Proteomic characterization of reversible thiol oxidations in proteomes and proteins

SIGNIFICANCE: Reactive oxygen species are produced during normal metabolism in cells, and their excesses have been implicated in protein damage and toxicity, as well as in the activation of signaling events. In particular, hydrogen peroxide participates in the regulation of different physiological processes as well as in the induction of antioxidant cascades, and often the redox molecular events triggering these pathways are based on reversible cysteine (Cys) oxidation. Recent Advances: Increases in peroxides can cause the accumulation of reversible Cys oxidations in proteomes, which may be either protecting thiols from irreversible oxidations or may just be reporters of future toxicity. It is also becoming clear, however, that only a few proteins, such as the bacterial OxyR or peroxidases, can suffer direct oxidation of their Cys residues by hydrogen peroxide and, therefore, may be the only true sensors initiating signaling events. CRITICAL ISSUES: We will in this study describe some of the methodologies used to characterize at the proteome level reversible thiol oxidations, specifically those combining gel-free approaches with mass spectrometry. In the second part of this review, we will summarize some of the electrophoretic and proteomic techniques used to monitor Cys oxidation at the protein level, needed to confirm that a protein contains redox Cys involved in signaling relays, using as examples some of the best characterized redox sensors such as bacterial OxyR or yeast Tpx1/Pap1. FUTURE DIRECTIONS: While Cys oxidations are often detected in proteomes and in specific proteins, major efforts have to be made to establish that they are physiologically relevant. Antioxid. Redox Signal. 26, 329-344This work was supported by the Spanish Ministry of Science and Innovation (BFU2015-68350-P, MINECO/FEDER, UE), and by 2014-SGR-154 from Generalitat de Catalunya (Spain) to E.H. A.D. is recipient of a predoctoral fellowship from Generalitat de Catalunya (Spain). E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain

Spatial sequestration of misfolded proteins as an active chaperone-mediated process during heat stress

Data de publicació electrónica: 01-01-2021Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degradation and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissolution. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates observed in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiological and heat-shock triggered PQC.This work is supported by the Ministerio de Ciencia, Innovación y Universidades (Spain), PLAN E and FEDER (BFU2016-75116-P to M.C. and PGC2018-093920-B-I00 to E.H.). The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539) and by Unidad de Excelencia María de Maeztu, funded by the AEI (CEX2018-000792-M) (Spain). M.C. is funded by the Ramon y Cajal program (MINECO-RYC2013-12858). E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain)

Proteomic characterization of reversible thiol oxidations in proteomes and proteins

SIGNIFICANCE: Reactive oxygen species are produced during normal metabolism in cells, and their excesses have been implicated in protein damage and toxicity, as well as in the activation of signaling events. In particular, hydrogen peroxide participates in the regulation of different physiological processes as well as in the induction of antioxidant cascades, and often the redox molecular events triggering these pathways are based on reversible cysteine (Cys) oxidation. Recent Advances: Increases in peroxides can cause the accumulation of reversible Cys oxidations in proteomes, which may be either protecting thiols from irreversible oxidations or may just be reporters of future toxicity. It is also becoming clear, however, that only a few proteins, such as the bacterial OxyR or peroxidases, can suffer direct oxidation of their Cys residues by hydrogen peroxide and, therefore, may be the only true sensors initiating signaling events. CRITICAL ISSUES: We will in this study describe some of the methodologies used to characterize at the proteome level reversible thiol oxidations, specifically those combining gel-free approaches with mass spectrometry. In the second part of this review, we will summarize some of the electrophoretic and proteomic techniques used to monitor Cys oxidation at the protein level, needed to confirm that a protein contains redox Cys involved in signaling relays, using as examples some of the best characterized redox sensors such as bacterial OxyR or yeast Tpx1/Pap1. FUTURE DIRECTIONS: While Cys oxidations are often detected in proteomes and in specific proteins, major efforts have to be made to establish that they are physiologically relevant. Antioxid. Redox Signal. 26, 329-344This work was supported by the Spanish Ministry of Science and Innovation (BFU2015-68350-P, MINECO/FEDER, UE), and by 2014-SGR-154 from Generalitat de Catalunya (Spain) to E.H. A.D. is recipient of a predoctoral fellowship from Generalitat de Catalunya (Spain). E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain

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)

Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry

Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labelingThis work was supported by the Spanish Ministry of Science and Innovation (nos.BFU2009-06933 and BFU2012-32045), by PLAN E and Fondo Europeo de Desarrollo Regional (FEDER), by the Spanish program Consolider-Ingenio 2010 (grant no. CSD 2007-0020) and by grant no. SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. and J.A. are recipients of Institució Catalana de Recerca i Estudis Avançats (ICREA) /nAcademia Awards (Generalitat de Catalunya

A systematic screen identifies Saf5 as a link between splicing and transcription in fission yeast

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5' splicing site (5'SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5'SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5'SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.This work was supported by grants BFU2018-PGC2018-097248-B-I00 and PID2022-136449NB-I00 funded by MICIU/AEI/10.13039/501100011033 and ERDF/EU to JA and by Unidad de Excelencia Maria de Maeztu (CEX2018-000792-M) to JA and EH. EH is a recipient of an ICREA Academia Award (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript