or trafficking of apoA-V in HepG2 and hepatocytes. Far U.V. circular dichroism spectroscopy will be used to characterize the free energy of unfolding and domain organization of lipid-free and lipid-associated apoA-V. Chemical cleavage of apoA-V will be performed to obtain fragments for examination of the hypothesis that the protein possesses independently folded domains. N- or C-terminal truncation variants will be engineered and characterized in terms of stability, lipid binding and biological effects. ApoA-V tertiary structure and lipid-induced conformational changes will be evaluated by fluorescence resonance energy transfer studies. Site directed mutagenesis will be performed to sequentially replace Trp residues in apoA-V, thereby generating a panel of discrete single Trp apoA-V variants. Cysteine 204 will be labeled with an extrinsic fluorescent probe and distance determinations between these donor/ acceptor pairs made. Taken together, proposed research will employ a combination of functional studies and structural characterization to improve our understanding of the mechanism whereby apoA-V modulates plasma TG levels.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL073061-02
Application #
6867399
Study Section
Metabolism Study Section (MET)
Program Officer
Applebaum-Bowden, Deborah
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
2
Fiscal Year
2005
Total Cost
$400,250
Indirect Cost
Name
Children's Hospital & Res Ctr at Oakland
Department
Type
DUNS #
076536184
City
Oakland
State
CA
Country
United States
Zip Code
94609
Forte, Trudy M; Ryan, Robert O (2015) Apolipoprotein A5: Extracellular and Intracellular Roles in Triglyceride Metabolism. Curr Drug Targets 16:1274-80
Sharma, Vineeta; Ryan, Robert O; Forte, Trudy M (2012) Apolipoprotein A-V dependent modulation of plasma triacylglycerol: a puzzlement. Biochim Biophys Acta 1821:795-9
Gin, Peter; Beigneux, Anne P; Voss, Constance et al. (2011) Binding preferences for GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. Arterioscler Thromb Vasc Biol 31:176-82
Shu, Xiao; Nelbach, Lisa; Ryan, Robert O et al. (2010) Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation. Biochim Biophys Acta 1801:605-8
Mauldin, Kasuen; Lee, Brian L; Oleszczuk, Marta et al. (2010) The carboxyl-terminal segment of apolipoprotein A-V undergoes a lipid-induced conformational change. Biochemistry 49:4821-6
Shu, Xiao; Nelbach, Lisa; Weinstein, Michael M et al. (2010) Intravenous injection of apolipoprotein A-V reconstituted high-density lipoprotein decreases hypertriglyceridemia in apoav-/- mice and requires glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1. Arterioscler Thromb Vasc Biol 30:2504-9
Wong-Mauldin, Kasuen; Raussens, Vincent; Forte, Trudy M et al. (2009) Apolipoprotein A-V N-terminal domain lipid interaction properties in vitro explain the hypertriglyceridemic phenotype associated with natural truncation mutants. J Biol Chem 284:33369-76
Forte, Trudy M; Shu, Xiao; Ryan, Robert O (2009) The ins (cell) and outs (plasma) of apolipoprotein A-V. J Lipid Res 50 Suppl:S150-5
Beigneux, Anne P; Gin, Peter; Davies, Brandon S J et al. (2008) Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase. J Lipid Res 49:1312-21
Gin, Peter; Yin, Liya; Davies, Brandon S J et al. (2008) The acidic domain of GPIHBP1 is important for the binding of lipoprotein lipase and chylomicrons. J Biol Chem 283:29554-62

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