The long range objective of the proposed research is to determine fundamental factors that regulate protein-lipid interactions within the cell. Specifically, we propose to examine the structure and ligand specificity of the lipid binding site(s) in recombinant fatty acid binding protein (FABP) [also called sterol carrier protein (SCP)], the role of FABP/SCP in fatty acid uptake and sterol uptake in intact cells, and the participation of FABP/SCP in fatty acid and sterol metabolism in vitro and in intact cells. The FABP/SCPs are ubiquitous proteins representing up to 14% of cell cytosolic protein. Although a massive amount of research into the FABP/SCPs in the past two decades has provided much circumstantial evidence for a role of FABP/SCP in intracellular fatty acid transport and utilization, conclusive evidence for their physiological function(s) is not yet available. Likewise, most of the information regarding the structure of the FABP/SCP ligand binding site has only been derived from comparative studies of amino acid sequence and secondary structure predictions. The approach is four-fold: 1) Isolate and resolve into isoforms recombinant liver FABP/SCP and intestinal FABP/SCP from E coli expressing the respective cDNAs. Phosphorylate the FABP/SCP either in vitro or in L-cell fibroblasts expressing the respective cDNA. 2) Determine the role of isoforms and phosphorylation on the structure of the FABP/SCP ligand binding site(s), ligand specificity (fatty acids, fatty acyl CoAs, cholesterol), and competitive ligand interactions with radiolabeled and fluorescent ligands, photoaffinity labels, phase fluorometry, and circular dichroism 3) Determine the ability of FABP/SCPs to alter metabolism of fatty acids, fatty acyl CoAs, and sterol in vitro and in vivo in transfected L cells. 4. Utilize fluorescent and radiolabeled fatty acids to examine the plasma membrane fatty acid transport system and its interaction with cytosolic FABP/SCP in L cell fibroblasts transfected with cDNA encoding liver or intestinal FABP/SCP. The unique application of structural and molecular biology in these experiments is designed to provide new insights as to how FABP/SCP bound ligands may modulate intracellular lipid metabolism and how fatty acids, fatty acyl CoAs, and sterols interact with intracellular lipid transfer proteins.
| 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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