RESUMEN
L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product. The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey. The complete cDNA, obtained by conventional library screening and 5' rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein. The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1. Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase. A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp. NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases. No similarity was found to the peroxisomal D-amino acid oxidases. The recombinant enzyme oxidized both L-pipecolic acid and sarcosine. However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Ácidos Pipecólicos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Haplorrinos , Humanos , Riñón/enzimología , Hígado/enzimología , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Trastorno Peroxisomal/metabolismo , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/metabolismo , Filogenia , Ácidos Pipecólicos/sangre , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcosina/sangre , Sarcosina-Oxidasa , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11alpha and PEX11beta. Overexpression of the human PEX11beta gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11beta induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11beta from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11alpha also induced peroxisome proliferation but at a much lower frequency than PEX11beta in our experimental system. The patterns of PEX11alpha and PEX11beta expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11beta mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11alpha mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11beta in the constitutive control of peroxisome abundance and suggest that PEX11alpha may regulate peroxisome abundance in response to extracellular stimuli.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Microcuerpos/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Cartilla de ADN , ADN Complementario , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/genética , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Peroxinas , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Peroxisomal matrix protein import requires the action of two AAA ATPases, PEX1 and PEX6. Mutations in either the PEX1 or PEX6 gene are the most common cause of the lethal neurologic disorders Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease and account for disease in 80% of all such patients. We report here that overexpression of PEX6 can suppress the phenotypes of certain PEX1-deficient cells, that overexpression of PEX1 can suppress the phenotypes of certain PEX6-deficient cells, and that these instances of suppression are allele-specific and require partial activity of the mutated gene. In addition to genetic evidence for interaction between PEX1 and PEX6, we find that the PEX1 and PEX6 proteins interact in the yeast two-hybrid assay and physically associate with one another in vitro. We previously identified a missense mutation in PEX1, G843D, which attenuates PEX1 function and is the most common cause of these diseases, present in one-third of all such patients. The G843D mutation attenuates the interaction between PEX1 and PEX6 in both the two-hybrid system and in vitro and appears to be suppressed by overexpression of PEX6. We conclude that PEX1 and PEX6 form a complex of central importance to peroxisome biogenesis and that mutations affecting this complex constitute the most common cause of the Zellweger syndrome spectrum of diseases.
Asunto(s)
Adenosina Trifosfatasas/genética , Adrenoleucodistrofia/genética , Glicoproteínas/genética , Proteínas de la Membrana , Trastorno Peroxisomal/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Glicoproteínas/metabolismo , Humanos , Recién Nacido , Fenotipo , Unión ProteicaRESUMEN
The peroxisome biogenesis disorders (PBDs) are a group of lethal autosomal-recessive diseases caused by defects in peroxisomal matrix protein import, with the concomitant loss of multiple peroxisomal enzyme activities. Ten complementation groups (CGs) have been identified for the PBDs, with CG1 accounting for 51% of all PBD patients. We identified the human orthologue of yeast PEX1, a gene required for peroxisomal matrix protein import. Expression of human PEX1 restored peroxisomal protein import in fibroblasts from 30 CG1 patients, and PEX1 mutations were detected in multiple CG1 probands. A common PEX1 allele, G843D, is present in approximately half of CG1 patients and has a deleterious effect on PEX1 activity. Phenotypic analysis of PEX1-deficient cells revealed severe defects in peroxisomal matrix protein import and destabilization of PEX5, the receptor for the type-1 peroxisomal targetting signal, even though peroxisomes were present in these cells and capable of importing peroxisomal membrane proteins. These data demonstrate an important role for PEX1 in peroxisome biogenesis and suggest that mutations in this gene are the most common cause of the PBDs.
Asunto(s)
Microcuerpos/genética , Mutación , Trastorno Peroxisomal/genética , Proteínas/genética , Alelos , Secuencia de Aminoácidos , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Endopeptidasa Neutra Reguladora de Fosfato PHEX , Biosíntesis de Proteínas , Proteínas/aislamiento & purificaciónRESUMEN
Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.