TRUSTEES OF BOSTON UNIVERSITY

The long-term objective of this project is to determine in molecular detail the energetic-structure-function relationship in plasma apolipoproteins and lipoproteins and thereby provide an insight into molecular mechanisms of lipoprotein action in the development of atherosclerosis and other lipoprotein-related diseases. Exchangeable apolipoproteins are soluble protein constituents of lipoproteins that mediate lipid and cholesterol transport and metabolism and play crucial roles in the pathogenesis of coronary artery disease, stroke and other major human disorders. Structural stability, compositional variability, enzymatic remodeling and oxidative modifications of plasma lipoproteins are essential for their functions and metabolism in normal and in disease states, and have to be understood in detail to elucidate molecular mechanisms of lipoprotein action. Our work addresses this goal through detailed studies of the structural stability, remodeling and fusion of various lipoproteins containing the smallest human apolipoprotein, apoC-1. ApoC-1 is an important constituent of high- and very low-density lipoproteins (HDL and VLDL); it delays the clearance of atherogenic triglyceride- rich VLDL, inhibits lipid transporters and activates enzymes that are central in cholesterol metabolism. Our studies of apoC-1-containing discoidal HDL revealed a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Interestingly, protein dissociation and lipoprotein fusion observed in our denaturation studies of HDL and VLDL mimic aspects of physiological lipoprotein reactions. These results led to a shift in the existing paradigm of lipoprotein stabilization and underscored the importance of kinetic barriers for lipoprotein metabolism. In the proposed work, detailed energetic and structural analyses of apoC-1-containing discoidal and spherical HDL varying in lipid composition and oxidation modifications will be carried out by using circular dichroism, fluorescence and absorption spectroscopy, electron microscopy, differential scanning and pressure perturbation calorimetry, and mass spectrometry. This analysis will identify critical determinants (such as lipid composition, electrostatic and hydrophobic interactions, oxidation, etc.) for the kinetic stabilization and fusion of HDL. These studies will be complemented by similar studies of VLDL stability, remodeling, oxidation and protein and lipid transfer between VLDL and HDL. The results will provide the first comprehensive analysis of lipoprotein fusion, which is an important yet not well-understood reaction in lipoprotein metabilism, and of the effects of protein and lipid oxidation on this reaction. Our work will also provide the energetic and structural basis for understanding molecular mechanisms of lipoprotein action and remodeling during metabolsim, and may help design lipoproteins with new improved properties. PUBLIC HEALTH RELEVANCE Plasma lipoproteins are large complexes of proteins and lipids that mediate cholesterol transport and metabolism and are central in the development of atherosclerosis, stroke and other human diseases. Structural stability and remodeling of these complexes are necessary for their functions. Our work will provide the molecular basis for understanding structural stability, metabolic remodeling and fusion of two major classes, high- and very low-density lipoproteins (also known as Good Cholesterol and precursors of Bad Cholesterol).

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