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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

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

References

  1. 1.

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

  2. 2.

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

  3. 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).

  4. 4.

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

  5. 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).

  6. 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).

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 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).

  11. 11.

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

  12. 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).

  13. 13.

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

  14. 14.

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

  15. 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).

  16. 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).

  17. 17.

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

  18. 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).

  19. 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).

  20. 20.

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

  21. 21.

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

  22. 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).

  23. 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).

  24. 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).

  25. 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).

  26. 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. 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).

  28. 28.

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

  29. 29.

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

  30. 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).

  31. 31.

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

  32. 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).

  33. 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).

  34. 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).

  35. 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).

  36. 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).

  37. 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).

  38. 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).

  39. 39.

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

  40. 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).

  41. 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).

  42. 42.

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

  43. 43.

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

  44. 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).

  45. 45.

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

  46. 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).

  47. 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).

  48. 48.

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

  49. 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).

  50. 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. 51.

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

  52. 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).

  53. 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).

  54. 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).

  55. 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).

  56. 56.

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

  57. 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).

  58. 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).

  59. 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).

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

Author notes

    • Mauro Lo Conte

    Present address: Novo Nordisk Research Center, Seattle, WA, USA

  1. These authors contributed equally: Salma Akter, Ling Fu.

Affiliations

  1. Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USA

    • Salma Akter
    • , Youngeun Jung
    • , Mauro Lo Conte
    •  & Kate S. Carroll
  2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing, China

    • Ling Fu
    • , Rui Sun
    • , Keke Liu
    •  & Jing Yang
  3. National Center for Protein Sciences–Beijing, Beijing, China

    • Ling Fu
    • , Rui Sun
    • , Keke Liu
    •  & Jing Yang
  4. Beijing Institute of Lifeomics, Beijing, China

    • Ling Fu
    • , Rui Sun
    • , Keke Liu
    •  & Jing Yang
  5. Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, USA

    • J. Reed Lawson
    •  & W. Todd Lowther
  6. Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC, USA

    • J. Reed Lawson
    •  & W. Todd Lowther

Authors

  1. Search for Salma Akter in:

  2. Search for Ling Fu in:

  3. Search for Youngeun Jung in:

  4. Search for Mauro Lo Conte in:

  5. Search for J. Reed Lawson in:

  6. Search for W. Todd Lowther in:

  7. Search for Rui Sun in:

  8. Search for Keke Liu in:

  9. Search for Jing Yang in:

  10. Search for Kate S. Carroll in:

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.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Jing Yang or Kate S. Carroll.

Electronic supplementary material

  1. Supplementary Text and Figures

    Supplementary Figures 1–22

  2. Reporting Summary

  3. Supplementary Note

    Synthetic procedures

  4. Supplementary Dataset 1

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

  5. Supplementary Dataset 2

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

  6. Supplementary Dataset 3

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

About this article

Publication history

Received

Accepted

Published

DOI

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

Further reading