The transfer of lipids, and particularly that of phospholipids to and from HDL, mediated by plasma transfer proteins is important for lipoprotein metabolism but the mechanisms of these processes are poorly understood. The objectives of this proposal are to address several broad questions related to the activities of lipid transfer proteins (LTPs) in human plasma. The primary emphasis will be on understanding the mechanism of LTP-2 transfer of phospholipids and the effects of this activity on the remodelling of HDL. There are 5 Specific Aims. One is to determine the molecular mechanisms by which plasma lipid transfer proteins transport lipids between lipid surfaces that are in contact with the plasma compartment. The second is to identify the structural and compositional determinants of plasma lipoproteins that regulate lipid transfer activity. A third objective is to identify the changes in lipid composition and structure that are mediated by lipid transfer proteins and to identify conditions under which phospholipid transfer might enhance neutral lipid transfer. Fourthly, using a combination of photo-affinity labeling and site-directed mutagenesis, the ligand-binding and surface-associating sites of the two major lipid transfer proteins of human plasma will be identified. Finally, the investigators will reconstitute the activities that remodel HDL using the appropriate combinations and concentrations of LTP-2, lecithin-cholesterol acyltransferase (LCAT), LTP-1, hepatic lipase, very low density lipoproteins (VLDL) and lipoprotein lipase. Successful completion of these aims will aid in the understanding of how these factors are altered in abnormal lipid metabolism. This project involves lipid synthesis, lipoprotein isolation and reassembly, kinetic and equilibrium analysis, photoactivation analysis, and membrane isolation.
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