IMMUNOCHEMICAL STRUCTURE/FUNCTION OF APOLIPOPROTEIN AI

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This proposal outlines classical protein structure/function studies. It tests the hypotheses that specific structural domains of a protein can be identified that confer a specific function, and that this function can, in specific instances, be mimicked by a smaller fragment of that protein. These studies will be performed on the important plasma protein, apolipoprotein (apo) A-I. Apo A-I is the major apoprotein of plasma high density lipoproteins (HDL). Individuals with high levels of circulating HDL are at lower risk for the complications of atherosclerosis. Multiple functions of HDL contribute to its beneficial properties. Apo A-I promotes reverse cholesterol transport by facilitating cholesterol efflux from foam cells of the atherosclerotic lesion and activating the plasma esterifying enzyme, lecithin:cholesterol acyltransferase (LCAT). Structure/function relationships of this important apoprotein will be examined using two complementary approaches: an immunochemical approach that will utilize a panel of 28 unique human apo A-I-specific monoclonal antibodies and 85 purified synthetic peptides, and a structural mutant approach that will study over 15 genetically engineered and expressed mutant forms of human apo A-I. The first aim tests the hypothesis that conformational changes occur in apo A-I as the lipid-free apoprotein is assembled onto lipid-containing discs or spheres. Because X-ray crystallography cannot be used to elucidate the structure of apo A-I when it is associated with lipid, an immunochemical approach can provide this otherwise unobtainable information. The second aim will test the hypothesis that apo A-I contains specific functional domains. Antibodies will be identified that modulate cholesterol efflux from macrophages and activation of LCAT. Likewise, mutant forms of apo A-I will be identified that have altered ability to modulate cellular cholesterol efflux or act as a cofactor for LCAT. In the third and fourth aims, key peptides will be tested for their ability to mimic these apo A-I functions in vitro and in vivo, respectively. The successful completion of these studies will provide: antibody reagents that can identify and isolate metabolically distinct HDL subpopulations, molecular information about the structural requirements for the protective functional properties of apo A-I, and peptides that mimic specific functions of apo A-I in vitro and in vivo so that the beneficial role of apo A-I can be exploited for the treatment of atherosclerosis.