Letter | Published:

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

Nature volume 533, pages 120124 (05 May 2016) | Download Citation

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Abstract

Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade1, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents2. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases3 or as enzymes that directly attack ubiquitin4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes5. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

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Change history

  • 04 May 2016

    Details for ref. 28 were updated.

References

  1. 1.

    & The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012)

  2. 2.

    , , & Ubiquitin in the immune system. EMBO Rep. 15, 28–45 (2014)

  3. 3.

    & Diversity of bacterial manipulation of the host ubiquitin pathways. Cell. Microbiol. 17, 26–34 (2015)

  4. 4.

    et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010)

  5. 5.

    & Cell biology of infection by Legionella pneumophila. Microbes Infect. 15, 157–167 (2013)

  6. 6.

    & Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl Acad. Sci. USA 101, 841–846 (2004)

  7. 7.

    et al. The E Block motif is associated with Legionella pneumophila translocated substrates. Cell. Microbiol. 13, 227–245 (2011)

  8. 8.

    et al. Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal. Proc. Natl Acad. Sci. USA 110, E707–E715 (2013)

  9. 9.

    et al. Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila. PLoS Pathog. 7, e1001289 (2011)

  10. 10.

    et al. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 (2012)

  11. 11.

    , , , & Icm/Dot-dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator. Cell. Microbiol. 16, 977–992 (2014)

  12. 12.

    et al. Legionella pneumophila effector RomA uniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication. Cell Host Microbe 13, 395–405 (2013)

  13. 13.

    et al. Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase. Proc. Natl Acad. Sci. USA 109, 13567–13572 (2012)

  14. 14.

    & Cell biology and immunology lessons taught by Legionella pneumophila. Sci. China Life Sci. 59, 3–10 (2016)

  15. 15.

    , & IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Mol. Microbiol. 56, 90–103 (2005)

  16. 16.

    , , , & Clostridium perfringens iota-toxin: structure and function. Toxins (Basel) 1, 208–228 (2009)

  17. 17.

    & The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases. Toxicon 39, 1647–1660 (2001)

  18. 18.

    , , , & Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites. J. Biol. Chem. 273, 7332–7337 (1998)

  19. 19.

    , & Novel bacterial ADP-ribosylating toxins: structure and function. Nature Rev. Microbiol. 12, 599–611 (2014)

  20. 20.

    & Toxicity and SidJ-mediated suppression of toxicity require distinct regions in the SidE family of Legionella pneumophila effectors. Infect. Immun. 83, 3506–3514 (2015)

  21. 21.

    , & Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ. PLoS Pathog. 11, e1004695 (2015)

  22. 22.

    , & Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination. Proc. Natl Acad. Sci. USA 108, 21212–21217 (2011)

  23. 23.

    & Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63, 3609–3620 (1995)

  24. 24.

    & The Legionella pneumophila effector SidJ is required for efficient recruitment of endoplasmic reticulum proteins to the bacterial phagosome. Infect. Immun. 75, 592–603 (2007)

  25. 25.

    & Rab-centric perspective of bacterial pathogen-occupied vacuoles. Cell Host Microbe 14, 256–268 (2013)

  26. 26.

    & Rab proteins of the endoplasmic reticulum: functions and interactors. Biochem. Soc. Trans. 40, 1426–1432 (2012)

  27. 27.

    et al. Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation. Mol. Biol. Cell 19, 2916–2925 (2008)

  28. 28.

    et al. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc. Natl Acad. Sci. USA 112, 15090–15095 (2015)

  29. 29.

    & Expanding the ubiquitin code through post-translational modification. EMBO Rep. 16, 1071–1083 (2015)

  30. 30.

    et al. The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse. Protein Sci. 11, 1657–1670 (2002)

  31. 31.

    & Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7, 7–19 (1993)

  32. 32.

    et al. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6, e1000822 (2010)

  33. 33.

    , , , & How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114, 4637–4650 (2001)

  34. 34.

    , & Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole. Cell. Microbiol. 7, 431–442 (2005)

  35. 35.

    , , , & Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors. Science 320, 1651–1654 (2008)

  36. 36.

    , & Mutations in the RNA polymerase II transcription machinery suppress the hyperrecombination mutant Hpr1Δ of Saccharomyces cerevisiae. Genetics 142, 749–759 (1996)

  37. 37.

    , , & Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360 (1995)

  38. 38.

    & Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism. Mol. Cell 12, 1333–1340 (2003)

  39. 39.

    & The Legionella pneumophila IcmR protein exhibits chaperone activity for IcmQ by preventing its participation in high-molecular-weight complexes. Mol. Microbiol. 40, 1113–1127 (2001)

  40. 40.

    , , & The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol. Microbiol. 48, 305–321 (2003)

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Acknowledgements

We thank P. Hollenbeck (Purdue University) for critical reading of the manuscript. J. Barbieri (Medical College of Wisconsin) for plasmids. This work was supported by National Institutes of Health grants R56AI103168, K02AI085403 and R21AI105714 (Z.-Q.L.), 2R01GM103401 (C.D.) and National Natural Science Foundation of China grants 21305006 and 21475005 (X.L.).

Author information

Author notes

    • Yunhao Tan

    Present address: Division of Gastroenterology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

Affiliations

  1. Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA

    • Jiazhang Qiu
    • , Yunhao Tan
    •  & Zhao-Qing Luo
  2. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, USA

    • Michael J. Sheedlo
    •  & Chittaranjan Das
  3. Institute of Analytical Chemistry and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

    • Kaiwen Yu
    •  & Xiaoyun Liu
  4. Biological Science Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA

    • Ernesto S. Nakayasu

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Contributions

J.Q. and Z.-Q.L. conceived the general ideas for this work. J.Q. and Z.-Q.L. planned, performed and interpreted experiments. Y.T. initiated the project, performed the bioinformatics analysis and determined the importance of the predicted mART motif in yeast toxicity. M.S., E.S.N., J.Q. and C.D. determined the reaction intermediates. K.Y., X.L. and E.S.N. performed mass spectrometric analyses. J.Q. and Z.-Q.L. wrote the manuscript and all authors provided editorial input.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Zhao-Qing Luo.

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DOI

https://doi.org/10.1038/nature17657

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