RESUMO
The ubiquitin-proteasome system is responsible for degradation of the majority of intracellular proteins in eukaryotic cells. The 26S proteasome proteolytic complex is composed of a 20S core particle responsible for protein degradation and the 19S lid which plays a role in the recognition of polyubiquitinated substrates. The 19S regulatory particle (Rps) is composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. In this study, we analyzed the expression profile of 19S Rpt subunits in the larvae and adult stage of the Schistosoma mansoni life cycle. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) revealed that the majority of the 19S Rpt subunits amplified at the expected molecular masses for various investigated stages. In addition, SmRpt1, SmRpt2, and SmRpt6 transcript levels were increased in 3 h-cultured schistosomula and reasonably maintained until 5 h in culture, as revealed by qRT-PCR. Phylogenetic analysis of 19S Rpt subunits showed high structural conservation in comparison to other Rpt orthologues. The mRNA expression profile of 19S Rpt subunits did not correlate with 26S proteasome proteolytic activity as judged by a (14)C-casein-degrading assay, in the early cultured schistosomula. Taken together, these results revealed a differential expression profile for 19S Rpt subunits whose transcript levels could not be directly associated to 26S proteasome activity.
Assuntos
Regulação da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/genética , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Adenosina Trifosfatases/genética , Animais , Sequência Conservada , Perfilação da Expressão Gênica , Humanos , Larva/enzimologia , Larva/genética , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Subunidades Proteicas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The human blood fluke Schistosoma mansoni is the primary cause of schistosomiasis, a debilitating disease that affects 200 million individuals in over 70 countries. The biogenic amine serotonin is essential for the survival of the parasite and serotonergic proteins are potential novel drug targets for treating schistosomiasis. Here we characterize two novel serotonin transporter gene transcripts, SmSERT-A and SmSERT-B, from S.mansoni. Southern blot analysis shows that the two mRNAs are the products of different alleles of a single SmSERT gene locus. The two SmSERT forms differ in three amino acid positions near the N-terminus of the protein. Both SmSERTs are expressed in the adult form and in the sporocyst form (infected snails) of the parasite, but are absent from all other stages of the parasite's complex life cycle. Heterologous expression of the two cDNAs in mammalian cells resulted in saturable, sodium-dependent serotonin transport activity with an apparent affinity for serotonin comparable to that of the human serotonin transporter. Although the two SmSERTs are pharmacologically indistinguishable from each other, efflux experiments reveal notably higher substrate selectivity for serotonin compared with their mammalian counterparts. Several well-established substrates for human SERT including (+/-)MDMA, S-(+)amphetamine, RU 24969, and m-CPP are not transported by SmSERTs, underscoring the higher selectivity of the schistosomal isoforms. Voltage-clamp recordings of SmSERT substrate-elicited currents confirm the substrate selectivity observed in efflux experiments and suggest that it may be possible to exploit the electrogenic nature of SmSERT to screen for compounds that target the parasite in vivo.
Assuntos
Alelos , Schistosoma mansoni/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Condutividade Elétrica , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Estágios do Ciclo de Vida/genética , Masculino , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismo , Xenopus laevis/fisiologiaRESUMO
DNA is often damaged by many environmental agents, which lead to the up-regulation of several genes involved in different repair pathways. Schistosoma mansoni has a complex life cycle, being exposed to a subset of DNA-damaging agents, such as those present in the environment and host immune response. Recently, studies showed that nucleotide excision repair (NER) is an indispensable mechanism for removing a broad spectrum of different DNA lesions. In the present report, we showed the gene expression of nucleotide excision repair factor 2 (NEF2) SmRad23 and SmRad4, in different developmental stages of S. mansoni, as well as the differential expression of these genes in S. mansoni adult worms treated with DNA-damaging agents. Furthermore, it was revealed the correlation of these genes with their orthologues in other eukaryotes. Our reports suggest that NER is an important repair pathway during the complex life cycle of S. mansoni.
