Peroxisomal palmitoyl-CoA oxidation in the Zucker rat.
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ABSTRACT:
SUBMITTER: Brady PS
PROVIDER: S-EPMC1153169 | biostudies-other | 1983 Jun
REPOSITORIES: biostudies-other
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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
Project description:Autophagy is a membrane trafficking pathway that sequesters proteins and organelles into autophagosomes. The selectivity of this pathway is determined by autophagy receptors, such as the Pichia pastoris autophagy-related protein 30 (Atg30), which controls the selective autophagy of peroxisomes (pexophagy) through the assembly of a receptor protein complex (RPC). However, how the pexophagic RPC is regulated for efficient formation of the phagophore, an isolation membrane that sequesters the peroxisome from the cytosol, is unknown. Here we describe a new, conserved acyl-CoA-binding protein, Atg37, that is an integral peroxisomal membrane protein required specifically for pexophagy at the stage of phagophore formation. Atg30 recruits Atg37 to the pexophagic RPC, where Atg37 regulates the recruitment of the scaffold protein, Atg11. Palmitoyl-CoA competes with Atg30 for Atg37 binding. The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy. We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.
| S-EPMC3926955 | biostudies-literature
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.
| S-EPMC3382043 | biostudies-literature
Project description:Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal beta-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp(249) to Arg(251)) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp(249) to Arg(251) and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.
| S-EPMC2924054 | biostudies-literature
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
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
Project description:Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via ?-oxidation, differences exist between the peroxisomal and mitochondrial ?-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the C? hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the K(m) values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.
| S-EPMC3436514 | biostudies-literature
Project description: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).
| S-EPMC1218062 | biostudies-other
Project description:1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation.
| S-EPMC1131695 | biostudies-other
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