Expression and Characterization of Recombinant Rat Acyl-CoA Synthetases 1, 4, and 5

Abstract

Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and-resistant acyl-CoA synthetases (ACS, EC 6.2.1.3) that are linked to specific metabolic pathways. In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the iso-forms present in liver and fat cells, expressed the iso-forms as ACS-Flag fusion proteins in Escherichia coli, and purified them by Flag affinity chromatography. The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme. Purified ACS1-, 4-, and 5-Flag isoforms differed in their apparent K m values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenyl-glyoxal. The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5. The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome prolif-erator-activated receptor (PPAR) ligands, troglita-zone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS4, with an IC 50 of less than 1.5 M. Troglitazone exhibited a mixed type inhibition of ACS4.-Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPAR ligand, GW1929, which is 7-fold more potent than rosigli-tazone, inhibited ACS1 and ACS4 poorly with an IC 50 of greater than 50 M, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition. Further, the PPAR li-gands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect on ACS1,-4, or-5. These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement. The results further suggest the possibility that the insulin-sensitizing effects of the thia-zolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPAR-independent manner. Acyl-CoA synthetase (ACS, 1 EC 6.2.1.3) catalyzes the liga-tion of long chain fatty acids with coenzyme A (CoA) to produce long chain acyl-CoAs (1). The resulting acyl-CoAs can be further metabolized in pathways of-oxidation, glycerolipid synthesis , cholesteryl ester (CE) synthesis, desaturation, elonga-tion, and protein acylation and can serve as signaling molecules (2-4). Although ACS was first believed to be a con-stitutive enzyme because the activity in liver was not altered by changes in nutritional status or hormonal stimuli (5-9), the cloning of ACS1 from rat liver in 1990 disclosed that hepatic ACS1 mRNA expression is sensitively regulated by fasting and refeeding as well as by specific nutrients provided as the energy source (10). Later, four additional rat ACS isoforms from different genes were cloned that differed in tissue distribution of their mRNA expression and in substrate preference (11-14). Of these, ACS2 and ACS3 mRNAs are abundantly expressed in brain, but are not detected in liver (13, 14). The mRNAs of ACS4 and ACS5 are highly expressed in steroidogenic tissues and in intestine, respectively, and are also present in liver (11, 12). ACS1 and ACS5 have a broad substrate specificity for saturated fatty acids of 12-18 carbon atoms and unsaturated fatty acids of 16-20 carbon atoms. In contrast, ACS4 has a marked preference for arachidonic acid and eicosapentaenoic acid. The presence of three ACS isoforms in liver suggests that liver ACS activity does not change with physiological alterations (5-9) because the different ACS isoenzymes compensate for each other. All ACS isoenzymes are members of the luciferase superfam-ily and have a common structure that consists of an N terminus , two luciferase-like regions, a linker connecting the two luciferase-like regions, and a C terminus. A highly conserved AMP-binding site and a predicted fatty acid binding site are located in the first and the second luciferase-like regions, respectively (10-14). ACS1, ACS2, and ACS5 are structurally similar, with more than 60% amino acid identity (11). ACS3 and ACS4 form a second subgroup with 30% homology to ACS1 and 68% identity to each other (12). Despite their high degree of homology, the expression of ACS1 and ACS5 mRNA is regulated independently. In liver, both ACS1 and ACS5 mRNAs are increased by high sucrose refeeding whereas high fat refeeding increases only ACS1 mRNA. ACS5 is the sole member of the ACS family whose mRNA decreases with fasting (11). In 3T3-L1 cells, ACS1 mRNA is detected only after adi-pocyte differentiation, whereas ACS5 mRNA is consistently expressed independent of differentiation status (10, 11). In the