Determining the Fatty Acid Substrate Preferences of Long-Chain Acyl-CoA Synthetase Isoforms

Before a fatty acid can be used in a cell, it must first be converted to its active form acyl-coenzyme A (acyl-CoA). This activation is catalyzed by a group of enzymes known as acyl-CoA synthetases, which use the energy of ATP to add a CoA group to the fatty acid to create fatty acyl-CoA. By controlling the synthesis of fatty acyl-CoAs, long-chain acyl-CoA synthetases (ACSL) can regulate fatty acid uptake and metabolism by selective activation of fatty acids. Activated fatty acids can be channeled to numerous downstream pathways after their conversion into acyl-CoA. The control over this fatty acid channeling towards different downstream pathways is not clear, but may vary depending on the isoform of the ACSL enzyme used to synthesize the acyl-CoA. Five different isoforms of ACSL (1,3,4,5,6) exist, each with varying roles in the body5. With each isoform, there is likely to be a distinct fatty acid preference and metabolic fate for the generated fatty acyl-CoA8,9,10,11. We expect each ACSL isoform to have differing chain-length and saturation preferences for its substrates. To better understand the substrate preferences of each isoform we used engineered expression vectors containing genes for each ACSL isoform along with a FLAG tag to produce purified recombinant enzyme. These expression vectors were transformed into E. coli and induced with IPTG to make recombinant protein. The FLAG-ACSL enzyme produced was affinity purified using a FLAG column and then used in an indirect spectrophotometric assay with different substrates to determine ACSL isoform substrate preference. The specific activity for each isoform was calculated with fatty acids of varying chain-length and saturation, to give quantitative values for the preferences of each isoform. Through troubleshooting and developing a protocol, we found that active isoforms were produced when induced at 25˚C for 16 hours. An indirect assay performed with purified ACSL5 showed activity with oleic acid and palmitic acid. ACSL5 showed a greater preference for palmitic acid, particularly at lower concentrations of purified protein. Due to time constrictions, and problems obtaining active ACSL isoforms, the protocol developed will have to be used in further studies to determine the substrate preferences of each ACSL isoform.Bachelor of Science in Public Healt

Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification