RESUMO
In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as ß-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite's glycosomes.
Assuntos
Microcorpos/química , Microcorpos/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Doença de Chagas/parasitologia , Estágios do Ciclo de Vida , Microcorpos/genética , Proteômica , Proteínas de Protozoários/genética , Trypanosoma cruzi/química , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanosomatids cause deadly diseases in humans. Of the various biochemical pathways in trypanosomatids, glycolysis, has received special attention because of being sequestered in peroxisome like organelles critical for the survival of the parasites. This study focuses on phosphoglycerate kinase (PGK) from Leishmania spp. which, exists in two isoforms, the cytoplasmic PGKB and glycosomal PGKC differing in their biochemical properties. Computational analysis predicted the likelihood of a transmembrane helix only in the glycosomal isoform PGKC, of approximate length 20 residues in the 62-residue extension, ending at, arginine residues R471 and R472. From experimental studies using circular dichroism and NMR with deuterated sodium dodecyl sulfate, we find that the transmembrane helix spans residues 448±2 to 476 in Leishmania mexicana PGKC. The significance of this observation is discussed in the context of glycosomal transport and substrate tunneling.
Assuntos
Leishmania mexicana/enzimologia , Peptídeos/química , Fosfoglicerato Quinase/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Dicroísmo Circular , Biologia Computacional , Citoplasma/enzimologia , Citoplasma/genética , Isoenzimas/química , Isoenzimas/genética , Leishmania mexicana/genética , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Micelas , Microcorpos/enzimologia , Microcorpos/genética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Solubilidade , Especificidade por SubstratoRESUMO
Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.
Assuntos
Malato Desidrogenase/análise , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia em Agarose/métodos , Reações Cruzadas/imunologia , Citosol/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes de Protozoários/genética , Isoenzimas/análise , Isoenzimas/imunologia , Malato Desidrogenase/genética , Malato Desidrogenase/imunologia , Microcorpos/enzimologia , Microcorpos/genética , Microcorpos/imunologia , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/imunologia , Ácido Oxaloacético/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Filogenia , Proteínas de Protozoários/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/métodos , Trypanosoma brucei brucei/imunologiaRESUMO
We describe the clinical, pathologic, and biochemical findings for two peroxisome-deficient patients in a newly identified complementation group. Both patients had biochemical findings typical of patients with peroxisome biogenesis disorders. However, whereas one patient had the typical clinicopathologic features of Zellweger syndrome, the other patient's phenotype was atypical.
Assuntos
Catalase/química , Microcorpos/química , Microcorpos/genética , Transtornos Peroxissômicos/diagnóstico , Transtornos Peroxissômicos/genética , Fusão Celular , Consanguinidade , Fibroblastos/química , Teste de Complementação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Plasma , Síndrome de Zellweger/diagnóstico , Síndrome de Zellweger/genéticaRESUMO
OBJECTIVE: To use the technique of complementation analysis to help define genotype and classify patients with clinical manifestations consistent with those of the disorders of peroxisome assembly, namely the Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). STUDY DESIGN: Clinical findings, peroxisomal function, and complementation groups were examined in 173 patients with the clinical manifestations of these disorders. RESULTS: In 37 patients (21%), peroxisome assembly was intact and isolated deficiencies of one of five peroxisomal enzymes involved in the beta-oxidation of fatty acids or plasmalogen biosynthesis were demonstrated. Ten complementation groups were identified among 93 patients (54%) with impaired peroxisome assembly and one of three phenotypes (ZS, NALD, or IRD) without correlation between complementation group and phenotype. Forty-three patients (25%) had impaired peroxisome assembly associated with the RCDP phenotype and belonged to a single complementation group. Of the 173 patients, 10 had unusually mild clinical manifestations, including survival to the fifth decade or deficits limited to congenital cataracts. CONCLUSIONS: At least 16 complementation groups, and hence genotypes, are associated with clinical manifestations of disorders of peroxisome assembly. The range of phenotype is wide, and some patients have mild involvement.