Lipid Transfer Mechanisms

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DESCRIPTION (provided by applicant): High-density lipoproteins (HDL) are a negative (protective) risk factor for cardiovascular disease. There has been great interest in how the remodeling of HDL by plasma factors might contribute to cardioprotection. A phospholipid transfer protein (PLTP) in human plasma is a key protein in the remodeling of HDL. Although recent studies have shown that PLTP modifies HDL structure and composition even in the absence of other plasma factors, the mechanism by which this occurs remains to be elucidated. We propose three aims that will test eight hypotheses concerning the remodeling mechanism. They are as follows: Aim 1: To identify the mechanism(s) by which PLTP remodels HDL, according to the following hypotheses: a] The remodeling of HDL is specific for apolipoprotein (apo) A-I. b] PLTP-independent HDL fusion occurs through a hydrophobic patch on the HDL surface. c] HDL3 fusion, which follows PLTP-mediated apo A-I release and patch formation, is independent of PLTP. d] Fusion but not lipid transfer is inhibited by a net positive or negative charge on HDL. e] The small HDL formed by PLTP are preferential acceptors for cholesterol and a preferred substrate for LCAT. f] Phospholipid transfer is an obligatory precursor of cholesteryl ester transfer. Aim 2: To identify the determinants of PLTP molecular transfer specificity. This aim will address the hypothesis that the molecular specificity of PLTP is determined by its affinity for and binding to monomeric lipid. Aim 3: To identify the effects of PLTP on the interaction of plasma lipoproteins with macrophage-derived lipoproteins. This aim will address the hypothesis that PLTP is a key mediator of the association of macrophage-derived lipoproteins with more mature forms of HDL. The major techniques to be employed are site-directed mutagenesis, assays of lecithin:cholesterol acyltransferase and phospholipase activity, tissue culture (P388D1 macrophages), equilibrium measurements and lipid and protein assays.