RESUMO
The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2-dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2. To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2-HIS3 fusion in the absence of H2O2. Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase. Northern blot and whole-genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2-inducible genes is elevated in Deltatrr1 mutants in the absence of external stress. In Deltatrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein-folding chaperones, are hyperinduced. However, despite the elevated expression of genes encoding antioxidant enzymes, Deltatrr1 mutants are extremely sensitive to H2O2. The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress-induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Análise de Sequência de DNA , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fatores de Transcrição/genéticaRESUMO
We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.
Assuntos
Meio Ambiente , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Carbono/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Diamida/farmacologia , Ditiotreitol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Calefação , Peróxido de Hidrogênio/farmacologia , Nitrogênio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Pressão Osmótica , Saccharomyces cerevisiae/efeitos dos fármacos , Reagentes de Sulfidrila/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Vitamina K/farmacologiaRESUMO
The glutathione- and thioredoxin-dependent reduction systems are responsible for maintaining the reduced environment of the Escherichia coli and Saccharomyces cerevisiae cytosol. Here we examine the roles of these two cellular reduction systems in the bacterial and yeast defenses against oxidative stress. The transcription of a subset of the genes encoding glutathione biosynthetic enzymes, glutathione reductases, glutaredoxins, thioredoxins, and thioredoxin reductases, as well as glutathione- and thioredoxin-dependent peroxidases is clearly induced by oxidative stress in both organisms. However, only some strains carrying mutations in single genes are hypersensitive to oxidants. This is due, in part, to the redundant effects of the gene products and the overlap between the two reduction systems. The construction of strains carrying mutations in multiple genes is helping to elucidate the different roles of glutathione and thioredoxin, and studies with such strains have recently revealed that these two reduction systems modulate the activities of the E. coli OxyR and SoxR and the S. cerevisiae Yap1p transcriptional regulators of the adaptive responses to oxidative stress.
Assuntos
Escherichia coli/fisiologia , Glutationa/metabolismo , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/fisiologia , Tiorredoxinas/metabolismo , OxirreduçãoRESUMO
The NhaA Na+/H+ antiporter is the main system responsible for adaptation to Na+ and alkaline pH (in the presence of Na+) in Escherichia coli and many other enteric bacteria. It is under intricate control. At the protein level it is regulated directly by pH, one of its regulatory signals. A pH shift from 7 to 8.5 activates the antiporter and, in a fashion correlated with the activity change, confers a conformation change that, in isolated membrane vesicles, is reflected in the exposure of trypsin-cleavable sites. H225 and G338 are essential for the pH response of NhaA. nhaA transcription is dependent on NhaR, a positive regulator of the LysR family, and is regulated by Na+, the other environmental signal. Na+ affects the NhaR/nhaA interaction directly by changing the footprint of NhaR on nhaA in a pH-dependent fashion. The expression of nhaA is also under global regulation of H-NS. We suggest that the pattern of regulation of nhaA found in E. coli is a paradigm for the response of proteins and genes to H+ and Na+, the most common ions that challenge every cell.
Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Prótons , Sódio , Fatores de Transcrição/metabolismoRESUMO
Escherichia coli NhaR controls expression of a sodium/proton (Na+/H+) antiporter, NhaA. The Vibrio cholerae NhaR protein shows over 60% identity to those of Escherichia coli and Salmonella enteritidis. V. cholerae NhaR complements an E. coli nhaR mutant for growth in 100 mM LiCl-33 mM NaCl, pH 7.6, and enhances the Na+-dependent induction of an E. coli chromosomal nhaA::lacZ fusion. These findings indicate functional homology to E. coli NhaR. Two V. cholerae nhaR mutants were constructed by using kanamycin resistance cartridge insertion at different sites to disrupt the gene. Both mutants showed sensitivity to growth in 120 mM LiCl, pH 9.2, compared with the wild-type strain and could be complemented by the introduction of V. cholerae nhaR on a low-copy-number plasmid. An nhaR mutation had no detectable effect on the virulence of the V. cholerae strain in the infant mouse model, suggesting that the antiporter system involved is not required in vivo, at least in this animal model.