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Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain–specific sensors

Abstract

Despite intensive research, there is a distinct lack of methodology for visualizing endogenous ubiquitination in living cells. In this protocol, we describe how unique properties of ubiquitin (Ub)-binding domains (UBDs) can be used to selectively detect, visualize and inhibit Ub-dependent processes in mammalian cells. The procedure deals with designing and validating the binding selectivity of GFP-tagged K63- and linear-linked sensors (TAB2 NZF and NEMO UBAN, respectively) in vitro. We describe how these moieties can be used to inhibit tumor necrosis factor (TNF)-mediated NF-κB signaling and to detect ubiquitinated cytosolic Salmonella in living cells, emphasizing a more flexible use compared with chain-specific antibodies. These chain-specific sensors can be used to detect Ub-like or autophagy-related modifiers and, in combination with mass spectrometry, to identify new Ub targets. These Ub (-like) sensors can be designed, constructed and tested in 2–3 weeks.

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Figure 1: Validation of UBD:Ub chain-interaction selectivity.
Figure 2: Expression of NEMO UBAN and TAB2 NZF inhibits TNF-induced NF-κB signaling.
Figure 3: mCherry-tagged NEMO UBAN is recruited to cytosolic Salmonella in living cells.

References

  1. 1

    Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series. EMBO Rep. 9, 536–542 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Iwai, K. & Tokunaga, F. Linear polyubiquitination: a new regulator of NF-B activation. EMBO Rep. 10, 706–713 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Ulrich, H.D. & Walden, H. Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11, 479–489 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Piper, R.C. & Lehner, P.J. Endosomal transport via ubiquitination. Trends Cell Biol. 21, 647–655 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Husnjak, K. & Dikic, I. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Dikic, I., Wakatsuki, S. & Walters, K.J. Ubiquitin-binding domains—from structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Randles, L. & Walters, K.J. Ubiquitin and its binding domains. Front. Biosci. 17, 2140–2157 (2012).

    Article  Google Scholar 

  10. 10

    Kulathu, Y., Akutsu, M., Bremm, A., Hofmann, K. & Komander, D. Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain. Nat. Struct. Mol. Biol. 16, 1328–1330 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Rahighi, S. et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-B activation. Cell 136, 1098–1109 (2009).

    CAS  Article  Google Scholar 

  12. 12

    Kulathu, Y. & Komander, D. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012).

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    Phu, L. et al. Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals. Mol. Cell Proteomics 10, M110 003756 (2011).

    Article  Google Scholar 

  15. 15

    Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).

    CAS  Article  Google Scholar 

  16. 16

    Haas, T.L. et al. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol. Cell 36, 831–844 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-B activity and apoptosis. Nature 471, 637–641 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Tokunaga, F. et al. SHARPIN is a component of the NF-B–activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Laney, J.D. & Hochstrasser, M. Analysis of protein ubiquitination. Curr. Protoc. Protein Sci. 66, 14.5.1–14.5.13 (2011).

    Article  Google Scholar 

  20. 20

    Argenzio, E. et al. Proteomic snapshot of the EGF-induced ubiquitin network. Mol. Syst. Biol. 7, 462 (2011).

    Article  Google Scholar 

  21. 21

    Dohmen, R.J. & Scheffner, M. Methods in Molecular Biology 832 (ed. Walker, J.M.) 1–660 (Humana Press, 2012).

    Article  Google Scholar 

  22. 22

    Kirkpatrick, D.S., Denison, C. & Gygi, S.P. Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat. Cell Biol. 7, 750–757 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Povlsen, L.K. et al. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat. Cell Biol. 14, 1089–1098 (2012).

    CAS  Article  Google Scholar 

  24. 24

    Wagner, S.A. et al. Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol. Cell Proteomics 11, 1578–1585 (2012).

    Article  Google Scholar 

  25. 25

    Xu, G., Paige, J.S. & Jaffrey, S.R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Wagner, S.A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell Proteomics 10, M111 013284 (2011).

    Article  Google Scholar 

  27. 27

    Emanuele, M.J. et al. Global identification of modular cullin-RING ligase substrates. Cell 147, 459–474 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Kirkpatrick, D.S., Gerber, S.A. & Gygi, S.P. The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35, 265–273 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Fujimuro, M. & Yokosawa, H. Production of antipolyubiquitin monoclonal antibodies and their use for characterization and isolation of polyubiquitinated proteins. Methods Enzymol. 399, 75–86 (2005).

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

    Matsumoto, M.L. et al. Engineering and structural characterization of a linear-polyubiquitin-specific antibody. J. Mol. Biol. 418, 144 (2011).

  32. 32

    Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

    CAS  Article  Google Scholar 

  33. 33

    Erpapazoglou, Z. et al. A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting. Mol. Biol. Cell 23, 2170–2183 (2012).

    CAS  Article  Google Scholar 

  34. 34

    van Wijk, S.J. et al. Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells. Mol. Cell 47, 797–809 (2012).

    CAS  Article  Google Scholar 

  35. 35

    Sims, J.J. et al. Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat. Methods 9, 303–309 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Chen, J. & Chen, Z.J. Regulation of NF-B by ubiquitination. Curr. Opin. Immunol. 25, 4–12 (2013).

    CAS  Article  Google Scholar 

  37. 37

    Grabbe, C., Husnjak, K. & Dikic, I. The spatial and temporal organization of ubiquitin networks. Nat. Rev. Mol. Cell Biol. 12, 295–307 (2011).

    CAS  Article  Google Scholar 

  38. 38

    Wild, P. et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 (2011).

    CAS  Article  Google Scholar 

  39. 39

    Thurston, T.L., Ryzhakov, G., Bloor, S., von Muhlinen, N. & Randow, F. The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat. Immunol. 10, 1215–1221 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Huett, A. et al. The LRR and RING domain protein LRSAM1 is an E3 ligase crucial for ubiquitin-dependent autophagy of intracellular Salmonella Typhimurium. Cell Host Microbe 12, 778–790 (2012).

    CAS  Article  Google Scholar 

  41. 41

    Bumann, D. & Valdivia, R.H. Identification of host-induced pathogen genes by differential fluorescence induction reporter systems. Nat. Protoc. 2, 770–777 (2007).

    CAS  Article  Google Scholar 

  42. 42

    van Wijk, S.J., Muller, S. & Dikic, I. Shared and unique properties of ubiquitin and SUMO interaction networks in DNA repair. Genes Dev. 25, 1763–1769 (2011).

    CAS  Article  Google Scholar 

  43. 43

    McEwan, D.G. & Dikic, I. The Three Musketeers of autophagy: phosphorylation, ubiquitylation and acetylation. Trends Cell Biol. 21, 195–201 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

    CAS  Article  Google Scholar 

  45. 45

    van de Linde, S., Heilemann, M. & Sauer, M. Live-cell super-resolution imaging with synthetic fluorophores. Annu. Rev. Phys. Chem. 63, 519–540 (2012).

    CAS  Article  Google Scholar 

  46. 46

    Nelson, D.E. et al. Oscillations in NF-B signaling control the dynamics of gene expression. Science 306, 704–708 (2004).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Bremm, D. McEwan and J. Lopez for critical reading of the manuscript. This work was supported by the Cluster of Excellence 'Macromolecular Complexes' of the Goethe University, Frankfurt am Main (EXC115); LOEWE Oncology Signaling Network and LOEWE Center for Cell and Gene Therapy, Frankfurt am Main; and a European Research Council Advance Grant (I.D).

Author information

Affiliations

Authors

Contributions

S.J.L.v.W. was involved in development of the protocol and prepared the manuscript. E.F. supported with Salmonella imaging and edited the manuscript. I.D. is the principal investigator who supervised the work and edited the manuscript.

Corresponding author

Correspondence to Ivan Dikic.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Schematic representation of potential sensors for Ub, UBLs and autophagy-like modifiers (PDF 598 kb)

Supplementary Figure 2

Schematic representation of mammalian Ub sensors used in this protocol (PDF 582 kb)

Supplementary Figure 3

Stably expressed GFP NEMO UBAN co localizes with Ub chain specific antibodies on Infected Salmonella (PDF 620 kb)

Supplementary Figure 4

Expression of TAB2 NZF E685A does not affect TNF induced NF-κB signalling (PDF 616 kb)

Supplementary Video 1

Recruitment of linear Ub chain-specific NEMO UBAN to cytosolic Salmonella in living cells (AVI 1951 kb)

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van Wijk, S., Fiškin, E. & Dikic, I. Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain–specific sensors. Nat Protoc 8, 1449–1458 (2013). https://doi.org/10.1038/nprot.2013.089

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