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  • Review Article
  • Published:

Atypical ubiquitylation — the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages

Nature Reviews Molecular Cell Biology volume 13, pages 508–523 (2012)Cite this article

Subjects

Key Points

  • The reversible conjugation of ubiquitin chains to protein substrates regulates almost every cellular process.

  • Ubiquitin chains can be assembled via one of the seven ubiquitin Lys residues (which are Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63) or via the amino terminus (Met1). Atypical ubiquitin chains are those not linked via canonical Lys48 linkages or Lys63 linkages.

  • Available structures of ubiquitin chains suggest that each linkage type adopts unique conformations. Ubiquitin-binding domains (UBDs) and deubiquitinases (DUBs) exploit the distinct features of polyubiquitin to achieve specificity.

  • All linkage types coexist in cells, and the abundance of particular atypical linkages changes in response to specific stimuli and can be altered in disease states.

  • The enzymatic assembly of ubiquitin chains requires the action of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-ligating enzymes. E2 enzymes and certain classes of E3 ligases determine the chain linkage type.

  • DUBs negatively regulate ubiquitylation by hydrolyzing ubiquitin chains. Although many DUBs are promiscuous, certain DUBs such as members of the ovarian tumour (OTU) family have evolved distinct mechanisms to achieve linkage selectivity.

  • New tools such as the application of chemical biology techniques to achieve site-specific ubiquitylation, the generation of ubiquitin polymers of defined linkage types, linkage-specific antibodies and ubiquitin sensors will advance the field of ubiquitin research.

  • Little is known about the biological roles of atypical ubiquitylation events. The reported physiological roles highlight the fact that the differently linked polyubiquitin chains are independent post-translational modifications.

Abstract

Ubiquitylation is one of the most abundant and versatile post-translational modifications (PTMs) in cells. Its versatility arises from the ability of ubiquitin to form eight structurally and functionally distinct polymers, in which ubiquitin moieties are linked via one of seven Lys residues or the amino terminus. Whereas the roles of Lys48- and Lys63-linked polyubiquitin in protein degradation and cellular signalling are well characterized, the functions of the remaining six 'atypical' ubiquitin chain types (linked via Lys6, Lys11, Lys27, Lys29, Lys33 and Met1) are less well defined. Recent developments provide insights into the mechanisms of ubiquitin chain assembly, recognition and hydrolysis and allow detailed analysis of the functions of atypical ubiquitin chains. The importance of Lys11 linkages and Met1 linkages in cell cycle regulation and nuclear factor-κB activation, respectively, highlight that the different ubiquitin chain types should be considered as functionally independent PTMs.

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Figure 1: Forms of ubiquitylation.
Figure 2: Mechanisms of ubiquitin chain assembly.
Figure 3: Mechanisms of ubiquitin chain hydrolysis.
Figure 4: Physiological roles of atypical ubiquitylation.
Figure 5: NF-κB signalling regulated by atypical linkages.

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References

  1. Ikeda, F. & Dikic, I. Atypical ubiquitin chains: new molecular signals. Protein modifications: beyond the usual suspects review series. EMBO Rep. 9, 536–542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Behrends, C. & Harper, J. W. Constructing and decoding unconventional ubiquitin chains. Nature Struct. Mol. Biol. 18, 520–528 (2011).

    Article  CAS  Google Scholar 

  3. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Dye, B. T. & Schulman, B. A. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 36, 131–150 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Winget, J. M. & Mayor, T. The diversity of ubiquitin recognition: hot spots and varied specificity. Mol. Cell 38, 627–635 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Reyes-Turcu, F. E., Ventii, K. H. & Wilkinson, K. D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Komander, D., Clague, M. J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nature Rev. Mol. Cell Biol. 10, 550–563 (2009).

    Article  CAS  Google Scholar 

  9. Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Virdee, S., Ye, Y., Nguyen, D. P., Komander, D. & Chin, J. W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nature Chem. Biol. 6, 750–757 (2010).

    Article  CAS  Google Scholar 

  11. Bremm, A. & Komander, D. Emerging roles for Lys11-linked polyubiquitin in cellular regulation. Trends Biochem. Sci. 36, 355–363 (2011).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  PubMed  Google Scholar 

  14. Cook, W. J., Jeffrey, L. C., Carson, M., Chen, Z. & Pickart, C. M. Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). J. Biol. Chem. 267, 16467–16471 (1992).

    CAS  PubMed  Google Scholar 

  15. Eddins, M. J., Varadan, R., Fushman, D., Pickart, C. M. & Wolberger, C. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH. J. Mol. Biol. 367, 204–211 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Datta, A. B., Hura, G. L. & Wolberger, C. The structure and conformation of Lys63-linked tetraubiquitin. J. Mol. Biol. 392, 1117–1124 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Varadan, R., Walker, O., Pickart, C. & Fushman, D. Structural properties of polyubiquitin chains in solution. J. Mol. Biol. 324, 637–647 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Rohaim, A., Kawasaki, M., Kato, R., Dikic, I. & Wakatsuki, S. Structure of a compact conformation of linear diubiquitin. Acta Crystallogr. D Biol. Crystallogr. 68, 102–108 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Sloper-Mould, K. E., Jemc, J. C., Pickart, C. M. & Hicke, L. Distinct functional surface regions on ubiquitin. J. Biol. Chem. 276, 30483–30489 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 1576–1583 (1989).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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). Discovery of Lys63-linked polyubiquitin and first implication of this chain type in DNA repair.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hofmann, R. M. & Pickart, C. M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Dammer, E. B. et al. Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease. J. Biol. Chem. 286, 10457–10465 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ziv, I. et al. A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis. Mol. Cell Proteomics 10, M111.009753 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Wagner, S. A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell Proteomics 10, M11.013284 (2011).

    Article  CAS  Google Scholar 

  30. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011). References 23 and 27–30 use mass spectrometry to study the abundance of differently linked ubiquitin polymers in cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bennett, E. J. et al. Global changes to the ubiquitin system in Huntington's disease. Nature 448, 704–708 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Ye, Y. & Rape, M. Building ubiquitin chains: E2 enzymes at work. Nature Rev. Mol. Cell Biol. 10, 755–764 (2009).

    Article  CAS  Google Scholar 

  33. Wenzel, D. M., Stoll, K. E. & Klevit, R. E. E2s: structurally economical and functionally replete. Biochem. J. 433, 31–42 (2010).

    Article  CAS  Google Scholar 

  34. Wenzel, D. M., Lissounov, A., Brzovic, P. S. & Klevit, R. E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011). Elegant biochemical experiments demonstrate that some E2s discharge their ubiquitin exclusively to Cys residues and reveal a thioester intermediate in RBR E3 ligases.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Alpi, A. F., Pace, P. E., Babu, M. M. & Patel, K. J. Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI. Mol. Cell 32, 767–777 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Longerich, S., San Filippo, J., Liu, D. & Sung, P. FANCI binds branched DNA and is monoubiquitinated by UBE2T–FANCL. J. Biol. Chem. 284, 23182–23186 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sato, K., Toda, K., Ishiai, M., Takata, M. & Kurumizaka, H. DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI. Nucleic Acids Res. 40, 4553–4561.

  38. Scaglione, K. M. et al. Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP. Mol. Cell 43, 599–612 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Williamson, A. et al. Regulation of ubiquitin chain initiation to control the timing of substrate degradation. Mol. Cell 42, 744–757 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jin, L., Williamson, A., Banerjee, S., Philipp, I. & Rape, M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133, 653–665 (2008). First report showing that the mammalian APC/C assembles Lys11 linkages, which requires a novel surface on ubiquitin termed the TEK box.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Windheim, M., Peggie, M. & Cohen, P. Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology. Biochem. J. 409, 723–729 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Duncan, L. M. et al. Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules. EMBO J. 25, 1635–1645 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Rodrigo-Brenni, M. C. & Morgan, D. O. Sequential E2s drive polyubiquitin chain assembly on APC targets. Cell 130, 127–139 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Moraes, T. F. et al. Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2–hUbc13. Nature Struct. Biol. 8, 669–673 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Wickliffe, K. E., Lorenz, S., Wemmer, D. E., Kuriyan, J. & Rape, M. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781 (2011). Shows that the Lys11-specific UBE2S E2 enzyme uses substrate-assisted catalysis to assemble ubiquitin polymers processively.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M. & Wolberger, C. Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nature Struct. Mol. Biol. 13, 915–920 (2006). First crystal structure of a ubiquitin ligation machinery poised for catalysis, explaining the Lys63-specificity of the UBE2N–UBE2V2 complex.

    Article  CAS  Google Scholar 

  48. David, Y., Ziv, T., Admon, A. & Navon, A. The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines. J. Biol. Chem. 285, 8595–8604 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Rotin, D. & Kumar, S. Physiological functions of the HECT family of ubiquitin ligases. Nature Rev. Mol. Cell Biol. 10, 398–409 (2009).

    Article  CAS  Google Scholar 

  50. Kim, H. C. & Huibregtse, J. M. Polyubiquitination by HECT E3s and the determinants of chain type specificity. Mol. Cell. Biol. 29, 3307–3318 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. You, J. & Pickart, C. M. A. HECT domain E3 enzyme assembles novel polyubiquitin chains. J. Biol. Chem. 276, 19871–19878 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Peng, D. J. et al. Noncanonical K27-linked polyubiquitination of TIEG1 regulates Foxp3 expression and tumor growth. J. Immunol. 186, 5638–5647 (2011).

    Article  CAS  PubMed  Google Scholar 

  53. Huang, H. et al. K33-linked polyubiquitination of T cell receptor-ζ regulates proteolysis-independent T cell signaling. Immunity 33, 60–70 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Chastagner, P., Israël, A. & Brou, C. Itch/AIP4 mediates deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep. 7, 1147–1153 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Huang, L. et al. Structure of an E6AP–UbcH7 complex: insights into ubiquitination by the E2–E3 enzyme cascade. Science 286, 1321–1326 (1999).

    Article  CAS  PubMed  Google Scholar 

  56. Kamadurai, H. B. et al. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin–HECTNEDD4L complex. Mol. Cell 36, 1095–1102 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. French, M. E., Kretzmann, B. R. & Hicke, L. Regulation of the RSP5 uiquitin ligase by an intrinsic ubiquitin-binding site. J. Biol. Chem. 284, 12071–12079 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Ogunjimi, A. A. et al. The ubiquitin binding region of the Smurf HECT domain facilitates polyubiquitylation and binding of ubiquitylated substrates. J. Biol. Chem. 285, 6308–6315 (2010).

    Article  CAS  PubMed  Google Scholar 

  59. Kim, H. C., Steffen, A. M., Oldham, M. L., Chen, J. & Huibregtse, J. M. Structure and function of a HECT domain ubiquitin-binding site. EMBO Rep. 12, 334–341 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Maspero, E. et al. Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation. EMBO Rep. 12, 342–349 (2011). Reveals, together with references 56 and 59, two binding sites for ubiquitin on NEDD4 family HECT domains.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Eisenhaber, B., Chumak, N., Eisenhaber, F. & Hauser, M.-T. The ring between ring fingers (RBR) protein family. Genome Biol. 8, 209 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Youle, R. J. & Narendra, D. P. Mechanisms of mitophagy. Nature Rev. Mol. Cell Biol. 12, 9–14 (2011).

    Article  CAS  Google Scholar 

  63. Tokunaga, F. & Iwai, K. LUBAC, a novel ubiquitin ligase for linear ubiquitination, is crucial for inflammation and immune responses. Microbes Infect. 14, 563–572 (2012).

    Article  CAS  PubMed  Google Scholar 

  64. Kirisako, T. et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 25, 4877–4887 (2006). Identification and biochemical characterization of LUBAC, the linear ubiquitin chain assembly complex.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Deshaies, R. J. & Joazeiro, C. A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    Article  CAS  PubMed  Google Scholar 

  66. Hatakeyama, S. TRIM proteins and cancer. Nature Rev. Cancer 11, 792–804 (2011).

    Article  CAS  Google Scholar 

  67. Clague, M. J., Coulson, J. M. & Urbe, S. Cellular functions of the DUBs. J. Cell Sci. 125, 277–286 (2012).

    Article  CAS  PubMed  Google Scholar 

  68. Shanmugham, A. & Ovaa, H. DUBs and disease: activity assays for inhibitor development. 11, 688–696 (2008).

  69. Cohen, P. & Tcherpakov, M. Will the ubiquitin system furnish as many drug targets as protein kinases? Cell 143, 686–693 (2010).

    Article  CAS  PubMed  Google Scholar 

  70. Faesen, A. C. et al. The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types. 18, 1550–1561 (2011).

  71. Sato, Y. et al. Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains. Nature 455, 358–362 (2008).

    Article  CAS  PubMed  Google Scholar 

  72. Cooper, E. M. et al. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1. EMBO J. 28, 621–631 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Popp, M. W., Artavanis-Tsakonas, K. & Ploegh, H. L. Substrate filtering by the active site crossover loop in UCHL3 revealed by sortagging and gain-of-function mutations. J. Biol. Chem. 284, 3593–3602 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Licchesi, J. D. F. et al. An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains. Nature Struct. Mol. Biol. 19, 62–71 (2011). Reveals the molecular basis for atypical chain cleavage by TRABID and identifies sites of accumulation of Lys29 and Lys33 chains in cells.

    Article  CAS  Google Scholar 

  75. Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009).

    Article  CAS  PubMed  Google Scholar 

  76. Tran, H., Hamada, F., Schwarz-Romond, T. & Bienz, M. Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains. Genes Dev. 22, 528–542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Wiener, R., Zhang, X., Wang, T. & Wolberger, C. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483, 618–622 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Juang, Y.-C. et al. OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function. Mol. Cell 45, 384–397 (2012). References 77 and 78 reveal the mechanisms of Lys48 specificity of OTUB and its mechanism to inhibit E2 enzymes and hence ubiquitin chain assembly.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Boname, J. M. et al. Efficient internalization of MHC I requires lysine-11 and lysine-63 mixed linkage polyubiquitin chains. Traffic 11, 210–220 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Wickliffe, K., Williamson, A., Jin, L. & Rape, M. The multiple layers of ubiquitin-dependent cell cycle control. Chem. Rev. 109, 1537–1548 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Wu-Baer, F., Lagrazon, K., Yuan, W. & Baer, R. The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin. J. Biol. Chem. 278, 34743–34746 (2003).

    Article  CAS  PubMed  Google Scholar 

  83. Nishikawa, H. et al. Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase. J. Biol. Chem. 279, 3916–3924 (2004).

    Article  CAS  PubMed  Google Scholar 

  84. Morris, J. R. & Solomon, E. BRCA1:BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair. Hum. Mol. Genet. 13, 807–817 (2004).

    Article  CAS  PubMed  Google Scholar 

  85. Sato, K. Nucleophosmin/B23 is a candidate substrate for the BRCA1–BARD1 ubiquitin ligase. J. Biol. Chem. 279, 30919–30922 (2004).

    Article  CAS  PubMed  Google Scholar 

  86. Yu, X. BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP. Genes Dev. 20, 1721–1726 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Wu, W. et al. BRCA1 ubiquitinates RPB8 in response to DNA damage. Cancer Res. 67, 951–958 (2007).

    Article  CAS  PubMed  Google Scholar 

  88. Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Wu-Baer, F., Ludwig, T. & Baer, R. The UBXN1 protein associates with autoubiquitinated forms of the BRCA1tumor suppressor and inhibits its enzymatic function. Mol. Cell. Biol. 30, 2787–2798 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Wickliffe, K. E., Williamson, A., Meyer, H.-J., Kelly, A. & Rape, M. K11-linked ubiquitin chains as novel regulators of cell division. Trends Cell Biol. 21, 656–663 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Williamson, A. et al. Identification of a physiological E2 module for the human anaphase-promoting complex. Proc. Natl Acad. Sci. USA 106, 18213–18218 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Pareja, F. et al. Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression. Oncogene 19 Dec 2011 (doi:10.1038/onc.2011.587).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  93. Goto, E. et al. Contribution of lysine 11-linked ubiquitination to MIR2-mediated major histocompatibility complex class I internalization. J. Biol. Chem. 285, 35311–35319 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Glauser, L., Sonnay, S., Stafa, K. & Moore, D. J. Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1. J. Neurochem. 118, 636–645 (2011).

    Article  CAS  PubMed  Google Scholar 

  95. Geisler, S. et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nature Cell Biol. 12, 119–131 (2010). Shows that parkin assembles Lys27 linkages on itself and on its substrates.

    Article  CAS  PubMed  Google Scholar 

  96. Durcan, T. M. et al. The Machado–Joseph disease-associated mutant form of ataxin-3 regulates parkin ubiquitination and stability. Hum. Mol. Genet. 20, 141–154 (2010).

    Article  CAS  PubMed  Google Scholar 

  97. Al-Hakim, A. K. et al. Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains. Biochem. J. 411, 249–260 (2008).

    Article  CAS  PubMed  Google Scholar 

  98. Hay-Koren, A., Caspi, M., Zilberberg, A. & Rosin-Arbesfeld, R. The EDD E3 ubiquitin ligase ubiquitinates and up-regulates beta-catenin. Mol. Biol. Cell 22, 399–411 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Ohshima, R. et al. Putative tumor suppressor EDD interacts with and up-regulates APC. Genes Cells 12, 1339–1345 (2007).

    Article  CAS  PubMed  Google Scholar 

  100. Metzger, M. B. & Weissman, A. M. Working on a chain: E3s ganging up for ubiquitylation. Nature Cell Biol. 12, 1124–1126 (2010).

    Article  CAS  PubMed  Google Scholar 

  101. Hwang, C.-S., Shemorry, A., Auerbach, D. & Varshavsky, A. The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases. Nature Cell Biol. 12, 1177–1185 (2010).

    Article  CAS  PubMed  Google Scholar 

  102. Koegl, M. et al. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644 (1999).

    Article  CAS  PubMed  Google Scholar 

  103. Locksley, R. M., Killeen, N. & Lenardo, M. J. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104, 487–501 (2001).

    Article  CAS  PubMed  Google Scholar 

  104. Wertz, I. E. & Dixit, V. M. Regulation of death receptor signaling by the ubiquitin system. Cell Death Differ. 17, 14–24 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Yamamoto, M. et al. Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling. Nature Immunol. 7, 962–970 (2006).

    Article  CAS  Google Scholar 

  108. Zotti, T. et al. TRAF7 protein promotes Lys-29-linked polyubiquitination of IκB kinase (IKKγ)/NF-κB essential modulator (NEMO) and p65/RelA protein and represses NF-κB activation. J. Biol. Chem. 286, 22924–22933 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  111. Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011). References 109, 110 and 111 identify sharpin as a component of LUBAC and provide genetic evidence for the importance of linear chains in TNFα signalling.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. 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). Identifies LUBAC as an endogenous component of the TNFR complex.

    Article  CAS  PubMed  Google Scholar 

  113. Seymour, R. E. et al. Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis. Genes Immun. 8, 416–421 (2007).

    Article  CAS  PubMed  Google Scholar 

  114. Kanayama, A. et al. TAB2 and TAB3 activate the NF-κB pathway through binding to polyubiquitin chains. Mol. Cell 15, 535–548 (2004).

    Article  CAS  PubMed  Google Scholar 

  115. Bloor, S. et al. Signal processing by its coil zipper domain activates IKKγ. Proc. Natl Acad. Sci. USA 105, 1279–1284 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Ea, C.-K., Deng, L., Xia, Z.-P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006).

    Article  CAS  PubMed  Google Scholar 

  117. Alam, S. L. et al. Ubiquitin interactions of NZF zinc fingers. EMBO J. 23, 1411–1421 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  119. Sato, Y., Yoshikawa, A., Yamashita, M., Yamagata, A. & Fukai, S. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by NZF domains of TAB2 and TAB3. EMBO J. 28, 3903–3909 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Sato, Y. et al. Specific recognition of linear ubiquitin chains by the Npl4 zinc finger (NZF) domain of the HOIL-1L subunit of the linear ubiquitin chain assembly complex. Proc. Natl Acad. Sci. USA 108, 20520–20525 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Schröfelbauer, B., Polley, S., Behar, M., Ghosh, G. & Hoffmann, A. NEMO ensures signaling specificity of the pleiotropic IKKβ by directing its kinase activity toward IκBα. Mol. Cell 23 May 2012 (doi:10.1016/j.molcel.2012.04.020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  123. Yoshikawa, A. et al. Crystal structure of the NEMO ubiquitin-binding domain in complex with Lys 63-linked di-ubiquitin. FEBS Lett. 583, 3317–3322 (2009).

    Article  CAS  PubMed  Google Scholar 

  124. Hadian, K. et al. NF-κB essential modulator (NEMO) interaction with linear and Lys-63 ubiquitin chains contributes to NF-κB activation. J. Biol. Chem. 286, 26107–26117 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Cordier, F. et al. The zinc finger of NEMO is a functional ubiquitin-binding domain. J. Biol. Chem. 284, 2902–2907 (2009).

    Article  CAS  PubMed  Google Scholar 

  126. Laplantine, E. et al. NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain. EMBO J. 28, 2885–2895 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Ivins, F. J. et al. NEMO oligomerization and its ubiquitin-binding properties. Biochem. J. 421, 243–251 (2009).

    Article  CAS  PubMed  Google Scholar 

  128. Lo, Y.-C. et al. Structural basis for recognition of diubiquitins by NEMO. Mol. Cell 33, 602–615 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nature Cell Biol. 11, 123–132 (2009).

    Article  CAS  PubMed  Google Scholar 

  130. Niu, J., Shi, Y., Iwai, K. & Wu, Z.-H. LUBAC regulates NF-κB activation upon genotoxic stress by promoting linear ubiquitination of NEMO. EMBO J. 30, 3741–3753 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Arimoto, K. I. et al. Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense. Proc. Natl Acad. Sci. USA 107, 15856–15861 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Ashida, H. et al. A bacterial E3 ubiquitin ligase IpaH9.8 targets NEMO/IKKγ to dampen the host NF-κB-mediated inflammatory response. Nature Cell Biol. 12, 66–73 (2009).

    Article  CAS  PubMed  Google Scholar 

  133. Jura, N., Scotto-Lavino, E., Sobczyk, A. & Bar-Sagi, D. Differential modification of Ras proteins by ubiquitination. Mol. Cell 21, 679–687 (2006).

    Article  CAS  PubMed  Google Scholar 

  134. Pierce, N. W., Kleiger, G., Shan, S.-O. & Deshaies, R. J. Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462, 615–619 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Thrower, J. S., Hoffman, L., Rechsteiner, M. & Pickart, C. M. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Lasker, K. et al. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc. Natl Acad. Sci. USA 109, 1380–1387 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Zhang, D. et al. Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor. Mol. Cell 36, 1018–1033 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Schaefer, J. B. & Morgan, D. O. Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes. J. Biol. Chem. 286, 45186–45196 (2011). Demonstrates how ubiquitin chain length can alter DUB activity.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Yin, L., Krantz, B., Russell, N. S., Deshpande, S. & Wilkinson, K. D. Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation. Biochemistry 39, 10001–10010 (2000).

    Article  CAS  PubMed  Google Scholar 

  141. Weikart, N. D. & Mootz, H. D. Generation of site-specific and enzymatically stable conjugates of recombinant proteins with ubiquitin-like modifiers by the CuI-catalyzed azide–alkyne cycloaddition. Chembiochem 11, 774–777 (2010).

    Article  CAS  PubMed  Google Scholar 

  142. Chatterjee, C., Mcginty, R. K., Fierz, B. & Muir, T. W. Disulfide-directed histone ubiquitylation reveals plasticity in hDot1L activation. Nature Chem. Biol. 6, 267–269 (2010).

    Article  CAS  Google Scholar 

  143. Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew. Chem. Int. Ed. 48, 9184–9187 (2009).

    Article  CAS  Google Scholar 

  144. Mcginty, R. K., Kim, J., Chatterjee, C., Roeder, R. G. & Muir, T. W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Chatterjee, C., Mcginty, R. K., Pellois, J.-P. & Muir, T. W. Auxiliary-mediated site-specific peptide ubiquitylation. Angew. Chem. Int. Ed. 46, 2814–2818 (2007).

    Article  CAS  Google Scholar 

  146. Yang, R., Pasunooti, K. K., Li, F., Liu, X.-W. & Liu, C.-F. Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine. Chem. Commun. 46, 7199–7201 (2010).

    Article  CAS  Google Scholar 

  147. Kumar, K. S. A., Spasser, L., Erlich, L. A., Bavikar, S. N. & Brik, A. Total chemical synthesis of di-ubiquitin chains. Angew. Chem. Int. Ed. 49, 9126–9131 (2010).

    Article  CAS  Google Scholar 

  148. Oualid, El, F. et al. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Ed. 49, 10149–10153 (2010).

    Article  CAS  Google Scholar 

  149. Ajish Kumar, K. S., Haj-Yahya, M., Olschewski, D., Lashuel, H. A. & Brik, A. Highly efficient and chemoselective peptide ubiquitylation. Angew. Chem. Int. Ed. 48, 8090–8094 (2009).

    Article  CAS  Google Scholar 

  150. Virdee, S. et al. Traceless and site-specific ubiquitination of recombinant proteins. J. Am. Chem. Soc. 133, 10708–10711 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Xu, P. & Peng, J. Dissecting the ubiquitin pathway by mass spectrometry. Biochim. Biophys. Acta 1764, 1940–1947 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Nielsen, M. L. et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 5, 459–460 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  154. Matsumoto, M. L. et al. Engineering and structural characterization of a linear-polyubiquitin-specific antibody. J. Mol. Biol. 418, 134–144 (2012).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Trempe, J.-F. et al. Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain. EMBO J. 24, 3178–3189 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. Wiggins, C. M. et al. BIMEL, an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation. J. Cell Sci. 124, 969–977 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. Keren-Kaplan, T. et al. Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria. EMBO J. 31, 378–390 (2012).

    Article  CAS  PubMed  Google Scholar 

  160. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin refined at 1.8Å resolution. J. Mol. Biol. 194, 531–544 (1987).

    Article  CAS  PubMed  Google Scholar 

  161. Xia, Z.-P. et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 461, 114–119 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Kirkpatrick, D. S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nature Cell Biol. 8, 700–710 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank their colleagues at the Medical Research Council (MRC) Laboratory of Molecular Biology for discussions and contributions to this Review, and apologize to authors whose work could not be cited due to space restrictions. Y.K. is supported by Marie Curie and EMBO long-term fellowships. D.K. is supported by the MRC (U105192732) and the EMBO Young Investigator Programme.

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    Yogesh Kulathu & David Komander

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David Komander is a consultant for Mission Therapeutics. Yogesh Kulathu declares no competing financial interests.

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Glossary

APC/C

(Anaphase promoting complex; also known as the cyclosome). A multi-subunit E3 ligase that targets proteins for proteasomal degradation. Substrates include many cell cycle proteins that need to be degraded during cell cycle progression.

RING domain

Domain of approximately 70 amino acids with conserved Cys and His residues that coordinate two zinc ions. U-box domains share the RING fold but do not bind zinc.

TEK box

A structural motif on the surface of ubiquitin that consists of Lys6, Lys11, Thr12, Thr14 and Glu34 and is required for UBE2C- and UBE2S-mediated assembly of Lys11-linked ubiquitin chains.

Coiled coil

Structural motifs that are formed by amino acids arranged in a heptad repeat in which positions 1 and 4 of the repeat are hydrophobic amino acids (Ile, Leu or Val). These domains are present in many proteins and confer them with dimerization or oligomerization propensities.

Cysteine proteases

Enzyme that use a nucleophilic Cys residue in the catalytic centre. The catalytic triad contains an adjacent His residue to deprotonate the Cys thiol and a polar residue to correctly position the His.

Zinc metalloproteases

Proteases that have a zinc-binding motif in the catalytic centre to coordinate zinc. During catalysis the zinc promotes nucleophilic attack.

p97

A hexameric AAA+ ATPase that exerts mechanical force on ubiquitylated cargo to, for example, dislocate proteins from membranes or disassemble complexes.

Mitophagy

The selective degradation of mitochondria by autophagy.

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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). https://doi.org/10.1038/nrm3394

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