RESUMEN
BACKGROUND: Entamoeba histolytica, an intestinal parasitic protozoan that usually lives and multiplies within the human gut, is the causative agent of amoebiasis. To date, de novo glutathione biosynthesis and its associated enzymes have not been identified in the parasite. Cysteine has been proposed to be the main intracellular thiol. METHODS: Using bioinformatics tools to search for glutaredoxin homologs in the E. histolytica genome database, we identified a coding sequence for a putative Grx-like small protein (EhGLSP) in the E. histolytica HM-1:IMSS genome. We produced the recombinant protein and performed its biochemical characterization. RESULTS: Through in vitro experiments, we observed that recombinant EhGLSP could bind GSH and L-Cys as ligands. However, the protein exhibited very low GSH-dependent disulfide reductase activity. Interestingly, via UV-Vis spectroscopy and chemical analysis, we detected that recombinant EhGLSP (freshly purified from Escherichia coli cells by IMAC) was isolated together with a redox-labile [FeS] bio-inorganic complex, suggesting that this protein could have some function linked to the metabolism of this cofactor. Western blotting showed that EhGLSP protein levels were modulated in E. histolytica cells exposed to exogenous oxidative species and metronidazole, suggesting that this protein cooperates with the antioxidant mechanisms of this parasite. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings support the existence of a new metabolic actor in this pathogen. To the best of our knowledge, this is the first report on this protein class in E. histolytica.
Asunto(s)
Entamoeba histolytica , Parásitos , Animales , Humanos , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Parásitos/metabolismo , Anaerobiosis , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron-sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Hierro-Azufre , Trypanosoma brucei brucei , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Ligandos , Unión Proteica , Pliegue de Proteína , Trypanosoma brucei brucei/metabolismoRESUMEN
The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.
Asunto(s)
Cianuros/metabolismo , Glutarredoxinas/metabolismo , Estrés Oxidativo , Sulfurtransferasas/metabolismo , Xylella/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glutarredoxinas/genética , Modelos Moleculares , Filogenia , Conformación Proteica , Sulfurtransferasas/genética , Tiosulfato Azufretransferasa/metabolismoRESUMEN
We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.
Asunto(s)
Citosol/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/aislamiento & purificación , Mitocondrias/metabolismo , Taenia solium/genética , Tiorredoxinas/genética , Tiorredoxinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cysticercus/genética , Cysticercus/aislamiento & purificación , Cysticercus/metabolismo , Citosol/química , Disulfuros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Cinética , Mitocondrias/química , Mitocondrias/genética , Sistemas de Lectura Abierta , Conejos , Taenia solium/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
SIGNIFICANCE: Major pathogenic enterobacteria and protozoan parasites from the phylum Euglenozoa, such as trypanosomatids, are endowed with glutathione (GSH)-spermidine (Sp) derivatives that play important roles in signaling and metal and thiol-redox homeostasis. For some Euglenozoa lineages, the GSH-Sp conjugates represent the main redox cosubstrates around which entire new redox systems have evolved. Several proteins underwent molecular adaptations to synthesize and utilize the new polyamine-based thiols. Recent Advances: The genomes of closely related organisms have recently been sequenced, which allows mining and analysis of gene sequences that belong to these peculiar redox systems. Similarly, the three-dimensional structures of several of these proteins have been solved, which allows for comparison with their counterparts in classical redox systems that rely on GSH/glutaredoxin and thioredoxin. CRITICAL ISSUES: The evolutionary and structural aspects related to the emergence and use of GSH-Sp conjugates in Euglenozoa are reviewed focusing on unique structural specializations that proteins developed to use N1,N8-bisglutathionylspermidine (trypanothione) as redox cosubstrate. An updated overview on the biochemical and biological significance of the major enzymatic activities is also provided. FUTURE DIRECTIONS: A thiol-redox system strictly dependent on trypanothione is a feature unique to trypanosomatids. The physicochemical properties of the polyamine-GSH conjugates were a major driving force for structural adaptation of proteins that use these thiols as ligand and redox cofactor. In fact, the structural differences of indispensable components of this system can be exploited toward selective drug development. Future research should clarify whether additional cellular processes are regulated by the trypanothione system. Antioxid. Redox Signal. 28, 463-486.
Asunto(s)
Glutarredoxinas/genética , Compuestos de Sulfhidrilo/química , Tiorredoxinas/genética , Trypanosomatina/metabolismo , Evolución Molecular , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Oxidación-Reducción , Poliaminas/química , Poliaminas/metabolismo , Espermidina/química , Espermidina/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Trypanosomatina/química , Trypanosomatina/genéticaRESUMEN
BACKGROUND/AIMS: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes. METHODS: Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile. RESULTS: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 â¼ 34 mM). CONCLUSION: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.
Asunto(s)
Cloruros/farmacología , Expresión Génica/efectos de los fármacos , Glutarredoxinas/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Secuencia de Aminoácidos , Aniones/química , Secuencia de Bases , Sitios de Unión , Línea Celular , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Glutarredoxinas/genética , Humanos , Ionóforos/análisis , Ionóforos/química , Metaloproteínas/genética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Ribosómicas/genética , Alineación de SecuenciaRESUMEN
The aim of the current study was to evaluate the effect of chronic dehydroepiandrosterone (DHEA) administration on steroid hormones and antioxidant parameters in aged rats. To this end, three groups of Sprague-Dawley rats were compared: young (3 months of age) untreated; aged (19 months old) untreated; and aged rats treated with 20 mg/kg DHEA for 8 weeks. Major organs of aged rats in the untreated group demonstrated physiological atrophy, compared to those of young rats; this effect appeared to have been partially reversed by DHEA treatment. Testosterone and estradiol contents were significantly decreased and aldosterone significantly increased in aged untreated, compared to young untreated rats. Steroid hormone levels were obviously reversed, however, in aged rats treated with DHEA. Additionally, superoxide dismutase activity in serum, brain, heart, and liver was decreased, and maleic dialdehyde content in heart was markedly increased in untreated aged, compared to young, rats. Importantly, these changes in brain and heart of aged rats were reversed by DHEA treatment. Heme oxygenase mRNA levels were increased and inducible nitric oxide synthase mRNA levels decreased in aged, compared to young, rats; DHEA treatment appeared to reverse these changes. These results indicate that chronic DHEA administration may have effects on steroid hormone levels and antioxidant parameters in aged rats and result in postponement of the aging process.
Asunto(s)
Envejecimiento/sangre , Deshidroepiandrosterona/farmacología , Aldosterona/sangre , Animales , Antioxidantes/metabolismo , Peso Corporal/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Estradiol/sangre , Expresión Génica/efectos de los fármacos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Masculino , Malondialdehído/sangre , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especificidad de Órganos , Oxidación-Reducción , Ratas Sprague-Dawley , Superóxido Dismutasa/sangre , Testosterona/sangreRESUMEN
BACKGROUND: Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. METHODS: We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. RESULTS AND CONCLUSIONS: We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. GENERAL SIGNIFICANCE: These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.
Asunto(s)
Asfixia Neonatal/enzimología , Encéfalo/enzimología , Glutarredoxinas/metabolismo , Hipoxia-Isquemia Encefálica/enzimología , Neuronas/enzimología , Tiorredoxinas/metabolismo , Animales , Asfixia Neonatal/patología , Encéfalo/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Glutarredoxinas/genética , Humanos , Hipoxia-Isquemia Encefálica/patología , Masculino , Neuronas/patología , Oxidación-Reducción , Oxígeno/metabolismo , Fenotipo , Interferencia de ARN , Ratas Sprague-Dawley , Transducción de Señal , Tiorredoxinas/genética , Factores de Tiempo , TransfecciónRESUMEN
In Trypanosoma cruzi, the modification of thiols by glutathionylation-deglutathionylation and its potential relation to protective, regulatory or signaling functions have been scarcely explored. Herein we characterize a dithiolic glutaredoxin (TcrGrx), a redox protein with deglutathionylating activity, having potential functionality to control intracellular homeostasis of protein and non-protein thiols. The catalytic mechanism followed by TcrGrx was found dependent on thiol concentration. Results suggest that TcrGrx operates as a dithiolic or a monothiolic Grx, depending on GSH concentration. TcrGrx functionality to mediate reduction of protein and non-protein disulfides was studied. TcrGrx showed a preference for glutathionylated substrates respect to protein disulfides. From in vivo assays involving TcrGrx overexpressing parasites, we observed the contribution of the protein to increase the general resistance against oxidative damage and intracellular replication of the amastigote stage. Also, studies performed with epimastigotes overexpressing TcrGrx strongly suggest the involvement of the protein in a cellular pathway connecting an apoptotic stimulus and apoptotic-like cell death. Novel information is presented about the participation of this glutaredoxin not only in redox metabolism but also in redox signaling pathways in T. cruzi. The influence of TcrGrx in several parasite physiological processes suggests novel insights about the protein involvement in redox signaling.
Asunto(s)
Glutarredoxinas/metabolismo , Redes y Vías Metabólicas , Proteínas Protozoarias/metabolismo , Tolueno/análogos & derivados , Trypanosoma cruzi/metabolismo , Apoptosis , Biocatálisis , Western Blotting , Citosol/enzimología , Glutarredoxinas/genética , Glutatión/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Oxidación-Reducción , Proteínas Protozoarias/genética , Especificidad por Sustrato , Tolueno/metabolismo , Trypanosoma cruzi/citología , Trypanosoma cruzi/genéticaRESUMEN
Mutations in Cu, Zn-superoxide dismutase (Sod1) have been associated with familial amyotrophic lateral sclerosis, an age-related disease. Because several studies suggest that oxidative stress plays a central role in neurodegeneration, we aimed to investigate the role of the antioxidant glutathione (GSH) in the activation of human A4V Sod1 during chronological aging. Transformation of wild-type and A4V hSod1 into a gsh null mutant and in its parental strain of Saccharomyces cerevisiae indicated that during aging, the number of viable cells was strongly influenced by A4V hSod1 mainly in cells lacking GSH. Activity of hSod1 increased in response to aging, although the increase observed in A4V hSod1 was almost 60% lower. Activation of hSod1 (A4V and WT) did not occur after aging, in cells lacking GSH, but could still be observed in the absence of Ccs1. Furthermore, no increase in activity could be seen in grx1 and grx2 null mutants, suggesting that glutathionylation is essential for hSod1 activation. The A4V mutation as well as the absence of GSH, reduced hSod1 activity, and increased oxidative damage after aging. In conclusion, our results point to a GSH requirement for hSod1 Ccs1-independent activation as well as for protection of hSod1 during the aging process.
Asunto(s)
Glutatión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Senescencia Celular/genética , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutatión/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Estrés Oxidativo/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.
Asunto(s)
Animales , Femenino , Ratones , Candida albicans/efectos de los fármacos , Candidiasis/microbiología , Diamida/farmacología , Glutarredoxinas/fisiología , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/fisiología , /farmacología , Candida albicans/enzimología , Candida albicans/genética , Modelos Animales de Enfermedad , Farmacorresistencia Fúngica/genética , Genotipo , Glutarredoxinas/genética , Ratones Endogámicos BALB C , Mutación , Fenotipo , Superóxido Dismutasa/genética , VirulenciaRESUMEN
To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.
Asunto(s)
Candida albicans/efectos de los fármacos , Candidiasis/microbiología , Diamida/farmacología , Glutarredoxinas/fisiología , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/fisiología , Vitamina K 3/farmacología , Animales , Candida albicans/enzimología , Candida albicans/genética , Modelos Animales de Enfermedad , Farmacorresistencia Fúngica/genética , Femenino , Genotipo , Glutarredoxinas/genética , Ratones , Ratones Endogámicos BALB C , Mutación , Fenotipo , Superóxido Dismutasa/genética , VirulenciaRESUMEN
It has been shown that the activation of cytosolic superoxide dismutase (Sod1) in Saccharomyces cerevisiae is only dependent on Ccs1, which is responsible for insertion of copper into the enzyme catalytic center, and that glutathione (GSH) is not necessary for this process. In this work, we addressed an important role of GSH in Sod1 activation by a Ccs1-dependent mechanism during oxidative stress and its role in yeast lifespan. Exponential cells of Saccharomyces cerevisiae, treated or not with 0.5 mM menadione for 1 h, were used for evaluation of the effect of a mild oxidative stress pre-treatment on chronological lifespan. The results showed that menadione induced a lifespan extension in the wild-type (WT) strain but this adaptive response was repressed in gsh1 and in sod1 strains. Interestingly, menadione treatment increased SOD1 and CCS1 gene expression in both WT and gsh1 strains. However, while these strains showed the same Sod1 activity before treatment, only the WT presented an increase of Sod1 activity after menadione exposure. Glutathionylation seems to be essential for Sod1 activation since no increase in activity was observed after menadione treatment in grx1 and grx2 null mutants. Our results suggest that GSH and glutathionylation are fundamental to protect Sod1 sulfhydryl residues under mild oxidative stress, enabling Sod1 activation and lifespan extension.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Glutatión/metabolismo , Saccharomyces cerevisiae/enzimología , Superóxido Dismutasa/metabolismo , Vitamina K 3/farmacología , Cronología como Asunto , Cobre/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Estrés Oxidativo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Glutaredoxins play an important role in cellular functionality. A putative dithiol glutaredoxin is encoded in the genome of Trypanosoma cruzi. We cloned the gene and obtained the recombinant protein, which behaved as a typical thioltransferase. Activity was variable and dependent on the nature of reducer or oxidant agent used, or both. Epimastigote extracts exhibited similar activity, suggesting the occurrence of the protein in the parasite. Results support a redox scenario in T. cruzi, with glutaredoxin being involved mainly in reduction of glutathione disulfide as well as in deglutathionylation of target proteins.
Asunto(s)
Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Clonación Molecular , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Trypanosoma cruzi/genéticaRESUMEN
Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K(M) for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 A, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.
Asunto(s)
Glutarredoxinas/química , Isoenzimas/química , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 muM CdSO(4), showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.
Asunto(s)
Apoptosis/genética , Compuestos de Cadmio/toxicidad , Glutarredoxinas/fisiología , Mutágenos/toxicidad , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Sulfatos/toxicidad , Glutarredoxinas/genética , Glutatión/metabolismo , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Resistance of Candida albicans to reactive oxygen species is thought to enhance its virulence in mammalian hosts. Genes such as SOD1, which encodes the anti-oxidant, superoxide dismutase, are known virulence factors. We disrupted the gene GRX2, which encodes a putative glutathione reductase (glutaredoxin) in C. albicans, and we compared the mutant with an sod1Deltamutant. In vitro, the grx2Deltastrain, but not the sod1Delta strain, was defective in hypha formation. The grx2Deltastrain, but not sod1Delta, was significantly more susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, both mutants were susceptible to 1 mM menadione, but grx2Deltanull alone was resistant to diamide. Both mutants were attenuated in a murine intravenous challenge model, and a GRX2 reintegrant regained partial virulence. Emphasis on the putative function of products of genes such as SOD1 and GRX2 in resistance to oxidative stress may oversimplify their functions in the virulence process, since the grx2Deltastrain also gave defective hypha formation. Both mutants were sensitive to menadione and were slow to form germ tubes, though growth rates matched controls once the lag phase was passed.