RESUMO
Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.
Assuntos
Metionina Sulfóxido Redutases , Trypanosoma cruzi , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/metabolismo , Trypanosoma cruzi/genética , Oxirredução , Cisteína/química , Metionina/metabolismoRESUMO
BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.
Assuntos
Citoplasma/química , Leptospira interrogans/genética , Metionina Sulfóxido Redutases/genética , Metionina/metabolismo , Sequência de Aminoácidos/genética , Catálise , Citoplasma/enzimologia , Genoma Bacteriano/genética , Humanos , Leptospira interrogans/enzimologia , Metionina/química , Metionina/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/ultraestrutura , Oxirredução , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por SubstratoRESUMO
To combat the deleterious effects that oxidation of the sulfur atom in methionine to sulfoxide may bring, aerobic cells express repair pathways involving methionine sulfoxide reductases (MSRs) to reverse the above reaction. Here, we show that Trypanosoma brucei, the causative agent of African trypanosomiasis, expresses two distinct trypanothione-dependent MSRs that can be distinguished from each other based on sequence, sub-cellular localisation and substrate preference. One enzyme found in the parasite's cytosol, shows homology to the MSRA family of repair proteins and preferentially metabolises the S epimer of methionine sulfoxide. The second, which contains sequence motifs present in MSRBs, is restricted to the mitochondrion and can only catalyse reduction of the R form of peptide-bound methionine sulfoxide. The importance of these proteins to the parasite was demonstrated using functional genomic-based approaches to produce cells with reduced or elevated expression levels of MSRA, which exhibited altered susceptibility to exogenous H2O2. These findings identify new reparative pathways that function to fix oxidatively damaged methionine within this medically important parasite.
Assuntos
Metionina Sulfóxido Redutases/genética , Metionina/análogos & derivados , Metionina/metabolismo , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Biocatálise , Citosol/efeitos dos fármacos , Citosol/enzimologia , Expressão Gênica , Teste de Complementação Genética , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Oxirredução , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologiaRESUMO
Porcine ear size is an important characteristic for distinguishing among pig breeds. In a previous genome-wide association study of porcine ear size, LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3), high mobility group AT-hook 2 (HMGA2), and Wnt inhibitory factor 1 (WIF1) were implicated as important candidate genes for ear size. This study investigated the expression levels of four candidate genes for ear size in Erhualian and Large White pigs. Ten Erhualian pigs with large ears and eight Large White pigs with small ears at 60 days of age were examined. The mRNA expression levels of the four candidate genes were quantified by real-time polymerase chain reaction. WIF1 mRNA expression was significantly higher in Large White than in Erhualian pigs (P < 0.05), whereas the expression levels of the other three genes were not significantly different between the two breeds. The protein expression levels of the four genes were analyzed using western blot. WIF1 protein expression was significantly higher in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 protein expression was significantly higher in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 is the main causal gene for ear size in pigs.
Assuntos
Orelha/crescimento & desenvolvimento , Locos de Características Quantitativas , Suínos/genética , Animais , Animais Endogâmicos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In the present study we evaluated the pre-treatment (priming) of Arabidopsis thaliana plants with sodium nitroprusside (SNP), a NO-donor, as an interesting approach for improving plant tolerance to cadmium stress. We focused on the cell redox balance and on the methionine sulfoxide reductases (MSR) family as a key component of such response. MSR catalyse the reversible oxidation of MetSO residues back to Met. Five MSRA genes and nine MSRB genes have been identified in A. thaliana, coding for proteins with different subcellular locations. After treating 20 days-old A. thaliana (Col 0) plants with 100 µM CdCl2, increased protein carbonylation in leaf tissue, lower chlorophyll content and higher levels of reactive oxygen species (ROS) in chloroplasts were detected, together with increased accumulation of all MSR transcripts evaluated. Further analysis showed reduction in guaiacol peroxidase activity (GPX) and increased catalase (CAT) activity, with no effect on ascorbate peroxidase (APX) activity. Pre-exposition of plants to 100 µM SNP before cadmium treatment restored redox balance; this seems to be linked to a better performance of antioxidant defenses. Our results indicate that NO priming may be acting as a modulator of plant antioxidant system by interfering in oxidative responses and by preventing up-regulation of MSR genes caused by metal exposure.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Cádmio/toxicidade , Metionina Sulfóxido Redutases/metabolismo , Nitroprussiato/farmacologia , Regulação para Cima/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/efeitos dos fármacos , Cloroplastos/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Metionina Sulfóxido Redutases/genética , Microscopia de Fluorescência , Família Multigênica , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/metabolismo , Oxirredução/efeitos dos fármacos , Peroxidase/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) carrying SCCmec type IV has emerged in hospitals worldwide. The aim of this study was to evaluate phenotypic and molecular characteristics of antimicrobial resistance in MRSA SCCmec IV isolates, presenting different genetic backgrounds, isolated from hospitals in Rio de Janeiro. The antimicrobial resistance of 128 S. aureus type IV isolates from 11 hospitals was characterized by the disk diffusion test and minimum inhibitory concentration (MIC) test. Mutations in parC gene, which encodes ciprofloxacin resistance, and genes associated with macrolide-lincosamide-streptogramin B (MLSb) resistance were also investigated. MRSA isolates belonging to USA400/ST1 (60 isolates), USA800/ST5 (40), USA1100/ST30 (13), and other 11 (15) lineages were mainly resistant to erythromycin (68%), ciprofloxacin (56%), and clindamycin (50%). The highest antimicrobial resistance rates were found among USA400 isolates (p < 0.05). The majority of them (90%) carried only the erm(C) gene and mainly presented two mutation types in the parC gene. The msr(A) gene was most frequently found among USA800 isolates (p < 0.05). Among MRSA type IV isolates from Rio de Janeiro hospitals, multiresistance, including mutations in parC gene, was associated to the USA400/ST1, while the msr(A) gene was associated with USA800/ST5 isolates, highlighting that these lineages could have more potential to persist in a hospital environment.
Assuntos
DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus Resistente à Meticilina/genética , Metionina Sulfóxido Redutases/genética , Metiltransferases/genética , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , DNA Topoisomerase IV/metabolismo , Hospitais , Humanos , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Metionina Sulfóxido Redutases/metabolismo , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Mutação , Quinolonas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Estreptogramina B/farmacologiaRESUMO
Promoters of genes encoding superoxide dismutase (sodA) and peptide methionine sulfoxide reductase (msrA) from Cory-nebacterium glutamicum were cloned and sequenced. Promoter region analysis of sodA-msrA was unable to identify putative sites of fixed eventual regulators except for possible sites of fixed OxyR and integra-tion host factor. A study of the regulation of these genes was performed using the lacZ gene of Escherichia coli as a reporter placed under the control of sequences downstream of sodA and msrA. In silico analysis was used to identify regulators in the genome of C. glutamicum, which revealed the absence of homologs of soxRS and arcA and the presence of inactive oxyR and putative candidates of the homologs of ahpC, ohrR, integration host factor, furA, IdeR, diphtheria toxin repressor, and mntR.
Assuntos
Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Metionina Sulfóxido Redutases/genética , Estresse Oxidativo/fisiologia , Superóxido Dismutase/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/efeitos da radiação , Metionina Sulfóxido Redutases/biossíntese , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Superóxido Dismutase/biossínteseRESUMO
Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.