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K63 polyubiquitination is a new modulator of the oxidative stress response

Nature Structural & Molecular Biology volume 22, pages 116123 (2015) | Download Citation

Abstract

Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.

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References

  1. 1.

    , , & Redox control and oxidative stress in yeast cells. Biochim. Biophys. Acta 1780, 1217–1235 (2008).

  2. 2.

    & Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399 (2004).

  3. 3.

    & The role of oxidative stress in carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 44, 239–267 (2004).

  4. 4.

    Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95 (2002).

  5. 5.

    & Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247 (2000).

  6. 6.

    , & The response to heat shock and oxidative stress in Saccharomyces cerevisiae. Genetics 190, 1157–1195 (2012).

  7. 7.

    Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–899 (2003).

  8. 8.

    & The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

  9. 9.

    , & Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009).

  10. 10.

    , & Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).

  11. 11.

    , , & The ubiquitin-proteasome system of Saccharomyces cerevisiae. Genetics 192, 319–360 (2012).

  12. 12.

    , , , & Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. USA 77, 1783–1786 (1980).

  13. 13.

    , , & A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 15, 1265–1273 (1995).

  14. 14.

    The emerging complexity of protein ubiquitination. Biochem. Soc. Trans. 37, 937–953 (2009).

  15. 15.

    & Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).

  16. 16.

    et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

  17. 17.

    et al. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol. Cell 39, 477–484 (2010).

  18. 18.

    , & K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway. J. Cell Biol. 185, 493–502 (2009).

  19. 19.

    , , , & Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation. EMBO Rep. 13, 331–338 (2012).

  20. 20.

    , , , & RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

  21. 21.

    et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).

  22. 22.

    et al. Bcl10 activates the NF-κB pathway through ubiquitination of NEMO. Nature 427, 167–171 (2004).

  23. 23.

    et al. Selectivity of the ubiquitin pathway for oxidatively modified proteins: relevance to protein precipitation diseases. FASEB J. 19, 1707–1709 (2005).

  24. 24.

    & Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins. J. Cell Biol. 182, 663–673 (2008).

  25. 25.

    , , & Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J. Biol. Chem. 278, 311–318 (2003).

  26. 26.

    et al. The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem. J. 432, 585–594 (2010).

  27. 27.

    , , , & Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol. Cell. Proteomics 11, 1475–1488 (2012).

  28. 28.

    , & Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat. Cell Biol. 7, 750–757 (2005).

  29. 29.

    et al. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184 (2010).

  30. 30.

    , , & The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1. J. Biol. Chem. 280, 9879–9886 (2005).

  31. 31.

    , , , & H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation. Mol. Cell 31, 57–66 (2008).

  32. 32.

    et al. Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11, 267–274 (2003).

  33. 33.

    , , & The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases. Nat. Cell Biol. 12, 1177–1185 (2010).

  34. 34.

    , , & The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme. Proc. Natl. Acad. Sci. USA 88, 7351–7355 (1991).

  35. 35.

    et al. Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS. Cell 131, 1084–1096 (2007).

  36. 36.

    & Bre1-associated protein, large 1 (Lge1), promotes H2B ubiquitylation during the early stages of transcription elongation. J. Biol. Chem. 285, 2361–2367 (2010).

  37. 37.

    , , , & The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p. J. Biol. Chem. 278, 34739–34742 (2003).

  38. 38.

    , , , & Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast. Proc. Natl. Acad. Sci. USA 109, 10837–10842 (2012).

  39. 39.

    & Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412, 553–557 (2001).

  40. 40.

    et al. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102, 67–76 (2000).

  41. 41.

    et al. Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Hum. Mol. Genet. 17, 431–439 (2008).

  42. 42.

    et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J. 28, 359–371 (2009).

  43. 43.

    Regulation of translation by hydrogen peroxide. Antioxid. Redox Signal. 15, 191–203 (2011).

  44. 44.

    , , & Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells. Nat. Commun. 4, 1568 (2013).

  45. 45.

    , , , & Deubiquitinases as a signaling target of oxidative stress. Cell Reports 2, 1475–1484 (2012).

  46. 46.

    , , & The deubiquitinating enzyme Ubp2 modulates Rsp5-dependent Lys63-linked polyubiquitin conjugates in Saccharomyces cerevisiae. J. Biol. Chem. 281, 36724–36731 (2006).

  47. 47.

    et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

  48. 48.

    et al. Reactive cysteine in proteins: protein folding, antioxidant defense, redox signaling and more. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 146, 180–193 (2007).

  49. 49.

    et al. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat. Commun. 5, 4763 (2014).

  50. 50.

    , & Ubiquitin Lys 63 chains - second-most abundant, but poorly understood in plants. Front. Plant Sci. 5, 15 (2014).

  51. 51.

    et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926 (2003).

  52. 52.

    Stationary phase in yeast. Curr. Opin. Microbiol. 5, 602–607 (2002).

  53. 53.

    & The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress. Microbiology 153, 247–253 (2007).

  54. 54.

    , , , & Activities of Escherichia coli ribosomes in IF3 and RMF change to prepare 100S ribosome formation on entering the stationary growth phase. Genes Cells 14, 271–280 (2009).

  55. 55.

    et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

  56. 56.

    et al. Global translational responses to oxidative stress impact upon multiple levels of protein synthesis. J. Biol. Chem. 281, 29011–29021 (2006).

  57. 57.

    , & Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress. Proc. Natl. Acad. Sci. USA 109, 17394–17399 (2012).

  58. 58.

    , & Protein expression regulation under oxidative stress. Mol. Cell Proteomics 10, M111.009217 (2011).

  59. 59.

    , , & Crystal structure of the eukaryotic ribosome. Science 330, 1203–1209 (2010).

  60. 60.

    , , & Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells. Biotechniques 42, 158,160,162 (2007).

  61. 61.

    , , , & eIF4GI links nutrient sensing by mTOR to cell proliferation and inhibition of autophagy. J. Cell Biol. 181, 293–307 (2008).

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Acknowledgements

We thank J.R. Cussiol (Cornell University) and M. Smolka (Cornell University) for the yeast deletion templates, support in yeast genetics and comments on the manuscript. We are grateful to G. Hsu and R. Schneider for technical support in polysome analysis, and V. Subramanian and A. Hochwagen in fluorescence-activated cell sorting analysis. We thank N. Brandt (New York University), D. Gresham (New York University), M. Hochstrasser (Yale University), M.A. Osley (University of New Mexico) and R. Ratan (Burke Medical Research Institute) for sharing yeast strains and HT22 cells. We thank J.R. Chapman for assistance with targeted MS, and the Pride Team (http://www.ebi.ac.uk/services/teams/pride) for assistance with MS data deposition. We are indebted to D. Gresham, J. Davis, E. Miraldi and T. Rock for feedback on the manuscript. This work was supported in part by US National Science Foundation EAGER grant MCB-1355462 (G.M.S. and C.V.), the Zegar Family Foundation Fund for Genomics Research at New York University (G.M.S. and C.V.) and US National Institutes of Health grant GM43601 (D.F.).

Author information

Affiliations

  1. Center for Genomics and Systems Biology, New York University, New York, New York, USA.

    • Gustavo M Silva
    •  & Christine Vogel
  2. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Daniel Finley

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Contributions

G.M.S. conceived of the project. G.M.S., D.F. and C.V. designed the experiments, and G.M.S. conducted the experiments. G.M.S. and C.V. wrote the manuscript. G.M.S., D.F. and C.V. discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gustavo M Silva or Christine Vogel.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Note

  2. 2.

    Supplementary Data Set 1

    Original gels and blots - Uncropped images of gels and blots used in the main figures of this study

Excel files

  1. 1.

    K63 ubiquitinated targets

    Description of the K63 targets identified by high-resolution mass spectrometry

  2. 2.

    K63 ubiquitinated core targets

    Description of the high-confidence K63 targets identified by two different search engines

  3. 3.

    Whole cell lysate protein abundance

    Description of protein abundance changes in the cellular lysate measured by high-resolution mass spectrometry

  4. 4.

    Yeast strains

    Table contains the genotypes and reference sources for the yeast Saccharomyces cerevisiae strains used in the study

  5. 5.

    Ubiquitin signature peptides for PRM analysis

    List of the signature peptides used in the parallel reaction monitoring analysis to quantify linkage specific polyubiquitination

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DOI

https://doi.org/10.1038/nsmb.2955

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