Studies on the effect of an heterologous fatty acid-binding protein on acyl-CoA oxidase induction in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Smaczynska I
PROVIDER: S-EPMC1137125 | biostudies-other | 1994 Jul
REPOSITORIES: biostudies-other
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Project description:In the present study, we have used DNA microarray and quantitative real-time PCR analysis to examine the transcriptional changes that occur in response to cellular depletion of the yeast acyl-CoA-binding protein, Acb1p. Depletion of Acb1p resulted in the differential expression of genes encoding proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), ion transport and uptake (e.g. ITR1 and HNM1) and stress response (e.g. HSP12, DDR2 and CTT1). In the present study, we show that transcription of the INO1 gene, which encodes inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline, and UAS(INO1) (inositol-sensitive upstream activating sequence)-driven transcription is enhanced in Acb1p-depleted cells. In addition, the reduction in inositol-mediated repression of INO1 transcription observed after depletion of Acb1p appeared to be independent of the transcriptional repressor, Opi1p. We also demonstrated that INO1 and OPI3 expression can be normalized in Acb1p-depleted cells by the addition of high concentrations of exogenous fatty acids, or by the overexpression of FAS1 or ACC1. Together, these findings revealed an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p mutant which is unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implied that gene expression is modulated either by the Acb1p-acyl-CoA ester complex directly or by its ability to donate acyl-CoA esters to utilizing systems.
| S-EPMC2049021 | biostudies-literature
Project description:Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.
| S-EPMC7573125 | biostudies-literature
Project description:Fatty acyl-coenzyme A oxidase 1 (ACOX1) knockout (ACOX1(-/-)) mice manifest hepatic metabolic derangements that lead to the development of steatohepatitis, hepatocellular regeneration, spontaneous peroxisome proliferation, and hepatocellular carcinomas. Deficiency of ACOX1 results in unmetabolized substrates of this enzyme that function as biological ligands for peroxisome proliferator-activated receptor-? (PPAR?) in liver. Here we demonstrate that sustained activation of PPAR? in ACOX1(-/-) mouse liver by these ACOX1 substrates results in endoplasmic reticulum (ER) stress. Overexpression of transcriptional regulator p8 and its ER stress-related effectors such as the pseudokinase tribbles homolog 3, activating transcription factor 4, and transcription factor CCAAT/-enhancer-binding protein homologous protein as well as phosphorylation of eukaryotic translation initiation factor 2?, indicate the induction of unfolded protein response signaling in the ACOX1(-/-) mouse liver. We also show here that, in the liver, p8 is a target for all three PPAR isoforms (-?, -?, and -?), which interact with peroxisome proliferator response elements in p8 promoter. Sustained activation of p8 and unfolded protein response-associated ER stress in ACOX1(-/-) mouse liver contributes to hepatocyte apoptosis and liver cell proliferation culminating in the development of hepatocarcinogenesis. We also demonstrate that human ACOX1 transgene is functional in ACOX1(-/-) mice and effectively prevents metabolic dysfunctions that lead to ER stress and carcinogenic effects. Taken together, our data indicate that progressive PPAR?- and p8-mediated ER stress contribute to the hepatocarcinogenesis in ACOX1(-/-) mice.
| S-EPMC3157234 | biostudies-literature
Project description:We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells and the size of the acyl-CoA pool. Thus ACBP can act as an intracellular acyl-CoA pool former. Possible physiological functions of ACBP in cells are discussed.
| S-EPMC1132282 | biostudies-other
Project description:BACKGROUND:Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. RESULTS:cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. CONCLUSION:The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.
| S-EPMC3265415 | biostudies-literature
Project description:The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.
| S-EPMC2943281 | biostudies-literature
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.
| S-EPMC10148872 | biostudies-literature
Project description:Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.
| S-EPMC1148584 | biostudies-other
Project description:BackgroundsAcyl-coenzyme A (CoA) esters are important intermediates in lipid metabolism with regulatory properties. Acyl-CoA-binding proteins bind and transport acyl-CoAs to fulfill these functions. RICE ACYL-COA-BINDING PROTEIN6 (OsACBP6) is currently the only one peroxisome-localized plant ACBP that has been proposed to be involved in β-oxidation in transgenic Arabidopsis. The role of the peroxisomal ACBP (OsACBP6) in rice (Oryza sativa) was investigated.ResultsHere, we report on the function of OsACBP6 in rice. The osacbp6 mutant showed diminished growth with reduction in root meristem activity and leaf growth. Acyl-CoA profiling and lipidomic analysis revealed an increase in acyl-CoA content and a slight triacylglycerol accumulation caused by the loss of OsACBP6. Comparative transcriptomic analysis discerned the biological processes arising from the loss of OsACBP6. Reduced response to oxidative stress was represented by a decline in gene expression of a group of peroxidases and peroxidase activities. An elevation in hydrogen peroxide was observed in both roots and shoots/leaves of osacbp6. Taken together, loss of OsACBP6 not only resulted in a disruption of the acyl-CoA homeostasis but also peroxidase-dependent reactive oxygen species (ROS) homeostasis. In contrast, osacbp6-complemented transgenic rice displayed similar phenotype to the wild type rice, supporting a role for OsACBP6 in the maintenance of the acyl-CoA pool and ROS homeostasis. Furthermore, quantification of plant hormones supported the findings observed in the transcriptome and an increase in jasmonic acid level occurred in osacbp6.ConclusionsIn summary, OsACBP6 appears to be required for the efficient utilization of acyl-CoAs. Disruption of OsACBP6 compromises growth and led to provoked defense response, suggesting a correlation of enhanced acyl-CoAs content with defense responses.
| S-EPMC7647982 | biostudies-literature
Project description:In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and carboxypeptidase Y is slightly delayed in Acb1p-depleted cells, whereas the maturation of alkaline phosphatase and Gas1p is unaffected. The fact that Gas1p maturation is unaffected by Acb1p depletion, despite the lowered ceramide content in these cells, indicates that ceramide synthesis in yeast could be compartmentalized. We suggest that the reduced ceramide synthesis in Acb1p-depleted cells leads to severely altered vacuole morphology, perturbed vacuole assembly and strong inhibition of homotypic vacuole fusion.
| S-EPMC1224232 | biostudies-other