Dataset Information

Phosphorylation and Acetylation of Acyl-CoA Synthetase- I.

ABSTRACT: Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post-translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 differentiated adipocytes, treated these cells with different hormones, and analyzed the resulting phosphorylated and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25 phosphorylation sites on ACSL1. Several unique acetylation and phosphorylation sites occurred under conditions in which fatty acid ?-oxidation is normally enhanced. Thirteen of the acetylated lysines had not previously been identified, and none of the phosphorylation sites had been previously identified. Site-directed mutagenesis was used to introduce mutations at three potential acetylation and phosphorylation sites believed to be important for ACSL1 function. At the ATP/AMP binding site and at a highly conserved site near the C terminus, modifications of Ser278 or Lys676, respectively, totally inhibited ACSL1 activity. In contrast, mutations of Lys285 that mimicked acetylation (Lys285Ala and Lys285Gln) reduced ACSL activity, whereas full activity was retained by Lys285Arg, suggesting that acetylation of Lys285 would be likely to decrease ACSL1 activity. These results indicate that ACSL1 is highly modified post-translationally. Several of these modifications would be expected to alter enzymatic function, but others may affect protein stability or protein-protein interactions.

SUBMITTER: Frahm JL

PROVIDER: S-EPMC3772793 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Phosphorylation and Acetylation of Acyl-CoA Synthetase- I.

Frahm Jennifer L JL Li Lei O LO Grevengoed Trisha J TJ Coleman Rosalind A RA

Journal of proteomics & bioinformatics 20110701 7

Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post-translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 diffe ...[more]

PMID: 24039348

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Project description:To investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C.The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes. Ni(2+)-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C. In addition, ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored. ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence. The ACSL assay mix included TrisHCl (pH 7.4), ATP, CoA, EDTA, DTT, MgCl(2), [9,10-(3)H] palmitic acid, and triton X-100. The 200 ?L reaction was initiated with the addition of solubilized, purified recombinant proteins or cellular lysates. Reactions were terminated after 10, 30 or 60 min of incubation with Doles medium.Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl beta-D-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni(2+)-affinity chromatography. Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5, as well as recombinant rat ACSL1 (sensitive control), but not recombinant rat ACSL5 (insensitive control). The IC50 for human ACSL5 was about 10 ?mol/L. The inhibitory triacsin C effect was similar for different incubation times (10, 30 and 60 min) and was not modified by the N- or C-terminal location of the 6xHis-tag. In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment, stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed. In both models, ACSL5 peak activity was found at pH 7.5 and pH 9.5, corresponding to the properties of recombinant human ACSL5 protein. In the presence of triacsin C (25 ?mol/L), total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.

Dataset Information

Phosphorylation and Acetylation of Acyl-CoA Synthetase- I.

Publications

Phosphorylation and Acetylation of Acyl-CoA Synthetase- I.

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