Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Chemical proteomics reveals new targets of cysteine sulfinic acid reductase

Abstract

Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1–4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species–associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Development of electrophilic nitrogen species for labeling sulfinic acids.
Fig. 2: Reactivity of electron-deficient diazenes toward recombinant-protein models.
Fig. 3: Detection of protein S-sulfinylation from cells with DiaFluo.
Fig. 4: Site-centric and quantitative chemoproteomic profiling of protein S-sulfinylation.
Fig. 5: Comparison of S-sulfenylome and S-sulfinylome sites and dynamic fold changes.
Fig. 6: Proteome-wide analysis of SRX-regulated changes in S-sulfinylation.

Similar content being viewed by others

References

  1. Paulsen, C. E. & Carroll, K. S. Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery. Chem. Rev. 113, 4633–4679 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gupta, V. & Carroll, K. S. Sulfenic acid chemistry, detection and cellular lifetime. Biochim. Biophys. Acta. 1840, 847–875 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Gupta, V., Yang, J., Liebler, D. C. & Carroll, K. S. Diverse redoxome reactivity profiles of carbon nucleophiles. J. Am. Chem. Soc. 139, 5588–5595 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yang, J. et al. Global, in situ, site-specific analysis of protein S-sulfenylation. Nat. Protoc. 10, 1022–1037 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yang, J., Gupta, V., Carroll, K. S. & Liebler, D. C. Site-specific mapping and quantification of protein S-sulphenylation in cells. Nat. Commun. 5, 4776 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Gould, N. S. et al. Site-specific proteomic mapping identifies selectively modified regulatory cysteine residues in functionally distinct protein networks. Cell Chem. Biol. 22, 965–975 (2015).

    CAS  Google Scholar 

  7. Depuydt, M. et al. A periplasmic reducing system protects single cysteine residues from oxidation. Science 326, 1109–1111 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Paulsen, C. E. et al. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat. Chem. Biol. 8, 57–64 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kulathu, Y. et al. Regulation of A20 and other OTU deubiquitinases by reversible oxidation. Nat. Commun. 4, 1569 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Seo, Y. H. & Carroll, K. S. Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies. Proc. Natl. Acad. Sci. USA 106, 16163–16168 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jacob, C., Holme, A. L. & Fry, F. H. The sulfinic acid switch in proteins. Org. Biomol. Chem. 2, 1953–1956 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Woo, H. A. et al. Reversible oxidation of the active site cysteine of peroxiredoxins to cysteine sulfinic acid: immunoblot detection with antibodies specific for the hyperoxidized cysteine-containing sequence. J. Biol. Chem. 278, 47361–47364 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Wood, Z. A., Poole, L. B. & Karplus, P. A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 300, 650–653 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Biteau, B., Labarre, J. & Toledano, M. B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 425, 980–984 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Chang, T. S. et al. Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine. J. Biol. Chem. 279, 50994–51001 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Woo, H. A. et al. Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins. J. Biol. Chem. 280, 3125–3128 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Lo Conte, M. & Carroll, K. S. The redox biochemistry of protein sulfenylation and sulfinylation. J. Biol. Chem. 288, 26480–26488 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Canet-Aviles, R. M. et al. The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc. Natl. Acad. Sci. USA 101, 9103–9108 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Blackinton, J. et al. Formation of a stabilized cysteine sulfinic acid is critical for the mitochondrial function of the parkinsonism protein DJ-1. J. Biol. Chem. 284, 6476–6485 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kil et al. Circadian oscillation of sulfiredoxin in the mitochondria. Mol. Cell. 59, 651–663 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Ramesh, A. et al. Role of sulfiredoxin in systemic diseases influenced by oxidative stress. Redox Biol. 2, 1023–1028 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dickinson, B. C. & Chang, C. J. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat. Chem. Biol. 7, 504–511 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wei, Q., Jiang, H., Matthews, C. P. & Colburn, N. H. Sulfiredoxin is an AP-1 target gene that is required for transformation and shows elevated expression in human skin malignancies. Proc. Natl. Acad. Sci. USA 105, 19738–19743 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wei, Q. et al. Sulfiredoxin-peroxiredoxin IV axis promotes human lung cancer progression through modulation of specific phosphokinase signaling. Proc. Natl. Acad. Sci. USA 108, 7004–7009 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kim, H. et al. Sulfiredoxin inhibitor induces preferential death of cancer cells through reactive oxygen species-mediated mitochondrial damage. Free Rad. Biol. Med. 91, 264–274 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. Woo, H. A. & Rhee, S. G. in Methods in Redox Signaling (ed. Das, D.) Ch. 4, 19–23 (Mary Ann Liebert, New Rochelle, NY, USA, 2010).

  27. Lee, C. F., Paull, T. T. & Person, M. D. Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM. J. Prot. Res. 12, 4302–4315 (2013).

    Article  CAS  Google Scholar 

  28. Kuo, Y. H. et al. Profiling protein S-sulfination with maleimide-linked probes. Chembiochem 18, 2028–2032 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lo Conte, M. & Carroll, K. S. Chemoselective ligation of sulfinic acids with aryl-nitroso compounds. Angew. Chem. Int. Ed. 51, 6502–6505 (2012).

    Article  CAS  Google Scholar 

  30. Lo Conte, M., Lin, J., Wilson, M. A. & Carroll, K. S. A chemical approach for the detection of protein sulfinylation. ACS Chem. Biol. 10, 1825–1830 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Majmudar, J. D. et al. Harnessing redox cross-reactivity to profile distinct cysteine modifications. J. Am. Chem. Soc. 138, 1852–1859 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mitroka, S. et al. Direct and nitroxyl (HNO)-mediated reactions of acyloxy nitroso compounds with the thiol-containing proteins glyceraldehyde 3-phosphate dehydrogenase and alkyl hydroperoxide reductase subunit C. J. Med. Chem. 56, 6583–6592 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schlick, T. L., Ding, Z., Kovacs, E. W. & Francis, M. B. Dual-surface modification of the tobacco mosaic virus. J. Am. Chem. Soc. 127, 3718–3723 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Delaunay, A., Pflieger, D., Barrault, M. B., Vinh, J. & Toledano, M. B. A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111, 471–481 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Baek, J. Y. et al. Sulfiredoxin protein is critical for redox balance and survival of cells exposed to low steady-state levels of H2O2. J. Biol. Chem. 287, 81–89 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. Kim, K. H., Lee, W. & Kim, E. E. Crystal structures of human peroxiredoxin 6 in different oxidation states. Biochem. Biophys. Res. Comm. 477, 717–722 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. van Montfort, R. L., Congreve, M., Tisi, D., Carr, R. & Jhoti, H. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 423, 773–777 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Mullen, L., Hanschmann, E. M., Lillig, C. H., Herzenberg, L. A. & Ghezzi, P. Cysteine oxidation targets peroxiredoxins 1 and 2 for exosomal release through a novel mechanism of redox-dependent secretion. Mol. Med. 21, 98–108 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Szabo-Taylor, K. et al. Oxidative and other posttranslational modifications in extracellular vesicle biology. Semin. Cell Dev. Biol. 40, 8–16 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Porta, C., Moroni, M., Guallini, P., Torri, C. & Marzatico, F. Antioxidant enzymatic system and free radicals pathway in two different human cancer cell lines. Anticancer. Res. 16, 2741–2747 (1996).

    CAS  PubMed  Google Scholar 

  41. Chauvin, J. R. & Pratt, D. A. On the reactions of thiols, sulfenic acids, and sulfinic acids with hydrogen peroxide. Angew. Chem. Int. Ed. 56, 6255–6259 (2017).

    Article  CAS  Google Scholar 

  42. Li, H. et al. Crystal structure and substrate specificity of PTPN12. Cell Rep. 15, 1345–1358 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. Jönsson, T. J. et al. Structural basis for the retroreduction of inactivated peroxiredoxins by human sulfiredoxin. Biochemistry 44, 8634–8642 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Klamt, F. et al. Oxidant-induced apoptosis is mediated by oxidation of the actin-regulatory protein cofilin. Nat. Cell Biol. 11, 1241–1246 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Cameron, J. M. et al. Polarized cell motility induces hydrogen peroxide to inhibit cofilin via cysteine oxidation. Curr. Biol. 25, 1520–1525 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hamann, M., Zhang, T., Hendrich, S. & Thomas, J. A. Quantitation of protein sulfinic and sulfonic acid, irreversibly oxidized protein cysteine sites in cellular proteins. Methods. Enzymol. 348, 146–156 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. White, M. D. et al. Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets. Nat. Commun. 8, 14690 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Schroder, K. NADPH oxidases in redox regulation of cell adhesion and migration. Antioxid. Redox Signal. 20, 2043–2058 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Li, X. et al. Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis. Mol. Cell 61, 705–719 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sun, T. et al. Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase. Cell 144, 703–718 (2011).

  51. Paulsen, C. E. & Carroll, K. S. Chemical dissection of an essential redox switch in yeast. Chem. Biol. 16, 217–225 (2009).

    Article  CAS  PubMed  Google Scholar 

  52. Cheng, H., Donahue, J. L., Battle, S. E., Ray, W. K. & Larson, T. J. Biochemical and genetic characterization of PspE and GlpE, two single-domain sulfurtransferases of Escherichia coli. Open Microbiol. J. 2, 18–28 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Chi, H. et al. pFind-Alioth: a novel unrestricted database search algorithm to improve the interpretation of high-resolution MS/MS data. J. Proteomics 125, 89–97 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Li, D. et al. pFind: a novel database-searching software system for automated peptide and protein identification via tandem mass spectrometry. Bioinformatics 21, 3049–3050 (2005).

    Article  CAS  PubMed  Google Scholar 

  55. Wang, L. H. et al. pFind 2.0: a software package for peptide and protein identification via tandem mass spectrometry. Rapid Commun. Mass Spectrom. 21, 2985–2991 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. Tan, D. et al. Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states. eLife 5, e12509 (2016).

  57. Liu, C. et al. pQuant improves quantitation by keeping out interfering signals and evaluating the accuracy of calculated ratios. Anal. Chem. 86, 5286–5294 (2014).

    Article  CAS  PubMed  Google Scholar 

  58. Ma, Y., McClatchy, D. B., Barkallah, S., Wood, W. W. & Yates, J. R. 3rd HILAQ: a novel strategy for newly synthesized protein quantification. J. Proteome Res. 16, 2213–2220 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Garcia, F. J. & Carroll, K. S. Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cells. Eur. J. Med. Chem. 17, 28–33 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Key R&D Program of China (2016YFA0501303 to J.Y.), the National Natural Science Foundation of China (31770885 to J.Y.), the Beijing Nova Program (Z171100001117014 to J.Y.) and the US National Institutes of Health (R01 GM102187 and R01 CA174864 to K.S.C. and R01 GM072866 to W.T.L.). This work was also supported by the Wake Forest Baptist Comprehensive Cancer Center (P30CA012197 to W.T.L.). We thank Q. Zhou and W. Leng (National Center for Protein Sciences–Beijing) for expert technical assistance, C. Liu and H. Chi (Institute of Computing Technology, CAS) for helpful discussions in proteomic informatics, K. Tallman and N. Porter (Vanderbilt University) for providing light and heavy Az–UV–biotin reagents, P. Wu (The Scripps Research Institute) for providing the BTTP click ligand, M. Wilson (University of Nebraska, Lincoln) for providing recombinant DJ-1, and S. G. Rhee (Yonsei University College of Medicine) and M. Toledano (Institut des Science du Vivant Frédérique Joliot) for providing Srx+/+ and Srx–/– MEFs.

Author information

Authors and Affiliations

Authors

Contributions

K.S.C. and J.Y. designed the experiments, analyzed data and wrote the manuscript, and all authors provided input. K.S.C. and M.L. devised the ENS concept for sulfinic acid detection. M.L. performed reactions of diazonium salts and diazenes with model sulfinic acids and fluorescence imaging. J.Y. developed the chemoproteomic method. L.F. and R.S. performed chemoproteomic experiments and validation of SRX substrates. S.A. verified and maintained cell lines; analyzed redox, time and dose dependence of DiaAlk proteome labeling; developed BioDiaAlk and SRX luminescence-based ATPase assays; and performed validation of SRX substrates. Y.J. synthesized probes; performed rate and adduct-stability studies; and characterized the N-Fmoc cysteine sulfur acid–DiaAlk adducts. J.R.L. and W.T.L. purified recombinant SRX and PRDX2. K.L. performed computational and bioinformatics analyses.

Corresponding authors

Correspondence to Jing Yang or Kate S. Carroll.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Text and Figures

Supplementary Figures 1–22

Reporting Summary

Supplementary Note

Synthetic procedures

Supplementary Dataset 1

S-Sulfinylated cysteines identified and quantified in response to exogenous oxidants in A549 and HeLa cells as shown in Fig. 4

Supplementary Dataset 2

S-Sulfenylated cysteines identified and quantified in response to exogenous oxidants in A549 and HeLa cells as shown in Fig. 5

Supplementary Dataset 3

S-Sulfinylated cysteines identified and quantified in Srx+/+ and Srx–/– MEF cells as shown in Fig. 6

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Akter, S., Fu, L., Jung, Y. et al. Chemical proteomics reveals new targets of cysteine sulfinic acid reductase. Nat Chem Biol 14, 995–1004 (2018). https://doi.org/10.1038/s41589-018-0116-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0116-2

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research