Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Peroxisomal acyl-coenzyme A (acyl-CoA) oxidase deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation due to a deficiency of straight-chain acyl-CoA oxidase (SCOX). The biochemical hallmark of this disorder is the accumulation of very long-chain fatty acids. Although some case reports and small series of patients have been published, a comprehensive overview of the clinical, biochemical, and mutational spectrum of this disorder is still lacking. For this reason, we report clinical information for a cohort of 22 patients with peroxisomal acyl-CoA oxidase deficiency and the results from biochemical and mutation analyses in fibroblasts of the patients. No clear genotype-phenotype correlation was observed. An intriguing mutation in the alternatively-spliced transcript encoding the isoform SCOX-exon 3II in a patient with normal expression of the transcript encoding the isoform SCOX-exon 3I, prompted us to characterize these two isoforms of human SCOX. The recombinant SCOX-exon 3I displayed activity toward medium-chain fatty acyl-CoAs and was not active with very long-chain fatty acyl-CoAs. In contrast, recombinant SCOX-exon 3II was capable of oxidizing a broad range of substrates, including very long-chain fatty acyl-CoAs. These results explain why this patient with a mutation in exon 3II of the ACOX1 gene, but with normal expression of exon 3I, was indistinguishable from other patients with peroxisomal acyl-CoA oxidase deficiency with respect to his clinical presentation and the biochemical abnormalities in his fibroblasts.
Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Peroxisomal acyl-coenzyme A (acyl-CoA) oxidase deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation due to a deficiency of straight-chain acyl-CoA oxidase (SCOX). The biochemical hallmark of this disorder is the accumulation of very long-chain fatty acids. Although some case reports and small series of patients have been published, a comprehensive overview of the clinical, biochemical, and mutational spectrum of this disorder is still lacking. For this reason, we report clinical information for a cohort of 22 patients with peroxisomal acyl-CoA oxidase deficiency and the results from biochemical and mutation analyses in fibroblasts of the patients. No clear genotype-phenotype correlation was observed. An intriguing mutation in the alternatively-spliced transcript encoding the isoform SCOX-exon 3II in a patient with normal expression of the transcript encoding the isoform SCOX-exon 3I, prompted us to characterize these two isoforms of human SCOX. The recombinant SCOX-exon 3I displayed activity toward medium-chain fatty acyl-CoAs and was not active with very long-chain fatty acyl-CoAs. In contrast, recombinant SCOX-exon 3II was capable of oxidizing a broad range of substrates, including very long-chain fatty acyl-CoAs. These results explain why this patient with a mutation in exon 3II of the ACOX1 gene, but with normal expression of exon 3I, was indistinguishable from other patients with peroxisomal acyl-CoA oxidase deficiency with respect to his clinical presentation and the biochemical abnormalities in his fibroblasts.
Peroxisomal acyl-coenzyme A (acyl-CoA) oxidase deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation due to a deficiency of straight-chain acyl-CoA oxidase (SCOX). The biochemical hallmark of this disorder is the accumulation of very long-chain fatty acids. Although some case reports and small series of patients have been published, a comprehensive overview of the clinical, biochemical, and mutational spectrum of this disorder is still lacking. For this reason, we report clinical information for a cohort of 22 patients with peroxisomal acyl-CoA oxidase deficiency and the results from biochemical and mutation analyses in fibroblasts of the patients. No clear genotype-phenotype correlation was observed. An intriguing mutation in the alternatively-spliced transcript encoding the isoform SCOX-exon 3II in a patient with normal expression of the transcript encoding the isoform SCOX-exon 3I, prompted us to characterize these two isoforms of human SCOX. The recombinant SCOX-exon 3I displayed activity toward medium-chain fatty acyl-CoAs and was not active with very long-chain fatty acyl-CoAs. In contrast, recombinant SCOX-exon 3II was capable of oxidizing a broad range of substrates, including very long-chain fatty acyl-CoAs. These results explain why this patient with a mutation in exon 3II of the ACOX1 gene, but with normal expression of exon 3I, was indistinguishable from other patients with peroxisomal acyl-CoA oxidase deficiency with respect to his clinical presentation and the biochemical abnormalities in his fibroblasts.
Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Peroxisomal acyl-coenzyme A (acyl-CoA) oxidase deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation due to a deficiency of straight-chain acyl-CoA oxidase (SCOX). The biochemical hallmark of this disorder is the accumulation of very long-chain fatty acids. Although some case reports and small series of patients have been published, a comprehensive overview of the clinical, biochemical, and mutational spectrum of this disorder is still lacking. For this reason, we report clinical information for a cohort of 22 patients with peroxisomal acyl-CoA oxidase deficiency and the results from biochemical and mutation analyses in fibroblasts of the patients. No clear genotype-phenotype correlation was observed. An intriguing mutation in the alternatively-spliced transcript encoding the isoform SCOX-exon 3II in a patient with normal expression of the transcript encoding the isoform SCOX-exon 3I, prompted us to characterize these two isoforms of human SCOX. The recombinant SCOX-exon 3I displayed activity toward medium-chain fatty acyl-CoAs and was not active with very long-chain fatty acyl-CoAs. In contrast, recombinant SCOX-exon 3II was capable of oxidizing a broad range of substrates, including very long-chain fatty acyl-CoAs. These results explain why this patient with a mutation in exon 3II of the ACOX1 gene, but with normal expression of exon 3I, was indistinguishable from other patients with peroxisomal acyl-CoA oxidase deficiency with respect to his clinical presentation and the biochemical abnormalities in his fibroblasts.
Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Peroxisomal acyl-coenzyme A (acyl-CoA) oxidase deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation due to a deficiency of straight-chain acyl-CoA oxidase (SCOX). The biochemical hallmark of this disorder is the accumulation of very long-chain fatty acids. Although some case reports and small series of patients have been published, a comprehensive overview of the clinical, biochemical, and mutational spectrum of this disorder is still lacking. For this reason, we report clinical information for a cohort of 22 patients with peroxisomal acyl-CoA oxidase deficiency and the results from biochemical and mutation analyses in fibroblasts of the patients. No clear genotype-phenotype correlation was observed. An intriguing mutation in the alternatively-spliced transcript encoding the isoform SCOX-exon 3II in a patient with normal expression of the transcript encoding the isoform SCOX-exon 3I, prompted us to characterize these two isoforms of human SCOX. The recombinant SCOX-exon 3I displayed activity toward medium-chain fatty acyl-CoAs and was not active with very long-chain fatty acyl-CoAs. In contrast, recombinant SCOX-exon 3II was capable of oxidizing a broad range of substrates, including very long-chain fatty acyl-CoAs. These results explain why this patient with a mutation in exon 3II of the ACOX1 gene, but with normal expression of exon 3I, was indistinguishable from other patients with peroxisomal acyl-CoA oxidase deficiency with respect to his clinical presentation and the biochemical abnormalities in his fibroblasts.
cDNA encoding the human peroxisomal acyl-coenzyme A oxidase (AOX) was cloned and sequenced. The longest cDNA insert isolated has 3083 bases and encodes the entire protein of 661-amino acids, including the carboxyl-terminal sequence (Ser-Lys-Leu) known as a minimal peroxisome-targeting signal. At the amino acid level, the significantly high homology (89%) to rat AOX was found. In the cDNA-expression experiment, significant amount of AOX was accumulated in human skin fibroblast and the expressed AOX was catalytically active, while only a limited amount was found in Zellweger syndrome patient's fibroblast not having normal peroxisomes.
We report on two new patients with straight-chain acyl-coenzyme A oxidase deficiency. Early onset hypotonia, seizures and psychomotor delay were observed in both cases. Plasma very-long-chain fatty acids were abnormal in both patients, whereas the plasma levels of phytanic acid, pristanic acid, the bile acid intermediates DHCA and THCA, and erythrocyte plasmalogen levels were normal. Studies in fibroblasts from the two patients revealed a deficiency of one of the two peroxisomal acyl-CoA oxidases, that is, straight-chain acyl-CoA oxidase (ACOX1). Subsequent molecular analysis of ACOX1 showed a homozygous deletion, which removes a large part of intron 3 and exons 4-14 in the first patient. Mutation analysis in the second patient revealed compound heterozygosity for two mutations, including: (1) a c.692 G > T (p.G231V) mutation and (2) skipping of exon 13 (c.1729_1935del (p.G577_E645del).
Evidence
4:
Inferred from Mutant PhenotypeUniProtKB
J. Biol. Chem. 270, 4908-4915 (1995)[PubMed:7876265]
Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.
Interacting selectively and non-covalently with the oxidized form, FAD, of flavin-adenine dinucleotide, the coenzyme or the prosthetic group of various flavoprotein oxidoreductase enzymes.
Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Interacting selectively and non-covalently with fatty acids, aliphatic monocarboxylic acids liberated from naturally occurring fats and oils by hydrolysis.
Interacting selectively and non-covalently with FAD, flavin-adenine dinucleotide, the coenzyme or the prosthetic group of various flavoprotein oxidoreductase enzymes, in either the oxidized form, FAD, or the reduced form, FADH2.
Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Interacting selectively and non-covalently with a protein N-terminus, the end of any peptide chain at which the 2-amino (or 2-imino) function of a constituent amino acid is not attached in peptide linkage to another amino-acid residue.
Evidence
1:
Inferred from Physical InteractionUniProtKB
Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in beta-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the beta-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology.
Interacting selectively and non-covalently with one or more specific sites on a receptor molecule, a macromolecule that undergoes combination with a hormone, neurotransmitter, drug or intracellular messenger to initiate a change in cell function.
Evidence
1:
Inferred from Physical InteractionUniProtKB
Most proteins are targeted to the peroxisomal matrix by virtue of a peroxisomal targeting signal-1 (PTS1), a short carboxy-terminal sequence specifically recognized by the PTS1 receptor Pex5p. We had previously developed a model that allowed the estimation of the affinities of many PTS1 sequences within the human proteome for Pex5p that revealed a wide range of predicted affinities. We have now experimentally determined the affinities of the PTS1-containing peptides from 42 proteins from the human proteome for Pex5p and show that these range over 4 orders of magnitude. These affinities correlate reasonably well with the predicted values and are substantially more precise. In an attempt to provide a possible explanation for the wide range of PTS1-Pex5p affinities, we compared these affinities with mRNA levels (as a proxy for rates of protein production) of the genes encoding these proteins in 79 human tissues and cell types. We note that high affinity PTS1-Pex5p interactions tend to correspond to proteins encoded by genes expressed at relatively low levels, whereas lower affinity PTS1-Pex5p interactions tend to correspond to proteins encoded by genes exhibiting higher levels and wider ranges of expression. Further analysis revealed that these relationships are consistent with the notion that a relatively uniform pool of protein-Pex5p complexes is maintained for appropriate peroxisome assembly.
A fatty acid beta-oxidation pathway in which the initial step, which converts an acyl-CoA to a trans-2-enoyl-CoA, is catalyzed by acyl-CoA oxidase; the electrons removed by oxidation pass directly to oxygen and produce hydrogen peroxide, which is cleaved by peroxisomal catalases. Fatty acid beta-oxidation begins with the addition of coenzyme A to a fatty acid, and ends when only two or three carbons remain (as acetyl-CoA or propionyl-CoA respectively).
We report on two new patients with straight-chain acyl-coenzyme A oxidase deficiency. Early onset hypotonia, seizures and psychomotor delay were observed in both cases. Plasma very-long-chain fatty acids were abnormal in both patients, whereas the plasma levels of phytanic acid, pristanic acid, the bile acid intermediates DHCA and THCA, and erythrocyte plasmalogen levels were normal. Studies in fibroblasts from the two patients revealed a deficiency of one of the two peroxisomal acyl-CoA oxidases, that is, straight-chain acyl-CoA oxidase (ACOX1). Subsequent molecular analysis of ACOX1 showed a homozygous deletion, which removes a large part of intron 3 and exons 4-14 in the first patient. Mutation analysis in the second patient revealed compound heterozygosity for two mutations, including: (1) a c.692 G > T (p.G231V) mutation and (2) skipping of exon 13 (c.1729_1935del (p.G577_E645del).
The removal of one or more electrons from a fatty acid, with or without the concomitant removal of a proton or protons, by reaction with an electron-accepting substance, by addition of oxygen or by removal of hydrogen.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
J. Biol. Chem. 270, 4908-4915 (1995)[PubMed:7876265]
Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.
Evidence
2:
Inferred from Genetic InteractionUniProtKB
Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.
The chemical reactions and pathways resulting in the formation of precursor metabolites, substances from which energy is derived, and any process involved in the liberation of energy from these substances.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
J. Biol. Chem. 270, 4908-4915 (1995)[PubMed:7876265]
Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.
Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.
Erratum in:
Lab Invest. 90(5), 808 (2010 May)
Evidence
2:
Inferred from Genetic InteractionUniProtKB
Evidence for Iso 1
Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.
The chemical reactions and pathways involving lipids, compounds soluble in an organic solvent but not, or sparingly, in an aqueous solvent. Includes fatty acids; neutral fats, other fatty-acid esters, and soaps; long-chain (fatty) alcohols and waxes; sphingoids and other long-chain bases; glycolipids, phospholipids and sphingolipids; and carotenes, polyprenols, sterols, terpenes and other isoprenoids.
cDNA encoding the human peroxisomal acyl-coenzyme A oxidase (AOX) was cloned and sequenced. The longest cDNA insert isolated has 3083 bases and encodes the entire protein of 661-amino acids, including the carboxyl-terminal sequence (Ser-Lys-Leu) known as a minimal peroxisome-targeting signal. At the amino acid level, the significantly high homology (89%) to rat AOX was found. In the cDNA-expression experiment, significant amount of AOX was accumulated in human skin fibroblast and the expressed AOX was catalytically active, while only a limited amount was found in Zellweger syndrome patient's fibroblast not having normal peroxisomes.
Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.
Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.
The chemical reactions and pathways involving prostaglandins, any of a group of biologically active metabolites which contain a cyclopentane ring due to the formation of a bond between two carbons of a fatty acid. They have a wide range of biological activities.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
J. Biol. Chem. 270, 4908-4915 (1995)[PubMed:7876265]
Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.
We report on two new patients with straight-chain acyl-coenzyme A oxidase deficiency. Early onset hypotonia, seizures and psychomotor delay were observed in both cases. Plasma very-long-chain fatty acids were abnormal in both patients, whereas the plasma levels of phytanic acid, pristanic acid, the bile acid intermediates DHCA and THCA, and erythrocyte plasmalogen levels were normal. Studies in fibroblasts from the two patients revealed a deficiency of one of the two peroxisomal acyl-CoA oxidases, that is, straight-chain acyl-CoA oxidase (ACOX1). Subsequent molecular analysis of ACOX1 showed a homozygous deletion, which removes a large part of intron 3 and exons 4-14 in the first patient. Mutation analysis in the second patient revealed compound heterozygosity for two mutations, including: (1) a c.692 G > T (p.G231V) mutation and (2) skipping of exon 13 (c.1729_1935del (p.G577_E645del).
Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.
Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.
Can reverse the Acox1 null phenotype in mouse which is characterized by severe microvesicular hepatic steatosis, sustained activation of Ppara, spontaneous massive peroxisome proliferation and eventual development of hepatocellular carcinomas.
CuratedUniProtKB
Biophysicochemical properties
Kinetic
parameters
KM
73 uM for palmitoyl-CoA (isoform 1)
KM
90 uM for palmitoyl-CoA (isoform 2)
Dependence
pH
Optimum pH is 8.5 for isoform 1 and 7.5-8.5 for isoform 2.
Temperature
Optimum temperature for isoform 1 at pH 7.5 is 40 degrees Celsius with no activity at 50 degrees Celsius. Optimum temperature for isoform 2 at pH 7.5 is 47.5 degrees Celsius with 57% activity retained at 50 degrees Celsius.
Protein involved in the biochemical reactions with fatty acids. Fatty acids are long chain organic acids of the general formula CH3(CnHx)COOH. They are constituents of lipids and can be saturated or unsaturated. The esterified forms are important both as energy storage molecules and structural molecules.
Protein involved in the biochemical reactions of lipids. Lipids are a diverse class of compounds which are insoluble in water but soluble in organic solvents. They include fats, oils, triacylglycerols, fatty acids, glycolipids, phospholipids and steroids.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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