RESUMO
Despite much evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Temperatura Baixa , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismoRESUMO
Despite the progress made in understanding the roles of proteasome polyubiquitin receptors, such as the subunits Rpn10 (regulatory particle non-ATPase 10) and Rpn13, and the transient interactors Rad23 (radiation sensitivity abnormal 23) and Dsk2 (dual-specificity protein kinase 2), the mechanisms involved in their regulation are virtually unknown. Rpn10, which is found in the cell in proteasome-bound and -unbound pools, interacts with Dsk2, and this interaction has been proposed to regulate the amount of Dsk2 that gains access to the proteasome. Rpn10 monoubiquitination has emerged as a conserved mechanism with a strong effect on Rpn10 function. In the present study, we show that functional yeast proteasomes have the capacity to associate and dissociate with Rpn10 and that Rpn10 monoubiquitination decreases the Rpn10-proteasome and Rpn10-Dsk2 associations. Remarkably, this process facilitates the formation of Dsk2-proteasomes in vivo. Therefore, Rpn10 monoubiquitination acts as mechanism that serves to switch the proteasome from an 'Rpn10 high/Dsk2 low' state to an 'Rpn10 low/Dsk2 high' state. Interestingly, Rpn10-ubiquitin, with an inactivated ubiquitin-interacting motif (UIM), and Dsk2(I45S), with an inactive ubiquitin-like domain (UBL), show temperature-dependent phenotypes with multiple functional interactions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Ubiquitinas/metabolismo , Proteínas de Ciclo Celular/genética , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética , Ubiquitinas/genéticaRESUMO
Despite being a common mechanism in eukaryotes, the process by which protein monoubiquitination is produced and regulated in vivo is not completely understood. We present here the analysis of the process of monoubiquitination of the proteasomal subunit Rpn10 (regulatory particle non-ATPase 10), involved in the recruitment of polyubiquitinated substrates. Rpn10 is monoubiquitinated in vivo by the Nedd4 (neural precursor cell expressed developmentally down-regulated 4) enzyme Rsp5 (reverses SPT-phenotype protein 5) and this modification impairs the interaction of Rpn10 with substrates, having a regulatory effect on proteasome function. Remarkably, a disordered region near the ubiquitin-interacting motif of Rpn10 plays a role in the restriction of the polyubiquitin extension activity of Rsp5. Mutations in this disordered region promote ubiquitin chain extension of Rpn10. Thus, our work sheds light on the molecular basis and the functional relevance of a type of monoubiquitination that is driven by the substrate. Moreover, we uncover a putative role for disordered regions in modulating ubiquitin-protein ligation.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , UbiquitinaçãoRESUMO
Either calorie restriction, loss-of-function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here, we show that either calorie restriction or inactivation of nutrient-dependent pathways induces lifespan extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 mitogen-activated protein (MAP) kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress programme, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue, Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase lifespan under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours lifespan extension.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Estresse Fisiológico/fisiologia , Fator 1 Ativador da Transcrição/metabolismo , Northern Blotting , Regulação Fúngica da Expressão Gênica , Glucose/farmacologia , Peróxido de Hidrogênio/farmacologia , Proteínas Quinases Ativadas por Mitógeno/genética , Consumo de Oxigênio , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Around 2 × 103-2.5 × 103 million years ago, a unicellular organism with radically novel features, ancestor of all eukaryotes, dwelt the earth. This organism, commonly referred as the last eukaryotic common ancestor, contained in its proteome the same functionally capable ubiquitin molecule that all eukaryotic species contain today. The fact that ubiquitin protein has virtually not changed during all eukaryotic evolution contrasts with the high expansion of the ubiquitin system, constituted by hundreds of enzymes, ubiquitin-interacting proteins, protein complexes, and cofactors. Interestingly, the simplest genetic arrangement encoding a fully-equipped ubiquitin signaling system is constituted by five genes organized in an operon-like cluster, and is found in archaea. How did ubiquitin achieve the status of central element in eukaryotic physiology? We analyze here the features of the ubiquitin molecule and the network that it conforms, and propose notions to explain the complexity of the ubiquitin signaling system in eukaryotic cells.
RESUMO
The ubiquitin-proteasome system has emerged in the last decades as a new paradigm in cell physiology. Ubiquitin is found in fundamental levels of cell regulation, as a target for degradation to the proteasome or as a signal that controls protein function in a complex manner. Even though many aspects of the ubiquitin system remain unexplored, the contributions on the field uncover that ubiquitin represents one of the most sophisticated codes in cellular biology. The proteasome is an ATP-dependent protease that degrades a large number of protein substrates in the cell. The proteasome recruits substrates by a number of receptors that interact with polyubiquitin. Recently, it has been shown that one of these receptors, Rpn10, is regulated by monoubiquitination. In this chapter, we show an overview of the central aspects of the pathway and describe the methodology to characterize in vitro the monoubiquitination of proteasome subunits.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Animais , Humanos , UbiquitinaçãoRESUMO
Oxidative stress constitutes the basis of physio-pathological situations such as neurodegenerative diseases and aging. However, sublethal exposure to toxic molecules such as reactive oxygen species can induce cellular responses that result in stress fitness. Studies in Schizosaccharomyces pombe have recently showed that the Sty1 MAP kinase, known to be activated by hydrogen peroxide and other cellular stressors, plays a pivotal role in promoting fitness and longevity when it becomes activated by calorie restriction, a situation which induces oxidative metabolism and reactive oxygen species production. Activation of the MAP kinase by calorie restriction during logarithmic growth induces a transcriptional anti-stress response including genes essential to promote lifespan extension. Importantly enough, the lifespan promotion exerted by deletion of the pka1 or sck2 genes, inactivating the two main nutrient-responsive pathways, is dependent on the presence of a functional Sty1 stress pathway, since double mutants also lacking Sty1 or its main substrate Atf1 do not display extended viability. In this Research Perspective, we review these findings in relation to previous reports and extend important aspects of the original study. We propose that moderate stress levels that are not harmful for cells can make them stronger.
Assuntos
Longevidade , Estresse Oxidativo , Schizosaccharomyces/fisiologia , Envelhecimento , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
BACKGROUND: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways. METHODOLOGY/PRINCIPAL FINDINGS: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H(2)O(2)-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. CONCLUSIONS/SIGNIFICANCE: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.
Assuntos
Adaptação Fisiológica , Cafeína/farmacologia , Genoma Fúngico , Schizosaccharomyces/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Mutação , Estresse Oxidativo , Proteínas Associadas a Pancreatite , Schizosaccharomyces/genéticaRESUMO
BACKGROUND: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. CONCLUSION/SIGNIFICANCE: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.
Assuntos
Mitocôndrias/patologia , Estresse Oxidativo , Schizosaccharomyces/fisiologia , Carbono/química , Respiração Celular , DNA Mitocondrial , Elétrons , Deleção de Genes , Peróxido de Hidrogênio/farmacologia , Modelos Biológicos , Mutação , Oxigênio/química , Consumo de Oxigênio , Espécies Reativas de Oxigênio , Fatores de TempoRESUMO
Schizosaccharomyces pombe triggers different signalling pathways depending on the severity of the oxidative stress exerted, the main ones being the Pap1 and the Sty1 pathways. The Pap1 transcription factor is more sensitive to hydrogen peroxide (H(2)O(2)) than the MAP kinase Sty1 pathway, and is designed to induce adaptation, rather than survival, responses. The peroxiredoxin Tpx1 acts as a H(2)O(2) sensor and the upstream activator of the Pap1 pathway. Therefore, sensitivity to H(2)O(2) depends on this thioredoxin peroxidase. In order to achieve maximal activation of the MAP kinase pathway, the concentration of H(2)O(2) needs to be at least fivefold higher than that to fully activate Pap1. Tpx1 is a H(2)O(2) scavenger, thus its peroxidase activity is essential for aerobic growth. As described for other eukaryotic peroxiredoxins, high doses of H(2)O(2) temporarily inactivate Tpx1 and delay Pap1 activation, whereas the Sty1 pathway remains fully functional under these conditions. As part of the Sty1-dependent transcriptional response, the expression of Srx1 is induced and this reductase re-activates the over-oxidised Tpx1. Therefore, the antioxidant pathways of the fission yeast are perfectly designed so that the transcriptional programs triggered by the different signalling pathways never overlap.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/fisiologia , Peroxidases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Transdução de Sinais/fisiologia , Proteínas Associadas a Pancreatite , PeroxirredoxinasRESUMO
Methylglyoxal, a toxic metabolite synthesized in vivo during glycolysis, inhibits cell growth. One of the mechanisms protecting eukaryotic cells against its toxicity is the glyoxalase system, composed of glyoxalase I and II (glo1 and glo2), which converts methylglyoxal into d-lactic acid in the presence of glutathione. Here we have shown that the two principal oxidative stress response pathways of Schizosaccharomyces pombe, Sty1 and Pap1, are involved in the response to methylglyoxal toxicity. The mitogen-activated protein kinase Sty1 is phosphorylated and accumulates in the nucleus following methylglyoxal treatment. Moreover, glo2 expression is induced by methylglyoxal and environmental stresses in a Sty1-dependent manner. The transcription factor Pap1 also accumulates in the nucleus, activating the expression of its target genes following methylglyoxal treatment. Our studies showed that the C-terminal cysteine-rich domain of Pap1 is sufficient for methylglyoxal sensing. Furthermore, the redox status of Pap1 is not changed by methylglyoxal. We propose that methylglyoxal treatment triggers Pap1 and Sty1 nuclear accumulation, and we describe the molecular basis of such activation mechanisms. In addition, we discuss the potential physiological significance of these responses to a natural toxic metabolite.