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Identification and characterization of the 2-enoyl-CoA hydratases involved in peroxisomal beta-oxidation in rat liver.

ABSTRACT: In this study we attempted to determine the number of 2-enoyl-CoA hydratases involved in peroxisomal beta-oxidation. We therefore separated peroxisomal proteins from rat liver on several chromatographic columns and measured hydratase activities on the eluates with different substrates. The results indicate that rat liver peroxisomes contain two hydratase activities: (1) a hydratase activity associated with multifunctional protein 1 (MFP-1) (2-enoyl-CoA hydratase/delta 3, delta 2-enoyl-CoA isomerase/L-3-hydroxyacyl-CoA dehydrogenase) and (2) a hydratase activity associated with MFP-2 (17 beta-hydroxysteroid dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase). MFP-1 forms and dehydrogenates L-3-hydroxyacyl-CoA species, whereas MFP-2 forms and dehydrogenates D-3-hydroxyacyl-CoA species. A portion of MFP-2 is proteolytically cleaved, most probably in the peroxisome, into a 34 kDa 17 beta-hydroxysteroid dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase and a 45 kDa D-specific 2-enoyl-CoA hydratase. Finally, the results confirm that MFP-1 is involved in the degradation of straight-chain fatty acids, whereas MFP-2 and its cleavage products seem to be involved in the degradation of the side chain of cholesterol (bile acid synthesis).

SUBMITTER: Dieuaide-Noubhani M

PROVIDER: S-EPMC1218062 | biostudies-other | 1997 Jan

REPOSITORIES: biostudies-other

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Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.

Project description:In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

| S-EPMC1218032 | biostudies-other

Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.

Project description:Ciprofibrate, a hypolipidemic drug that acts as a peroxisome proliferator, induces the transcription of genes encoding peroxisomal beta-oxidation enzymes. To identify cis-acting promoter elements involved in this induction, 5.8 kilobase pairs of promoter sequence from the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EC 4.2.1.17/EC 1.1.1.35) was inserted upstream of a luciferase reporter gene. Transfection of this expression vector into rat hepatoma H4IIEC3 cells in the presence of ciprofibrate resulted in a 5- to 10-fold, cell type-specific increase in luciferase activity as compared to cells transfected in the absence of drug. A peroxisome proliferator-responsive element (PPRE) was localized to a 196-nucleotide region centered at position -2943 from the transcription start site. This PPRE conferred ciprofibrate responsiveness on a heterologous promoter and functioned independently of orientation or position. Gel retardation analysis with nuclear extracts demonstrated that ciprofibrate-treated or untreated H4IIEC3 cells, but not HeLa cells or monkey kidney cells, contained sequence-specific DNA binding factors that interact with the PPRE. These results have implications for understanding the mechanisms of coordinated transcriptional induction of genes encoding peroxisomal proteins by hypolipidemic agents and other peroxisome proliferators.

| S-EPMC49746 | biostudies-other

Peroxisomal palmitoyl-CoA oxidation in the Zucker rat.

Project description:The effects of 3 or 6 days of starvation on hepatic peroxisomal palmitoyl-CoA oxidation were examined in adult lean and obese Zucker rats. When expressed either per mg of DNA or per total liver, obese rats had almost 2-fold higher oxidation rates than the lean rats. Within 6 days of starvation rates fell by 50% among both phenotypes. When data were expressed per 100 g body wt., lean and obese rats had similar rates, falling from a mean of 0.57 to 0.28 mumol/min per 100 g body wt. within 6 days of starvation. Peroxisomal oxidative changes paralleled mitochondrial beta-oxidative changes.

| S-EPMC1153169 | biostudies-other

Characterization of the stimulatory effect of high-fat diets on peroxisomal beta-oxidation in rat liver.

Project description:1. The effect on rat liver peroxisomal beta-oxidation of feeding diets containing various amounts of dietary oils was investigated. With increasing amounts (5-25%, w/w) of soya-bean oil an apparent, but not statistically significant, increase of 1.5-fold was found both in specific activity, and in total liver activity. Increasing amounts of partially hydrogenated marine oil revealed a sigmoidal dose-response-curve, giving a 4-6-fold increase in the peroxisomal beta-oxidation activity at 20% or more of this oil in the diet. 2. Addition of small amounts of soya-bean oil to the marine-oil diet had no effect on the peroxisomal beta-oxidation activity, but decreased the C20:3(5,8,11) fatty acid/C20:4(5,8,11,14) fatty acid ratio in liver phospholipids from 0.74 to 0.01. 3. Starvation for 2 days led to a 1.5-1.8-fold increase in the peroxisomal beta-oxidation activity in rats previously fed on a standard pelleted diet, but had no effect in rats given high-fat diets. 4. Feeding partially hydrogenated marine oil or partially hydrogenated rape-seed oil resulted in higher activities than the corresponding unhydrogenated oils. 5. No significant differences in the effect on peroxisomal beta-oxidation could be detected between diets containing rape-seed oils with 15 or 45% erucic acid respectively. 6. These findings are discussed in relation to the possible effects of C22:1 and trans fatty acids in the process leading to increased peroxisomal beta-oxidation activity in the liver.

| S-EPMC1158574 | biostudies-other

Acetyl-CoA Derived from Hepatic Peroxisomal β-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.

Project description:Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal β-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.

| S-EPMC7335356 | biostudies-literature

Induction of peroxisomal beta-oxidation in rat liver by high-fat diets.

Project description:Liver peroxisomes were prepared by using a Percoll gradient in a vertical rotor. beta-Oxidation was measured in peroxisomes isolated from livers of rats fed on either high-(15% by wt.) or low- (5% by wt.) fat diets. The feeding of high-fat diets gave a 1.4-2.4-fold increase in total liver peroxisomal beta-oxidation, and a similar increase in specific activity. A 1.5-4.5-fold increase was seen in the specific activity of purified peroxisomal preparations. The reasons for these increases are discussed.

| S-EPMC1161539 | biostudies-other

Estimation of peroxisomal beta-oxidation in rat heart by a direct assay of acyl-CoA oxidase.

Project description:The contribution of peroxisomes to palmitate beta-oxidation in rat heart was estimated by either inhibiting mitochondrial beta-oxidation or measuring the activity of acyl-CoA oxidase. When respiratory inhibitors such as KCN or antimycin plus rotenone, or inhibitors of mitochondrial fatty acid uptake such as 2-tetradecylglycidic acid or 2-bromopalmitate, were used, degrees of inhibitions ranging from 24% to 87% were observed for palmitate beta-oxidation by a rat heart homogenate. Although the oxidation of palmitoyl-L-carnitine by coupled rat heart mitochondria was almost completely (94%) inhibited by KCN, the inhibition by antimycin plus rotenone was incomplete (77%) and was stimulated by L-carnitine. A direct assay of acyl-CoA oxidase, based on the spectrophotometric measurement at 300 nm of 2,4-decadienoyl-CoA formation from 4-trans-decenoyl-CoA, was evaluated with the aim of obtaining reliable values for the activity of this enzyme, which is presumed to catalyse the rate-limiting step of peroxisomal beta-oxidation. Activities determined by use of this assay were much higher than activities obtained by a coupled assay [Small, Burdett and Connock (1985) Biochem. J. 227, 205-210] commonly used to measure the activity of acyl-CoA oxidase. However, both methods yielded the same relative activities with different tissue homogenates. Based on an estimated palmitoyl-CoA oxidase activity of 0.3 nmol/min per mg of protein, the contribution of peroxisomes to palmitate beta-oxidation in a rat heart homogenate would optimally be 4%, and most likely is several-fold lower.

| S-EPMC1137186 | biostudies-other

Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal beta-oxidation.

Project description:Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10-C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4-C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 microM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3-8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570-8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal beta-oxidation are discussed.

| S-EPMC1217973 | biostudies-other

Acquired deficiency of the peroxisomal bi-functional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is a metabolic vulnerability in murine hepatoblastoma

Project description:Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. However, capitalizing on this therapeutically has been only moderately successful due to the relatively small magnitude of these differences and because cancers may re-program their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bi-functional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate dodecanedioic acid (DDDA), a toxic C12 metabolite that causes hepatocyte necrosis and acute liver failure. In a murine model of pediatric hepatoblastoma (HB), down-regulation of Ehhadh also occurs in combination with a more general suppression of mitochondrial β- and peroxisomal ω-fatty acid oxidation (FAO) pathways. HB-bearing mice provided with C12 and/or DDDA-supplemented diets survived significantly longer than those on standard diets. The tumors also developed massive necrosis in response to short-term DDDA supplementation. Reduced Ehhadh was noted in murine hepatocellular carcinomas (HCCs) and in substantial subsets of human cancers, including HCCs. Acquired DDDA resistance was not associated with Ehhadh re-expression but was associated with 129 transcript differences ~90% of which were down-regulated in DDDA-resistant tumors and ~two-thirds of which correlated with survival in several human cancers. These transcripts often encoded components of the extracellular matrix suggesting that DDDA resistance arises from its reduced intracellular transport. Our results demonstrate the feasibility of a metabolic intervention that is non-toxic, inexpensive and likely compatible with traditional therapies. C12 and/or DDDA-containing diets could potentially be used to supplement other treatments or as alternative therapeutic choices.

2021-03-23 | GSE156545 | GEO

Factors influencing palmitoyl-CoA oxidation by rat liver peroxisomal fractions. Substrate concentration, organelle integrity and ATP.

Project description:1. The first dehydrogenation step of peroxisomal beta-oxidation involves the reduction of O2 to H2O2. Production rates of H2O2 and acetyl units by purified rat liver peroxisomes oxidizing palmitoyl-CoA were equal, indicating that H2O2 production is a reliable index for the release of acetyl units during peroxisomal fatty-acid oxidation. 2. Measurements of H2O2 and acid-soluble oxidation products during [1-14C]palmitoyl-CoA oxidation by purified peroxisomes revealed that the number of acetyl units released per molecule of palmitoyl-CoA oxidized rapidly decreased with increasing unbound palmitoyl-CoA concentrations. Structural damage to the peroxisomes caused by detergents or other treatments also decreased the number of acetyl units released. Under conditions where oxidation proceeded linearly with time the theoretical maximum of 5 acetyl units released per molecule of palmitoyl-CoA oxidized [Lazarow (1978) J. Biol. Chem. 253, 1522--1528] was never reached. 3. Expressed in terms of acetyl units produced and measured at low unbound-palmitoyl-CoA concentrations, mitochondrial oxidation was 10--20-fold higher than peroxisomal oxidation. 4. ATP stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. The ATP effect required the presence of Mg2+ and was lost when peroxisomal membranes were disrupted by Triton X-100 or high concentrations of unbound palmitoyl-CoA. 5. Disruption of peroxisomes by detergents, freeze--thawing, osmotic or mechanical treatment did not stimulate palmitoyl-CoA oxidation in the presence of ATP, indicating that peroxisomal fatty-acid-CoA oxidation was not latent. In the absence of ATP, Triton X-100 stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold.

| S-EPMC1162123 | biostudies-other

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