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.
| Milligan, Sherrelle; Martin, Gregory G; Landrock, Danilo et al. (2018) Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice. Biochim Biophys Acta Mol Cell Biol Lipids 1863:323-338 |
| Martin, Gregory G; Seeger, Drew R; McIntosh, Avery L et al. (2018) Scp-2/Scp-x ablation in Fabp1 null mice differentially impacts hepatic endocannabinoid level depending on dietary fat. Arch Biochem Biophys 650:93-102 |
| Martin, Gregory G; Landrock, Danilo; Chung, Sarah et al. (2017) Fabp1 gene ablation inhibits high-fat diet-induced increase in brain endocannabinoids. J Neurochem 140:294-306 |
| Storey, Stephen M; Huang, Huan; McIntosh, Avery L et al. (2017) Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice. J Lipid Res 58:1153-1165 |
| McIntosh, Avery L; Storey, Stephen M; Huang, Huan et al. (2017) Sex-dependent impact of Scp-2/Scp-x gene ablation on hepatic phytol metabolism. Arch Biochem Biophys 635:17-26 |
| Landrock, Danilo; Milligan, Sherrelle; Martin, Gregory G et al. (2017) Effect of Fabp1/Scp-2/Scp-x Ablation on Whole Body and Hepatic Phenotype of Phytol-Fed Male Mice. Lipids 52:385-397 |
| Huang, Huan; McIntosh, Avery L; Martin, Gregory G et al. (2016) FABP1: A Novel Hepatic Endocannabinoid and Cannabinoid Binding Protein. Biochemistry 55:5243-55 |
| Huang, Huan; McIntosh, Avery L; Landrock, Kerstin K et al. (2015) Human FABP1 T94A variant enhances cholesterol uptake. Biochim Biophys Acta 1851:946-55 |
| Martin, Gregory G; Landrock, Danilo; Landrock, Kerstin K et al. (2015) Relative contributions of L-FABP, SCP-2/SCP-x, or both to hepatic biliary phenotype of female mice. Arch Biochem Biophys 588:25-32 |
| Klipsic, Devon; Landrock, Danilo; Martin, Gregory G et al. (2015) Impact of SCP-2/SCP-x gene ablation and dietary cholesterol on hepatic lipid accumulation. Am J Physiol Gastrointest Liver Physiol 309:G387-99 |
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