RESUMEN
2-Hydroxyphytanoyl-CoA lyase (abbreviated as 2-HPCL), renamed to 2-hydroxyacyl-CoA lyase (abbreviated as HACL1), is the first peroxisomal enzyme in mammals that has been found to be dependent on TPP (thiamin pyrophosphate). It was discovered in 1999, when studying alpha-oxidation of phytanic acid. HACL1 has an important role in at least two pathways: (i) the degradation of 3-methyl-branched fatty acids like phytanic acid and (ii) the shortening of 2-hydroxy long-chain fatty acids. In both cases, HACL1 catalyses the cleavage step, which involves the splitting of a carbon-carbon bond between the first and second carbon atom in a 2-hydroxyacyl-CoA intermediate leading to the production of an (n-1) aldehyde and formyl-CoA. The latter is rapidly converted into formate and subsequently to CO(2). HACL1 is a homotetramer and has a PTS (peroxisomal targeting signal) at the C-terminal side (PTS1). No deficiency of HACL1 has been described yet in human, but thiamin deficiency might affect its activity.
Asunto(s)
Ácidos Grasos/metabolismo , Liasas/metabolismo , Peroxisomas/metabolismo , Animales , Ácidos Grasos/química , Tiamina/metabolismoRESUMEN
Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear. Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs, (ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Peroxisomas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas de Saccharomyces cerevisiae/química , Transferasas Alquil y Aril/metabolismo , Secuencias de Aminoácidos , Animales , Células CHO , Línea Celular Transformada , Secuencia de Consenso , Cricetinae , Cricetulus , Fibroblastos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ácido Oléico/metabolismo , Peroxisomas/ultraestructura , Prenilación de Proteína/fisiología , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Eliminación de Secuencia , Relación Estructura-Actividad , TransfecciónRESUMEN
The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the alpha-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and alpha-oxidation has not been studied, nor have possible adverse effects of deficient alpha-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that alpha-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired alpha-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their alpha-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished.
Asunto(s)
Anemia Megaloblástica/metabolismo , Ácidos Grasos/metabolismo , Ácido Fitánico/metabolismo , Deficiencia de Tiamina/metabolismo , Tiamina Pirofosfato/metabolismo , Animales , Liasas de Carbono-Carbono/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Ratones , Oxidación-Reducción , Ratas , Ratas Wistar , Tiamina/metabolismoRESUMEN
The purpose of this study was to investigate whether deficient peroxisomal beta-oxidation is causally involved in the neuronal migration defect observed in Pex5 knockout mice. These mice are models for Zellweger syndrome, a peroxisome biogenesis disorder. Neocortical development was evaluated in mice carrying a partial or complete defect of peroxisomal beta-oxidation at the level of the second enzyme of the pathway, namely, the hydratase-dehydrogenase multifunctional/bifunctional enzymes MFP1/L-PBE and MFP2/D-PBE. In contrast to patients with multifunctional protein 2 deficiency who present with neocortical dysgenesis, impairment of neuronal migration was not observed in the single MFP2 or in the double MFP1/MFP2 knockout mice. At birth, the double knockout pups displayed variable growth retardation and about one half of them were severely hypotonic, whereas the single MFP2 knockout animals were all normal in the perinatal period. These results indicate that in the mouse, defective peroxisomal beta-oxidation does not cause neuronal migration defects by itself. This does not exclude that the inactivity of this metabolic pathway contributes to the brain pathology in mice and patients with complete absence of functional peroxisomes.
Asunto(s)
Movimiento Celular/fisiología , Neuronas/metabolismo , Peroxisomas/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Síndrome de Zellweger/enzimología , Animales , Química Encefálica , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Oxidación-Reducción , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Receptores Citoplasmáticos y Nucleares/metabolismo , Síndrome de Zellweger/genética , Síndrome de Zellweger/fisiopatologíaRESUMEN
Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S. Biochem. J., 1999, 340, 561-568). To identify other unknown peroxisomal enzymes, we further exploited this promising approach. Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1). Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family. However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate. The recombinant protein interacted with HsPex5p, the human PTS1-binding protein. Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted. Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1. These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe.
Asunto(s)
Bacteriófago M13/enzimología , Ácido Graso Desaturasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Peroxisomas/enzimología , Proteínas Virales de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Escherichia coli/metabolismo , Técnicas In Vitro , Datos de Secuencia Molecular , Conejos , Saccharomyces cerevisiae/metabolismoRESUMEN
Based on the primary structure of the rat peroxisomal 2,4-dienoyl-CoA reductase (M. Fransen, P.P. Van Veldhoven, S. Subramani, Biochem. J. 340 (1999) 561-568), the cDNA of the human counterpart was cloned. It contained an open reading frame of 878 bases encoding a protein of 291 amino acids (calculated molecular mass 30778 Da), being 83% identical to the rat reductase. The gene, encompassing nine exons, is located at chromosome 16p13. Bacterially expressed poly(His)-tagged reductase was active not only towards short and medium chain 2,4-dienoyl-CoAs, but also towards 2,4,7,10,13,16,19-docosaheptaenoyl-CoA. Hence, the reductase does not seem to constitute a rate limiting step in the peroxisomal degradation of docosahexaenoic acid. The reduction of docosaheptaenoyl-CoA, however, was severely decreased in the presence of albumin.
Asunto(s)
Ácido Graso Desaturasas/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Peroxisomas/enzimología , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/química , Ácidos Docosahexaenoicos/metabolismo , Ácido Graso Desaturasas/biosíntesis , Ácido Graso Desaturasas/química , Humanos , Cinética , Datos de Secuencia MolecularRESUMEN
Based on peroxin protein 5 (Pex5p) homology searches in the expressed sequence tag database and sequencing of large full-length cDNA inserts, three novel and related human cDNAs were identified. The brain-derived cDNAs coded for two related proteins that differ only slightly at their N-terminus, and exhibit 39.8% identity to human PEX5p. The shorter liver-derived cDNA coded for the C-terminal tetratricopeptide repeat-containing domain of the brain cDNA-encoded proteins. Since these three proteins specifically bind to various C-terminal peroxisome-targeting signals in a manner indistinguishable from Pex5p and effectively compete with Pex5p in an in vitro peroxisome-targeting signal 1 (PTS1)-binding assay, we refer to them as 'Pex5p-related proteins' (Pex5Rp). In contrast to Pex5p, however, human PEX5Rp did not bind to Pex14p or to the RING finger motif of Pex12p, and could not restore PTS1 protein import in Pex5(-/-) mouse fibroblasts. Immunofluorescence analysis of epitope-tagged PEX5Rp in Chinese hamster ovary cells suggested an exclusively cytosolic localization. Northern-blot analysis showed that the PEX5R gene, which is localized to chromosome 3q26.2--3q27, is expressed preferentially in brain. Mouse PEX5Rp was also delineated. In addition, experimental evidence established that the closest-related yeast homologue, YMR018wp, did not bind PTS1. Based on its subcellular localization and binding properties, Pex5Rp may function as a regulator in an early step of the PTS1 protein import process.
Asunto(s)
Encéfalo/metabolismo , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetinae , Citosol/metabolismo , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Conejos , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: 2-Methylacyl-CoA racemase interconverts the 2-methyl group of pristanoyl-CoA or the 25-methyl group of hydroxylated cholestanoyl-CoAs, allowing further peroxisomal desaturation of these compounds in man by the branched chain acyl-CoA oxidase, which recognise only the S-isomers. Hence, oxidation studies in fibroblasts, currently based on the use of racemic substrates such as [1-14C] pristanic acid, do not allow us to distinguish between a deficient racemase or an impaired oxidase. DESIGN: To evaluate the racemase activity directly, the 2R-isomer of[1-14C] pristanic acid, as well as the 2R-isomer of 2-methyl-[1-14C] hexadecanoic, a synthetic pristanic acid substitute, were prepared and their degradation by cultured human skin fibroblasts was compared to that of the racemic substrates. RESULTS: In fibroblasts in a young girl, presenting with elevated urinary levels of trihydroxycholestanoic acid metabolites but normal plasma levels of very long chain fatty acids, a partial deficient degradation of racemic [1-14C] pristanic acid was observed. Incorporation of 2R-[1-14C] pristanic acid in glycerolipids of the patient's fibroblasts proceeded normally, but breakdown was impaired. Similar findings were seen with the 2R-isomer of 2-methyl-[1-14C] hexadecanoic. These data, combined with the fact that the branched chain acyl-CoA oxidase, catalyzing the first oxidation step of pristanic acid and bile acid intermediates in man, appeared normal, suggested a peroxisomal beta-oxidation defect in the patient at the level of 2-methylacyl-CoA racemase. CONCLUSION: Carboxy-labelled 2R-methyl branched chain fatty acids might be useful tools to document cases of racemase deficiencies. Because a brother of the patient died with a diagnosis of vitamin K deficiency, an impaired racemase might be responsible for other cases of unexplicable malabsorption.
Asunto(s)
Síndromes de Malabsorción/etiología , Trastorno Peroxisomal/enzimología , Peroxisomas/enzimología , Racemasas y Epimerasas/deficiencia , Deficiencia de Vitamina K/etiología , Células Cultivadas , Ácidos Grasos/síntesis química , Ácidos Grasos/metabolismo , Femenino , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Recién Nacido , Isomerismo , Oxidación-Reducción , Ácidos Palmíticos/síntesis química , Ácidos Palmíticos/metabolismo , Piel/citologíaRESUMEN
The goal of this study was to clarify the mechanism responsible for the catabolism of alpha-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 microM of alpha-tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an omega-acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that alpha-tocopherol is omega-oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O2. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).
Asunto(s)
Microsomas Hepáticos/metabolismo , alfa-Tocoferol/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Espectroscopía de Resonancia Magnética , Masculino , NADP/farmacología , Oxidación-Reducción , Oxígeno/farmacología , Ratas , Ratas WistarRESUMEN
The molecular machinery underlying peroxisomal membrane biogenesis is not well understood. The observation that cells deficient in the peroxins Pex3p, Pex16p, and Pex19p lack peroxisomal membrane structures suggests that these molecules are involved in the initial stages of peroxisomal membrane formation. Pex19p, a predominantly cytosolic protein that can be farnesylated, binds multiple peroxisomal integral membrane proteins, and it has been suggested that it functions as a soluble receptor for the targeting of peroxisomal membrane proteins (PMPs) to the peroxisome. An alternative view proposes that Pex19p functions as a chaperone at the peroxisomal membrane. Here, we show that the peroxisomal sorting determinants and the Pex19p-binding domains of a number of PMPs are distinct entities. In addition, we extend the list of peroxins with which human Pex19p interacts to include the PMP Pex16p and show that Pex19p's CaaX prenylation motif is an important determinant in the affinity of Pex19p for Pex10p, Pex11pbeta, Pex12p, and Pex13p.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Fraccionamiento Celular , Cricetinae , Genes Reporteros/genética , Humanos , Proteínas de la Membrana/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Peroxisomas/química , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Mammalian peroxisomes degrade fatty carboxylates via two pathways, beta-oxidation and, as shown more recently, alpha-oxidation. The latter process consists of an activation step, followed by a hydroxylation at position 2 and cleavage of the 2-hydroxyacyl-CoA, generating formyl-CoA (precursor of formate/CO(2)) and, in case of phytanic acid as substrate, pristanal (precursor of pristanic acid). The stereochemistry of the overall pathway, cofactor requirements and substrate specificity of the hydroxylase and the cleavage enzyme, which is homologous with bacterial oxalyl-CoA decarboxylases, will be discussed. With regard to beta-oxidation, peroxisomes contain different acyl-CoA oxidases, multifunctional proteins and thiolases. Based on substrate spectra and stereospecificities of these enzymes, a model was proposed whereby straight chain and branched compounds are degraded by separate pathways. The biochemical findings in mice lacking the D-specific multifunctional protein, however, do not fully support this model. These animals, together with the Pex5(-/-) mice, might be useful to pinpoint the pathological factors contributing to the brain abnormalities in Zellweger patients. Apparently, the deficit in docosahexaenoic acid, presumably formed via peroxisomal beta-oxidation, is not the major cause.
Asunto(s)
Metabolismo de los Lípidos , Peroxisomas/metabolismo , Animales , Dióxido de Carbono/metabolismo , Humanos , Lípidos/química , Ratones , Ratones Noqueados , Modelos Animales , Oxidación-Reducción , Oxidorreductasas/metabolismo , Peroxisomas/enzimologíaRESUMEN
The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: alpha-oxidation, beta-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5(-/-) knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C(26:0) was observed in comparison with control littermates, indicating that ether phospholipid synthesis and beta-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the beta-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the alpha-oxidation pathway suggested that alpha-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in alpha-oxidation and beta-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal beta-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.
Asunto(s)
Peroxisomas/metabolismo , Fosfolípidos/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Desarrollo Embrionario y Fetal , Femenino , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones Noqueados , Oxidación-Reducción , Peroxisomas/enzimología , EmbarazoRESUMEN
The introduction of the double bond in the sphingoid backbone of sphingolipids occurs at the level of dihydroceramide via an NADPH-dependent desaturase, as discovered in permeabilized rat hepatocytes. In the rat, the enzyme activity, which has now been further characterized, appeared to be mostly enriched in liver and Harderian gland. By means of subcellular fractionation of rat liver homogenates and density gradient separation of microsomal fractions, the desaturase was localized to the endoplasmic reticulum. Various detergents were inhibitory to the enzyme, and maximal activities were obtained in the presence of NADPH and when the substrate was complexed to albumin. In the presence of albumin, the chain length of the fatty acid of the truncated dihydroceramides hardly affected the activity. Finally, in view of a likely evolutionary relationship between desaturases and hydroxylases, the formation of hydroxylated intermediates was analyzed. No evidence for their presence was found under our assay conditions.
Asunto(s)
Oxidorreductasas/química , Oxidorreductasas/farmacocinética , Albúminas/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Ceramidas/biosíntesis , Ceramidas/química , Detergentes/farmacología , Retículo Endoplásmico/metabolismo , Glándula de Harder/metabolismo , Hígado/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Modelos Químicos , NADP/metabolismo , Ratas , Ratas Wistar , Fracciones Subcelulares/metabolismo , Especificidad por Sustrato , Distribución TisularRESUMEN
2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal beta-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C(27) bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85.Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.
Asunto(s)
Hígado/ultraestructura , Mitocondrias Hepáticas/enzimología , Peroxisomas/enzimología , Racemasas y Epimerasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cricetinae , Proteínas Fluorescentes Verdes , Humanos , Hígado/enzimología , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Racemasas y Epimerasas/química , Racemasas y Epimerasas/genética , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia , TransfecciónRESUMEN
Sphingosine-1-phosphate lyase catalyzes the last step in sphingolipid breakdown, the cleavage of phosphorylated sphingoid bases such as sphingenine-1-phosphate. The latter lipid is not only a catabolite, but can influence as an inter- and/or intracellular second messenger many cellular processes. To allow for the diagnosis of human disorders that might be linked to a deficient lyase, the human sphingosine-1-phosphate lyase cDNA was cloned. The obtained cDNA encoded a protein of 568 amino acids with a calculated molecular mass of 63492 Da. Hydropathy plots revealed the presence of one membrane span near the amino-terminal which is however not required for enzyme activity since recombinant poly-His-tagged lyase, lacking this membrane span, was functionally active. Site-directed mutagenesis disclosed the importance of the cysteine residues 218 and 317 for the cleavage reaction. Northern analysis showed the presence of rare large-sized mRNAs of 6.7, 5.8 and 4 kb and the highest expression in liver. By fluorescent in situ hybridization, the gene was mapped to chromosome 10q22.
Asunto(s)
Aldehído-Liasas/genética , Cromosomas Humanos Par 10 , Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
In a search for possible endogenous ligands of nuclear receptors that are activated by peroxisome proliferators (PPARs), a solid phase binding assay was developed employing recombinant mouse PPAR-alpha, containing a myc-epitope, a histidine repeat and a kinase A domain. After in vitro labelling with 32P-gamma-ATP, the binding of purified 32P-PPAR-alpha to a panel of different natural and synthetic lipids, immobilized on silica layers, was evaluated. Autoradiographs of the silica layers revealed binding to two main classes of lipophilic compounds. A first class comprised (poly)unsaturated fatty acids. Compounds belonging to a second class were characterized by the presence of an overall positive charge such as long chain amines, sphingoid bases (sphingenine), and lysoglycosphingolipids (psychosine). PPAR-alpha did not bind to N-acylated sphingoid bases (ceramides) or to sphingenine phosphorylated at the primary hydroxy group (sphingenine-1-phosphate). The binding of PPAR-alpha to sphingoid bases might be of interest given the role of PPAR-alpha and sphingolipids in various cellular processes.
Asunto(s)
Metabolismo de los Lípidos , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Núcleo Celular/metabolismo , Ceramidas/metabolismo , Cromatografía en Capa Delgada , Epítopos , Ácidos Grasos Insaturados/metabolismo , Histidina/química , Ligandos , Ratones , Fosfotransferasas/química , Unión Proteica , Estructura Terciaria de Proteína , Psicosina/química , Psicosina/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Esfingosina/química , Esfingosina/metabolismoRESUMEN
Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/aislamiento & purificación , Hígado/enzimología , Peroxisomas/enzimología , Animales , Colesterol/metabolismo , Cromatografía en Agarosa , Cromatografía Líquida de Alta Presión , Citosol/enzimología , Concentración de Iones de Hidrógeno , Immunoblotting , Cetonas/metabolismo , Cinética , Masculino , Ratas , Ratas WistarRESUMEN
According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/genética , Enoil-CoA Hidratasa/genética , Ácidos Grasos/metabolismo , Complejos Multienzimáticos/genética , 3-Hidroxiacil-CoA Deshidrogenasas/deficiencia , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Ácidos y Sales Biliares/química , Ácidos y Sales Biliares/metabolismo , Dieta , Enoil-CoA Hidratasa/deficiencia , Enoil-CoA Hidratasa/metabolismo , Fibroblastos , Crecimiento/genética , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones Noqueados , Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/metabolismo , Peroxisomas/enzimología , Peroxisomas/metabolismo , Fitol/metabolismoRESUMEN
Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway.
Asunto(s)
Acilcoenzima A/metabolismo , Adenosina Trifosfato/metabolismo , Guanosina Trifosfato/metabolismo , Magnesio/metabolismo , Oxigenasas de Función Mixta/metabolismo , Cationes Bivalentes/metabolismo , Compuestos Ferrosos/metabolismo , Humanos , Hidroxilación , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/genética , Ácidos Palmíticos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
In the present report we describe a method for the complete purification of native sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase) from normal rat liver peroxisomes. The isolation procedure is based on the alteration in chromatographic properties of the enzyme in the presence of low concentrations of CoA. The purified preparation of SCP-2/thiolase consisted of 58- and 46-kDa polypeptides. Peroxisomes prepared freshly from normal rat liver contained three SCP-2/thiolase isoforms, separable by conventional chromatography. Immunochemical, molecular sieving, and chemical cross-linking experiments indicated that these isoforms represent thiolytically active homo- and heterodimeric combinations of the 46- and 58-kDa subunits (2 x 58, 58-46, and 2 x 46-kDa proteins).