PROJECT 2: STRUCTURAL BASIS OF THE ANTI-ATHEROGENIC PROPERTIES OF APO A-l The goal of this project is to elucidate the molecular mechanisms underlying the functions of apolipoprotein (apo) A-l in reverse cholesterol transport (RCT). ApoA-l is the major protein of plasma highdensity lipoprotein (HDL) and the functions of this molecule underlie the anti-atherogenic properties of the lipoprotein. Specific Aim 1 is to define the key properties of the two tertiary structure domains of the apoA-l molecule, and to better define lipid-free human apoA-l structure in dilute solution at physiological concentrations. The influence of the properties of the two apoA-l structural domains on the affinity and mechanism of lipid binding will also be investigated. These objectives will be accomplished by using engineered apoA-l molecules and a range of physical-biochemical methods. Specific Aim 2 is to establish the mechanistic basis for the biogenesis of heterogeneous nascent HDL particles via the ATP-binding cassette transporter Al (ABCA1 )-mediated efflux of cellular lipids to apoA-l. The role of apoA-l structure and influence of cell type will be determined by measuring the efflux of phospholipid and cholesterol molecules from macrophages, fibroblasts and liver cells growing in culture to engineered apoA-l molecules. Specific Aim 3 is to define the effects of the properties of the tertiary structural domains of apoA-l on the protein's functionality in cholesterol transport using adeno-associated virus-induced expression of natural human apoA-l variants in mice to assess the effects on RCT and atherosclerosis (in collaboration with Project 3). The functionalities of the mouse HDL particles containing the apoA-l mutations in mediating cholesterol transport into and out of cells will be determined in collaboration with Project 1. The incidence of premature coronary artery disease is reduced in human populations with elevated levels of plasma HDL cholesterol and apoA-l. The reasons for this protective effect are not understood fully and this project seeks to uncover the molecular mechanisms underlying the beneficial properties of apoA-l.