The high density lipoproteins (HDL) have received considerable attention due to the inverse correlation of HDL-cholesterol levels and cardiovascular disease. In human plasma, the flux of cholesterol through the HDL system depends on the interrelationships of lecithin:cholesterol acyltransferase (LCAT), which converts free cholesterol and phosphatidylcholine to cholesteryl ester, and the lipid transfer proteins which provide the substrate, phospholipid transfer protein (PTP), and remove the product, cholesteryl ester transfer protein (CETP), of the reaction. The kinetics, mechanism, and specificities of these lipid transfer proteins remain undefined and their regulation of cholesterol flux in normal and diseased states uncertain. The objectives of this proposal are to isolate and characterize CETP and PTP from human plasma, to determine the compositional and structural features of the lipoprotein substrate which regulate their activity, to determine the molecular mechanisms of PTP and CETP, to characterize native and model HDL modified by PTP as substrates for LCAT and acceptors of cellular cholesterol, and to characterize the role of acceptor lipoproteins on the CETP removal of product inhibition of LCAT. These studies will use model lipoproteins with known surface composition and properties to allow critical examination of the rate-controlling factors in lipid transfer. Well-characterized apolipoprotein-phospholipid-cholesterol recombinants and apolipoprotein-phospholipid-triglyceride microemulsions will be used. Kinetic analysis will utilize the fluorescent properties of pyrenyl lipids which allows measurements to be made without the physical separation of the lipoprotein particles. This investigation will involve protein isolation, lipid synthesis, fluorescent and radioactive assays of lipid transfer, lipoprotein assembly, equilibrium binding techniques and lipid physical chemistry. The long-term goal of this research is to delineate the structural features of plasma lipoproteins which regulate the action of lipid transfer proteins and lipolytic enzymes. Understanding the individual contributions of these proteins is important in interpreting the role of HDL in cholesterol flux in human plasma.
Showing the most recent 10 out of 12 publications
