STRUCTURE/FUNCTION RELATIONSHIPS OF APOLIPOPROTEIN A (APOA-1)

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The incidence of premature coronary atherosclerosis in the human population is highly correlated to decreased concentrations of high density lipoprotein (HDL) and its major apoprotein, apo A-I found in the blood. Transgenic and knockout animal studies have shown conclusively that the "protective effect" of circulating HDL is primarily a function of its unique ability to accept and organize cholesterol. It is also a function of its ability to activate the enzyme lecithin: cholesterol acyltransferase (LCAT) for cholesterol acyltransferase (LCAT) for cholesterol to cholesterol ester conversion in the plasma compartment. The directional movement of cholesterol from the artery wall and peripheral tissues towards its only site of catabolism, the live, involves a number of well studied steps. Apo-AI appears to be to be plays a key role in each of these steps. Apo A-I is the primary acceptor for effluxed cholesterol from peripheral cells. Together with phospholipid, apo A-I and cholesterol form nascent discoidal HDL which is the preferred substrate for the plasma LCAT. This enzyme is responsible for converting newly effluxed cholesterol to cholesterol ester. Accumulation of the hydrophobic cholesterol ester as a lipid droplet in the core of spherical HDL and its ultimate delivery of cholesterol ester to the live completes the "reverse cholesterol transport" pathway. In this research proposal. we will investigate the molecular basis for the "activation of the enzyme LCAT by apo A-I. This important enzymatic pathway is known to be defective in humans who carry certain mutations within the apo A-I coding sequence. However, it is not known "how" the apo A-I protein on the surface of a nascent discoidal HDL particle co-activate this catalytic process. Therefore, to elucidate the molecular mechanism of this process we will construct a series of specific amino acid mutants using PCR mutagenesis, then produce these proteins in milligram quantities using our baculoviral Sf-9 cell system. The mutant apo A-I proteins will be extensively studied using both biochemical and biophysical techniques to determine which key structural features are responsible for properly orienting the nascent HDL phospholipid acyl chain for LCAT catalysis.