RESUMEN
Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.
Asunto(s)
Peroxirredoxinas , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Cisteína/metabolismo , Disulfuros , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic 2-Cys peroxiredoxins (Prx) are abundant antioxidant enzymes whose thioredoxin peroxidase activity plays an important role in protecting against oxidative stress, aging, and cancer. Paradoxically, this thioredoxin peroxidase activity is highly sensitive to inactivation by peroxide-induced Prx hyperoxidation. However, any possible advantage in preventing Prx from removing peroxides under oxidative stress conditions has remained obscure. Here we demonstrate that, in cells treated with hydrogen peroxide, the Prx Tpx1 is a major substrate for thioredoxin in the fission yeast Schizosaccharomyces pombe and, as such, competitively inhibits thioredoxin-mediated reduction of other oxidized proteins. Consequently, we reveal that the hyperoxidation of Tpx1 is critical to allow thioredoxin to act on other substrates ensuring repair of oxidized proteins and cell survival following exposure to toxic levels of hydrogen peroxide. We conclude that the inactivation of the thioredoxin peroxidase activity of Prx is important to maintain thioredoxin activity and cell viability under oxidative stress conditions.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Peroxirredoxinas/antagonistas & inhibidores , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Tiorredoxinas/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Disulfuros/metabolismo , Técnicas de Inactivación de Genes , Peróxido de Hidrógeno/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Viabilidad Microbiana , Oxidación-Reducción , Estrés Oxidativo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/genética , Tiorredoxinas/genéticaRESUMEN
The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the 'p38-related' Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.
Asunto(s)
Candida albicans/fisiología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Candida albicans/enzimología , Candida albicans/genética , Candida albicans/patogenicidad , Regulación hacia Abajo , Femenino , Proteínas Fúngicas/genética , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Biológicos , Fenotipo , Fosforilación , Estrés Fisiológico , VirulenciaRESUMEN
Transcriptional control is exerted by the antagonistic activities of activator and repressor proteins. In Saccharomyces cerevisiae, transcription factor complexes containing the MADS box protein Mcm1p are key regulators of cell cycle-dependent transcription at both the G2/M and M/G1 transitions. The homeodomain repressor protein Yox1p acts in a complex with Mcm1p to control the timing of gene expression. Here, we show that Yox1p interacts with Mcm1p through a motif located N terminally to its homeodomain. Yox1p functions as a transcriptional repressor by competing with the forkhead transcription activator protein Fkh2p for binding to Mcm1p through protein-protein interactions at promoters of a subset of Mcm1p-regulated genes. Importantly, this competition is not through binding the same DNA site that is commonly observed. Thus, this study describes a different mechanism for determining the timing of cell cycle-dependent gene expression that involves competition between short peptide motifs in repressor and activator proteins for interaction with a common binding partner.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteína 1 de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
As a more selectively reactive oxygen species, H2O2 (hydrogen peroxide) has been co-opted as a signalling molecule, but high levels can still lead to lethal amounts of cell damage. 2-Cys Prxs (peroxiredoxins) are ubiquitous thioredoxin peroxidases which utilize reversibly oxidized catalytic cysteine residues to reduce peroxides. As such, Prxs potentially make an important contribution to the repertoire of cell defences against oxidative damage. Although the abundance of eukaryotic 2-Cys Prxs suggests an important role in maintaining cell redox, the surprising sensitivity of their thioredoxin peroxidase activity to inactivation by H2O2 has raised questions as to their role as an oxidative stress defence. Indeed, work in model yeast has led the way in revealing that Prxs do much more than simply remove peroxides and have even uncovered circumstances where their thioredoxin peroxidase activity is detrimental. In the present paper, we focus on what we have learned from studies in the fission yeast Schizosaccharomyces pombe about the different roles of 2-Cys Prxs in responses to H2O2 and discuss the general implications of these findings for other systems.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Peroxirredoxinas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
Quiescence (G0) is a reversible non-dividing state that facilitates cellular survival in adverse conditions. Here, we demonstrate that the HIRA histone chaperone complex is required for the reversibility and longevity of nitrogen starvation-induced quiescence in Schizosaccharomyces pombe. The HIRA protein, Hip1 is not required for entry into G0 or the induction of autophagy. Although hip1Δ cells retain metabolic activity in G0, they rapidly lose the ability to resume proliferation. After a short period in G0 (1 day), hip1Δ mutants can resume cell growth in response to the restoration of a nitrogen source but do not efficiently reenter the vegetative cell cycle. This correlates with a failure to induce the expression of MBF transcription factor-dependent genes that are critical for S phase. In addition, hip1Δ G0 cells rapidly progress to a senescent state in which they can no longer re-initiate growth following nitrogen source restoration. Analysis of a conditional hip1 allele is consistent with these findings and indicates that HIRA is required for efficient exit from quiescence and prevents an irreversible cell cycle arrest.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Chaperonas de Histonas/genética , División Celular , Proteínas de Ciclo Celular/metabolismo , Nitrógeno/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Polo kinases have crucial conserved functions in controlling the eukaryotic cell cycle through orchestrating several events during mitosis. An essential element of cell cycle control is exerted by altering the expression of key regulators. Here we show an important function for the polo kinase Cdc5p in controlling cell-cycle-dependent gene expression that is crucial for the execution of mitosis in the model eukaryote Saccharomyces cerevisiae. In particular, we find that Cdc5p is temporally recruited to promoters of the cell-cycle-regulated CLB2 gene cluster, where it targets the Mcm1p-Fkh2p-Ndd1p transcription factor complex, through direct phosphorylation of the coactivator protein Ndd1p. This phosphorylation event is required for the normal temporal expression of cell-cycle-regulated genes such as CLB2 and SWI5 in G2/M phases. Furthermore, severe defects in cell division occur in the absence of Cdc5p-mediated phosphorylation of Ndd1p. Thus, polo kinase is required for the production of key mitotic regulators, in addition to previously defined roles in controlling other mitotic events.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Ciclina B/genética , Modelos Genéticos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina/metabolismo , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
Oxidative damage caused by reactive oxygen species (ROS) is implicated in many diseases and in aging. Removal of ROS by antioxidant enzymes plays an important part in limiting this damage. For instance, peroxiredoxins (Prx) are conserved, abundant, thioredoxin peroxidase enzymes that function as tumor suppressors. In addition to detoxifying peroxides, studies in single-cell systems have revealed that Prx act as chaperones and redox sensors. However, it is unknown in what manner the different activities of Prx influence stress resistance or longevity in the context of whole animals. Here, we reveal three distinct roles for the 2-Cys Prx, PRDX-2, in the stress resistance of the nematode worm Caenorhabditis elegans. (i) The thioredoxin peroxidase activity of PRDX-2 protects against hydrogen peroxide. (ii) Consistent with a chaperone activity for hyperoxidized PRDX-2, peroxide-induced oxidation of PRDX-2 increases resistance to heat stress. (iii) Unexpectedly, loss of PRDX-2 increases the resistance of C. elegans to some oxidative stress-causing agents, such as arsenite, apparently through a signaling mechanism that increases the levels of other antioxidants and phase II detoxification enzymes. Despite their increased resistance to some forms of oxidative stress, prdx-2 mutants are short-lived. Moreover, intestinal expression of PRDX-2 accounts for its role in detoxification of exogenous peroxide, but not its influence on either arsenite resistance or longevity, suggesting that PRDX-2 may promote longevity and protect against environmental stress through different mechanisms. Together the data reveal that in metazoans Prx act through multiple biochemical activities, and have tissue-specific functions in stress resistance and longevity.
Asunto(s)
Caenorhabditis elegans/fisiología , Respuesta al Choque Térmico , Mucosa Intestinal/metabolismo , Longevidad , Peroxirredoxinas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans , Respuesta al Choque Térmico/genética , Peróxido de Hidrógeno/farmacología , Longevidad/genética , Oxidación-Reducción , Peroxirredoxinas/genética , Especies Reactivas de Oxígeno/metabolismo , Distribución TisularRESUMEN
Peroxiredoxins are ubiquitous proteins that are found from bacteria to humans. Until recently they were thought to solely act as antioxidants catalysing the reduction of peroxides through their associated thioredoxin peroxidase activity. However, recent work has begun to uncover hitherto unsuspected roles for one group of these proteins, the typical 2-Cys peroxiredoxins (2-Cys Prx). For example, typical 2-Cys Prxs have been found to have roles in the model organisms Schizosaccharomvces pombe and Saccharomyces cerevisiae in regulating signal transduction, in DNA damage responses and as molecular chaperones. There is increasing evidence that H2O2 is utilised as a signalling molecule to regulate a range of important cellular processes. As abundant and ubiquitous peroxidase enzymes the peroxidase activity of typical 2-Cys Prxs is important in the regulation of these functions. Significantly, studies in yeast suggest that the regulation of the thioredoxin peroxidase and chaperone activities of these multifunction enzymes is an important aspect of H2O2-mediated signal transduction and consequently have provided important insight into the roles of these proteins in higher eukaryotes.
Asunto(s)
Peroxirredoxinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Tiorredoxinas/metabolismo , Antioxidantes/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Catálisis , Daño del ADN/fisiología , Humanos , Peróxido de Hidrógeno/metabolismo , Chaperonas Moleculares/metabolismo , Transducción de Señal/fisiologíaRESUMEN
SIGNIFICANCE: In 2003, structural studies revealed that eukaryotic 2-Cys peroxiredoxins (Prx) have evolved to be sensitive to inactivation of their thioredoxin peroxidase activity by hyperoxidation (sulfinylation) of their peroxide-reacting catalytic cysteine. This was accompanied by the unexpected discovery, that the sulfinylation of this cysteine was reversible in vivo and the identification of a new enzyme, sulfiredoxin, that had apparently co-evolved specifically to reduce hyperoxidized 2-Cys Prx, restoring their peroxidase activity. Together, these findings have provided the impetus for multiple studies investigating the purpose of this reversible, Prx hyperoxidation. Recent Advances: It has been suggested that inhibition of the thioredoxin peroxidase activity by hyperoxidation can both promote and inhibit peroxide signal transduction, depending on the context. Prx hyperoxidation has also been proposed to protect cells against reactive oxygen species (ROS)-induced damage, by preserving reduced thioredoxin and/or by increasing non-peroxidase chaperone or signaling activities of Prx. CRITICAL ISSUES: Here, we will review the evidence in support of each of these proposed functions, in view of the in vivo contexts in which Prx hyperoxidation occurs, and the role of sulfiredoxin. Thus, we will attempt to explain the basis for seemingly contradictory roles for Prx hyperoxidation in redox signaling. FUTURE DIRECTIONS: We provide a rationale, based on modeling and experimental studies, for why Prx hyperoxidation should be considered a suitable, early biomarker for damaging levels of ROS. We discuss the implications that this has for the role of Prx in aging and the detection of hyperoxidized Prx as a conserved feature of circadian rhythms. Antioxid. Redox Signal. 28, 574-590.
Asunto(s)
Catálisis , Peróxido de Hidrógeno/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Cisteína/química , Cisteína/metabolismo , Chaperonas Moleculares/química , Oxidación-Reducción , Peróxidos/metabolismo , Peroxirredoxinas/química , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Recent studies in Saccharomyces cerevisiae by using global approaches have significantly enhanced our knowledge of the components involved in the transcriptional regulation of the cell cycle. The Mcm1p-Fkh2p complex, in combination with the coactivator Ndd1p, plays an important role in the cell cycle-dependent expression of the CLB2 gene cluster during the G2 and M phases ([4-7]; see [8-10]for reviews). Fkh2p is phosphorylated in a cell cycle-dependent manner, and peak phosphorylation occurs coincidentally with maximal expression of Mcm1p-Fkh2p-dependent gene expression. However, the mechanism by which this complex is activated in a cell cycle-dependent manner is unknown. Here, we demonstrate that the forkhead-associated (FHA) domain of Fkh2p directs cell cycle-regulated transcription and that the activity of this domain is dependent on the coactivator Ndd1p. Ndd1p was found to be phosphorylated in a cell cycle-dependent manner by Cdc28p-Clb2p, and, importantly, this phosphorylation event promotes interactions between Ndd1p and the FHA domain of Fkh2p. Furthermore, mutation of the FHA domain blocks these phosphorylation-dependent interactions and abolishes transcriptional activity. Our data therefore link the transcriptional activity of the FHA domain with cell cycle-dependent phosphorylation of the coactivator Ndd1p and reveal a mechanism that permits precise temporal activation of the Mcm1p-Fkh2p complex.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Ciclina B/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Western Blotting , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Factores de Transcripción Forkhead , Componentes del Gen , Modelos Biológicos , Fosforilación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The forkhead transcription factor Fkh2p acts in a DNA-bound complex with Mcm1p and the coactivator Ndd1p to regulate cell cycle-dependent expression of the CLB2 gene cluster in Saccharomyces cerevisiae. Here, we demonstrate that Fkh2p is a target of cyclin-dependent protein kinases and that phosphorylation of Fkh2p promotes interactions between Fkh2p and the coactivator Ndd1p. These phosphorylation-dependent changes in the Fkh2p-Ndd1p complex play an important role in the cell cycle-regulated expression of the CLB2 cluster. Our data therefore identify an important regulatory target for cyclin-dependent kinases in the cell cycle and further our molecular understanding of the key cell cycle regulatory transcription factor Fkh2p.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/genética , Factores de Transcripción Forkhead , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Sustancias Macromoleculares , Modelos Biológicos , Familia de Multigenes , Mutagénesis , Fosforilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
The yeast MADS-box transcription factor Mcm1p plays an important regulatory role in several diverse cellular processes. In common with a subset of other MADS-box transcription factors, Mcm1p elicits substantial DNA bending. However, the role of protein-induced bending by MADS-box proteins in eukaryotic gene regulation is not understood. Here, we demonstrate an important role for Mcm1p-mediated DNA bending in determining local promoter architecture and permitting the formation of ternary transcription factor complexes. We constructed mutant mcm1 alleles that are defective in protein-induced bending. Defects in nuclear division, cell growth or viability, transcription, and gene expression were observed in these mutants. We identified one likely cause of the cell growth defects as the aberrant formation of the cell cycle-regulatory Fkh2p-Mcm1p complex. Microarray analysis confirmed the importance of Mcm1p-mediated DNA bending in maintaining correct gene expression profiles and revealed defects in Mcm1p-mediated repression of Ty elements and in the expression of the cell cycle-regulated YFR and CHS1 genes. Thus, we discovered an important role for DNA bending by MADS-box proteins in the formation and function of eukaryotic transcription factor complexes.
Asunto(s)
ADN/química , ADN/metabolismo , Proteína 1 de Mantenimiento de Minicromosoma/genética , Proteína 1 de Mantenimiento de Minicromosoma/metabolismo , Alelos , Secuencia de Bases , Sitios de Unión , División Celular , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Genes Reporteros , Glutatión Transferasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Factores de Tiempo , Transcripción GenéticaRESUMEN
In Schizosaccharomyces pombe, the Sty1 mitogen-activated protein kinase and the Atf1 transcription factor control transcriptional induction in response to elevated salt concentrations. Herein, we demonstrate that two repressors, Tup11 and Tup12, and the Prr1 transcription factor also function in the response to salt shock. We find that deletion of both tup genes together results in hypersensitivity to elevated cation concentrations (K(+) and Ca(2+)) and we identify cta3(+), which encodes an intracellular cation transporter, as a novel stress gene whose expression is positively controlled by the Sty1 pathway and negatively regulated by Tup repressors. The expression of cta3(+) is maintained at low levels by the Tup repressors, and relief from repression requires the Sty1, Atf1, and Prr1. Prr1 is also required for KCl-mediated induction of several other Sty1-dependent genes such as gpx1(+) and ctt1(+). Surprisingly, the KCl-mediated induction of cta3(+) expression occurs independently of Sty1 in a tup11Delta tup12Delta mutant and so the Tup repressors link induction to the Sty1 pathway. We also report that in contrast to a number of other Sty1- and Atf1-dependent genes, the expression of cta3(+) is induced only by high salt concentrations. However, in the absence of the Tup repressors this specificity is lost and a range of stresses induces cta3(+) expression.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Sales (Química)/farmacología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Transporte Biológico , Cationes , Relación Dosis-Respuesta a Droga , Regulación Fúngica de la Expresión Génica , Iones , Modelos Biológicos , Fenotipo , Plásmidos/metabolismo , Potasio/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Previous work has implicated the Hog1 stress-activated protein kinase (SAPK) in osmotic and oxidative stress responses in the human pathogen Candida albicans. In this study, we have characterized the role of Hog1 in mediating these and other stress responses in C. albicans. We provide evidence that a SAPK-dependent core stress response exists in this pathogen. The Hog1 SAPK is phosphorylated and it accumulates in the nucleus in response to diverse stress conditions. In addition, we have identified Hog1-regulated genes that are induced in response to stress conditions that activate Hog1. These analyses reveal both activator and repressor functions for the Hog1 SAPK. Our results also demonstrate that stress cross-protection, a classical hallmark of the core stress response, occurs in C. albicans between stresses that activate the Hog1 SAPK. Importantly, we find that the core stress response in C. albicans has adapted to the environmental niche of this human pathogen. This niche specificity is reflected by the specific environmental conditions that drive the Hog1-regulated core stress response in C. albicans and by differences in the molecular circuitry that control this response.
Asunto(s)
Candida albicans/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Secuencia de Bases , Candida albicans/genética , Candida albicans/patogenicidad , Núcleo Celular/enzimología , ADN de Hongos/genética , Activación Enzimática , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Prueba de Complementación Genética , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Biológicos , Datos de Secuencia Molecular , Presión Osmótica , Estrés Oxidativo , Fenotipo , Fosforilación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidad de la EspecieRESUMEN
The signaling pathways that sense adverse stimuli and communicate with the nucleus to initiate appropriate changes in gene expression are central to the cellular stress response. Herein, we have characterized the role of the Sty1 (Spc1) stress-activated mitogen-activated protein kinase pathway, and the Pap1 and Atf1 transcription factors, in regulating the response to H(2)O(2) in the fission yeast Schizosaccharomyces pombe. We find that H(2)O(2) activates the Sty1 pathway in a dose-dependent manner via at least two sensing mechanisms. At relatively low levels of H(2)O(2), a two component-signaling pathway, which feeds into either of the two stress-activated mitogen-activated protein kinase kinase kinases Wak1 or Win1, regulates Sty1 phosphorylation. In contrast, at high levels of H(2)O(2), Sty1 activation is controlled predominantly by a two-component independent mechanism and requires the function of both Wak1 and Win1. Individual transcription factors were also found to function within a limited range of H(2)O(2) concentrations. Pap1 activates target genes primarily in response to low levels of H(2)O(2), whereas Atf1 primarily controls the transcriptional response to high concentrations of H(2)O(2). Our results demonstrate that S. pombe uses a combination of stress-responsive regulatory proteins to gauge and effect the appropriate transcriptional response to increasing concentrations of H(2)O(2).
Asunto(s)
Peróxido de Hidrógeno/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Transcripción Activador 1 , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Modelos Biológicos , Oxidantes/metabolismo , Oxidantes/farmacología , Estrés Oxidativo , Proteínas Asociadas a Pancreatitis , Peroxidasas/genética , Fosforilación , Schizosaccharomyces/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiologíaRESUMEN
The MADS-box transcription factor Mcm1p and forkhead (FKH) transcription factor Fkh2p act in a DNA-bound complex to regulate cell-cycle dependent expression of the CLB2 cluster in Saccharomyces cerevisiae. Binding of Fkh2p requires prior binding by Mcm1p. Here we have investigated the molecular determinants governing the formation of the Mcm1p- Fkh2p complex. Fkh2p exhibits cooperativity in complex formation with Mcm1p and we have mapped a small region of Fkh2p located immediately upstream of the FKH DNA binding domain that is required for this cooperativity. This region is lacking in the related protein Fkh1p that cannot form ternary complexes with Mcm1p. A second region is identified that inhibits Mcm1p-independent DNA binding by Fkh2p. The spacing between the Mcm1p and Fkh2p binding sites is also a critical determinant for complex formation. We also show that Fkh2p can form ternary complexes with the human counterpart of Mcm1p, serum response factor (SRF). Mutations at analogous positions in Mcm1p, which are known to affect SRF interaction with its partner protein Elk-1, abrogate complex formation with Fkh2p, demonstrating evolutionary conservation of coregulatory protein binding surfaces. Our data therefore provide molecular insights into the mechanisms of Mcm1p- Fkh2p complex formation and more generally aid our understanding of MADS-box protein function.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Fúngicas , Proteína 1 de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead , Proteína 1 de Mantenimiento de Minicromosoma/genética , Mutación , Regiones Promotoras Genéticas/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
SQSTM1/p62 (sequestosome 1) selectively targets polyubiquitinated proteins for degradation via macroautophagy and the proteasome. Additionally, SQSTM1 shuttles between the cytoplasmic and nuclear compartments, although its role in the nucleus is relatively unknown. Here, we report that SQSTM1 dynamically associates with DNA damage foci (DDF) and regulates DNA repair. Upon induction of DNA damage SQSTM1 interacts with FLNA (filamin A), which has previously been shown to recruit DNA repair protein RAD51 (RAD51 recombinase) to double-strand breaks and facilitate homologous recombination (HR). SQSTM1 promotes proteasomal degradation of FLNA and RAD51 within the nucleus, resulting in reduced levels of nuclear RAD51 and slower DNA repair. SQSTM1 regulates the ratio between HR and nonhomologous end joining (NHEJ) by promoting the latter at the expense of the former. This SQSTM1-dependent mechanism mediates the effect of macroautophagy on DNA repair. Moreover, nuclear localization of SQSTM1 and its association with DDF increase with aging and are prevented by life-span-extending dietary restriction, suggesting that an imbalance in the mechanism identified here may contribute to aging and age-related diseases.
Asunto(s)
Autofagia , Reparación del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismo , Animales , Núcleo Celular/metabolismo , Daño del ADN , Filaminas , Cinética , Ratones Endogámicos C57BL , Modelos Biológicos , Transporte de Proteínas , Proteolisis , Recombinasa Rad51/metabolismoRESUMEN
Many proteins involved in autophagy have been identified in the yeast Saccharomyces cerevisiae. For example, Atg3 and Atg10 are two E2 enzymes that facilitate the conjugation of the ubiquitin-like proteins (Ubls) Atg8 and Atg12, respectively. Here, we describe the identification and characterization of the predicted Atg10 homolog (SpAtg10) of the evolutionarily distant Schizosaccharomyces pombe. Unexpectedly, SpAtg10 is not essential for autophagy. Instead, we find that SpAtg10 is essential for normal cell cycle progression, and for responses to various stress conditions that perturb the cell cycle, independently of Atg12 conjugation. Taken together, our data indicate that autophagic Ubl conjugation pathways differ between eukaryotes and, furthermore, that enzymes such as Atg10 may have additional functions in controlling key cellular processes such as cell cycle progression. Atg10-related proteins are found from yeast to humans, and, thus, this study has implications for understanding the functions of this protein family in Ubl conjugation in eukaryotes.
Asunto(s)
Autofagia/fisiología , Ciclo Celular/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Western Blotting , Ciclo Celular/genética , Cartilla de ADN/genética , Microscopía Fluorescente , Schizosaccharomyces/fisiologíaRESUMEN
H2O2 can cause oxidative damage associated with age-related diseases such as diabetes and cancer but is also used to initiate diverse responses, including increased antioxidant gene expression. Despite significant interest, H2O2-signaling mechanisms remain poorly understood. Here, we present a mechanism for the propagation of an H2O2 signal that is vital for the adaptation of the model yeast, Schizosaccharomyces pombe, to oxidative stress. Peroxiredoxins are abundant peroxidases with conserved antiaging and anticancer activities. Remarkably, we find that the only essential function for the thioredoxin peroxidase activity of the Prx Tpx1(hPrx1/2) in resistance to H2O2 is to inhibit a conserved thioredoxin family protein Txl1(hTxnl1/TRP32). Thioredoxins regulate many enzymes and signaling proteins. Thus, our discovery that a Prx amplifies an H2O2 signal by driving the oxidation of a thioredoxin-like protein has important implications, both for Prx function in oxidative stress resistance and for responses to H2O2.