This project describes the chemistry component of a Program Project designed to gain understanding of cellular cholesterol efflux and formation of high density lipoprotein (HDL). Photoaffinity labeling is a powerful tool for the analysis of lipid-protein and lipid-lipid interactions in biologically active complexes and living cells, and in this project a variety of lipids, including sterol, phosphatidylcholine, and sphingomyelin analogs containing photoactivable benzophenone groups at various defined positions within the structures, will be synthesized. Analogs of cholesterol will be synthesized with the photophore at strategically designed positions extending from ring A at one end of the sterol to the ring D side chain. Phosphatidylcholine analogs will be synthesized with benzophenone groups at various positions within the 1- or 2-acyl side chains, or in the choline moiety. Sphingomyelin analogs will be synthesized bearing benzophenone groups within the N-acyl chain or in the choline moiety. Finally, benzophenone-containing oxysterols may be required to explore the basis of their ability to displace FC from its protein binding sites. Syntheses of selected tritium-labeled lipids among these compounds will be required, and these will also be prepared in this project. These synthetic lipids are readily incorporated into living cells, and in particular into cell surface caveolae. In the presence of apolipoprotein A-1 (apo A-1, the major protein of plasma high density lipoprotein, HDL) cellular cholesterol and phospholipids, and their synthesized photoactivable analogs, are transferred out of the cell to form lipoprotein complexes. The organization of lipids in these complexes will be stabilized by covalent crosslinks caused by photoactivation of the complexed analogs. Analyses of the photolabeled, crosslinked products by chromatographic methods and electrospray mass spectrometry will provide novel information on the organization of lipids and proteins in caveolae and in newly formed HDL. Since there is clear evidence that HDL is a major protective factor against atherosclerosis, the insights into HDL genesis provided by these experiments may suggest new approaches for combating human heart disease.
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