FATTY ACID BINDING PROTEINS-LIGAND SPECIFICITY

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The long range objective of the proposed research is to determine fundamental factors that regulate protein-lipid interactions within the cell. Specifically, the binding site(s) of fatty acids and sterols in fatty acid binding protein (FABP) [also called sterol carrier protein (SCP)] and the function of FABP in cholesterol esterification will be examined. This ubiquitous protein is present in microorganisms, plants, and animals, accounting for up to 14% of cytosolic protein. Despite this abundance little is know regarding 1) the function, 2) the binding specificities, affinities stoichiometries, and competition between exogenous ligands, and 3) the structures of the FABP/SCP proteins. A function in cellular fatty acid and/or sterol uptake and esterification has been proposed. Using fluorescent cholesterol analogues and time resolved fluorescence spectroscopy, we demonstrated for the first time that FABP/SCP binds sterols in vivo and in vitro. the approach is three-fold: 1) Isolate pure liver FABP/SCP and intestinal FABP/SCP which have two and one fatty acid binding sites, respectively. The coding region of the full length liver FABP and intestinal FABP cDNA will be expressed in E. coli and large quantities of the proteins will be isolated therefrom; 2) Use pure fluorescent sterols (cholestatrienol and dehydroergosterol), and fluorescent fatty acids (trans- and cis-parinaric acid) to characterize: a) the sterol binding site, b) the fatty acid binding site(s) and c) the competitive binding of sterols and fatty acids for the same or different binding sites on the FABP/SCP. since 60% of cytosolic free fatty acids are bound to FABP/SCP and FABP/SCP has similar Kd's for both fatty acids and fluorescent sterols, it seems highly likely that the cholesterol and the fatty acid carrying ability of FABP/SCP are mutually interdepending. 3) Determine the ability of these proteins to stimulate acyl-CoA Cholesteryl Acyl Transferase (ACAT) activity in vitro and in vivo under fatty acid free and fatty acid loaded conditions. The in vivo experiments will be performed with L cell fibroblasts transfected with the above protein(s). Results of these experiments should provide insights as to how a FABP/SCP bound lipid may modulate intracellular function. In addition, basic information regarding fatty acid interactions with a pure protein will be obtained.