Epidemiologic studies have shown a strong inverse relationship between high density lipoprotein cholesterol (HDL-C) levels and the risk of coronary artery disease. HDL-C levels are inversely correlated with plasma very low density lipoprotein triglyceride levels indicating a metabolic relationship between these two lipoprotein classes. HDL-C metabolism consists of several protein-mediated physical-chemical and biochemical steps. Lecithin:cholesterol acyl transferase (LCAT) and apoA-I form cholesteryl esters from free cholesterol and phosphatidycholine. The cholesteryl ester transfer protein (CETP), a phospholipid transfer protein (PLTP), and apolipoprotein D (apoD) mediate the transfer of the substrates and products of the LCAT reaction among HDL subclasses, different lipoprotein classes, and from cells. Lipid transfer protein kinetics, mechanism(s), and specificities remain undefined and their regulation of cholesterol flux in normal and diseased states is uncertain. The objectives of this proposal are (a) to determine the molecular mechanisms and the factors that regulate the activity of apoD, CETP, and PLTP using purified proteins and well-characterized model systems, (b) to determine the roles of apoD, CETP, PLTP, and LCAT on the in vitro modeling of HDL subclasses and HDL-mediated efflux of cholesterol from cells, and (c) to investigate the function of these proteins in regulating cholesterol metabolism in the golden Syrian hamster. The experimental methods will utilize well characterized model and native lipoproteins, physical/chemical studies of protein structure/function, protein purification and generation of recombinant proteins in the insect cell-baculovirus expression system, immunological techniques for protein and lipoprotein characterization, isolation, and quantitation, and plasma lipoprotein turnover and metabolism in the hamster. The long term goal of this research is to delineate the structural features of plasma lipoproteins which regulate the action of lipid transfer proteins and lipolytic enzymes. Understanding the individual contributions of these proteins is important in interpreting the role of HDL in cholesterol flux through plasma.
