The incidence of premature coronary atherosclerosis in the human population is highly correlated to decreased concentrations of high density lipoprotein and its major apolipoprotein constituent, apoA-I. One hypothesis describing the protective effect of HDL is based on its purported role in a process called """"""""reverse cholesterol transport"""""""". This hypothesis assumes that efflux of free cholesterol from cellular membranes is driven by its utilization in an esterification reaction catalyzed by lecithin:cholesterol acyltransferase (LCAT) during the synthesis of HDL cholesteryl esters. This process can potentially facilitate efflux of cholesterol from peripheral tissues to HDL and consequently to the liver where cholesterol is eliminated by the body. Thus, apoA-I has two important physiological functions, it is a key structural component of HDL as a result of its unique lipid binding capacity and secondly it acts as structural component of HDL as a result of its unique lipid binding capacity and secondly it acts as cofactor for the catalytic conversion of HDL cholesterol to cholesteryl ester by LCAT. In order to gain a clearer understanding of the key structural features of apoA-I that determine the dual functionality of this important apolipoprotein, we have designed studies to identify structure:function relationships within apoA-I. These studies will potentially provide a mechanistic view of apoA-I's activation of the phospholipid substrate and to specifically determine which structural domains of apoA-I are most important in the activation of LCAT. Our approach will utilize the construction nd characterization of mutant apoA-I proteins generated by site-directed mutagenesis and a complete analysis of their lipid binding affinity and LCAT activation properties. ApoA-I amphipathic alpha- helices showing the highest inter-species conservation of the 22 amino acid amphipathic domains within exon 4 have been identified and are targeted for mutation analysis. Mutants will be made which will allow for the assessment of the importance of amphipathic helix inter-facial charge density and helix hydrophobic moment on lipid binding and LCAT activation. The hypothesis that will be directly tested by the studies proposed in this application is that multiple amphipathic alpha-helices are involved in stabilizing lipid:protein interaction required for cholesterol esterification and that one or more of these same helices provide the proper interfacial orientation of boundary phospholipid of LCAT. In this model specific apoA-I amphipathic helices interact with the phospholipid bilayer and interact with the boundary lipid molecules for enzyme catalyses by LCAT. It is possible that amphipathic peptides penetrate the phospholipid surface to a certain critical depth and increase the accessibility of the sn-2 fatty acid carboxyl group to enzymatic attack by modulating the physical properties of the acyl chain. In summary, these studies will provide for the first time a correlation of protein secondary structure, representing the entire apoA-I molecule, to the individual functional domains within this polypeptide, thereby aiding our understanding of apoA-I's role in regulating cholesterol homeostasis.
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