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Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling


Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin–binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1β and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.

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Figure 1: An avidity-based design strategy yielded tUIM peptides with high affinity and linkage specificity.
Figure 2: Designed proteins localized to linkage-specific structures inside cells.
Figure 3: Vx3K0-EGFP was recruited to mitochondria after uncoupling and the translocation of the E3 ubiquitin-ligase Parkin.
Figure 4: tUIM sensor proteins inhibited linkage-specific functions of cellular polyubiquitin.
Figure 5: Differential inhibition of NF-κB activation reveals distinct roles for Lys63-polyUb in diverse ligand-dependent signaling pathways.


  1. 1

    Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).

    CAS  Article  Google Scholar 

  2. 2

    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 

  3. 3

    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 

  4. 4

    Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. EMBO Rep. 9, 536–542 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Finley, D. et al. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol. Cell. Biol. 14, 5501–5509 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Spence, J., Sadis, S., Haas, A.L. & Finley, D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 15, 1265–1273 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Xu, M., Skaug, B., Zeng, W. & Chen, Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta. Mol. Cell 36, 302–314 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Pickart, C.M. & Cohen, R.E. Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Messick, T.E. & Greenberg, R.A. The ubiquitin landscape at DNA double-strand breaks. J. Cell Biol. 187, 319–326 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Kraft, C., Peter, M. & Hofmann, K. Selective autophagy: ubiquitin-mediated recognition and beyond. Nat. Cell Biol. 12, 836–841 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Lauwers, E., Erpapazoglou, Z., Haguenauer-Tsapis, R. & Andre, B. The ubiquitin code of yeast permease trafficking. Trends Cell Biol. 20, 196–204 (2010).

    CAS  Article  Google Scholar 

  13. 13

    Liu, S. & Chen, Z.J. Expanding role of ubiquitination in NF-kappaB signaling. Cell Res. 21, 6–21 (2011).

    Article  Google Scholar 

  14. 14

    Sims, J.J. & Cohen, R.E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80. Mol. Cell 33, 775–783 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Sims, J.J., Haririnia, A., Dickinson, B.C., Fushman, D. & Cohen, R.E. Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains. Nat. Struct. Mol. Biol. 16, 883–889 (2009).

    CAS  Article  Google Scholar 

  16. 16

    Swanson, K.A., Kang, R.S., Stamenova, S.D., Hicke, L. & Radhakrishnan, I. Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J. 22, 4597–4606 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Kaiser, S.E. et al. Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools. Nat. Methods 8, 691–696 (2011).

    CAS  Article  Google Scholar 

  18. 18

    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 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

    Geisler, S. et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119–131 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Narendra, D., Kane, L.A., Hauser, D.N., Fearnley, I.M. & Youle, R.J. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy 6, 1090–1106 (2010).

    CAS  Article  Google Scholar 

  22. 22

    Narendra, D., Tanaka, A., Suen, D.F. & Youle, R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Arnason, T. & Ellison, M.J. Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Mol. Cell. Biol. 14, 7876–7883 (1994).

    CAS  Article  Google Scholar 

  24. 24

    Skaug, B., Jiang, X. & Chen, Z.J. The role of ubiquitin in NF-kappaB regulatory pathways. Annu. Rev. Biochem. 78, 769–796 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Dynek, J.N. et al. c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J. 29, 4198–4209 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Saitoh, T. et al. TWEAK induces NF-kappaB2 p100 processing and long lasting NF-kappaB activation. J. Biol. Chem. 278, 36005–36012 (2003).

    CAS  Article  Google Scholar 

  27. 27

    Feltham, R. et al. Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding. J. Biol. Chem. 285, 17525–17536 (2010).

    CAS  Article  Google Scholar 

  28. 28

    Hjerpe, R. et al. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep. 10, 1250–1258 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117–120 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Wang, H. et al. Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. Proc. Natl. Acad. Sci. USA 105, 20197–20202 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    CAS  Article  Google Scholar 

  32. 32

    Pickart, C.M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Bremm, A., Freund, S.M. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Millard, B.L., Niepel, M., Menden, M.P., Muhlich, J.L. & Sorger, P.K. Adaptive informatics for multifactorial and high-content biological data. Nat. Methods 8, 487–493 (2011).

    CAS  Article  Google Scholar 

  35. 35

    Janes, K.A. et al. The response of human epithelial cells to TNF involves an inducible autocrine cascade. Cell 124, 1225–1239 (2006).

    CAS  Article  Google Scholar 

  36. 36

    Janes, K.A. et al. A systems model of signaling identifies a molecular basis set for cytokine-induced apoptosis. Science 310, 1646–1653 (2005).

    CAS  Article  Google Scholar 

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We thank S. Beese-Sims for constructing the leucine-auxotrophic version of the single-ubiquitin yeast strains, P. Sorger and B. Millard for helpful discussions about the manuscript and technical assistance on NF-κB activation experiments, A. Sliva for assistance with yeast experiments, B. Schmitt for assistance with binding experiments, A. Hess for help with statistical analyses and T. Yao for many helpful discussions. J.J.S. is supported by a fellowship from the Damon Runyon Cancer Research Foundation (DRG#2073-11). This work was supported, in part, by US National Institutes of Health (NIH) Common Fund grant RR020839 (J.D.B.), NIH grant P01 CA139980 (P. Sorger), US National Institute of Neurologial Disorders and Stroke Intramural Program (R.J.Y.), and NIH grants RC1 GM091424 (R.E.C.) and 1R01 GM097452 (R.E.C.).

Author information




J.J.S. developed the tUIM design strategy; J.J.S. and R.E.C. conceived of the study; J.J.S., E.M.C., F.S., L.A.K. and R.E.C. performed the experiments; J.J.S., E.M.C., F.S., L.A.K., R.E.C., R.J.Y. and J.D.B. contributed to experimental design and data analysis; J.J.S. and R.E.C. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Joshua J Sims or Robert E Cohen.

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Competing interests

A US patent application (12/815,74) describing design and applications of linkage-specific polyubiquitin binding proteins has been filed by Johns Hopkins University on behalf of J.J.S. and R.E.C.

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Supplementary Figures 1–6 and Supplementary Tables 1–2 (PDF 2595 kb)

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

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