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Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes*

  • Author Footnotes
    ‡‡ These authors contributed equally to this work.
    Ling Fu
    Footnotes
    ‡‡ These authors contributed equally to this work.
    Affiliations
    From the State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Author Footnotes
    ‡‡ These authors contributed equally to this work.
    Keke Liu
    Footnotes
    ‡‡ These authors contributed equally to this work.
    Affiliations
    From the State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Author Footnotes
    ‡‡ These authors contributed equally to this work.
    Mingan Sun
    Footnotes
    ‡‡ These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, China;
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  • Caiping Tian
    Affiliations
    From the State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
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  • Rui Sun
    Affiliations
    From the State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;

    State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 211198, China;
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  • Carlos Morales Betanzos
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232;
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  • Keri A. Tallman
    Affiliations
    Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232.
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  • Ned A. Porter
    Affiliations
    Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232.
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  • Yong Yang
    Affiliations
    State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, Center for New Drug Safety Evaluation and Research, China Pharmaceutical University, Nanjing 211198, China;
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  • Dianjing Guo
    Affiliations
    State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, China;
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  • Daniel C. Liebler
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232;
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  • Jing Yang
    Correspondence
    To whom correspondence should be addressed:National Center for Protein Science, 38 Life Science Park Road, Beijing 102206 China. Tel.:86-10-61777114
    Affiliations
    From the State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China;
    Search for articles by this author
  • Author Footnotes
    * Part of this work was supported by the National Key R&D Program of China (No. 2016YFA0501303), the National Natural Science Foundation of China (No. 31500666 and No. 81573395), the Beijing Natural Science Foundation (No.5162009), and Beijing Nova Program (No. Z171100001117014) to J.Y. and the National Institutes of Health (U24CA159988) to D.C.L.
    This article contains supplemental material.
    ‡‡ These authors contributed equally to this work.
Open AccessPublished:August 21, 2017DOI:https://doi.org/10.1074/mcp.RA117.000108
      Protein cysteinyl residues are the mediators of hydrogen peroxide (H2O2)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward H2O2 in human cells. We identified >900 H2O2-sensitive cysteines, which are defined as the H2O2-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the H2O2-dependent redoxome varies dramatically between different cells. Structural analyses reveal that H2O2-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.
      Hydrogen peroxide (H2O2)
      The abbreviations used are: H2O2, hydrogen peroxide; ACN, acetonitrile; Az-UV-biotin, azido biotin with a photocleavable linker; BCA, bicinchoninic acid; CuAAC, copper catalyzed azide-alkyne cycloaddition; DMEM, Dulbecco's modified eagle's medium; DTT, dithiothreitol; FDR, false discovery rate; HCD, high energy collisional dissociation; GDH, glucose dehydrogenase; GSNO, S-nitrosoglutathione; GSH, reduced glutathione; GSSG, oxidized glutathione; GO, gene ontology; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDS, lithium dodecyl sulfate; IAM, iodoacetamide; IPM, Iodo-N-(prop-2-yn-1-yl) acetamide; isoTOP-ABPP, isotopic tandem orthogonal proteolysis-activity based protein profiling; NEM, N-ethylmaleimide; PBS, phosphate buffered saline; PTMs, posttranslational modifications; PVDF, polyvinylidene difluoride; RT, room temperature; SCX, strong cation exchange; TBST, tris-buffered saline plus 0.05% Tween-20 (v/v); TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.
      1The abbreviations used are: H2O2, hydrogen peroxide; ACN, acetonitrile; Az-UV-biotin, azido biotin with a photocleavable linker; BCA, bicinchoninic acid; CuAAC, copper catalyzed azide-alkyne cycloaddition; DMEM, Dulbecco's modified eagle's medium; DTT, dithiothreitol; FDR, false discovery rate; HCD, high energy collisional dissociation; GDH, glucose dehydrogenase; GSNO, S-nitrosoglutathione; GSH, reduced glutathione; GSSG, oxidized glutathione; GO, gene ontology; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDS, lithium dodecyl sulfate; IAM, iodoacetamide; IPM, Iodo-N-(prop-2-yn-1-yl) acetamide; isoTOP-ABPP, isotopic tandem orthogonal proteolysis-activity based protein profiling; NEM, N-ethylmaleimide; PBS, phosphate buffered saline; PTMs, posttranslational modifications; PVDF, polyvinylidene difluoride; RT, room temperature; SCX, strong cation exchange; TBST, tris-buffered saline plus 0.05% Tween-20 (v/v); TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.
      generated in a wide range of physiological and pathological processes can promote cell damage, but also can activate cell regulatory and signaling pathways as a signaling molecule (
      • Giorgio M.
      • Trinei M.
      • Migliaccio E.
      • Pelicci P.G.
      Hydrogen peroxide: A metabolic by-product or a common mediator of ageing signals?.
      ,
      • Holmstrom K.M.
      • Finkel T.
      Cellular mechanisms and physiological consequences of redox-dependent signalling.
      ). Site-specific modification of cysteinyl thiols on H2O2-sensitive proteins represents a unique molecular mechanism for transducing oxidant signals into biological responses (
      • Paulsen C.E.
      • Carroll K.S.
      Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery.
      ,
      • Dickinson B.C.
      • Chang C.J.
      Chemistry and biology of reactive oxygen species in signaling or stress responses.
      ). In species from yeast to human, the evolutionarily conserved catalytic cysteines of peroxiredoxins have the attributes to act as the most reactive sensors of H2O2 (
      • Wood Z.A.
      • Schroder E.
      • Robin Harris J.
      • Poole L.B.
      Structure, mechanism and regulation of peroxiredoxins.
      ). The mechanism of H2O2-sensing and transduction by peroxiredoxins is becoming well understood (
      • Rhee S.G.
      • Kil I.S.
      Multiple functions and regulation of mammalian peroxiredoxins.
      ). Approximately 20 other proteins, including protein tyrosine phosphatases, metabolic enzymes, and stress sensors, have been characterized in the last two decades as targets of H2O2 with functionally important roles (
      • Karisch R.
      • Fernandez M.
      • Taylor P.
      • Virtanen C.
      • St-Germain J.R.
      • Jin L.L.
      • Harris I.S.
      • Mori J.
      • Mak T.W.
      • Senis Y.A.
      • Ostman A.
      • Moran M.F.
      • Neel B.G.
      Global proteomic assessment of the classical protein-tyrosine phosphatome and “redoxome”.
      ,
      • Anastasiou D.
      • Poulogiannis G.
      • Asara J.M.
      • Boxer M.B.
      • Jiang J.K.
      • Shen M.
      • Bellinger G.
      • Sasaki A.T.
      • Locasale J.W.
      • Auld D.S.
      • Thomas C.J.
      • Vander Heiden M.G.
      • Cantley L.C.
      Inhibition of pyruvate kinase M2 By reactive oxygen species contributes to cellular antioxidant responses.
      ,
      • Guo Z.
      • Kozlov S.
      • Lavin M.F.
      • Person M.D.
      • Paull T.T.
      Atm activation by oxidative stress.
      ). However, recent advances suggest that hundreds of cysteines are oxidation-sensitive (
      • Deng X.
      • Weerapana E.
      • Ulanovskaya O.
      • Sun F.
      • Liang H.
      • Ji Q.
      • Ye Y.
      • Fu Y.
      • Zhou L.
      • Li J.
      • Zhang H.
      • Wang C.
      • Alvarez S.
      • Hicks L.M.
      • Lan L.
      • Wu M.
      • Cravatt B.F.
      • He C.
      Proteome-wide quantification and characterization of oxidation-sensitive cysteines in pathogenic bacteria.
      ,
      • Rosenwasser S.
      • Graff Van Creveld S.
      • Schatz D.
      • Malitsky S.
      • Tzfadia O.
      • Aharoni A.
      • Levin Y.
      • Gabashvili A.
      • Feldmesser E.
      • Vardi A.
      Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment.
      ,
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      ). This suggests that the scope of the redox-sensitive proteome is considerably broader than previously imagined and raises important new questions. Is H2O2 redox reactivity an intrinsic feature of certain cysteines in specific sequences and conserved across different cellular proteomes? Does the cell-specific redox environment affect the reactivity of cysteines toward H2O2? Are the relative reactivity and functional contributions of oxidation-sensitive cysteines in H2O2 sensing conserved in different cellular contexts? A fundamental barrier to answering these questions is the lack of methodology to globally quantify site-specific H2O2-dependent cysteine oxidations across native proteomes.
      Here we describe a chemoproteomic strategy, called quantitative thiol reactivity profiling (QTRP), which we applied to systematically and quantitatively determine the reactivities of 6566 cysteines on 3557 proteins toward different levels of H2O2 stimulation in multiple human cell lines. This large-scale redox proteomic survey provides a rank order of redox reactivity of these cysteines in cells and reveals—surprisingly—that most cysteines sense H2O2 in a cell-specific manner, thereby presenting a new conceptual framework onto which to build models of cellular redox control. Systems level analyses of this data set uncover the functional contributions of cell-specific redoxomes in H2O2 sensing and the downstream cellular responses. Structural analyses reveal that H2O2-sensitive cysteines are mostly unconserved, but display distinct sequence motifs, structural features, and potential crosstalk with sequence-adjacent lysine modifications. In addition, the data demonstrate potential utility of a redox chemoproteomic strategy in predicting oxidation-triggered protein conformational changes.

      DISCUSSION

      Here we have described a quantitative chemoproteomic strategy called QTRP (Fig. 1) to systematically investigate the reactions between H2O2 and proteomic cysteines in human cells. The analyses enabled discovery of a cohort of known and novel H2O2-sensitive cysteines and a proteome-wide comparison of their redox behavior in cells. Our most notable finding is that the H2O2-sensitive redoxome is highly variable between different cells. The cell redoxomes we analyzed shared a relatively small set of H2O2 sensors, consisting primarily of well-known antioxidants (peroxiredoxin and thioredoxin family) and metabolic enzymes (GAPDH, PKM2, BCAT2, and ALDH5A1). We hypothesize that this conserved redox-responsive antioxidant- and metabolism-regulating network combines with a much larger, more cell type-specific set of redox-sensitive cysteines to constitute the H2O2-sensitive redoxome. This hypothesis is consistent with the requirement that different cells must develop distinct redox sensing mechanisms to cope with diverse and dynamic environmental or metabolic stress conditions while accommodating a variety of differentiated cellular functions. This hypothesis is supported by our observation that most of H2O2-sensitive cysteines are not evolutionarily conserved and only a few cysteines with key roles in antioxidant and metabolic functions have been fixed during evolution.
      Our analyses also suggest that the cysteine-mediated H2O2 sensing events in human cells are more complicated than previously anticipated. Because different cells usually display different phenotypic responses upon H2O2 stimulation, there are longstanding debates on the role of H2O2 in regulation of redox adaptive responses, such as oxidation reduction and proteostasis (
      • Mitozo P.A.
      • De Souza L.F.
      • Loch-Neckel G.
      • Flesch S.
      • Maris A.F.
      • Figueiredo C.P.
      • Dos Santos A.R.
      • Farina M.
      • Dafre A.L.
      A study of the relative importance of the peroxiredoxin-, catalase-, and glutathione-dependent systems in neural peroxide metabolism.
      ,
      • Hanzen S.
      • Vielfort K.
      • Yang J.
      • Roger F.
      • Andersson V.
      • Zamarbide-Fores S.
      • Andersson R.
      • Malm L.
      • Palais G.
      • Biteau B.
      • Liu B.
      • Toledano M.B.
      • Molin M.
      • Nystrom T.
      Lifespan control by redox-dependent recruitment of chaperones to misfolded proteins.
      ). It is noteworthy that the oxidation-sensitive proteins involved in these biological processes may function in seemingly contradictory ways in cells treated with H2O2. For example, antioxidant enzymes, such as PRDX5 and TXN2, can be deactivated by overdose of H2O2. Meanwhile, H2O2-dependent inhibition of key metabolic enzymes (GAPDH, PKM2) may elevate cellular antioxidant responses by regulating the production of reducing equivalents such as NADPH (
      • Anastasiou D.
      • Poulogiannis G.
      • Asara J.M.
      • Boxer M.B.
      • Jiang J.K.
      • Shen M.
      • Bellinger G.
      • Sasaki A.T.
      • Locasale J.W.
      • Auld D.S.
      • Thomas C.J.
      • Vander Heiden M.G.
      • Cantley L.C.
      Inhibition of pyruvate kinase M2 By reactive oxygen species contributes to cellular antioxidant responses.
      ,
      • Deng X.
      • Liang H.
      • Ulanovskaya O.A.
      • Ji Q.
      • Zhou T.
      • Sun F.
      • Lu Z.
      • Hutchison A.L.
      • Lan L.
      • Wu M.
      • Cravatt B.F.
      • He C.
      Steady-state hydrogen peroxide induces glycolysis in staphylococcus aureus and Pseudomonas aeruginosa.
      ). Likewise, in the process of ubiquitin-mediated proteolysis, the deubiquitinating enzymes can be inhibited by H2O2, which results in accumulation of ubiquitinated proteins upon H2O2 treatment (
      • Lee J.G.
      • Baek K.
      • Soetandyo N.
      • Ye Y.
      Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells.
      ,
      • Kulathu Y.
      • Garcia F.J.
      • Mevissen T.E.
      • Busch M.
      • Arnaudo N.
      • Carroll K.S.
      • Barford D.
      • Komander D.
      Regulation of A20 and other Otu deubiquitinases by reversible oxidation.
      ,
      • Cotto-Rios X.M.
      • Bekes M.
      • Chapman J.
      • Ueberheide B.
      • Huang T.T.
      Deubiquitinases As a signaling target of oxidative stress.
      ), whereas dissociation of the 26S proteasome mediated by H2O2 may compromise proteolysis of these ubiquitinated proteins (
      • Wang X.
      • Yen J.
      • Kaiser P.
      • Huang L.
      Regulation of the 26s proteasome complex during oxidative stress.
      ). Thus, the phenotypic outcome of H2O2 stress in cells reflects the net effect of H2O2 on multiple sensors. We found that most H2O2 sensors exhibit quite different reactivities toward H2O2 in different cells, which results in the cell-specific phenotypic changes upon H2O2 treatment.
      Besides many well-known redox sensors identified in the data set (supplemental Fig. S8B), our data contain many interesting leads for future functional studies. For example, we found that C779 of PSMD2, a regulatory subunit of the 26S proteasome, displays varying sensitivity to H2O2 in different cell contexts (Fig. 2D), suggesting a potential role of this site in regulation of H2O2-induced dissociation and inactivation of the 26S proteasome complex (
      • Wang X.
      • Yen J.
      • Kaiser P.
      • Huang L.
      Regulation of the 26s proteasome complex during oxidative stress.
      ). Another ubiquitous target of H2O2 in human cells is C290 of C-terminal Src Kinase (CSK), which is an important regulator of the oncoprotein Src (Fig. 2D). CSK can interact with Src through an intermolecular disulfide bond in vitro (
      • Kemble D.J.
      • Sun G.
      Direct and specific inactivation of protein tyrosine kinases in the Src and Fgfr Families By Reversible Cysteine Oxidation.
      ). However, the biological purpose of this disulfide bond formation is largely unknown. Whether the H2O2-dependent C290 of CSK transduces this redox signal to Src or other downstream effectors in cells through intermolecular disulfide bond remains to be determined.
      To better understand H2O2 sensing at a systems level, we performed bioinformatics and structural analysis of the data set, which uncovered previously unknown regulatory functions, crosstalk and distinct target preference of H2O2 (Fig. 2, Fig. 3). Of interest, the analysis identified the potential crosstalk between H2O2-dependent cysteine oxidation and lysine acetylation by sequence proximity. In accordance with this finding, H2O2 was found to preferentially target bromodomains of several histone acetyltransferases, which is involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins (
      • Fujisawa T.
      • Filippakopoulos P.
      Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.
      ). Moreover, this potential crosstalk could be further confirmed by the observation of H2O2-induced perturbation on several histone lysine acetylation events in U2OS cells. H2O2 may also stimulate dynamic interplay between cysteine oxidation and other chemical modifications at the same site, such as S-ADP-ribosylation (
      • Westcott N.P.
      • Fernandez J.P.
      • Molina H.
      • Hang H.C.
      Chemical proteomics reveals ADP-ribosylation of small gtpases during oxidative stress.
      ) and S-fatty-acylation (
      • Burgoyne J.R.
      • Haeussler D.J.
      • Kumar V.
      • Ji Y.
      • Pimental D.R.
      • Zee R.S.
      • Costello C.E.
      • Lin C.
      • Mccomb M.E.
      • Cohen R.A.
      • Bachschmid M.M.
      Oxidation of Hras cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis.
      ). Another important feature of this study is that QTRP provides a new opportunity to predict conformational changes on certain H2O2 sensors, as illustrated by our evidence for an unexpected mode for avoidance of overoxidation of GAPDH. In combination with simple chemical tools that preferentially and stably label reactive groups of proteins, this chemoproteomics approach should be adapted to provide global and site-specific assessment of protein surface accessibility for studying dynamic structural change and interaction of proteins.
      In conclusion, our quantitative chemoproteomic strategy has substantially expanded the scope of the observable redoxome and suggests a change in functional paradigm from a small set of conserved switches to a much larger, adaptable and cell type specific system. Our data set provides a basis for greatly expanded exploration of the complex networks controlled by H2O2-induced redox sensing, transduction and cellular adaptive responses. Recent advances in chemoproteomic strategy include the development of novel chemical probes for in vivo and/or organelle-specific labeling of thiol redox proteome (
      • Abo M.
      • Weerapana E.
      A caged electrophilic probe for global analysis of cysteine reactivity in living cells.
      ,
      • Abo M.
      • Bak D.W.
      • Weerapana E.
      Optimization of caged electrophiles for improved monitoring of cysteine reactivity in living cells.
      ), as well as advances in mass spectrometry-based proteomics to increase sensitivity and throughput for analyzing probe-modified peptides. We foresee this strategy being used to further explore the dynamics of reactive thiol proteomes in numerous physiological and pathophysiological contexts.

      Data Availability

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Vizcaino J.A.
      • Csordas A.
      • Del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 Update of the pride database and its related tools.
      ) partner repository with the data set identifier PXD007153 and 10.6019/PXD007153.

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