ApoA-I is the major protein component of HDL and its physiological functions are of major biological and medical importance. In this application we propose to use in vitro and in vivo approaches to elucidate the structure and functions of apoA-I. It is our hypothesis based on our recent finding, that hydrophobic residues in the carboxyl- terminal domain (residues 208-243) are essential for binding to lipids and formation of HDL. It is also our hypothesis that charged residues in the kink regions that separate the apoA-I helices may contribute to binding to SR-Bl. For in vitro studies we will capitalize on existing cell lines expressing variant forms of apoA-I that have been generated in the previous grant period and the methods we have developed for large-scale growth of cells, purification and analysis of apoA-I. Gene transfer and transgenic methodologies will be used for in vivo studies.
Our specific aims are: 1) To utilize existing permanent cell lines expressing apoA-I forms generated in the previous grant period as cell factories for synthesis of apoA-I and apoA-I containing lipoproteins. Normal and mutant apoA-I forms produced from these cell lines will be purified and characterized using established methodologies and will be utilized to study the physicochemical and functional properties of apoA- I. The physicochemical analyses are designed to map specific domains and residues of apoA-I involved in intra-and inter-molecular interactions in solution and when bound to lipids that are responsible for stabilizing the conformation and structure of apoA-I. Functional analyses will include LCAT activation and cholesterol efflux properties of the mutants. 2a) To map the domains and/or residues of apoA-I that are important for binding to the scavenger SR-BI receptor using existing as well as new apoA-I variants. The rationale of these studies is based on recent collaborative work with Dr. Krieger showing that rHDL particles compete for the binding of 125I HDL to the SRBI receptor. 2b) To map the residues of apoA-I within the carboxyl-terminal domain that are involved in binding to lipids and lipoproteins that may be essential for the assembly of HDL subclasses, using existing as well as new apoA-I variants. Epidemiological and genetic data combined with recent transgenic experiments suggest that increased apoA-I and HDL levels protect from atherosclerosis. In contrast, low apoA-I and HDL levels predispose to coronary artery disease (CAD), a leading cause of mortality worldwide. Understanding the biological functions of apoA-I and HDL which are relevant to the development of CAD may lead to new pharmacological approaches to prevent and/or treat these conditions.
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