The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6-amino acid substitution into the putative active site (amino acids 249 -254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464 -523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.The murine fatty acid transport protein (FATP1) 1 was identified and cloned by Schaffer and Lodish (1) from a 3T3-L1 adipocyte cDNA expression library and is localized to the plasma and other membranes of adipocytes and other target tissues such as brain, skeletal muscle, heart, and kidney (1). The FATP1 gene is conserved widely in biology from bacteria to mammals and is one of several putative transporters of fatty acids (1-4). Factors that control FATP1 gene expression have been investigated by a number of laboratories and reveal regulation by several effector systems: up-regulation during preadipocyte differentiation (1, 5) by peroxisome proliferator-activated receptors (6 -8) and by fasting (5) and down-regulation by insulin (9). Despite a growing body of knowledge relating to the control of FATP1 gene expression, studies on the FATP1 protein and its mechanistic role in fatty acid uptake have been limited (10).FATP1 exhibits broad-based amino acid similarity to a family of very long chain coenzyme A synthetases but exhibits only limited sequence similarity to the multigene family of long chain coenzyme A synthetases. Disruption of Saccharomyces cerevisiae FAT1, the yeast homologue to mammalian FATP1 (2), results in decreased fatty acid uptake, a substantial reduction in very long chain fatty acyl-CoA synthetase activity, and the accumulation of very long chain fatty acids (11, 12). Moreover, in animal cells, fatty acid uptake is diminished in cell lines overexpressing FATP1 if either endogenous ATP levels are depleted or if FATP1 is mutated (S250A) at a putative covalent AMP bin...

Fatty acid transport protein 1 (FATP1) is an ϳ63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16 -24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the K m values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.

. interacts with the activated, phosphorylated HSL and that the association is likely to be regulatory; either delivering FA to inhibit HSL (facilitating feedback inhibition) or affecting multicomponent complex formation on the droplet surface. Adipocyte fatty acid-binding protein (AFABP)2 , also known as aP2, is the predominant member of the multigene family of intracellular lipid-binding proteins found in adipose tissue (1). The fatty acid-binding proteins (FABPs) are low molecular weight (15 kDa) abundant, ubiquitously expressed proteins that bind and sequester hydrophobic ligands such as free fatty acids and/or closely related compounds facilitating their solubilization and trafficking between various aqueous compartments of the cell. Whereas the members of the multigene family share only 20 -70% sequence identity, they fold into a structurally conserved ␤-barrel that produces an internal water-filled cavity that functions as the fatty acid binding site (1).Lipolysis in adipocytes (defined as the regulated release of fatty acids from the cell) is facilitated via a series of regulatory phosphorylation events linking receptor-mediated increases in cAMP to the phosphorylation of several key proteins including perilipin A and the hormone-sensitive lipase (HSL) resulting in an increase in triacylglycerol, diacylglycerol, and monoacylglycerol hydrolysis and efflux of fatty acid from the adipocyte (2). Perilipin A phosphorylation results in a dynamic restructuring of the lipid droplet surface, potentially providing access to the lipid droplets for lipases (3,4). Recent structure-function studies have indicated that diacylglycerol is the predominant glyceride substrate for HSL in vivo and is provided by the action of the upstream adipose triglyceride lipase (5). HSL function is under multidimensional regulation by hormones and catecholamines, and several investigators have shown that protein kinase A-dependent phosphorylation of Ser 659 and Ser 660 is required for both the translocation of HSL to the lipid droplet and increased hydrolytic activity (6,7,8). In addition, AMPKdependent phosphorylation of HSL (Ser 565 ) also results in activation of the lipase and is necessary for translocation (7,9). Concomitant with HSL phosphorylation, the phosphorylation of perilipin facilitates the translocation of HSL from the cytosol to the lipid droplet and access to the substrate (10). As such, lipolysis is a multicomponent highly regulated process linking a number of hydrolytic events leading toward fatty acid release from the adipocyte.AFABP/aP2-null mice exhibit decreased basal and hormone-stimulated lipolysis both in situ and in vivo, but demonstrate rates of fatty acid influx identical to those of wildtype mice, suggesting that the protein facilitates diffusion of fatty acids from the site of lipid hydrolysis (droplet surface) to the plasma membrane as part of lipid efflux from the cell (11). Importantly, AFABP/aP2-null mice accumulate intracellular FFA, suggesting that lipid release (lipolysis) from adipocytes is attenuat...

Molecular disruption of the lipid carrier AFABP/aP2 in mice results in improved insulin sensitivity and protection from atherosclerosis. Since small molecule inhibitors may be efficacious in defining the mechanism(s) of AFABP/aP2 action, a chemical library was screened and identified 1 (HTS01037) as a pharmacologic ligand capable of displacing the fluorophore 1-anilinonaphthalene 8-sulfonic acid from the lipid binding cavity. The X-ray crystal structure of 1 bound to AFABP/aP2 revealed that the ligand binds at a structurally similar position to a long-chain fatty acid. Similar to AFABP/aP2 knockout mice, 1 inhibits lipolysis in 3T3-L1 adipocytes and reduces LPS-stimulated inflammation in cultured macrophages. 1 acts as an antagonist of the protein-protein interaction between AFABP/aP2 and hormone sensitive lipase but does not activate PPARγ in macrophage or CV-1 cells. These results identify 1 as an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by AFABP/aP2.

Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.