It is well known that plasma levels of apolipoprotein (apo)AI and high density lipoprotein (HDL) are inversely correlated with the risk of cardiovascular disease (CVD). CVDs, which include heart attacks, stroke and high blood pressure, are estimated to shorten the average American life expectancy by about 10 years. Unfortunately, the mechanisms that protect the body from the pathological accumulation of lipid and cholesterol that cause CVD are not well understood. However, recent studies have shown that lipid-poor forms of apoAI may be particularly effective at promoting cholesterol removal from the periphery by a mechanism that is distinct from that used by the bulk of plasma HDL. The long-term goal of this research is to determine how the structure of lipid-poor modulates its ability to remove cholesterol from peripheral cells such as those that comprise the vessel wall. A secondary goal is to develop variant forms of apoAI that vary in their ability to interact with cell surfaces and to promote cholesterol efflux. These mutants will not only provide valuable information on the mechanism of cholesterol removal by lipid-poor apoAI, but will also be useful for future transgenic mouse and gene therapy studies designed to improve the effectiveness of HDL in the prevention of CVD. The approaches will include the use of sophisticated fluorescence studies combined with mutants of apoAI that contain single tryptophan residues to study the structure of apoAI in the lipid- free form and in various states of HDL particle maturation. This will provide information on apoAI dynamics that is not yet been possible by X-ray crystallography or NMR. Mutagenesis techniques will also be used to modulate the ability of apoAI to interact with lipids and to cell surface proteins. These mutants will be examined using detailed lipid binding and cell surface association assays. Finally, the effect of those modifications on the ability of apoAI to promote cholesterol removal from cells will be determined in cell culture-based studies.
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