Sphingolipids are important constituents of mammalian plasma membranes and lipoproteins, and also provide key molecules for signal transduction processes. In this application, synthetic sphingolipids are proposed for study of a range of intermolecular interactions in order to obtain information about processes that affect lipid metabolism, virus infectivity, and membrane-related diseases such as sphingolipidosis, septicemia, and inflammation. Studies of molecular interactions between membrane components have functional relevance in normal membranes and in membranes associated with many disease processes, such as atherosclerosis.
The aims are: (1) to analyze the interaction of cholesterol with synthetic sphingomyelins and phosphatidylcholines modified in the acyl chains, by extending preliminary studies which show that these interactions can be probed by kinetics with cholesterol oxidase in monolayers, quantitative infrared measurements of CD2 rocking modes and CH2 wagging modes in bilayers, and magic-angle spinning nuclear magnetic resonance 1H linewidths and spinning sideband intensities and 13C linewidths and spin-relaxation time measurements; (2) to map the active site of recombinant human acid sphingomyelinase purified to homogeneity by using synthetic substrate and product analogs, as well as photoactivatable sphingolipids; (3) to examine the transmembrane target that becomes activated by lipopolysaccharide (LPS) in cells containing CD14 by using novel ceramide-lipid A synthetic conjugates that are anticipated to possess lipid A-like activity and, if the target for the action of LPS is a ceramide-activated membrane protein, to eventually interfere with LPS signalling via structural modification of the glycosphingolipid conjugate; and (4) to extend the finding that sphingolipids are specifically involved in fusion of Semliki Forest virus with target membranes by examining what molecular features are involved in its interactions with the fusion-active conformation of the viral spike, and to explore the possible target for the design of a new class of antiviral drugs. Thus, these studies with synthetic sphingolipids will impact several areas involving events related to cell surfaces and disease states.
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