Acyl-CoA synthetase 6 regulates long-chain polyunsaturated fatty acid composition of membrane phospholipids in spermatids and supports normal spermatogenic processes in mice.
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ABSTRACT:
SUBMITTER: Shishikura K
PROVIDER: S-EPMC6894091 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 β-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.
| S-EPMC3772793 | biostudies-literature
Project description:Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.
| S-EPMC4700060 | biostudies-literature
Project description:Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.
| S-EPMC3196225 | biostudies-literature
Project description:The contribution of lipid metabolic pathways to malignancy is poorly understood. Expression of the fatty acyl-CoA synthetase ACSVL3 was found to be markedly elevated in clinical malignant glioma specimens but nearly undetectable in normal glia. ACSVL3 levels correlated with the malignant behavior of human glioma cell lines and glioma cells propagated as xenografts. ACSVL3 expression was induced by the activation of oncogenic receptor tyrosine kinases (RTK) c-Met and epidermal growth factor receptor. Inhibiting c-Met activation with neutralizing anti-hepatocyte growth factor monoclonal antibodies reduced ACSVL3 expression concurrent with tumor growth inhibition in vivo. ACSVL3 expression knockdown using RNA interference, which decreased long-chain fatty acid activation, inhibited anchorage-dependent and anchorage-independent glioma cell growth by approximately 70% and approximately 90%, respectively. ACSVL3-depleted cells were less tumorigenic than control cells, and subcutaneous xenografts grew approximately 60% slower than control tumors. Orthotopic xenografts produced by ACSVL3-depleted cells were 82% to 86% smaller than control xenografts. ACSVL3 knockdown disrupted Akt function as evidenced by RTK-induced transient decreases in total and phosphorylated Akt, as well as glycogen synthase kinase 3beta, via a caspase-dependent mechanism. Expressing constitutively active myr-Akt rescued cells from the anchorage-dependent and anchorage-independent growth inhibitory effects of ACSVL3 depletion. These studies show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function.
| S-EPMC2795097 | biostudies-literature
Project description:ObjectiveAcyl-CoA synthetase short-chain family member 2 (ACSS2) activity provides a major source of acetyl-CoA to drive histone acetylation. This study aimed to unravel the ACSS2 expression during mouse spermatogenesis, where a dynamic and stage-specific genome-wide histone hyperacetylation occurs before histone eviction.Materials and methodsIn this experimental study, ACSS2 expression levels during spermatogenesis were verified by Immunodetection. Testis paraffin-embedded sections were used for IHC staining with anti-H4 pan ac and anti-ACSS2. Co-detection of ACSS2 and H4K5ac was performed on testis tubular sections by immunofluorescence. Proteins extracts from fractionated male germ cells were subjected to western-blotting and immunoblot was probed with anti- ACSS2 and anti-actin.ResultsThe resulting data showed that the commitment of progenitor cells into meiotic divisions leads to a robust accumulation of ACSS2 in the cell nucleus, especially in pachytene spermatocytes (P). However, ACSS2 protein drastically declines during post-meiotic stages, when a genome-wide histone hyperacetylation is known to occur.ConclusionThe results of this study are in agreement with the idea that the major function of ACSS2 is to recycle acetate generated after histone deacetylation to regenerate acetyl-CoA which is required to maintain the steady state of histone acetylation. Thus, it is suggested that in spermatogenic cells, nuclear activity of ACSS2 maintains the acetate recycling until histone hyperacetylation, but disappears before the acetylation-dependent histone degradation.
| S-EPMC6791068 | biostudies-literature
Project description:Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥.
| S-EPMC5454516 | biostudies-literature
Project description:Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [(14)C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.
| S-EPMC3963287 | biostudies-literature
Project description:The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.
| S-EPMC3311324 | biostudies-literature
Project description:A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23-28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.
| S-EPMC34396 | biostudies-literature
Project description:AimTo investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C.MethodsThe 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 μL 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.ResultsExpression 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 μmol/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 μmol/L), total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.ConclusionThe data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.
| S-EPMC3235631 | biostudies-literature