RESUMO
Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.
Assuntos
Animais , Endopeptidases/genética , Schistosoma mansoni/enzimologia , Ubiquitina Tiolesterase/genética , Cercárias/enzimologia , Cercárias/genética , Sequência Conservada/genética , Evolução Molecular , Expressão Gênica , Genoma Helmíntico/genética , Genoma/genética , Estágios do Ciclo de Vida/genética , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alinhamento de Sequência , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Transcriptoma/fisiologia , Transcitose/fisiologia , Ubiquitina Tiolesterase/classificação , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/fisiologiaRESUMO
Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite's genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.
Assuntos
Endopeptidases/genética , Schistosoma mansoni/enzimologia , Ubiquitina Tiolesterase/genética , Animais , Cercárias/enzimologia , Cercárias/genética , Sequência Conservada/genética , Evolução Molecular , Expressão Gênica , Genoma/genética , Genoma Helmíntico/genética , Estágios do Ciclo de Vida/genética , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Alinhamento de Sequência , Transcriptoma/fisiologia , Transcitose/fisiologia , Ubiquitina Tiolesterase/classificação , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/fisiologiaRESUMO
UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.
