The proposed research will examine fundamental factors that regulate cholesterol distribution within the cell. To this end both the domain structure of cholesterol within membranes and the transfer of cholesterol between membranes will be examined. specifically the work will focus on the role of the sterol carrier proteins, SCP and SCP-2, in regulation membrane cholesterol domain structure and in conferring specificity to intermembrane cholesterol transfer. Preliminary evidence indicates that these proteins differ markedly in their ability to enhance sterol transfer between membranes and apparently do so by entirely different mechanisms. The effect of SCPs on membrane sterol domains and on exchange of sterols between biological membranes will be examined in vitro using SCPs isolated from rat liver and recombinant SCPs obtained from E.coli, expressing the cloned SCP genes. Membrane sterol domains and sterol exchange will be measured with a fluorescent cholesterol analogue, a new assay not requiring separation of donor and acceptor membranes, monochromatic He/Cd laser excitation, and computerized multifrequency phase and modulation fluorometry.
The specific aims are to examine: 1) Role of SCPs in regulating the domain structure of cholesterol within membranes. 2) Specificity of spontaneous and SCP-mediated sterol transfer. 3) Mechanism(s) (carrier, collisional transfer, fusion, or direct effect on membranes) whereby SCPs may enhance and/or confer specificity to sterol movement. Understanding the role of SCPs in intracellular sterol movement requires a knowledge of the domain structure of cholesterol in membranes. This domain structure may have important consequences for cholesterol transport and the regulation of microsomal enzymes such as acyl CoA cholesteryl acyl transferase. The long term goal of this research is to examine the role of SCPs in the intracellular movement of cholesterol and how they may be involved in the process of cholesterol accumulation. Knowledge of these factors should be helpful in increasing our understanding of a number of diseases involving abnormal sterol trafficking and/or accumulation in cells (spur-cell anemia, neoplasia, atherosclerosis, and aging).
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