A novel acyl-CoA-binding protein from bovine liver. Effect on fatty acid synthesis.
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ABSTRACT:
SUBMITTER: Mogensen IB
PROVIDER: S-EPMC1147542 | biostudies-other | 1987 Jan
REPOSITORIES: biostudies-other
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Project description:Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi-dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O₂ consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.
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Project description:The diffuse nature of Glioblastoma (GBM) tumors poses a challenge to current therapeutic options. We have previously shown that Acyl-CoA Binding Protein (ACBP, also known as DBI) regulates lipid metabolism in GBM cells, favoring fatty acid oxidation (FAO). Here we show that ACBP downregulation results in wide transcriptional changes affecting invasion-related genes. In vivo experiments using patient-derived xenografts combined with in vitro models demonstrated that ACBP sustains GBM invasion via binding to fatty acyl-CoAs. Blocking FAO mimics ACBPKD-induced immobility, a cellular phenotype that can be rescued by increasing FAO rates. Further investigation into ACBP-downstream pathways served to identify Integrin beta-1, a gene downregulated upon inhibition of either ACBP expression or FAO rates, as a mediator for ACBP's role in GBM invasion. Altogether, our findings highlight a role for FAO in GBM invasion and reveal ACBP as a therapeutic vulnerability to stall FAO and subsequent cell invasion in GBM tumors.
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Project description:Oxylipins are crucial components in plant wound responses that are mobilised via the plant vasculature. Previous studies have shown that the overexpression of an Arabidopsis acyl-CoA-binding protein, AtACBP3, led to an accumulation of oxylipin-containing galactolipids, and AtACBP3pro::BETA-GLUCURONIDASE (GUS) was expressed in the phloem of transgenic Arabidopsis. To investigate the role of AtACBP3 in the phloem, reverse transcription-polymerase chain reaction and western blot analysis of phloem exudates from the acbp3 mutant and wild type revealed that the AtACBP3 protein, but not its mRNA, was detected in the phloem sap. Furthermore, micrografting demonstrated that AtACBP3 expressed from the 35S promoter was translocated from shoot to root. Subsequently, AtACBP3 was localised to the companion cells, sieve elements and the apoplastic space of phloem tissue by immunogold electron microscopy using anti-AtACBP3 antibodies. AtACBP3pro::GUS was induced locally in Arabidopsis leaves upon wounding, and the expression of wound-responsive jasmonic acid marker genes (JASMONATE ZIM-DOMAIN10, VEGETATIVE STORAGE PROTEIN2, and LIPOXYGENASE2) increased more significantly in both locally wounded and systemic leaves of the wild type in comparison to acbp3 and AtACBP3-RNAi. Oxylipin-related fatty acid (FA) (C18:2-FA, C18:3-FA and methyl jasmonate) content was observed to be lower in acbp3 and AtACBP3-RNAi than wild-type phloem exudates using gas chromatography-mass spectrometry. Experiments using recombinant AtACBP3 in isothermal titration calorimetry analysis showed that medium- and long-chain acyl-CoA esters bind (His)6-AtACBP3 with KD values in the micromolar range. Taken together, these results suggest that AtACBP3 is likely to be a phloem-mobile protein that affects the FA pool and jasmonate content in the phloem, possibly by its binding to acyl-CoA esters.
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Project description:Thirteen homologous proteins comprise the long-chain acyl-CoA synthetase (ACSL), fatty acid transport protein (FATP), and bubblegum (ACSBG) subfamilies that activate long-chain and very-long-chain fatty acids to form acyl-CoAs. Gain- and loss-of-function studies show marked differences in the ability of these enzymes to channel fatty acids into different pathways of complex lipid synthesis. Further, the ability of the ACSLs and FATPs to enhance cellular FA uptake does not always require these proteins to be present on the plasma membrane; instead, FA uptake can be increased by enhancing its conversion to acyl-CoA and its metabolism in downstream pathways. Since altered fatty acid metabolism is a hallmark of numerous metabolic diseases and pathological conditions, the ACSL, FATP and ACSBG isoforms are likely to play important roles in disease etiology.
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Project description:Acetyl-CoA carboxylases (ACCs) are enzyme complexes generally composed of three catalytic domains and distributed in all organisms. In prokaryotes and plastids of most plants, these domains are encoded in distinct subunits forming heteromeric complexes. Distinctively, cytosolic ACCs from eukaryotes and plastids of graminaceous monocots, are organized in a single multidomain polypeptide. Until now, no multidomain ACCs had been discovered in bacteria. Here, we show that a putative multidomain ACC in Saccharopolyspora erythraea is encoded by the sace_4237 gene, representing the first prokaryotic ACC homodimeric multidomain complex described. The SACE_4237 complex has both acetyl-CoA and propionyl-CoA carboxylase activities. Importantly, we demonstrate that sace_4237 is essential for S. erythraea survival as determined by the construction of a sace_4237 conditional mutant. Altogether, our results show that this prokaryotic homodimeric multidomain ACC provides malonyl-CoA for de novo fatty acid biosynthesis. Furthermore, the data presented here suggests that evolution of these enzyme complexes, from single domain subunits to eukaryotic multidomain ACCs, occurred in bacteria through domain fusion.
| S-EPMC6491548 | biostudies-literature
Project description:In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25-35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1(L)(-/-) liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [(14)C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [(14)C]oleate suggested that hepatic ACSL1 is important for mitochondrial beta-oxidation of long chain fatty acids. Because the Acsl1(L)(-/-) mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance.
| S-EPMC2788832 | biostudies-literature
Project description:BackgroundThe absorption of dietary long chain fatty acids (LCFA) largely occurs in the jejunum. LCFA are activated via conjugation with Coenzyme A (CoA), a reaction catalyzed by Acyl-CoA synthetases (ACS). Acyl-CoA sythesis is critical for dietary LCFA absorption; yet, the jejunal ACS enzymes that catalyze the reaction are largely unknown.FindingsHigh throughput mRNA sequencing of the mouse jejunum revealed that the expression of acyl-CoA synthetase 5 (Acsl5) and fatty-acid transport protein 4 (Fatp4) largely exceeded all other annotated ACS genes that activate LCFA. Interestingly, Acsl5 knockout (KO) mice displayed a decrease of 60% in jejunal total long chain acyl-CoA synthesis rate. Nevertheless, and despite of this decrease, dietary LCFA absorption and body-weight gain in response to high fat diet remained unaffected.ConclusionAcsl5 is a major activator of dietary LCFA, yet in Acsl5 KO mice residual ACS activity is sufficient for maintaining a normal LCFA absorption. Our findings provide further evidence for a robust small intestine LCFA absorption capacity.
| S-EPMC3699395 | biostudies-literature
Project description:Bovine and rat liver acyl-CoA-binding proteins (ACBP) were found to exhibit a much higher affinity for long-chain acyl-CoA esters than both bovine hepatic and cardiac fatty-acid-binding proteins (hFABP and cFABP respectively). In the Lipidex 1000- as well as the liposome-binding assay, bovine and rat hepatic ACBP effectively bound long-chain acyl-CoA ester, h- and c-FABP were, under identical conditions, unable to bind significant amounts of long-chain acyl-CoA esters. When FABP, ACBP and [1-14C]hexadecanoyl-CoA were mixed, hexadecanoyl-CoA could be shown to be bound to ACBP only. The experimental results give strong evidence that ACBP, and not FABP, is the predominant carrier of acyl-CoA in liver.
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Project description:Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitutions. In comparison with human ACBP, yeast ACBPs exhibit 48% (type 1) and 49% (type 2) conservation of amino acid residues. The amino acid sequence of S. carlsbergensis ACBP type 1 was found to be identical with the one ACBP present in Saccharomyces cerevisiae. A recombinant form of this protein was expressed in Escherichia coli and S. cerevisiae, purified, and its acyl-CoA-binding properties were characterized by isoelectric focusing and microcalorimetric analyses. The yeast ACBP was found to bind acyl-CoA esters with high affinity (Kd 0.55 x 10(-10) M). Overexpression of yeast ACBP in S. cerevisiae resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis.
| S-EPMC1137253 | biostudies-other
Project description:Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.
| S-EPMC1222666 | biostudies-other