Abstract
Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
£14.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
£139.00 per year
only £11.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Seet, B. T., Dikic, I., Zhou, M. M. & Pawson, T. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 7, 473–483 (2006).
Zheng, N. & Shabek, N. Ubiquitin ligases: structure, function, and regulation. Annu. Rev. Biochem. 86, 129–157 (2017).
Harper, J. W. & Schulman, B. A. Cullin-RING ubiquitin ligase regulatory circuits: a quarter century beyond the F-Box hypothesis. Annu. Rev. Biochem. 90, 403–429 (2021).
Wang, Y., Argiles-Castillo, D., Kane, E. I., Zhou, A. & Spratt, D. E. HECT E3 ubiquitin ligases-emerging insights into their biological roles and disease relevance. J. Cell Sci. 133, jcs258087 (2020).
van Wijk, S. J. L., Fiškin, E. & Dikic, I. Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain-specific sensors. Nat. Protoc. 8, 1449–1458 (2013).
Sims, J. J. et al. Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Nat. Methods 9, 303–309 (2012).
Mattiroli, F. & Sixma, T. K. Lysine-targeting specificity in ubiquitin and ubiquitin-like modification pathways. Nat. Struct. Mol. Biol. 21, 308–316 (2014).
Ciechanover, A. & Ben-Saadon, R. N-terminal ubiquitination: more protein substrates join in. Trends Cell Biol. 14, 103–106 (2004).
Tatham, M. H., Plechanovová, A., Jaffray, E. G., Salmen, H. & Hay, R. T. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini. Biochem. J. 453, 137–145 (2013).
Dittmar, G. & Winklhofer, K. F. Linear ubiquitin chains: cellular functions and strategies for detection and quantification. Front. Chem. 7, 915 (2019).
Nakatsuka, T., Sasaki, T. & Kaiser, E. T. Peptide segment synthesis catalyzed by the semisynthetic enzyme thiolsubtilisin. J. Am. Chem. Soc. 109, 3808–3810 (1987).
Chu, S.-H. & Mautner, H. G. Analogs of neuroeffectors. V. Neighboring-group effects in the reactions of esters, thiolesters, and selenolesters. The hydrolysis and aminolysis of benzoylcholine, benzoylthiolcholine, benzoylselenolcholine, and of their dimethylamino analogs. J. Org. Chem. 31, 308–312 (1966).
Wang, X. et al. Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3. J. Cell Biol. 177, 613–624 (2007).
Cadwell, K. & Coscoy, L. Biochemistry: ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Science 309, 127–130 (2005).
Shimizu, Y., Okuda-Shimizu, Y. & Hendershot, L. M. Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids. Mol. Cell 40, 917–926 (2010).
Ishikura, S., Weissman, A. M. & Bonifacino, J. S. Serine residues in the cytosolic tail of the T-cell antigen receptor alpha-chain mediate ubiquitination and endoplasmic reticulum-associated degradation of the unassembled protein. J. Biol. Chem. 285, 23916–23924 (2010).
Carvalho, A. F. et al. Ubiquitination of mammalian Pex5p, the peroxisomal import receptor. J. Biol. Chem. 282, 31267–31272 (2007).
Williams, C., van den Berg, M., Sprenger, R. R. & Distel, B. A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p. J. Biol. Chem. 282, 22534–22543 (2007).
Léon, S. & Subramani, S. A conserved cysteine residue of Pichia pastoris Pex20p is essential for its recycling from the peroxisome to the cytosol. J. Biol. Chem. 282, 7424–7430 (2007).
Vosper, J. M. D. et al. Ubiquitylation on canonical and non-canonical sites targets the transcription factor neurogenin for ubiquitin-mediated proteolysis. J. Biol. Chem. 284, 15458–15468 (2009).
Pao, K. C. et al. Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity. Nature 556, 381–385 (2018). By coupling activity-based probe technology with mass spectrometry, this study discovers that MYCBP2 not only uses a previously unknown RCR mechanism but also targets Thr instead of Lys for ubiquitylation.
Pao, K. C. et al. Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation. Nat. Chem. Biol. 12, 324–331 (2016).
Mabbitt, P. D. et al. Structural basis for RING-Cys-relay E3 ligase activity and its role in axon integrity. Nat. Chem. Biol. 16, 1227–1236 (2020).
Virdee, S. An atypical ubiquitin ligase at the heart of neural development and programmed axon degeneration. Neural Regen. Res. 17, 2347–2350 (2022).
Kelsall, I. R., Zhang, J., Knebel, A., Arthur, S. J. C. & Cohen, P. The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells. Proc. Natl Acad. Sci. USA 116, 13293–13298 (2019).
Kelsall, I. R. et al. HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation. EMBO J. 41, e109700 (2022). Beginning with the observation that HOIL1 deficiency causes toxic polyglucosan deposits in human tissues, this study shows that HOIL1 is able to ubiquitylate carbohydrates and is allosterically activated by Met1-linked or Lys63-linked oligomeric ubiquitin.
Yang, C. S. et al. Ubiquitin modification by the E3 ligase/ADP-ribosyltransferase Dtx3L/Parp9. Mol. Cell 66, 503–516 (2017).
Chatrin, C. et al. Structural insights into ADP-ribosylation of ubiquitin by Deltex family E3 ubiquitin ligases. Sci. Adv. 6, eabc0418 (2020).
Ahmed, S. F. et al. DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination. Sci. Adv. 6, eabc0629 (2020). Yang et al. (2017), Chatrin et al. (2020) and Ahmed et al. (2020) show that ubiquitin can be ADP-ribosylated on Gly76 by the histone E3 ligase DELTEX and illustrate how PTM of ubiquitin can contribute to protein regulation during DNA repair.
Li, Y. & Reverter, D. Molecular mechanisms of dubs regulation in signaling and disease. Int. J. Mol. Sci. 22, 986 (2021).
Cho, J., Park, J., Kim, E. E. & Song, E. J. Assay systems for profiling deubiquitinating activity. Int. J. Mol. Sci. 21, 5638 (2020).
de Cesare, V. et al. Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases. Proc. Natl Acad. Sci. USA 118, e2006947118 (2021).
Dang, L. C., Melandri, F. D. & Stein, R. L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry 37, 1868–1879 (1998).
Maculins, T., Fiskin, E., Bhogaraju, S. & Dikic, I. Bacteria-host relationship: ubiquitin ligases as weapons of invasion. Cell Res. 26, 499–510 (2016).
Lange, S. M., Armstrong, L. A. & Kulathu, Y. Deubiquitinases: from mechanisms to their inhibition by small molecules. Mol. Cell 82, 15–29 (2022).
Qiu, J. et al. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature 533, 120–124 (2016).
Bhogaraju, S. et al. Phosphoribosylation of ubiquitin promotes serine ubiquitination and impairs conventional ubiquitination. Cell 167, 1636–1649 (2016).
Akturk, A. et al. Mechanism of phosphoribosyl-ubiquitination mediated by a single legionella effector. Nature 557, 729–745 (2018).
Kalayil, S. et al. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination. Nature 557, 734–738 (2018).
Shin, D. et al. Regulation of phosphoribosyl-linked serine ubiquitination by deubiquitinases DupA and DupB. Mol. Cell 77, 164–179 (2020).
Liu, Y. et al. Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection. Cell Death Differ. 10, 2957–2969 (2021).
Kotewicz, K. M. et al. A single legionella effector catalyzes a multistep ubiquitination pathway to rearrange tubular endoplasmic reticulum for replication. Cell Host Microbe 21, 169–181 (2017). Qiu et al. (2016), Bhogaraju et al. (2016) and Kotewicz et al. (2017) describe a novel type of ubiquityltion, in L. pneumophila, in which SidE effectors attach ubiquitin phosphoribosylated at Arg42 to Ser residues of substrates.
de Leon, J. A. et al. Positive and negative regulation of the master metabolic regulator mTORC1 by two families of legionella pneumophila effectors. Cell Rep. 21, 2031–2038 (2017).
Wan, M. et al. Deubiquitination of phosphoribosyl-ubiquitin conjugates by phosphodiesterase-domain-containing Legionella effectors. Proc. Natl Acad. Sci. USA 116, 23518–23526 (2019).
Bhogaraju, S. et al. Inhibition of bacterial ubiquitin ligases by SidJ–calmodulin catalysed glutamylation. Nature 572, 382–386 (2019).
Black, M. H. et al. Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases. Science 364, 787–792 (2019).
Gan, N. et al. Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase. Nature 572, 387–391 (2019).
Taieb, F., Nougayrède, J., Philippe & Oswald, E. Cycle inhibiting factors (Cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells. Toxins 3, 356–368 (2011).
Cui, J. et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010).
Jubelin, G. et al. Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways. PLoS Pathog. 6, e1001128 (2010).
Morikawa, H. et al. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of cullin1. Biochem. Biophys. Res. Commun. 401, 268–274 (2010).
Yu, C. et al. Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8. Nat. Commun. 6, 10053 (2015).
Baek, K. et al. NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly. Nature 578, 461–466 (2020).
Horn-Ghetko, D. et al. Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly. Nature 590, 671–676 (2021).
Kostrhon, S. et al. CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation. Nat. Chem. Biol. 17, 1075–1083 (2021).
Valleau, D. et al. Discovery of ubiquitin deamidases in the pathogenic arsenal of Legionella pneumophila. Cell Rep. 23, 568–583 (2018).
Gan, N., Nakayasu, E. S., Hollenbeck, P. J. & Luo, Z. Q. Legionella pneumophila inhibits immune signalling via MavC-mediated transglutaminase-induced ubiquitination of UBE2N. Nat. Microbiol. 4, 134–143 (2019).
Mu, Y. et al. Structural insights into the mechanism and inhibition of transglutaminase-induced ubiquitination by the Legionella effector MavC. Nat. Commun. 11, 1774 (2020).
Wang, Y. et al. Insights into catalysis and regulation of non-canonical ubiquitination and deubiquitination by bacterial deamidase effectors. Nat. Commun. 11, 2751 (2020).
Guan, H. et al. Molecular basis of ubiquitination catalyzed by the bacterial transglutaminase MavC. Adv. Sci. 7, 2000871 (2020).
Deng, L. 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).
Gan, N. et al. Legionella pneumophila regulates the activity of UBE2N by deamidase-mediated deubiquitination. EMBO J. 39, e102806 (2020).
Otten, E. G. et al. Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection. Nature 594, 111–116 (2021). The host’s E3 ligase RNF213 targets a specific lipid of the bacterial outer membrane for attachment of ubiquitin, which in turn serves for further chain elongation and recruitment of antibacterial effector proteins.
Noad, J. et al. LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-κB. Nat. Microbiol. 2, 17063 (2017).
Takeda, M. et al. Moyamoya disease patient mutations in the RING domain of RNF213 reduce its ubiquitin ligase activity and enhance NFκB activation and apoptosis in an AAA+ domain-dependent manner. Biochem. Biophys. Res. Commun. 525, 668–674 (2020).
Wang, C. et al. RNF213 gene silencing upregulates transforming growth factor β1 expression in bone marrow-derived mesenchymal stem cells and is involved in the onset of Moyamoya disease. Exp. Ther. Med. 22, 1024 (2021).
Liu, W. et al. Identification of RNF213 as a susceptibility gene for moyamoya disease and its possible role in vascular development. PLoS ONE 6, e22542 (2011).
Thery, F. et al. Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity. Nat. Commun. 12, 5772 (2021).
Ahel, J. et al. Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism. Elife 9, e56185 (2020).
Ahel, J. et al. E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP. Preprint at bioRxiv https://doi.org/10.1101/2021.05.10.443411 (2021).
Der, S. D., Zhou, A., Williams, B. R. G. & Silverman, R. H. Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays. Proc. Natl Acad. Sci. USA 95, 15623–15628 (1998).
Loeb, K. R. & Haas, A. L. The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. J. Biol. Chem. 267, 7806–7813 (1992).
Minakawa, M., Sone, T., Takeuchi, T. & Yokosawa, H. Regulation of the nuclear factor (NF)-κB pathway by ISGylation. Biol. Pharm. Bull. 31, 2223–2227 (2008).
Durfee, L. A., Lyon, N., Seo, K. & Huibregtse, J. M. The ISG15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of ISG15. Mol. Cell 38, 722–732 (2010).
Werneke, S. W. et al. ISG15 is critical in the control of chikungunya virus infection independent of UbE1l mediated conjugation. PLoS Pathog. 7, e1002322 (2011).
D’Cunha, J., Knight, E., Haas, A. L., Truitt, R. L. & Borden, E. C. Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc. Natl Acad. Sci. USA 93, 211–215 (1996).
Swaim, C. D. et al. Modulation of extracellular ISG15 signaling by pathogens and viral effector proteins. Cell Rep. 31, 107772 (2020).
Yuan, W. & Krug, R. M. Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J. 20, 362–371 (2001).
Yuan, W., Aramini, J. M., Montelione, G. T. & Krug, R. M. Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus. Virology 304, 291–301 (2002).
Zhao, C. et al. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins. Nat. Commun. 7, 12754 (2016).
Mielech, A. M., Kilianski, A., Baez-Santos, Y. M., Mesecar, A. D. & Baker, S. C. MERS-CoV papain-like protease has deISGylating and deubiquitinating activities. Virology 450–451, 64–70 (2014).
Medina, G. N. et al. Impairment of the DeISGylation activity of foot-and-mouth disease virus Lpro causes attenuation in vitro and in vivo. J. Virol. 94, e00341-20 (2020).
Shin, D. et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature 587, 657–662 (2020). This is the first article describing the dual role of PL(pro) of SARS-Cov-2, which cleaves viral polypeptides and also deconjugates ISG15 and ubiquitin. Structural work indicates differences in affinity and specificity of PL(pro) for ISG15 versus ubiquitin, thereby regulating antiviral immune responses.
Frias-Staheli, N. et al. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe 2, 404–416 (2007).
Swatek, K. N. et al. Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies. Proc. Natl Acad. Sci. USA 115, 2371–2376 (2018).
Klemm, T. et al. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. EMBO J. 39, e106275 (2020).
Békés, M. et al. Recognition of Lys48-linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease. Mol. Cell 62, 572–585 (2016).
Freitas, B. T. et al. Characterization and noncovalent inhibition of the deubiquitinase and deISGylase activity of SARS-CoV-2 papain-like protease. ACS Infect. Dis. 6, 2099–2109 (2020).
Báez-Santos, Y. M., st. John, S. E. & Mesecar, A. D. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds. Antivir. Res. 115, 21–38 (2015).
Rut, W. et al. Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: a framework for anti–COVID-19 drug design. Sci. Adv. 6, eabd4596 (2020).
Patchett, S. et al. A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease. Cell Rep. 36, 109754 (2021).
Tay, M. Z., Poh, C. M., Rénia, L., MacAry, P. A. & Ng, L. F. P. The trinity of COVID-19: immunity, inflammation and intervention. Nat. Rev. Immunol. 20, 363–374 (2020).
Munnur, D. et al. Altered ISGylation drives aberrant macrophage-dependent immune responses during SARS-CoV-2 infection. Nat. Immunol. 22, 1416–1427 (2021).
Harper, J. W. & Schulman, B. A. Structural complexity in ubiquitin recognition. Cell 124, 1133–1136 (2006).
Hurley, J. H., Lee, S. & Prag, G. Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006).
Husnjak, K. & Dikic, I. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322 (2012).
Haakonsen, D. L. & Rape, M. Branching out: improved signaling by heterotypic ubiquitin chains. Trends Cell Biol. 29, 704–716 (2019).
Oh, E., Akopian, D. & Rape, M. Principles of ubiquitin- dependent signaling. Annu. Rev. Cell Dev. Biol. 34, 137–162 (2018).
Grumati, P. & Dikic, I. Ubiquitin signaling and autophagy. J. Biol. Chem. 293, 5404–5413 (2018).
Jackson, S. P. & Durocher, D. Regulation of DNA damage responses by ubiquitin and SUMO. Mol. Cell 49, 795–807 (2013).
García-Rodríguez, N., Wong, R. P. & Ulrich, H. D. Functions of ubiquitin and SUMO in DNA replication and replication stress. Front. Genet. 7, 87 (2016).
Chen, X., Htet, Z. M., López-Alfonzo, E., Martin, A. & Walters, K. J. Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies. FEBS J. 288, 5231–5251 (2021).
Davis, C., Spaller, B. L. & Matouschek, A. Mechanisms of substrate recognition by the 26S proteasome. Curr. Opin. Struct. Biol. 67, 161–169 (2021).
Wu, X. & Rapoport, T. A. Mechanistic insights into ER-associated protein degradation. Curr. Opin. Cell Biol. 53, 22–28 (2018).
Worden, E. J. & Wolberger, C. Activation and regulation of H2B-Ubiquitin-dependent histone methyltransferases. Curr. Opin. Struct. Biol. 59, 98–106 (2019).
Armstrong, A. A., Mohideen, F. & Lima, C. D. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature 483, 59–65 (2012).
Swatek, K. N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).
Rennie, M. L., Chaugule, V. K. & Walden, H. Modes of allosteric regulation of the ubiquitination machinery. Curr. Opin. Struct. Biol. 62, 189–196 (2020).
Khago, D., Fucci, I. J. & Byrd, R. A. The role of conformational dynamics in the recognition and regulation of ubiquitination. Molecules 25, 5933 (2020).
de la Peña, A. H., Goodall, E. A., Gates, S. N., Lander, G. C. & Martin, A. Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation. Science 362, eaav0725 (2018).
Ding, Z. et al. Structural snapshots of 26S proteasome reveal tetraubiquitin-induced conformations. Mol. Cell 73, 1150–1161 (2019).
Chen, X. et al. Cryo-EM reveals unanchored M1-ubiquitin chain binding at hRpn11 of the 26S proteasome. Structure 28, 1206–1217 (2020).
Dong, Y. et al. Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome. Nature 565, 49–55 (2018). De la Peña et al. (2018) and Dong et al. (2018) trap a proteasomal substrate within the hexameric ATP motor to determine its structure in the cryogenic electron microscope. They show three successive conformational states comprising ATP binding, hydrolysis and phosphate release, which are coordinated among the subunits of the motor and govern the transport of the substrate through the proteasome.
Mao, Y. Structure, dynamics and function of the 26S proteasome. Subcell. Biochem. 96, 1–151 (2021).
Bard, J. A. M. et al. Structure and function of the 26S proteasome. Annu. Rev. Biochem. 87, 697–724 (2018).
Sakata, E., Eisele, M. R. & Baumeister, W. Molecular and cellular dynamics of the 26S proteasome. Biochim. Biophys. Acta Proteins Proteom. 1869, 140583 (2021).
Martinez-Fonts, K. et al. The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates. Nat. Commun. 11, 477 (2020).
Jonsson, E., Htet, Z. M., Bard, J. A. M., Dong, K. C. & Martin, A. Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation. Preprint at bioRxiv https://doi.org/10.1101/2021.08.18.456915 (2021).
Berner, N., Reutter, K. R. & Wolf, D. H. Protein quality control of the endoplasmic reticulum and ubiquitin-proteasome-triggered degradation of aberrant proteins: yeast pioneers the path. Annu. Rev. Biochem. 87, 751–782 (2018).
Stach, L. & Freemont, P. S. The AAA+ ATPase p97, a cellular multitool. Biochem. J. 474, 2953–2976 (2017).
Bays, N. W. & Hampton, R. Y. Cdc48-Ufd1-Npl4: stuck in the middle with Ub. Curr. Biol. 12, R366–R371 (2002).
Olszewski, M. M., Williams, C., Dong, K. C. & Martin, A. The Cdc48 unfoldase prepares well-folded protein substrates for degradation by the 26S proteasome. Commun. Biol. 2, 29 (2019).
Buchberger, A., Schindelin, H. & Hänzelmann, P. Control of p97 function by cofactor binding. FEBS Lett. 589, 2578–2589 (2015).
Ji, Z. et al. Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase. Mol. Cell 82, 570–584 (2021).
Cooney, I. et al. Structure of the Cdc48 segregase in the act of unfolding an authentic substrate. Science 365, 502–505 (2019).
Pan, M. et al. Mechanistic insight into substrate processing and allosteric inhibition of human p97. Nat. Struct. Mol. Biol. 28, 614–p625 (2021).
Sato, Y. et al. Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4. Nat. Commun. 10, 5708 (2019).
Twomey, E. C. et al. Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding. Science 365, eaax1033 (2019).
Ciehanover, A., Hod, Y. & Hershko, A. A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105 (1978).
Sun, H. et al. Diverse fate of ubiquitin chain moieties: the proximal is degraded with the target, and the distal protects the proximal from removal and recycles. Proc. Natl Acad. Sci. USA 116, 7805–7812 (2019).
Fujisawa, R. & Labib, K. P. M. Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase. Preprint at bioRxiv https://doi.org/10.1101/2022.01.13.476277 (2022).
Meyer, H. J. & Rape, M. Enhanced protein degradation by branched ubiquitin chains. Cell 157, 910–921 (2014).
Fuchs, G. & Oren, M. Writing and reading H2B monoubiquitylation. Biochim. Biophys. Acta 1839, 694–701 (2014).
Xue, H. et al. Structural basis of nucleosome recognition and modification by MLL methyltransferases. Nature 573, 445–449 (2019).
Worden, E. J., Zhang, X. & Wolberger, C. Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome. Elife 9, e53199 (2020).
Hsu, P. L. et al. Structural basis of H2B ubiquitination-dependent H3K4 methylation by COMPASS. Mol. Cell 76, 712–723.e4 (2019).
Bilokapic, S. & Halic, M. Nucleosome and ubiquitin position Set2 to methylate H3K36. Nat. Commun. 10, 3795 (2019).
Worden, E. J., Hoffmann, N. A., Hicks, C. W. & Wolberger, C. Mechanism of cross-talk between H2B ubiquitination and H3 methylation by Dot1L. Cell 176, 1490–1501 (2019). By replacing the DOTL1 methylation substrate H3 Lys79 with the unnatural amino acid norleucine, the authors are able to visualize remarkable structural remodelling that activates DOT1L in complex with a monoubiquitylated H2B Lys120 nucleosome.
Anderson, C. J. et al. Structural basis for recognition of ubiquitylated nucleosome by Dot1L methyltransferase. Cell Rep. 26, 1681–1690 (2019).
Valencia-Sánchez, M. I. et al. Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Mol. Cell 74, 1010–1019 (2019).
Yao, T. et al. Structural basis of the crosstalk between histone H2B monoubiquitination and H3 lysine 79 methylation on nucleosome. Cell Res. 29, 330–333 (2019).
Hoeller, D. et al. Regulation of ubiquitin-binding proteins by monoubiquitination. Nat. Cell Biol. 8, 163–169 (2006).
Flick, K. et al. Proteolysis-independent regulation of the transcription factor Met4 by a single Lys 48-linked ubiquitin chain. Nat. Cell Biol. 6, 634–641 (2004).
Baba, D. et al. Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature 435, 979–982 (2005).
Rusnac, D. V. & Zheng, N. Structural Biology of CRL Ubiquitin Ligases. Adv. Exp. Med. Biol. 1217, 9–31 (2020).
Wang, K., Deshaies, R. J. & Liu, X. Assembly and regulation of CRL ubiquitin ligases. Adv. Exp. Med. Biol. 1217, 33–46 (2020).
Sakata, E. et al. Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity. Nat. Struct. Mol. Biol. 14, 167–168 (2007).
Kelsall, I. R. et al. TRIAD1 and HHARI bind to and are activated by distinct neddylated cullin-RING ligase complexes. EMBO J. 32, 2848–2860 (2013).
Hospenthal, M. K., Freund, S. M. V. & Komander, D. Assembly, analysis and architecture of atypical ubiquitin chains. Nat. Struct. Mol. Biol. 20, 555–565 (2013).
McClellan, A. J., Laugesen, S. H. & Ellgaard, L. Cellular functions and molecular mechanisms of non-lysine ubiquitination. Open. Biol. 9, 190147 (2019).
Yau, R. & Rape, M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586 (2016).
Swaney, D. L., Rodríguez-Mias, R. A. & Villén, J. Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover. EMBO Rep. 16, 1131–1144 (2015).
Wauer, T. et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO J. 34, 307–325 (2015).
Gladkova, C. et al. An invisible ubiquitin conformation is required for efficient phosphorylation by PINK 1. EMBO J. 36, 3555–3572 (2017).
Gersch, M. et al. Mechanism and regulation of the Lys6-selective deubiquitinase USP30. Nat. Struct. Mol. Biol. 24, 920–930 (2017).
Ohtake, F. et al. Ubiquitin acetylation inhibits polyubiquitin chain elongation. EMBO Rep. 16, 192–201 (2015).
Lacoursiere, R. E. & Shaw, G. S. Acetylated ubiquitin modulates the catalytic activity of the E1 enzyme Uba1. Biochemistry 60, 1276–1285 (2021).
Kienle, S. M. et al. Electrostatic and steric effects underlie acetylation-induced changes in ubiquitin structure and function. Nat. Commun. 13, 5435 (2022).
Herhaus, L. & Dikic, I. Expanding the ubiquitin code through post-translational modification. EMBO Rep. 16, 1071–1083 (2015).
Wang, Y. & Wang, F. Post-translational modifications of deubiquitinating enzymes: expanding the ubiquitin code. Front. Pharmacol. 12, 685011 (2021).
Mevissen, T. E. T. & Komander, D. Mechanisms of deubiquitinase specificity and regulation. Annu. Rev. Biochem. 86, 159–192 (2017).
Scott, D. C., Monda, J. K., Bennett, E. J., Harper, J. W. & Schulman, B. A. N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex. Science 334, 674–678 (2011). This is the report to show how acetylation of a protein’s amino terminus may direct crucial interactions within multiprotein complexes, thereby promoting distinctive activities.
Pilli, M. et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity 37, 223–234 (2012).
Richter, B. et al. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc. Natl Acad. Sci. USA 113, 4039–4044 (2016).
Wild, P. et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 (2011).
Matsumoto, G., Wada, K., Okuno, M., Kurosawa, M. & Nukina, N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol. Cell 44, 279–289 (2011).
Denuc, A., Bosch-Comas, A., Gonzàlez-Duarte, R. & Marfany, G. The UBA-UIM domains of the USP25 regulate the enzyme ubiquitination state and modulate substrate recognition. PLoS ONE 4, e5571 (2009).
Meulmeester, E., Kunze, M., Hsiao, H. H., Urlaub, H. & Melchior, F. Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25. Mol. Cell 30, 610–619 (2008).
Vere, G., Kealy, R., Kessler, B. M. & Pinto-Fernandez, A. Ubiquitomics: an overview and future. Biomolecules 10, 1453 (2020).
Wang, Y. S., Wu, K. P., Jiang, H. K., Kurkute, P. & Chen, R. H. Branched ubiquitination: detection methods, biological functions and chemical synthesis. Molecules 25, 5200 (2020).
Henneberg, L. T. & Schulman, B. A. Decoding the messaging of the ubiquitin system using chemical and protein probes. Cell Chem. Biol. 28, 889–902 (2021).
Wang, G. P., Khatoon, S., Iqbal, K. & Grundke-Iqbal, I. Brain ubiquitin is markedly elevated in Alzheimer disease. Brain Res. 566, 146–151 (1991).
Takada, K. et al. Serum concentrations of free ubiquitin and multiubiquitin chains. Clin. Chem. 43, 1188–1195 (1997).
Knight, E. & Cordova, B. IFN-induced 15-kDa protein is released from human lymphocytes and monocytes. J. Immunol. 146, 2280–2284 (1991).
Radoshevich, L. et al. ISG15 counteracts Listeria monocytogenes infection. Elife 4, e06848 (2015).
Napolitano, A. et al. Cysteine-reactive free ISG15 generates IL-1β–producing CD8α+ dendritic cells at the site of infection. J. Immunol. 201, 604–614 (2018).
Swaim, C. D., Scott, A. F., Canadeo, L. A. & Huibregtse, J. M. Extracellular ISG15 signals cytokine secretion through the LFA-1 integrin receptor. Mol. Cell 68, 581–590 (2017).
Recht, M., Borden, E. C. & Knight, E. A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma. J. Immunol. 147, 2617–2623 (1991).
Villarreal, D. O. et al. Ubiquitin-like molecule ISG15 acts as an immune adjuvant to enhance antigen-specific CD8 T-cell tumor immunity. Mol. Ther. 23, 1653–1662 (2015).
Bogunovic, D. et al. Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency. Science 337, 1684–1688 (2012).
Eduardo-Correia, B., Martinez-Romero, C., Garcia-Sastre, A. & Guerra, S. ISG15 is counteracted by vaccinia virus E3 protein and controls the proinflammatory response against viral infection. J. Virol. 88, 2312–2318 (2014).
Malakhova, O. A. & Zhang, D. E. ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response. J. Biol. Chem. 283, 8783–8787 (2008).
Okumura, A., Pitha, P. M. & Harty, R. N. ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc. Natl Acad. Sci. USA 105, 3974–3979 (2008).
Nakashima, H., Nguyen, T., Goins, W. F. & Chiocca, E. A. Interferon-stimulated gene 15 (ISG15) and ISG15-linked proteins can associate with members of the selective autophagic process, histone deacetylase 6 (HDAC6) and SQSTM1/p62. J. Biol. Chem. 290, 1485–1495 (2015).
Burks, J., Reed, R. E. & Desai, S. D. Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective. Oncotarget 6, 7221–7231 (2015).
Chen, R. H. et al. Tumor cell-secreted ISG15 promotes tumor cell migration and immune suppression by inducing the macrophage M2-like phenotype. Front. Immunol. 11, 594775 (2020).
Huang, Y. F., Wee, S., Gunaratne, J., Lane, D. P. & Bulavin, D. V. Isg15 controls p53 stability and functions. Cell Cycle 13, 2199–2209 (2014).
Li, C. et al. Interferon-stimulated gene 15 (ISG15) is a trigger for tumorigenesis and metastasis of hepatocellular carcinoma. Oncotarget 5, 8429–8441 (2014).
Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).
Rajsbaum, R. et al. Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase-mediated antiviral response. Immunity 40, 880–895 (2014).
Zeng, W. et al. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315–330 (2010).
Kumar, S. K. et al. Carfilzomib or bortezomib in combination with lenalidomide and dexamethasone for patients with newly diagnosed multiple myeloma without intention for immediate autologous stem-cell transplantation (ENDURANCE): a multicentre, open-label, phase 3, randomised, controlled trial. Lancet Oncol. 21, 1317–1330 (2020).
de Cesare, V. et al. The MALDI-TOF E2/E3 ligase assay as universal tool for drug discovery in the ubiquitin pathway. Cell Chem. Biol. 25, 1117–1127 (2018).
Gutierrez-Diaz, B. T., Gu, W. & Ntziachristos, P. Deubiquitinases: pro-oncogenic activity and therapeutic targeting in blood malignancies. Trends Immunol. 41, 327–340 (2020).
Kannt, A. & Đikić, I. Expanding the arsenal of E3 ubiquitin ligases for proximity-induced protein degradation. Cell Chem. Biol. 28, 1014–1031 (2021).
Jevtić, P., Haakonsen, D. L. & Rapé, M. An E3 ligase guide to the galaxy of small-molecule-induced protein degradation. Cell Chem. Biol. 28, 1000–1013 (2021).
Hanzl, A. et al. Charting functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders. Preprint at bioRxiv https://doi.org/10.1101/2022.04.14.488316 (2022).
Khan, S. et al. Proteolysis targeting chimeras (PROTACs) as emerging anticancer therapeutics. Oncogene 39, 4909 (2020).
Li, W., Elhassan, R. M., Hou, X. & Fang, H. Recent advances in small molecule PROTACs for the treatment of cancer. Curr. Med. Chem. 28, 4893–4909 (2021).
Takahashi, D. et al. AUTACs: cargo-specific degraders using selective autophagy. Mol. Cell 76, 797–810 (2019).
Li, Z., Zhu, C., Ding, Y., Fei, Y. & Lu, B. ATTEC: a potential new approach to target proteinopathies. Autophagy 16, 185–187 (2020).
Ahn, G. et al. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat. Chem. Biol. 17, 937–946 (2021).
Cotton, A. D., Nguyen, D. P., Gramespacher, J. A., Seiple, I. B. & Wells, J. A. Development of antibody-based PROTACs for the degradation of the cell-surface immune checkpoint protein PD-L1. J. Am. Chem. Soc. 143, 593–598 (2021).
Acknowledgements
The authors thank D. Höller for critical reading, text editing and figure preparations, and C. Wolberger and A. Martin for helpful comments. This work was supported by the Deutsche Forschungsgemeinschaft (project number 259130777-SFB 1177), the Cluster Project ENABLE funded by the Hessian Ministry for Science and the Arts and European Research Council grant H2020 UbBAC 742720 to I.D., and by the Max Planck Gesellschaft, European Research Council grant H2020 NEDD8Activate 789016 to B.A.S. and the Leibniz Prize from the Deutsche Forschungsgemeinschaft (SCHU 3196/1-1) to B.A.S.
Author information
Authors and Affiliations
Contributions
The authors contributed equally to all aspects of the article.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Reviews Molecular Cell Biology thanks Yogesh Kulathu and the other, anonymous, reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Glossary
- Ubiquitin-like proteins
-
(UBLs). A family of proteins that share key structural features with ubiquitin and can be either conjugated to substrates (type I) such ubiquitin or exist as protein domains (type II) in larger proteins.
- Really interesting new gene (RING) domain
-
A zinc-finger protein domain that serves as a docking site for ubiquitin-loaded E2 enzymes and allosterically activates the thioester bond between E2 and ubiquitin to facilitate ubiquitin transfer.
- Homologous to the E6-AP carboxy terminus (HECT) domain
-
A protein domain that contains a conserved Cys that accepts ubiquitin from E2–ubiquitin and then transfers it directly to the substrate Lys.
- RING-between-RING (RBR) domain
-
A protein domain present in E3s that utilize a really interesting new gene (RING) domain–homologous to the E6-AP carboxy terminus (HECT) domain hybrid mechanism for ubiquitin conjugation.
- Nuclear magnetic resonance spectroscopy
-
A structural biology technique that allows monitoring interactions and perturbations of NMR-active nuclei. This method allows identification through bond and through space interactions to determine structures of organic molecules, including proteins.
- Myddosome
-
Large intracellular signalling complexes also called ‘supramolecular organizing centres’ assembled after recognition of microorganisms that function as a signalling platform.
- ADP-ribose
-
(ADPR). An ester formed between the aldehydic carbon of ribose and the terminal phosphate of adenosine diphosphate that either can be attached to proteins as a post-translational modification regulating, for example, DNA repair processes or can function as a second messenger on its own, for example by activating cation channels.
- Legionnaires’ disease
-
A severe form of pneumonia first described after an outbreak among attendants at a convention of the American Legion in 1976. It is caused by the bacterium Legionella pneumophila inhaled from contaminated water or soil droplets and can lead to life-threatening complications.
- Isgylation
-
Ubiquitylation-like process in which the ubiquitin-like modifier interferon-stimulated gene 15 (ISG15) is covalently conjugated to Lys residues of substrate proteins.
- Single-molecule fluorescence resonance energy transfer
-
A means of measuring distances at the 1–10-nm scale in a single biomolecule allowing the quantification and characterization of binding events, intramolecular transitions (for example, during protein folding), and kinetics and dwell times for single molecules.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Dikic, I., Schulman, B.A. An expanded lexicon for the ubiquitin code. Nat Rev Mol Cell Biol 24, 273–287 (2023). https://doi.org/10.1038/s41580-022-00543-1
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41580-022-00543-1
This article is cited by
-
Multi-omics integration highlights the role of ubiquitination in endometriosis fibrosis
Journal of Translational Medicine (2024)
-
Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes
Communications Biology (2024)
-
Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis
Cell Death & Differentiation (2024)
-
Understanding ubiquitination in neurodevelopment by integrating insights across space and time
Nature Structural & Molecular Biology (2024)
-
Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1
Nature Structural & Molecular Biology (2024)