RESUMO
In the eukaryotic model yeast Saccharomyces cerevisiae, arsenic (As) detoxification is regulated by two transcriptional factors, Yap8 and Yap1. Yap8 specifically controls As extrusion from the cell, whether Yap1 avoids arsenic-induced oxidative damages. Accordingly, cells lacking both Yap1 and Yap8 are more sensitive to arsenate than cells lacking each regulator individually. Strikingly enough, the same sensitivity pattern was observed under anoxia, suggesting that Yap1 role in As detoxification might not be restricted to the regulation of the oxidative stress response. This finding prompted us to study the transcriptomic profile of wild-type and yap1 mutant cells exposed to arsenate. Interestingly, we found that, under such conditions, several genes involved in the biogenesis of FeS proteins were upregulated in a Yap1-dependent way. In line with this observation, arsenate treatment decreases the activity of the mitochondrial aconitase, Aco1, an FeS cluster-containing enzyme, this effect being even more pronounced in the yap1 mutant. Reinforcing the relevance of FeS cluster biogenesis in arsenate detoxification, the overexpression of several ISC and CIA machinery genes alleviates the deleterious effect of arsenate caused by the absence of Yap1 and Yap8. Altogether our data suggest that the upregulation of FeS biogenesis genes regulated by Yap1 might work as a cellular shield against arsenate toxicity.
Assuntos
Arseniatos/toxicidade , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Ferro-Enxofre/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteínas Ferro-Enxofre/efeitos dos fármacos , Proteínas Ferro-Enxofre/genética , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake.
Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Ligação ao Ferro/biossíntese , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Transporte Biológico/genética , Cádmio/toxicidade , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cobre , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Mutação , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Response to hyperosmotic stress in the yeast Saccharomyces cerevisiae involves the participation of the general stress response mediated by Msn2/4 transcription factors and the HOG pathway. One of the transcription factors activated through this pathway is Hot1, which contributes to the control of the expression of several genes involved in glycerol synthesis and flux, or in other functions related to adaptation to adverse conditions. This work provides new data about the interaction mechanism of this transcription factor with DNA. By means of one-hybrid and electrophoretic mobility assays, we demonstrate that the C-terminal region, which corresponds to amino acids 610-719, is the DNA-binding domain of Hot1. We also describe how this domain recognizes sequence 5'-GGGACAAA-3' located in the promoter of gene STL1. The bioinformatics analysis carried out in this work allowed the identification of identical or similar sequences (with up to two mismatches) in the promoter of other Hot1 targets, where central element GGACA was quite conserved among them. Finally, we found that small variations in the sequence recognized by Hot1 may influence its ability to recognize its targets in vivo.
Assuntos
DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Sequência Conservada , DNA Fúngico/genética , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Osmorregulação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
In this paper I describe the main aspects of my career and focus on the retrospective on my life and my work.
Assuntos
Pesquisa Biomédica/história , Microbiologia/história , História do Século XX , História do Século XXIRESUMO
The potential of sulfate-reducing bacteria (SRB) as biocatalysts for H2 production from formate was recently demonstrated, but the electron transfer pathways involved were not described. In the present work, we analyzed the H2 production capacity of five Desulfovibrio strains: Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio fructosivorans, and Desulfovibrio gigas. D. vulgaris showed the highest H2 productivity (865 mL Lmedium (-1)), and D. gigas the lowest one (374 mL Lmedium (-1) of H2). The electron transfer pathways involved in formate-driven H2 production by these two organisms were further investigated through the study of deletion mutants of hydrogenases (Hases) and formate dehydrogenases (Fdhs). In D. vulgaris, the periplasmic FdhAB is the key enzyme for formate oxidation and two pathways are apparently involved in the production of H2 from formate: a direct one only involving periplasmic enzymes and a second one that involves transmembrane electron transfer and may allow energy conservation. In the presence of selenium, the Hys [NiFeSe] Hase is the main periplasmic enzyme responsible for H2 production, and the cytoplasmic Coo Hase is apparently involved in the ability of D. vulgaris to grow by converting formate to H2, in sparging conditions. Contrary to D. vulgaris, H2 production in D. gigas occurs exclusively by the direct periplasmic route and does not involve the single cytoplasmic Hase, Ech. This is the first report of the metabolic pathways involved in formate metabolism in the absence of sulfate in SRB, revealing that the electron transfer pathways are species-specific.
Assuntos
Desulfovibrio/metabolismo , Transporte de Elétrons , Formiatos/metabolismo , Hidrogênio/metabolismo , Biotransformação , Deleção de Genes , Redes e Vias Metabólicas/genéticaRESUMO
BACKGROUND: Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1 mediates tolerance to cobalt toxicity. METHODS: Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. RESULTS: Cobalt activates Yap1 under aerobiosis and anaerobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we show that Yap1 is a repressor of the low affinity iron transporter, FET4, which is also known to transport cobalt. CONCLUSIONS: Cobalt activates Yap1 that alleviates the oxidative damage caused by this metal. Yap1 partially controls cobalt cellular uptake via the regulation of PHO84. Although FET4 repression by Yap1 has no effect on cobalt uptake, it may be its first line of defense against other toxic metals. GENERAL SIGNIFICANCE: Our results emphasize the important role of Yap1 in mediating cobalt-induced oxidative damages and reveal new routes for cell protection provided by this regulator.
Assuntos
Cobalto/toxicidade , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Cobalto/metabolismo , Proteínas de Transporte de Cobre , Proteínas de Ligação ao Ferro/fisiologia , Fosfatos/metabolismo , Simportadores de Próton-Fosfato/fisiologia , Saccharomyces cerevisiae/metabolismo , Superóxidos/metabolismoRESUMO
Arsenic is a double-edge sword. On the one hand it is powerful carcinogen and on the other it is used therapeutically to treat acute promyelocytic leukemia. Here we report that arsenic activates the iron responsive transcription factor, Aft1, as a consequence of a defective high-affinity iron uptake mediated by Fet3 and Ftr1, whose mRNAs are drastically decreased upon arsenic exposure. Moreover, arsenic causes the internalization and degradation of Fet3. Most importantly, fet3ftr1 mutant exhibits increased arsenic resistance and decreased arsenic accumulation over the wild-type suggesting that Fet3 plays a role in arsenic toxicity. Finally we provide data suggesting that arsenic also disrupts iron uptake in mammals and the link between Fet3, arsenic and iron, can be relevant to clinical applications.
Assuntos
Arseniatos , Ferro/metabolismo , Saccharomyces cerevisiae , Animais , Arseniatos/efeitos adversos , Arseniatos/metabolismo , Arseniatos/uso terapêutico , Ceruloplasmina/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Mamíferos , Proteínas de Membrana Transportadoras/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio gigas/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Hidrogenase/classificação , Hidrogenase/metabolismo , Proteínas de Bactérias/genética , Desulfovibrio gigas/genética , Desulfovibrio gigas/metabolismo , Fermentação , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/fisiologia , Hidrogênio/metabolismo , Hidrogenase/genética , Ácido Láctico/metabolismo , Dados de Sequência Molecular , Ácido Pirúvico/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. gigas mutants Δroo and ΔnorR1-like is impaired. In order to confirm that D. gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ΔnorR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ΔnorR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. gigas subjected to nitrosative stress. It is identified in δ-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.
Assuntos
Proteínas de Bactérias/fisiologia , Desulfovibrio gigas/genética , Desulfovibrio gigas/fisiologia , Regulação Bacteriana da Expressão Gênica , Nitratos/fisiologia , Estresse Fisiológico/genética , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Teste de Complementação Genética , Genoma Bacteriano , Dados de Sequência Molecular , Nitrosação , Oxirredutases , Proteínas PII Reguladoras de Nitrogênio/classificação , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/fisiologia , Filogenia , Fatores de Transcrição/classificação , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Aluminium (Al) toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. Being a trait governed by multiple genes, the identification and characterization of novel transcription factors (TFs) regulating the expression of entire response networks is a very promising approach. Therefore, the aim of the present study was to clone, localize, and characterize the TaSTOP1 gene, which belongs to the zinc finger family (Cys2His2 type) transcription factor, at molecular level in bread wheat. RESULTS: TaSTOP1 loci were cloned and localized on the long arm of homoeologous group 3 chromosomes [3AL (TaSTOP1-A), 3BL (TaSTOP1-B) and 3DL (TaSTOP1-D)] in bread wheat. TaSTOP1 showed four potential zinc finger domains and the homoeologue TaSTOP1-A exhibited transactivation activity in yeast. Expression profiling of TaSTOP1 transcripts identified the predominance of homoeologue TaSTOP1-A followed by TaSTOP1-D over TaSTOP1-B in root and only predominance of TaSTOP1-A in shoot tissues of two diverse bread wheat genotypes. Al and proton (H(+)) stress appeared to slightly modulate the transcript of TaSTOP1 homoeologues expression in both genotypes of bread wheat. CONCLUSIONS: Physical localization of TaSTOP1 results indicated the presence of a single copy of TaSTOP1 on homoeologous group 3 chromosomes in bread wheat. The three homoeologues of TaSTOP1 have similar genomic structures, but showed biased transcript expression and different response to Al and proton (H(+)) toxicity. These results indicate that TaSTOP1 homoeologues may differentially contribute under Al or proton (H(+)) toxicity in bread wheat. Moreover, it seems that TaSTOP1-A transactivation potential is constitutive and may not depend on the presence/absence of Al at least in yeast. Finally, the localization of TaSTOP1 on long arm of homoeologous group 3 chromosomes and the previously reported major loci associated with Al resistance at chromosome 3BL, through QTL and genome wide association mapping studies suggests that TaSTOP1 could be a potential candidate gene for genomic assisted breeding for Al tolerance in bread wheat.
Assuntos
Alumínio/toxicidade , Proteínas de Plantas/metabolismo , Triticum/efeitos dos fármacos , Triticum/metabolismo , Cromossomos de Plantas/genética , Proteínas de Plantas/genética , Prótons , Triticum/genéticaRESUMO
Although arsenic is notoriously poisonous to life, its utilization in therapeutics brings many benefits to human health, so it is therefore essential to discover the molecular mechanisms underlying arsenic stress responses in eukaryotic cells. Aiming to determine the contribution of Ca(2+) signalling pathways to arsenic stress responses, we took advantage of the use of Saccharomyces cerevisiae as a model organism. Here we show that Ca(2+) enhances the tolerance of the wild-type and arsenic-sensitive yap1 strains to arsenic stress in a Crz1-dependent manner, thus providing the first evidence that Ca(2+) signalling cascades are involved in arsenic stress responses. Moreover, our results indicate that arsenic shock elicits a cytosolic Ca(2+) burst in these strains, without the addition of exogenous Ca(2+) sources, strongly supporting the notion that Ca(2+) homeostasis is disrupted by arsenic stress. In response to an arsenite-induced increase of Ca(2+) in the cytosol, Crz1 is dephosphorylated and translocated to the nucleus, and stimulates CDRE-driven expression of the lacZ reporter gene in a Cnb1-dependent manner. The activation of Crz1 by arsenite culminates in the induction of the endogenous genes PMR1, PMC1 and GSC2. Taken together, these data establish that activation of Ca(2+) signalling pathways and the downstream activation of the Crz1 transcription factor contribute to arsenic tolerance in the eukaryotic model organism S. cerevisiae.
Assuntos
Arsênio/toxicidade , Cálcio/metabolismo , Proteínas de Ligação a DNA/biossíntese , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Fatores de Transcrição/biossíntese , Cátions Bivalentes/metabolismo , Perfilação da Expressão Gênica , Saccharomyces cerevisiae/genética , Ativação TranscricionalRESUMO
Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.
Assuntos
Desulfovibrio vulgaris/enzimologia , Formiato Desidrogenases/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Molibdênio/farmacologia , Tungstênio/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desulfovibrio vulgaris/metabolismo , Formiato Desidrogenases/genética , Formiatos/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.
RESUMO
The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gene expression when exposed to several environmental challenges. Homeostasis is achieved through a highly coordinated mechanism of transcription regulation involving several transcription factors, each one acting singly or in combination to perform specific functions. Here, we review our current knowledge of the function of the Yap transcription factors in stress response. They belong to b-ZIP proteins comprising eight members with specificity at the DNA-binding domain distinct from that of the conventional yeast AP-1 factor, Gcn4. We finish with new insights into the links of transcriptional networks controlling several cellular processes. The data reviewed in this article illustrate how much our comprehension of the biology of Yap family involved in stress response has advanced, and how much research is still needed to unravel the complexity of the role of these transcriptional factors. The complexities of these regulatory interactions, as well as the dynamics of these processes, are important to understand in order to elucidate the control of stress response, a highly conserved process in eukaryotes.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Yap4 is a nuclear-resident transcription factor induced in Saccharomyces cerevisiae when exposed to several stress conditions, which include mild hyperosmotic and oxidative stress, temperature shift or metal exposure. This protein is also phosphorylated. Here we report that this modification is driven by PKA and GSK3. In order to ascertain whether Yap4 is directly or indirectly phosphorylated by PKA, we searched for stress and PKA-related kinases that could phosphorylate Yap4. We show that phosphorylation is independent of the kinases Rim15, Yak1, Sch9, Slt2, Ste20 and Ptk2. In addition, we showed that Yap4 phosphorylation is also abrogated in the triple GSK3 mutant mck1 rim11 yol128c. Furthermore, our data reveal that Yap4 nuclear localization is independent of its phosphorylation state. This protein has several putative phosphorylation sites, but only the mutation of residues T192 and S196 impairs its phosphorylation under different stress conditions. The ability of the non-phosphorylated forms of Yap4 to partially rescue the hog1 severe sensitivity phenotype is not affected, suggesting that Yap4 activity is maintained in the absence of phosphorylation. However, this modification seems to be required for stability of the protein, as the non-phosphorylated form has a shorter half-life than the phosphorylated one.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Primers do DNA/genética , Genes Fúngicos , Quinase 3 da Glicogênio Sintase/genética , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
In the budding yeast Saccharomyces cerevisiae, arsenic detoxification involves the activation of Yap8, a member of the Yap (yeast AP-1-like) family of transcription factors, which in turn regulates ACR2 and ACR3, genes encoding an arsenate reductase and a plasma-membrane arsenite-efflux protein respectively. In addition, Yap1 is involved in the arsenic adaptation process through regulation of the expression of the vacuolar pump encoded by YCF1 (yeast cadmium factor 1 gene) and also contributing to the regulation of ACR genes. Here we show that Yap1 is also involved in the removal of ROS (reactive oxygen species) generated by arsenic compounds. Data on lipid peroxidation and intracellular oxidation indicate that deletion of YAP1 and YAP8 triggers cellular oxidation mediated by inorganic arsenic. In spite of the increased amounts of As(III) absorbed by the yap8 mutant, the enhanced transcriptional activation of the antioxidant genes such as GSH1 (gamma- glutamylcysteine synthetase gene), SOD1 (superoxide dismutase 1 gene) and TRX2 (thioredoxin 2 gene) may prevent protein oxidation. In contrast, the yap1 mutant exhibits high contents of protein carbonyl groups and the GSSG/GSH ratio is severely disturbed on exposure to arsenic compounds in these cells. These results point to an additional level of Yap1 contribution to arsenic stress responses by preventing oxidative damage in cells exposed to these compounds. Transcriptional profiling revealed that genes of the functional categories related to sulphur and methionine metabolism and to the maintenance of cell redox homoeostasis are activated to mediate adaptation of the wild-type strain to 2 mM arsenate treatment.
Assuntos
Arsênio/farmacologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Northern Blotting , Western Blotting , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Yeast adaptation to stress has been extensively studied. It involves large reprogramming of genome expression operated by many, more or less specific, transcription factors. Here, we review our current knowledge on the function of the eight Yap transcription factors (Yap1 to Yap8) in Saccharomyces cerevisiae, which were shown to be involved in various stress responses. More precisely, Yap1 is activated under oxidative stress, Yap2/Cad1 under cadmium, Yap4/Cin5 and Yap6 under osmotic shock, Yap5 under iron overload and Yap8/Arr1 by arsenic compounds. Yap3 and Yap7 seem to be involved in hydroquinone and nitrosative stresses, respectively. The data presented in this article illustrate how much knowledge on the function of these Yap transcription factors is advanced. The evolution of the Yap family and its roles in various pathogenic and non-pathogenic fungal species is discussed in the last section.
RESUMO
In the yeast Saccharomyces cerevisiae Aft1, the low iron-sensing transcription factor is known to regulate the expression of the FET3 gene. However, we found that a strain-lacking FET3 is more sensitive to copper excess than a strain-lacking AFT1, and accordingly, FET3 expression is not fully compromised in the latter. These findings suggest that, under such conditions, another regulator comes into play and controls FET3 expression. In this work, we identify Ace1, the regulator of copper detoxification genes, as a regulator of FET3. We suggest that the activation of FET3 by Ace1 prevents the hyper activation of Aft1, possibly by assuring the adequate functioning of mitochondrial iron-sulfur cluster biogenesis. While reinforcing the link between iron and copper homeostasis, this work unveils a novel protection mechanism against copper toxicity mediated by Ace1, which relies in the activation of FET3 and results in the restriction of Aft1 activity as a means to prevent excessive copper accumulation.
Assuntos
Ceruloplasmina/metabolismo , Cobre/metabolismo , Proteínas de Ligação a DNA/fisiologia , Inativação Metabólica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Ceruloplasmina/genética , Cobre/toxicidade , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Homeostase , Ferro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Towards elucidating the function of Yap2, which remains unclear, we have taken advantage of the C-terminal homology between Yap1 and Yap2. Swapping domains experiments show that the Yap2 C-terminal domain functionally substitutes for the homologous Yap1 domain in the response to Cd, but not to H2O2. We conclude that specificity determinants of the Cd response are encoded within both Yap1 and Yap2 C-terminus, whereas those required for H2O2 response are only present in the Yap1 C-terminus. Furthermore, our results identify FRM2 as Cd-responsive Yap2 target and indicate a possible role of this protein in regulating a metal stress response.
Assuntos
Cádmio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cádmio/toxicidade , Cisteína/química , Cisteína/genética , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Carioferinas/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional , Proteína Exportina 1RESUMO
Flavoredoxin participates in Desulfovibrio gigas thiosulfate reduction pathway. Its 3-dimensional model was generated allowing the oxidized riboflavin-5'-phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein. Comparison of K(d) values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were correlated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavoredoxin.