Mitochondrial Dysfunction Increases Oxidative Stress and Decreases Chronological Life Span in Fission Yeast

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.This work was supported by Dirección General de Investigación of Spain Grant BFU2006-02610, and by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020 to E.H

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Isabel A. Calvo et al...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 H2O2-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.This work was supported by Direccion General de Investigacion of Spain Grant BFU2006-02610, and by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020, to E.H.Peer reviewe

Control of redox homeostasis : environmental and genetic regulation of oxidative protein damage in Schizosaccharomyces pombe

Oxidation of specific cysteines in proteins by hydrogen peroxide (H2O2) is a cellular mechanism that triggers signalling pathways, such as the antioxidant responses in microbial systems. However, H2O2 can be transformed to the more reactive radical hydroxyl OH▪ species, through the Fenton reaction, the main causative of protein carbonylations. These modifications can not be repaired, and constitute classical marks of oxidative damage. Along this thesis we were interested in studying and understand the consequences of reactive oxygen species (ROS) reactivity on proteins. Thus, we developed protocols for studying cysteine oxidations at the proteome level, upon treatment with H2O2 or in strains devoid of antioxidant systems, to characterize the more sensitive to oxidation and therefore the more prone to participate in redox events. We tried protocols for the identification of protein carbonyls, however we were unsuccessful due to the complexity of these type of modifications. Also, we studied the role of protein quality control pathways in the clearance of carbonylated proteins (protein aggregation results toxic for cells). Finally, we identified and characterized the main H2O2 scavengers in S. pombe and the identified the free methionine as a primary barrier in the defence against H2O2 stress.La oxidación específica de cisteinas in proteinas por peróxido de hidrógeno (H2O2) es un mecanismo de activación de rutas de señalización. Sin embargo, el H2O2 puede también convertirse en radical hidroxilo (OH▪). Ésta es una especie reactiva de oxígeno (ERO) más reactiva que el H2O2, que da lugar a la formación de grupos carbonilo en proteinas. Los grupos carbonilo no pueden ser reparados y constituyen marcas de daño oxidativo. En esta tesis hemos estudiado las consecuencias de la reactividad de las ERO en proteínas. Así, hemos desarrollado protocolos para estudiar oxidaciones de cisteínas, tras tratamiento con H2O2 o en cepas manipuladas genéticamente, a nivel proteómico, caracterizando aquellas más reactivas, y por lo tanto más adecuadas para participar en señalización redox. Intentamos protocolos para identificar proteinas carboniladas, aunque sin éxito debido a la complejidad de este tipo de modificaciones. También hemos estudiado el papel de componentes que actuan en el control de calidad de proteinas en la degradación de proteinas carboniladas (la agregación y acumulación de proteinas es tóxica para la célula). Finalmente, hemos caracterizado los principales detoxificadores de H2O2 en S. pombe y hemos identificado la metionina como una barrera primera en la defensa contra el estrés por H2O2

Analysis of reversible and irreversible protein modifications upon oxidative stress in Schizosaccharomyces pombe by proteomic approach

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Gel-free proteomic methodologies to study reversible cysteine oxidation and irreversible protein carbonyl formation

Oxidative modifications in proteins have been traditionally considered as hallmarks of damage by oxidative stress and aging. However, oxidants can generate a huge variety of reversible and irreversible modifications in amino acid side chains as well as in the protein backbones, and these post-translational modifications can contribute to the activation of signal transduction pathways, and also mediate the toxicity of oxidants. Among the reversible modifications, the most relevant ones are those arising from cysteine oxidation. Thus, formation of sulfenic acid or disulfide bonds is known to occur in many enzymes as part of their catalytic cycles, and it also participates in the activation of signaling cascades. Furthermore, these reversible modifications have been usually attributed with a protective role, since they may prevent the formation of irreversible damage by scavenging reactive oxygen species. Among irreversible modifications, protein carbonyl formation has been linked to damage and death, since it cannot be repaired and can lead to protein loss-of-function and to the formation of protein aggregates. This review is aimed at researchers interested on the biological consequences of oxidative stress, both at the level of signaling and toxicity. Here we are providing a concise overview on current mass-spectrometry-based methodologies to detect reversible cysteine oxidation and irreversible protein carbonyl formation in proteomes. We do not pretend to impose any of the different methodologies, but rather to provide an objective catwalk on published gel-free approaches to detect those two types of modifications, from a biologist's point of view.This work was supported by the Spanish Ministry of Science and Innovation (BFU2012-32045), PLAN E and FEDER, and by SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. J.A. and E. H. are recipients of ICREA Academia Awards (Generalitat de Catalunya)

Gel-free proteomic methodologies to study reversible cysteine oxidation and irreversible protein carbonyl formation

Oxidative modifications in proteins have been traditionally considered as hallmarks of damage by oxidative stress and aging. However, oxidants can generate a huge variety of reversible and irreversible modifications in amino acid side chains as well as in the protein backbones, and these post-translational modifications can contribute to the activation of signal transduction pathways, and also mediate the toxicity of oxidants. Among the reversible modifications, the most relevant ones are those arising from cysteine oxidation. Thus, formation of sulfenic acid or disulfide bonds is known to occur in many enzymes as part of their catalytic cycles, and it also participates in the activation of signaling cascades. Furthermore, these reversible modifications have been usually attributed with a protective role, since they may prevent the formation of irreversible damage by scavenging reactive oxygen species. Among irreversible modifications, protein carbonyl formation has been linked to damage and death, since it cannot be repaired and can lead to protein loss-of-function and to the formation of protein aggregates. This review is aimed at researchers interested on the biological consequences of oxidative stress, both at the level of signaling and toxicity. Here we are providing a concise overview on current mass-spectrometry-based methodologies to detect reversible cysteine oxidation and irreversible protein carbonyl formation in proteomes. We do not pretend to impose any of the different methodologies, but rather to provide an objective catwalk on published gel-free approaches to detect those two types of modifications, from a biologist's point of view.This work was supported by the Spanish Ministry of Science and Innovation (BFU2012-32045), PLAN E and FEDER, and by SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. J.A. and E. H. are recipients of ICREA Academia Awards (Generalitat de Catalunya)

Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction

Activation of redox cascades through hydrogen peroxide-mediated reversible cysteine oxidation is a major mechanism for intracellular signaling. Understanding why some cysteine residues are specifically oxidized, in competition with other proximal cysteine residues and in the presence of strong redox buffers, is therefore crucial for understanding redox signaling. In this review, we explore the recent advances in thiol-redox chemistry linked to signaling. We describe the last findings in the field of redox sensors, those that are naturally present in different model organisms as well as those that have been engineered to quantify intracellular hydrogen peroxide concentrations. Finally, we provide a summary of the newest approaches developed to study reversible cysteine oxidation at the proteomic level.This work was supported by the Spanish Ministry of Science and Innovation (BFU2012-32045), PLAN E and FEDER, by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020, and by Grant SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. is the recipient of an ICREA Academia Award (Generalitat de Catalunya)

Methionine sulphoxide reductases revisited: free methionine as a primary target of H2O2 stress in auxotrophic fission yeast

Amino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over-oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H2 O2 sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H2 O2 stress decreases by 100-fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H2 O2 stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid.This work was supported by the Spanish Ministry of Science and Innovation (BFU2009-06933, BFU2012-32045), PLAN E and FEDER, by the Spanish program Consolider-Ingenio 2010 Grant CSD 2007-0020 and by SGR 2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. and J.A. are recipients of ICREA Academia Awards (Generalitat de Catalunya). The authors declare no conflict of interests

Identification of ubiquitin-proteasome system components affecting the degradation of the transcription factor Pap1

Signaling cascades respond to specific inputs, but also require active interventions to be maintained in their basal/inactive levels in the absence of the activating signal(s). In a screen to search for protein quality control components required for wild-type tolerance to oxidative stress in fission yeast, we have isolated eight gene deletions conferring resistance not only to H2O2 but also to caffeine. We show that dual resistance acquisition is totally or partially dependent on the transcription factor Pap1. Some gene products, such as the ribosomal-ubiquitin fusion protein Ubi1, the E2 conjugating enzyme Ubc2 or the E3 ligase Ubr1, participate in basal ubiquitin labeling of Pap1, and others, such as Rpt4, are non-essential constituents of the proteasome. We demonstrate here that basal nucleo-cytoplasmic shuttling of Pap1, occurring even in the absence of stress, is sufficient for the interaction of the transcription factor with nuclear Ubr1, and we identify a 30 amino acids peptide in Pap1 as the degron for this important E3 ligase. The isolated gene deletions increase only moderately the concentration of the transcription factor, but it is sufficient to enhance basal tolerance to stress, probably by disturbing the inactive stage of this signaling cascade.This work is supported by the Ministerio de Economía y Competitividad (Spain), PLAN E and FEDER (BFU2015-68350-P and PGC2018-093920 to E.H) and by Unidad de Excelencia María de Maeztu (MDM-2014-0370). The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539). E. H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain)