Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.
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ABSTRACT:
SUBMITTER: Vamecq J
PROVIDER: S-EPMC1148584 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:Peroxisomes carry out many essential lipid metabolic functions. Nearly all of these functions require that an acyl group-either a fatty acid or the acyl side chain of a steroid derivative-be thioesterified to coenzyme A (CoA) for subsequent reactions to proceed. This thioesterification, or "activation", reaction, catalyzed by enzymes belonging to the acyl-CoA synthetase family, is thus central to cellular lipid metabolism. However, despite our rather thorough understanding of peroxisomal metabolic pathways, surprisingly little is known about the specific peroxisomal acyl-CoA synthetases that participate in these pathways. Of the 26 acyl-CoA synthetases encoded by the human and mouse genomes, only a few have been reported to be peroxisomal, including ACSL4, SLC27A2, and SLC27A4. In this review, we briefly describe the primary peroxisomal lipid metabolic pathways in which fatty acyl-CoAs participate. Then, we examine the evidence for presence and functions of acyl-CoA synthetases in peroxisomes, much of which was obtained before the existence of multiple acyl-CoA synthetase isoenzymes was known. Finally, we discuss the role(s) of peroxisome-specific acyl-CoA synthetase isoforms in lipid metabolism.
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Project description:A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.
| S-EPMC1144827 | biostudies-other
Project description:It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043--2046; Lazarow (1978) J. Biol. Chem. 253, 1522--1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system.
| S-EPMC1161412 | biostudies-other
Project description:Fatty acid transport protein (FATP)2, a member of the FATP family of fatty acid uptake mediators, has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). Here we address whether FATP2 is 1) a peroxisomal enzyme, 2) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or 3) a multifunctional protein. We found that, in mouse livers, only a minor fraction of FATP2 localizes to peroxisomes, where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2, while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Adeno-associated virus 8-based short hairpin RNA expression vectors were used to achieve liver-specific FATP2 knockdown, which significantly reduced hepatosteatosis in the face of continued high-fat feeding, concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to peroxisomal (V)LACS activity and hepatic fatty acid uptake, suggesting FATP2 as a potential novel target for the treatment of nonalcoholic fatty liver disease.
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Project description:The specificity of import of peroxisomal matrix proteins is dependent on the targeting signals encoded within their amino acid sequences. Two known import signals, peroxisomal targeting signal 1 (PTS1), positioned at the C-termini and PTS2 located close to N-termini of these proteins are recognized by the Pex5p and Pex7p receptors, respectively. However, in several yeast species, including Saccharomyces cerevisiae, proteins exist that are efficiently imported into peroxisomes despite having neither PTS1 nor PTS2 and for which no other import signal has been determined. An example of such a protein is S. cerevisiae acyl-CoA oxidase (AOx) encoded by the POX1 gene. While it is known that its import is driven by its interaction with the N-terminal segment of Pex5p, which is separate from its C-terminal PTS1-recognizing tetratricopeptide domain, to date, no AOx polypeptide region has been implicated as critical for this interaction, and thus would constitute the long-sought PTS3 signal. Using random mutagenesis combined with a two-hybrid screen, we identified single amino acid residues within the AOx polypeptide that are crucial for this interaction and for the peroxisomal import of this protein. Interestingly, while scattered throughout the primary sequence, these amino acids come close to each other within two domains of the folded AOx. Although the role of one or both of these regions as the PTS3 signal is not finally proven, our data indicate that the signal guiding AOx into peroxisomal matrix is not a linear sequence but a signal patch.
| S-EPMC7135854 | biostudies-literature
Project description:We have cloned the cDNA encoding human peroxisomal acyl-CoA oxidase, the first enzyme in the peroxisomal beta-oxidation of very long chain fatty acids. Its nucleotide sequence was found to be highly homologous (85%) to the rat cDNA counterpart. An 88% homology between rat and human was found in the COOH-terminal end of the cDNA which includes the Ser-Lys-Leu peroxisomal targeting signal common to many peroxisomal proteins. The gene spans approximately 30-40 kb and is poorly polymorphic. Southern blot analyses were performed in two previously reported siblings with an isolated peroxisomal acyl-CoA oxidase deficiency (pseudoneonatal adrenoleukodystrophy). A deletion of at least 17 kb, starting down-stream from exon 2 and extending beyond the 3' end of the gene, was observed in the two patients. These observations provide a molecular basis for the observed acyl-CoA oxidase deficiency in our family. In addition, our study will enable the characterization of the genetic defect in unrelated families with suspected acyl-CoA oxidase disorders.
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Project description:Human ABC transporters ABCD1-3 are all localized on the peroxisomal membrane and participate in the β-oxidation of fatty acyl-CoAs, but they differ from each other in substrate specificity. The transport of branched-chain fatty acids from cytosol to peroxisome is specifically driven by ABCD3, dysfunction of which causes severe liver diseases such as hepatosplenomegaly. Here we report two cryogenic electron microscopy (cryo-EM) structures of ABCD3 bound to phytanoyl-CoA and ATP at resolutions of 2.9 Å and 3.2 Å, respectively. A pair of phytanoyl-CoA molecules were observed in ABCD3, each binding to one transmembrane domain (TMD), which is distinct from our previously reported structure of ABCD1, where each fatty acyl-CoA molecule strongly crosslinks two TMDs. Upon ATP binding, ABCD3 exhibits a conformation that is open towards the peroxisomal matrix, leaving two extra densities corresponding to two CoA molecules deeply embedded in the translocation cavity. Structural analysis combined with substrate-stimulated ATPase activity assays indicated that the present structures might represent two states of ABCD3 in the transport cycle. These findings advance our understanding of fatty acid oxidation and the molecular pathology of related diseases.
| S-EPMC11369193 | biostudies-literature
Project description:Rhizomelic chondrodysplasia punctata (RCDP) is a group of disorders with overlapping clinical features including rhizomelia, chondrodysplasia punctata, coronal clefts, cervical dysplasia, congenital cataracts, profound postnatal growth retardation, severe intellectual disability, and seizures. Mutations in PEX7, GNPAT, and AGPS, all involved in the plasmalogen-biosynthesis pathway, have been described in individuals with RCDP. Here, we report the identification of mutations in another gene in plasmalogen biosynthesis, fatty acyl-CoA reductase 1 (FAR1), in two families affected by severe intellectual disability, early-onset epilepsy, microcephaly, congenital cataracts, growth retardation, and spasticity. Exome analyses revealed a homozygous in-frame indel mutation (c.495_507delinsT [p.Glu165_Pro169delinsAsp]) in two siblings from a consanguineous family and compound-heterozygous mutations (c.[787C>T];[1094A>G], p.[Arg263(?)];[Asp365Gly]) in a third unrelated individual. FAR1 reduces fatty acids to their respective fatty alcohols for the plasmalogen-biosynthesis pathway. To assess the pathogenicity of the identified mutations, we transfected human embryonic kidney 293 cells with plasmids encoding FAR1 with either wild-type or mutated constructs and extracted the lipids from the cells. We screened the lipids with gas chromatography and mass spectrometry and found that all three mutations abolished the reductase activity of FAR1, given that no fatty alcohols could be detected. We also observed reduced plasmalogens in red blood cells in one individual to a range similar to that seen in individuals with RCDP, further supporting abolished FAR1 activity. We thus expand the spectrum of clinical features associated with defects in plasmalogen biosynthesis to include FAR1 deficiency as a cause of syndromic severe intellectual disability with cataracts, epilepsy, and growth retardation but without rhizomelia.
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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: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