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Deubiquitinating enzymes (DUBs): Regulation, homeostasis, and oxidative stress response

Open AccessPublished:August 12, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101077
      Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein–protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.

      Keywords

      Abbreviations:

      A20 (tumor necrosis factor alpha–induced protein 3), AMSH (associated molecule with the SH3 domain of STAM), ATXN (ataxin), BAP1 (BRCA1-associated protein 1), BER (base excision repair), CK2 (casein kinase 2), CYLD (conserved cylindromatosis), DTT (dithiothreitol), DUB (deubiquitinating enzyme or deubiquitinase), EGFR (epidermal growth factor receptor), ESCRT (endosomal sorting complexes required for transport), FOXO4 (forkhead box O4), ISR (integrated stress response), MDM2 (Mouse double minute 2), MIC-CAP (microcephaly-capillary malformation), MINDY (motif interacting with Ub-containing novel DUB), MJD (Machado–Josephin domain), NLS (nuclear localization signal), OTU (ovarian tumor domain), OTUB (varian tumor deubiquitinase, ubiquitin aldehyde binding), OTUD (ovarian tumor deubiquitinase), PCNA (proliferating cell nuclear antigen), Polβ (DNA polymerase beta), PTM (posttranslational modification), QC (quality control), ROS (reactive oxygen species), RPN11 (regulatory particle non-ATPase 11), RQC (ribosome-associated quality control), RTU (redox control of translation by ubiquitin), SUMO (small ubiquitin-like modifier), TGF-β (transforming growth factor beta), TNF (tumor necrosis factor), TRAF2 (tumor necrosis factor (TNF) receptor associated factor 2), TRAF6 (tumor necrosis factor (TNF) receptor associated factor 6), Ubp (ubiquitin-binding protein), UCH (ubiquitin C-terminal hydrolase), UIM (ubiquitin interacting motif), UPS (ubiquitin-proteasome system), USP (ubiquitin-specific protease), WDR48 (WD repeat domain 48)
      When the subject of ubiquitination arises, most people think of the canonical pathway of ubiquitin-dependent proteasomal targeting. This, however, only represents a fraction of the functional diversity of ubiquitin. Ubiquitin is a small (76 amino acid) and highly conserved eukaryotic protein that acts as a posttranslational protein modifier (
      • Watson D.C.
      • Levy W.B.
      • Dixon G.H.
      Free ubiquitin is a non-histone protein of trout testis chromatin.
      ). Ubiquitin signaling is a very robust and diverse process where a series of ubiquitin conjugases and ligases act in a coordinated process to covalently modify targets with one or more ubiquitin molecules in the form of a chain (
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      ). Each of these different ubiquitin chains has a unique structure, thus providing the opportunity for this single protein to influence numerous pathways, other than proteasomal degradation, from DNA damage repair to protein translation and trafficking (
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      ). In most of these pathways, including protein degradation, ubiquitin is removed from its substrates in a reversible fashion in a process achieved through dynamic regulation of ubiquitin hydrolases known as deubiquitinating enzymes (DUBs) (
      • Komander D.
      Mechanism, specificity and structure of the deubiquitinases.
      ,
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      • Komander D.
      Mechanisms of deubiquitinase specificity and regulation.
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      • Wilkinson K.D.
      Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.
      ). Due to the highly adaptive and reversible nature of ubiquitin signaling, DUBs are frequently utilized to regulate protein function in response to environmental changes and stress (
      • Fang N.N.
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      • Wu K.P.
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      Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress.
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      • Gao C.
      • Long Y.
      • Xu Y.
      CYLD deubiquitinase negatively regulates high glucose induced NF-kappaB inflammatory signaling in mesangial cells.
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      Deubiquitinases as a signaling target of oxidative stress.
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      • Xiao Z.D.
      • You M.J.
      • Li W.
      • Su X.
      • Gan B.
      BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response.
      ).
      In the canonical ubiquitination pathway (Fig. 1), an E1 ubiquitin activating enzyme is responsible for activating ubiquitin in an ATP-dependent manner and charging the E2 ubiquitin conjugase with the ubiquitin (
      • Schulman B.A.
      • Harper J.W.
      Ubiquitin-like protein activation by E1 enzymes: The apex for downstream signalling pathways.
      ). The E2 then either transfers the ubiquitin to the substrate or to an E3 ubiquitin ligase that will then transfer the ubiquitin molecule to the substrate (
      • Ye Y.
      • Rape M.
      Building ubiquitin chains: E2 enzymes at work.
      ). In the former case, the substrate is brought into proximity of the E2 through an associated E3 ubiquitin ligase (
      • Ye Y.
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      Building ubiquitin chains: E2 enzymes at work.
      ). A number of E2 and E3 ubiquitin enzymes exist (∼40 E2s and over 600 E3s in humans and 12 E2s and ∼80 E3s in yeast), and the pairing of these enzymes determines the specificity of substrate that is ubiquitinated (
      • George A.J.
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      • Charles A.J.
      • Zhu Y.
      • Mabb A.M.
      A comprehensive atlas of E3 ubiquitin ligase mutations in neurological disorders.
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      • Stewart M.D.
      • Ritterhoff T.
      • Klevit R.E.
      • Brzovic P.S.
      E2 enzymes: More than just middle men.
      ). This pairing is also responsible for determining how the ubiquitin chains are assembled (
      • Ye Y.
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      Building ubiquitin chains: E2 enzymes at work.
      ,
      • Metzger M.B.
      • Pruneda J.N.
      • Klevit R.E.
      • Weissman A.M.
      RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination.
      ).
      Figure thumbnail gr1
      Figure 1Cycle of ubiquitin signaling. Ubiquitin (ub) is synthesized as polymers or fusions of ribosomal proteins that are cleaved into ubiquitin monomers by deubiquitinating enzymes (DUBs). Ubiquitin monomers are then used by E1 ubiquitin-activating enzymes to charge E2 ubiquitin conjugases, which work with or without E3 ubiquitin ligases to attach ubiquitin to targets. The conformation of K-linked ubiquitin chains (e.g., K48 or K63) determines the fate of the targets, whether they undergo ubiquitin signaling, or if they are sent to the proteasome for degradation. In either case, DUBs are responsible for removal of the ubiquitin and replenishment of the ubiquitin monomer pool. Reactive oxygen species (ROS)-sensitive steps are labeled in red. Structures depicted include ubiquitin (PDB: 1UBQ), and representatives of two forms of di-ubiquitin, K48 (PDB: 3AUL) and K63 (PDB: 3H7P).
      Protein ubiquitination most commonly occurs via an isopeptide bond between the C-terminus of ubiquitin and a lysine of the substrate (
      • Rechsteiner M.
      Ubiquitin-mediated pathways for intracellular proteolysis.
      ). Other forms of nonlysine ubiquitination are rare but exist, including peptide bonds with the N-terminal methionine (M1), thioester bonds with a cysteine residue, and hydroxyester bonds with a serine or threonine residue (
      • McClellan A.J.
      • Laugesen S.H.
      • Ellgaard L.
      Cellular functions and molecular mechanisms of non-lysine ubiquitination.
      ). Furthermore, ubiquitin has recently been shown to be conjugated to nonprotein surfaces such as lipopolysaccharides (
      • Otten E.G.
      • Werner E.
      • Crespillo-Casado A.
      • Boyle K.B.
      • Dharamdasani V.
      • Pathe C.
      • Santhanam B.
      • Randow F.
      Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection.
      ). Once the first ubiquitin molecule is conjugated to a substrate, a second round of conjugation occurs linking two ubiquitin molecules together in the form of a chain (
      • Nonhoff U.
      • Ralser M.
      • Welzel F.
      • Piccini I.
      • Balzereit D.
      • Yaspo M.L.
      • Lehrach H.
      • Krobitsch S.
      Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.
      ). Ubiquitin is linked into polyubiquitin chains through any of the seven lysine residues in ubiquitin (K6, K11, K27, K29, K33, K48, or K63) as well as through the amino group of M1 (
      • Akutsu M.
      • Dikic I.
      • Bremm A.
      Ubiquitin chain diversity at a glance.
      ). Each of these linkage sites provides a unique chain archetype, and while many of these chains can signal for proteasome-dependent substrate degradation to some extent, others serve an array of regulatory roles (
      • Akutsu M.
      • Dikic I.
      • Bremm A.
      Ubiquitin chain diversity at a glance.
      ).
      Proteomics data demonstrate that while most ubiquitin linkage types increase upon inhibition of the proteasome, K63-linked polyubiquitin does not, suggesting that it is involved almost exclusively in nonproteasomal pathways (
      • Kim W.
      • Bennett E.J.
      • Huttlin E.L.
      • Guo A.
      • Li J.
      • Possemato A.
      • Sowa M.E.
      • Rad R.
      • Rush J.
      • Comb M.J.
      • Harper J.W.
      • Gygi S.P.
      Systematic and quantitative assessment of the ubiquitin-modified proteome.
      ,
      • Xu P.
      • Duong D.M.
      • Seyfried N.T.
      • Cheng D.
      • Xie Y.
      • Robert J.
      • Rush J.
      • Hochstrasser M.
      • Finley D.
      • Peng J.
      Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.
      ). Each linkage forms unique topologies that may be bound and recognized by distinct proteins to generate different signals and functions (
      • Husnjak K.
      • Dikic I.
      Ubiquitin-binding proteins: Decoders of ubiquitin-mediated cellular functions.
      ). Additionally, ubiquitin can form both homotypic chains, where each ubiquitin is linked through the same lysine residue (e.g., K48-linked chains or K63-linked chains) and heterotypic chains, where multiple linkage types are utilized to form the chain (
      • Yau R.
      • Rape M.
      The increasing complexity of the ubiquitin code.
      ). Furthermore, ubiquitin can be assembled to produce branched chains and can themselves be modified through PTMs such as phosphorylation or SUMOylation, further expanding the signaling possibilities (
      • Yau R.
      • Rape M.
      The increasing complexity of the ubiquitin code.
      ,
      • Ohtake F.
      • Tsuchiya H.
      The emerging complexity of ubiquitin architecture.
      ).
      While the regulation of ubiquitin conjugation has been more widely studied, significant advancement in the study of the family of enzymes responsive for removal of ubiquitin has only recently occurred. Humans encode ∼100 different DUBs that regulate ubiquitin signaling by removing ubiquitin and thus disassembling the chains and thereby their signals, while recycling ubiquitin for further conjugation (
      • Komander D.
      • Clague M.J.
      • Urbe S.
      Breaking the chains: Structure and function of the deubiquitinases.
      ). This can be done by cleaving single ubiquitin monomers from the distal end of a chain or by cleaving entire chains by breaking the bond between the proximal ubiquitin and the substrate (
      • Reyes-Turcu F.E.
      • Ventii K.H.
      • Wilkinson K.D.
      Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.
      ).
      Similar to ubiquitin ligases and conjugases, DUBs usually have specific ubiquitin linkage types that they bind as substrates (
      • Mevissen T.E.T.
      • Komander D.
      Mechanisms of deubiquitinase specificity and regulation.
      ). Since DUBs serve as direct antagonists of ubiquitin conjugation, this results in a switch-like system (
      • Mevissen T.E.T.
      • Komander D.
      Mechanisms of deubiquitinase specificity and regulation.
      ). The levels of ubiquitin conjugation can therefore be determined by these two competing enzymatic systems, which can themselves be regulated at expression, subcellular location, or activity levels through mechanisms controlled by protein–protein interactions and posttranslational modifications (PTMs) (Fig. 2) (
      • Sahtoe D.D.
      • Sixma T.K.
      Layers of DUB regulation.
      ,
      • Zheng N.
      • Shabek N.
      Ubiquitin ligases: Structure, function, and regulation.
      ).
      Figure thumbnail gr2
      Figure 2DUB regulatory mechanisms. Examples of posttranslational DUB regulatory mechanisms are depicted. These regulatory mechanisms include: (A) posttranslational modification (e.g., ubiquitination, SUMOylation, phosphorylation), (B) protein binding, (C) redox regulation, and (D) subcellular localization.
      E2s, E3s, and DUBs can respond to environmental cues that induce the prioritization of the generation or removal of signals (
      • Kliza K.
      • Husnjak K.
      Resolving the complexity of ubiquitin networks.
      ). DUBs in particular are highly sensitive to environmental stresses, and this review will discuss the main mechanisms of DUB regulation, in particular those that result from oxidative stress, which directly targets and inhibits the active site of a large number of DUBs (
      • Lee J.G.
      • Baek K.
      • Soetandyo N.
      • Ye Y.
      Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells.
      ).
      The ability of DUBs to regulate such a dynamic signaling system also makes them an interesting target for therapeutics. Indeed, dysregulation or malfunction of DUBs has been implicated in a number of human diseases, including cancers and neurological disorders, usually resulting from aberrant signaling within the cell (
      • Kowalski J.R.
      • Juo P.
      The role of deubiquitinating enzymes in synaptic function and nervous system diseases.
      ,
      • Fraile J.M.
      • Quesada V.
      • Rodriguez D.
      • Freije J.M.
      • Lopez-Otin C.
      Deubiquitinases in cancer: New functions and therapeutic options.
      ). Due to their physiological importance, a number of small-molecule inhibitors of selective DUBs are being developed with the goal of therapeutic utilization as treatments for these diseases (
      • Harrigan J.A.
      • Jacq X.
      • Martin N.M.
      • Jackson S.P.
      Deubiquitylating enzymes and drug discovery: Emerging opportunities.
      ). Recent insights into the mechanisms and importance of DUB regulation, combined with the development of drugs with therapeutic potential, have marked the importance of this newly burgeoning field and provided directions for future studies to come.

      WHY do we need DUBs?

      As we learn more about the importance and diversity of ubiquitin chains in cellular processes, we also gain emphasis on the importance of the DUBs that regulate these signals. In addition to regulating the ubiquitin signals generated by E2s and E3s, DUBs are essential to maintain the supply of ubiquitin by recycling and breaking down newly synthesized ubiquitin fusion proteins (Fig. 1) (
      • Pickart C.M.
      • Rose I.A.
      Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides.
      ,
      • Hanna J.
      • Meides A.
      • Zhang D.P.
      • Finley D.
      A ubiquitin stress response induces altered proteasome composition.
      ). Constant maintenance of a free ubiquitin pool is necessary for cellular homeostasis but also required, to rapidly generate signals to respond to changes in the cellular environment or to biomolecular damage incurred during stress (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ). This section will highlight the classes of DUBs and the mechanisms and functional roles they play in eukaryotic cells. Table 1 summarizes the class, family, and functional roles of all DUBs specifically discussed in this review.
      Table 1List of DUBs mentioned in this review and their classifications
      DUB (known to be redox-sensitive)ClassFamilyKnown ub linkage specificityKnown pathways affected
      A20Cysteine proteaseoutK63NF-κB signaling (
      • Shembade N.
      • Harhaj E.W.
      Regulation of NF-kappaB signaling by the A20 deubiquitinase.
      )
      AMSHMetalloproteaseJAMM/MPN+K63Endocytosis/sorting (
      • Davies C.W.
      • Paul L.N.
      • Das C.
      Mechanism of recruitment and activation of the endosome-associated deubiquitinase AMSH.
      )
      AMSH-LPMetalloproteaseJAMM/MPN+K63Endocytosis/sorting (
      • Davies C.W.
      • Paul L.N.
      • Das C.
      Mechanism of recruitment and activation of the endosome-associated deubiquitinase AMSH.
      )
      ATXN3Cysteine proteaseMJDK48, K63Protein homeostasis (
      • Durcan T.M.
      • Kontogiannea M.
      • Thorarinsdottir T.
      • Fallon L.
      • Williams A.J.
      • Djarmati A.
      • Fantaneanu T.
      • Paulson H.L.
      • Fon E.A.
      The Machado-Joseph disease-associated mutant form of ataxin-3 regulates parkin ubiquitination and stability.
      ,
      • Blount J.R.
      • Tsou W.L.
      • Ristic G.
      • Burr A.A.
      • Ouyang M.
      • Galante H.
      • Scaglione K.M.
      • Todi S.V.
      Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23.
      ); ER-associated degradation (
      • Zhong X.
      • Pittman R.N.
      Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates.
      ); transcription regulation (
      • Evert B.O.
      • Araujo J.
      • Vieira-Saecker A.M.
      • de Vos R.A.
      • Harendza S.
      • Klockgether T.
      • Wullner U.
      Ataxin-3 represses transcription via chromatin binding, interaction with histone deacetylase 3, and histone deacetylation.
      ); cytoskeletal regulation (
      • Rodrigues A.J.
      • do Carmo Costa M.
      • Silva T.L.
      • Ferreira D.
      • Bajanca F.
      • Logarinho E.
      • Maciel P.
      Absence of ataxin-3 leads to cytoskeletal disorganization and increased cell death.
      ); DNA repair (
      • Pfeiffer A.
      • Luijsterburg M.S.
      • Acs K.
      • Wiegant W.W.
      • Helfricht A.
      • Herzog L.K.
      • Minoia M.
      • Bottcher C.
      • Salomons F.A.
      • van Attikum H.
      • Dantuma N.P.
      Ataxin-3 consolidates the MDC1-dependent DNA double-strand break response by counteracting the SUMO-targeted ubiquitin ligase RNF4.
      )
      ATXN3LCysteine proteaseMJDK48, K63Protein homeostasis (
      • Ge F.
      • Chen W.
      • Qin J.
      • Zhou Z.
      • Liu R.
      • Liu L.
      • Tan J.
      • Zou T.
      • Li H.
      • Ren G.
      • Chen C.
      Ataxin-3 like (ATXN3L), a member of the Josephin family of deubiquitinating enzymes, promotes breast cancer proliferation by deubiquitinating Kruppel-like factor 5 (KLF5).
      )
      BAP1Cysteine proteaseUCHK48DNA repair/transcription (
      • Yu H.
      • Pak H.
      • Hammond-Martel I.
      • Ghram M.
      • Rodrigue A.
      • Daou S.
      • Barbour H.
      • Corbeil L.
      • Hebert J.
      • Drobetsky E.
      • Masson J.Y.
      • Di Noia J.M.
      • Affar El B.
      Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair.
      ,
      • Okino Y.
      • Machida Y.
      • Frankland-Searby S.
      • Machida Y.J.
      BRCA1-associated protein 1 (BAP1) deubiquitinase antagonizes the ubiquitin-mediated activation of FoxK2 target genes.
      )
      BRCC36MetalloproteaseJAMM/MPN+K63DNA repair/cell cycle (
      • Wang B.
      • Hurov K.
      • Hofmann K.
      • Elledge S.J.
      NBA1, a new player in the Brca1 A complex, is required for DNA damage resistance and checkpoint control.
      ,
      • Yan K.
      • Li L.
      • Wang X.
      • Hong R.
      • Zhang Y.
      • Yang H.
      • Lin M.
      • Zhang S.
      • He Q.
      • Zheng D.
      • Tang J.
      • Yin Y.
      • Shao G.
      The deubiquitinating enzyme complex BRISC is required for proper mitotic spindle assembly in mammalian cells.
      )
      CezanneCysteine proteaseoutK11, K48, K63NF-κB signaling (
      • Hu H.
      • Brittain G.C.
      • Chang J.H.
      • Puebla-Osorio N.
      • Jin J.
      • Zal A.
      • Xiao Y.
      • Cheng X.
      • Chang M.
      • Fu Y.X.
      • Zal T.
      • Zhu C.
      • Sun S.C.
      OTUD7B controls non-canonical NF-kappaB activation through deubiquitination of TRAF3.
      )
      CSN5MetalloproteaseJAMM/MPN+K63DNA repair (
      • Pan Y.
      • Yang H.
      • Claret F.X.
      Emerging roles of Jab1/CSN5 in DNA damage response, DNA repair, and cancer.
      ); cell cycle (
      • Shackleford T.J.
      • Claret F.X.
      JAB1/CSN5: A new player in cell cycle control and cancer.
      ); protein sorting (
      • Liu Y.
      • Shah S.V.
      • Xiang X.
      • Wang J.
      • Deng Z.B.
      • Liu C.
      • Zhang L.
      • Wu J.
      • Edmonds T.
      • Jambor C.
      • Kappes J.C.
      • Zhang H.G.
      COP9-associated CSN5 regulates exosomal protein deubiquitination and sorting.
      )
      CYLDCysteine proteaseoutK63, M1Cell cycle (
      • Wickstrom S.A.
      • Masoumi K.C.
      • Khochbin S.
      • Fassler R.
      • Massoumi R.
      CYLD negatively regulates cell-cycle progression by inactivating HDAC6 and increasing the levels of acetylated tubulin.
      ); NF-κB/WNT signaling (
      • Komander D.
      CYLD tidies up dishevelled signaling.
      ,
      • Sun S.C.
      CYLD: A tumor suppressor deubiquitinase regulating NF-kappaB activation and diverse biological processes.
      )
      JOSD1Cysteine proteaseMJDK48, K63Endocytosis; membrane sorting (
      • Seki T.
      • Gong L.
      • Williams A.J.
      • Sakai N.
      • Todi S.V.
      • Paulson H.L.
      JosD1, a membrane-targeted deubiquitinating enzyme, is activated by ubiquitination and regulates membrane dynamics, cell motility, and endocytosis.
      )
      JOSD2Cysteine proteaseMJDK48, K63Metabolism (
      • Krassikova L.
      • Zhang B.
      • Nagarajan D.
      • Queiroz A.L.
      • Kacal M.
      • Samakidis E.
      • Vakifahmetoglu-Norberg H.
      • Norberg E.
      The deubiquitinase JOSD2 is a positive regulator of glucose metabolism.
      )
      MINDY1Cysteine proteaseMINDYK48Self-renewal of stem cells (
      • James C.
      • Zhao T.Y.
      • Rahim A.
      • Saxena P.
      • Muthalif N.A.
      • Uemura T.
      • Tsuneyoshi N.
      • Ong S.
      • Igarashi K.
      • Lim C.Y.
      • Dunn N.R.
      • Vardy L.A.
      MINDY1 is a downstream target of the polyamines and promotes embryonic stem cell self-renewal.
      )
      MINDY2Cysteine proteaseMINDYNon-specific
      MINDY3Cysteine proteaseMINDYK48
      MINDY4Cysteine proteaseMINDYK48
      MINDY4BCysteine proteaseMINDYK48
      MYSM1MetalloproteaseJAMM/MPN+K63Transcription (
      • Zhu P.
      • Zhou W.
      • Wang J.
      • Puc J.
      • Ohgi K.A.
      • Erdjument-Bromage H.
      • Tempst P.
      • Glass C.K.
      • Rosenfeld M.G.
      A histone H2A deubiquitinase complex coordinating histone acetylation and H1 dissociation in transcriptional regulation.
      ); immune signaling (
      • Jiang X.X.
      • Chou Y.
      • Jones L.
      • Wang T.
      • Sanchez S.
      • Huang X.F.
      • Zhang L.
      • Wang C.
      • Chen S.Y.
      Epigenetic regulation of antibody responses by the histone H2A deubiquitinase MYSM1.
      )
      OTUB1Cysteine proteaseoutK48DNA repair (
      • Wu Q.
      • Huang Y.
      • Gu L.
      • Chang Z.
      • Li G.M.
      OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination.
      ); immune signaling (
      • Zhou X.
      • Yu J.
      • Cheng X.
      • Zhao B.
      • Manyam G.C.
      • Zhang L.
      • Schluns K.
      • Li P.
      • Wang J.
      • Sun S.C.
      The deubiquitinase Otub1 controls the activation of CD8(+) T cells and NK cells by regulating IL-15-mediated priming.
      )
      OTUB2Cysteine proteaseoutK11, K48, K63DNA repair (
      • Kato K.
      • Nakajima K.
      • Ui A.
      • Muto-Terao Y.
      • Ogiwara H.
      • Nakada S.
      Fine-tuning of DNA damage-dependent ubiquitination by OTUB2 supports the DNA repair pathway choice.
      ); protein homeostasis (
      • Li X.Y.
      • Mao X.F.
      • Tang X.Q.
      • Han Q.Q.
      • Jiang L.X.
      • Qiu Y.M.
      • Dai J.
      • Wang Y.X.
      Regulation of Gli2 stability by deubiquitinase OTUB2.
      ); translation (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      )
      OTUD1Cysteine proteaseoutK63Translation (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      ); immune signaling (
      • Lu D.
      • Song J.
      • Sun Y.
      • Qi F.
      • Liu L.
      • Jin Y.
      • McNutt M.A.
      • Yin Y.
      Mutations of deubiquitinase OTUD1 are associated with autoimmune disorders.
      )
      OTUD2Cysteine proteaseoutK11, K27, K29, K33, K48, K63Protein sorting; ER unfolded protein response (
      • Ernst R.
      • Mueller B.
      • Ploegh H.L.
      • Schlieker C.
      The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER.
      )
      OTUD3Cysteine proteaseoutK6, K11, K27, K48Translation (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      ); immune regulation (
      • Zhang Z.
      • Fang X.
      • Wu X.
      • Ling L.
      • Chu F.
      • Li J.
      • Wang S.
      • Zang J.
      • Zhang B.
      • Ye S.
      • Zhang L.
      • Yang B.
      • Lin S.
      • Huang H.
      • Wang A.
      • et al.
      Acetylation-dependent deubiquitinase OTUD3 controls MAVS activation in innate antiviral immunity.
      )
      OTUD5Cysteine proteaseoutK48, K63Cell signaling (
      • Cho J.H.
      • Kim K.
      • Kim S.A.
      • Park S.
      • Park B.O.
      • Kim J.H.
      • Kim S.Y.
      • Kwon M.J.
      • Han M.H.
      • Lee S.B.
      • Park B.C.
      • Park S.G.
      • Kim J.H.
      • Kim S.
      Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.
      ,
      • Park S.Y.
      • Choi H.K.
      • Choi Y.
      • Kwak S.
      • Choi K.C.
      • Yoon H.G.
      Deubiquitinase OTUD5 mediates the sequential activation of PDCD5 and p53 in response to genotoxic stress.
      ); immune regulation (
      • Guo Y.
      • Jiang F.
      • Kong L.
      • Wu H.
      • Zhang H.
      • Chen X.
      • Zhao J.
      • Cai B.
      • Li Y.
      • Ma C.
      • Yi F.
      • Zhang L.
      • Liu B.
      • Zheng Y.
      • Zhang L.
      • et al.
      OTUD5 promotes innate antiviral and antitumor immunity through deubiquitinating and stabilizing STING.
      )
      OtulinCysteine proteaseoutM1NF-κB/WNT signaling (
      • Damgaard R.B.
      • Walker J.A.
      • Marco-Casanova P.
      • Morgan N.V.
      • Titheradge H.L.
      • Elliott P.R.
      • McHale D.
      • Maher E.R.
      • McKenzie A.N.J.
      • Komander D.
      The deubiquitinase OTULIN is an essential negative regulator of inflammation and autoimmunity.
      )
      RPN11MetalloproteaseJAMM/MPN+Proteasome (
      • Verma R.
      • Aravind L.
      • Oania R.
      • McDonald W.H.
      • Yates 3rd, J.R.
      • Koonin E.V.
      • Deshaies R.J.
      Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome.
      )
      Ubp2Cysteine proteaseUSPK63DNA repair (
      • Alvarez V.
      • Vinas L.
      • Gallego-Sanchez A.
      • Andres S.
      • Sacristan M.P.
      • Bueno A.
      Orderly progression through S-phase requires dynamic ubiquitylation and deubiquitylation of PCNA.
      ); translation (
      • Silva G.M.
      • Finley D.
      • Vogel C.
      K63 polyubiquitination is a new modulator of the oxidative stress response.
      ); protein sorting (
      • Kee Y.
      • Lyon N.
      • Huibregtse J.M.
      The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme.
      )
      Ubp3Cysteine proteaseUSPProtein sorting (
      • Wang P.
      • Ye Z.
      • Banfield D.K.
      A novel mechanism for the retention of Golgi membrane proteins mediated by the Bre5p/Ubp3p deubiquitinase complex.
      ); translation (
      • Takehara Y.
      • Yashiroda H.
      • Matsuo Y.
      • Zhao X.
      • Kamigaki A.
      • Matsuzaki T.
      • Kosako H.
      • Inada T.
      • Murata S.
      The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation.
      )
      UCHL1Cysteine proteaseUCHMAP kinase pathway (
      • Wang W.
      • Zou L.
      • Zhou D.
      • Zhou Z.
      • Tang F.
      • Xu Z.
      • Liu X.
      Overexpression of ubiquitin carboxyl terminal hydrolase-L1 enhances multidrug resistance and invasion/metastasis in breast cancer by activating the MAPK/Erk signaling pathway.
      ); translation (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      )
      UCHL3Cysteine proteaseUCHK48Insulin signaling (
      • Suzuki M.
      • Setsuie R.
      • Wada K.
      Ubiquitin carboxyl-terminal hydrolase l3 promotes insulin signaling and adipogenesis.
      ); protein sorting (
      • Butterworth M.B.
      • Edinger R.S.
      • Ovaa H.
      • Burg D.
      • Johnson J.P.
      • Frizzell R.A.
      The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel.
      )
      UCHL5Cysteine proteaseUCHK48DNA repair (
      • Nishi R.
      • Wijnhoven P.
      • le Sage C.
      • Tjeertes J.
      • Galanty Y.
      • Forment J.V.
      • Clague M.J.
      • Urbe S.
      • Jackson S.P.
      Systematic characterization of deubiquitylating enzymes for roles in maintaining genome integrity.
      ); proteasome (
      • Yao T.
      • Song L.
      • Xu W.
      • DeMartino G.N.
      • Florens L.
      • Swanson S.K.
      • Washburn M.P.
      • Conaway R.C.
      • Conaway J.W.
      • Cohen R.E.
      Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1.
      )
      USP1Cysteine proteaseUSPmonoubiquitinDNA repair (
      • Nijman S.M.
      • Huang T.T.
      • Dirac A.M.
      • Brummelkamp T.R.
      • Kerkhoven R.M.
      • D'Andrea A.D.
      • Bernards R.
      The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.
      )
      USP4Cysteine proteaseUSPK48, K63RNA splicing (
      • Song E.J.
      • Werner S.L.
      • Neubauer J.
      • Stegmeier F.
      • Aspden J.
      • Rio D.
      • Harper J.W.
      • Elledge S.J.
      • Kirschner M.W.
      • Rape M.
      The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome.
      ); immune response (
      • Yang J.
      • Xu P.
      • Han L.
      • Guo Z.
      • Wang X.
      • Chen Z.
      • Nie J.
      • Yin S.
      • Piccioni M.
      • Tsun A.
      • Lv L.
      • Ge S.
      • Li B.
      Cutting edge: Ubiquitin-specific protease 4 promotes Th17 cell function under inflammation by deubiquitinating and stabilizing RORgammat.
      ); signaling (
      • Zhang L.
      • Zhou F.
      • Drabsch Y.
      • Gao R.
      • Snaar-Jagalska B.E.
      • Mickanin C.
      • Huang H.
      • Sheppard K.A.
      • Porter J.A.
      • Lu C.X.
      • ten Dijke P.
      USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-beta type I receptor.
      ,
      • Yun S.I.
      • Kim H.H.
      • Yoon J.H.
      • Park W.S.
      • Hahn M.J.
      • Kim H.C.
      • Chung C.H.
      • Kim K.K.
      Ubiquitin specific protease 4 positively regulates the WNT/beta-catenin signaling in colorectal cancer.
      ,
      • Xiao N.
      • Li H.
      • Luo J.
      • Wang R.
      • Chen H.
      • Chen J.
      • Wang P.
      Ubiquitin-specific protease 4 (USP4) targets TRAF2 and TRAF6 for deubiquitination and inhibits TNFalpha-induced cancer cell migration.
      )
      USP5Cysteine proteaseUSPK48DNA repair (
      • Nakajima S.
      • Lan L.
      • Wei L.
      • Hsieh C.L.
      • Rapic-Otrin V.
      • Yasui A.
      • Levine A.S.
      Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.
      ); immune response (
      • Kummari E.
      • Alugubelly N.
      • Hsu C.Y.
      • Dong B.
      • Nanduri B.
      • Edelmann M.J.
      Activity-based proteomic profiling of deubiquitinating enzymes in salmonella-infected macrophages leads to identification of putative function of UCH-L5 in inflammasome regulation.
      )
      USP7Cysteine proteaseUSPK48, K63Autophagy (
      • Cui L.
      • Song W.
      • Zeng Y.
      • Wu Q.
      • Fan Z.
      • Huang T.
      • Zeng B.
      • Zhang M.
      • Ni Q.
      • Li Y.
      • Wang T.
      • Li D.
      • Mao X.
      • Lian T.
      • Yang D.
      • et al.
      Deubiquitinase USP7 regulates Drosophila aging through ubiquitination and autophagy.
      ); protein transport (
      • Hao Y.H.
      • Fountain Jr., M.D.
      • Fon Tacer K.
      • Xia F.
      • Bi W.
      • Kang S.H.
      • Patel A.
      • Rosenfeld J.A.
      • Le Caignec C.
      • Isidor B.
      • Krantz I.D.
      • Noon S.E.
      • Pfotenhauer J.P.
      • Morgan T.M.
      • Moran R.
      • et al.
      USP7 acts as a molecular rheostat to promote WASH-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder.
      ); DNA repair (
      • Pozhidaeva A.
      • Bezsonova I.
      USP7: Structure, substrate specificity, and inhibition.
      ); signaling (
      • An T.
      • Gong Y.
      • Li X.
      • Kong L.
      • Ma P.
      • Gong L.
      • Zhu H.
      • Yu C.
      • Liu J.
      • Zhou H.
      • Mao B.
      • Li Y.
      USP7 inhibitor P5091 inhibits Wnt signaling and colorectal tumor growth.
      )
      USP8Cysteine proteaseUSPK48, K63Protein sorting (
      • Alexopoulou Z.
      • Lang J.
      • Perrett R.M.
      • Elschami M.
      • Hurry M.E.
      • Kim H.T.
      • Mazaraki D.
      • Szabo A.
      • Kessler B.M.
      • Goldberg A.L.
      • Ansorge O.
      • Fulga T.A.
      • Tofaris G.K.
      Deubiquitinase Usp8 regulates alpha-synuclein clearance and modifies its toxicity in Lewy body disease.
      ); cell cycle (
      • Martins C.S.
      • Camargo R.C.
      • Coeli-Lacchini F.B.
      • Saggioro F.P.
      • Moreira A.C.
      • de Castro M.
      USP8 mutations and cell cycle regulation in corticotroph adenomas.
      ); DNA repair (
      • Ge C.
      • Che L.
      • Ren J.
      • Pandita R.K.
      • Lu J.
      • Li K.
      • Pandita T.K.
      • Du C.
      BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1.
      )
      USP10Cysteine proteaseUSPAutophagy (
      • Yuan J.
      • Luo K.
      • Zhang L.
      • Cheville J.C.
      • Lou Z.
      USP10 regulates p53 localization and stability by deubiquitinating p53.
      ); DNA repair (
      • Emami S.
      Interplay between p53-family, their regulators, and PARPs in DNA repair.
      ); signaling (
      • Lim R.
      • Sugino T.
      • Nolte H.
      • Andrade J.
      • Zimmermann B.
      • Shi C.
      • Doddaballapur A.
      • Ong Y.T.
      • Wilhelm K.
      • Fasse J.W.D.
      • Ernst A.
      • Kaulich M.
      • Husnjak K.
      • Boettger T.
      • Guenther S.
      • et al.
      Deubiquitinase USP10 regulates Notch signaling in the endothelium.
      ); translation (
      • Meyer C.
      • Garzia A.
      • Morozov P.
      • Molina H.
      • Tuschl T.
      The G3BP1-family-USP10 deubiquitinase complex rescues ubiquitinated 40S subunits of ribosomes stalled in translation from lysosomal degradation.
      )
      USP12Cysteine proteaseUSPAutophagy (
      • Aron R.
      • Pellegrini P.
      • Green E.W.
      • Maddison D.C.
      • Opoku-Nsiah K.
      • Oliveira A.O.
      • Wong J.S.
      • Daub A.C.
      • Giorgini F.
      • Muchowski P.
      • Finkbeiner S.
      Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.
      ); cell cycle (
      • Tang L.J.
      • Li Y.
      • Liu Y.L.
      • Wang J.M.
      • Liu D.W.
      • Tian Q.B.
      USP12 regulates cell cycle progression by involving c-Myc, cyclin D2 and BMI-1.
      )
      USP14Cysteine proteaseUSPDNA repair (
      • Sharma A.
      • Alswillah T.
      • Singh K.
      • Chatterjee P.
      • Willard B.
      • Venere M.
      • Summers M.K.
      • Almasan A.
      USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination.
      ); proteasome (
      • Hu M.
      • Li P.
      • Song L.
      • Jeffrey P.D.
      • Chenova T.A.
      • Wilkinson K.D.
      • Cohen R.E.
      • Shi Y.
      Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14.
      ); chemotaxis (
      • Mines M.A.
      • Goodwin J.S.
      • Limbird L.E.
      • Cui F.F.
      • Fan G.H.
      Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK ativation.
      )
      USP19Cysteine proteaseUSPK63Cell cycle (
      • Lu Y.
      • Adegoke O.A.
      • Nepveu A.
      • Nakayama K.I.
      • Bedard N.
      • Cheng D.
      • Peng J.
      • Wing S.S.
      USP19 deubiquitinating enzyme supports cell proliferation by stabilizing KPC1, a ubiquitin ligase for p27Kip1.
      ); DNA repair (
      • Wu M.
      • Tu H.Q.
      • Chang Y.
      • Tan B.
      • Wang G.
      • Zhou J.
      • Wang L.
      • Mu R.
      • Zhang W.N.
      USP19 deubiquitinates HDAC1/2 to regulate DNA damage repair and control chromosomal stability.
      ); ER-associated degradation (
      • Hassink G.C.
      • Zhao B.
      • Sompallae R.
      • Altun M.
      • Gastaldello S.
      • Zinin N.V.
      • Masucci M.G.
      • Lindsten K.
      The ER-resident ubiquitin-specific protease 19 participates in the UPR and rescues ERAD substrates.
      )
      USP21Cysteine proteaseUSPCell cycle (
      • Arceci A.
      • Bonacci T.
      • Wang X.
      • Stewart K.
      • Damrauer J.S.
      • Hoadley K.A.
      • Emanuele M.J.
      FOXM1 deubiquitination by USP21 regulates cell cycle progression and paclitaxel sensitivity in basal-like breast cancer.
      ); transcription (
      • Heride C.
      • Rigden D.J.
      • Bertsoulaki E.
      • Cucchi D.
      • De Smaele E.
      • Clague M.J.
      • Urbe S.
      The centrosomal deubiquitylase USP21 regulates Gli1 transcriptional activity and stability.
      ); translation (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      )
      USP25Cysteine proteaseUSPK48, K63ER-associated degradation (
      • Blount J.R.
      • Burr A.A.
      • Denuc A.
      • Marfany G.
      • Todi S.V.
      Ubiquitin-specific protease 25 functions in endoplasmic reticulum-associated degradation.
      ); signaling (
      • Zhong B.
      • Liu X.
      • Wang X.
      • Liu X.
      • Li H.
      • Darnay B.G.
      • Lin X.
      • Sun S.C.
      • Dong C.
      Ubiquitin-specific protease 25 regulates TLR4-dependent innate immune responses through deubiquitination of the adaptor protein TRAF3.
      ,
      • Nino C.A.
      • Wollscheid N.
      • Giangreco G.
      • Maspero E.
      • Polo S.
      USP25 regulates EGFR fate by modulating EGF-induced ubiquitylation dynamics.
      ); trafficking (
      • Sadler J.B.A.
      • Lamb C.A.
      • Welburn C.R.
      • Adamson I.S.
      • Kioumourtzoglou D.
      • Chi N.W.
      • Gould G.W.
      • Bryant N.J.
      The deubiquitinating enzyme USP25 binds tankyrase and regulates trafficking of the facilitative glucose transporter GLUT4 in adipocytes.
      )
      USP28Cysteine proteaseUSPDNA repair (
      • Zhang D.
      • Zaugg K.
      • Mak T.W.
      • Elledge S.J.
      A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response.
      ); cell cycle (
      • Bassermann F.
      • Frescas D.
      • Guardavaccaro D.
      • Busino L.
      • Peschiaroli A.
      • Pagano M.
      The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint.
      )
      USP32Cysteine proteaseUSPCell proliferation (
      • Nakae A.
      • Kodama M.
      • Okamoto T.
      • Tokunaga M.
      • Shimura H.
      • Hashimoto K.
      • Sawada K.
      • Kodama T.
      • Copeland N.G.
      • Jenkins N.A.
      • Kimura T.
      Ubiquitin specific peptidase 32 acts as an oncogene in epithelial ovarian cancer by deubiquitylating farnesyl-diphosphate farnesyltransferase 1.
      ); trafficking (
      • Sapmaz A.
      • Berlin I.
      • Bos E.
      • Wijdeven R.H.
      • Janssen H.
      • Konietzny R.
      • Akkermans J.J.
      • Erson-Bensan A.E.
      • Koning R.I.
      • Kessler B.M.
      • Neefjes J.
      • Ovaa H.
      USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7.
      )
      USP37Cysteine proteaseUSPK11, K48Cell cycle (
      • Yeh C.
      • Coyaud E.
      • Bashkurov M.
      • van der Lelij P.
      • Cheung S.W.
      • Peters J.M.
      • Raught B.
      • Pelletier L.
      The deubiquitinase USP37 regulates chromosome cohesion and mitotic progression.
      ); DNA replication (
      • Hernandez-Perez S.
      • Cabrera E.
      • Amoedo H.
      • Rodriguez-Acebes S.
      • Koundrioukoff S.
      • Debatisse M.
      • Mendez J.
      • Freire R.
      USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.
      )
      USP46Cysteine proteaseUSPSignaling (
      • Hodul M.
      • Dahlberg C.L.
      • Juo P.
      Function of the deubiquitinating enzyme USP46 in the nervous system and its regulation by WD40-repeat proteins.
      ,
      • Wang W.
      • Chen M.
      • Xu H.
      • Lv D.
      • Zhou S.
      • Yang H.
      USP46 inhibits cell proliferation in lung cancer through PHLPP1/AKT pathway.
      )
      USP47Cysteine proteaseUSPDNA repair (
      • Parsons J.L.
      • Dianova I.I.
      • Khoronenkova S.V.
      • Edelmann M.J.
      • Kessler B.M.
      • Dianov G.L.
      USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase beta.
      ); cell cycle (
      • Peschiaroli A.
      • Skaar J.R.
      • Pagano M.
      • Melino G.
      The ubiquitin-specific protease USP47 is a novel beta-TRCP interactor regulating cell survival.
      )
      USP9XCysteine proteaseUSPK29, K33, K48Cell cycle (
      • Li X.
      • Song N.
      • Liu L.
      • Liu X.
      • Ding X.
      • Song X.
      • Yang S.
      • Shan L.
      • Zhou X.
      • Su D.
      • Wang Y.
      • Zhang Q.
      • Cao C.
      • Ma S.
      • Yu N.
      • et al.
      USP9X regulates centrosome duplication and promotes breast carcinogenesis.
      ); signaling (
      • Dietachmayr M.
      • Rathakrishnan A.
      • Karpiuk O.
      • von Zweydorf F.
      • Engleitner T.
      • Fernandez-Saiz V.
      • Schenk P.
      • Ueffing M.
      • Rad R.
      • Eilers M.
      • Gloeckner C.J.
      • Clemm von Hohenberg K.
      • Bassermann F.
      Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival.
      )
      ZUFSPCysteine proteaseZUFSPK11, K48, K63DNA repair and replication (
      • Kwasna D.
      • Abdul Rehman S.A.
      • Natarajan J.
      • Matthews S.
      • Madden R.
      • De Cesare V.
      • Weidlich S.
      • Virdee S.
      • Ahel I.
      • Gibbs-Seymour I.
      • Kulathu Y.
      Discovery and characterization of ZUFSP/ZUP1, a distinct deubiquitinase class important for genome stability.
      )
      Abbreviations: BRCC36, BRCA1/BRCA2-containing complex subunit 3; CSN5, COP9 signalosome complex subunit 5; JOSD, Josephin domain-containing; MYSM1, Myb-like, SWIRM and MPN domains 1; ZUFSP, zinc finger with UFM1-specific peptidase domain.

      DUB classification

      There are two major classes of DUBs, cysteine proteases and metalloproteases (
      • Ambroggio X.I.
      • Rees D.C.
      • Deshaies R.J.
      JAMM: A metalloprotease-like zinc site in the proteasome and signalosome.
      ,
      • Amerik A.Y.
      • Hochstrasser M.
      Mechanism and function of deubiquitinating enzymes.
      ). The former of these classes is further broken down into six families of proteins, based on sequence conservation and domain architecture (
      • Li Y.
      • Reverter D.
      Molecular mechanisms of DUBs regulation in signaling and disease.
      ). However, all DUBs in this class utilize a catalytic triad composed of an active site cysteine residue, along with a histidine and (in most cases) an asparagine or aspartate, to catalyze the hydrolysis of the ubiquitin linkages (
      • Amerik A.Y.
      • Hochstrasser M.
      Mechanism and function of deubiquitinating enzymes.
      ). Because of this catalytic cysteine, this class of DUBs can be regulated by reactive oxygen species (ROS) during oxidative stress, adding to the complexity of mechanisms by which DUBs may be regulated (
      • Cotto-Rios X.M.
      • Békés M.
      • Chapman J.
      • Ueberheide B.
      • Huang T.T.
      Deubiquitinases as a signaling target of oxidative stress.
      ,
      • Lee J.G.
      • Baek K.
      • Soetandyo N.
      • Ye Y.
      Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells.
      ,
      • Kulathu Y.
      • Garcia F.J.
      • Mevissen T.E.
      • Busch M.
      • Arnaudo N.
      • Carroll K.S.
      • Barford D.
      • Komander D.
      Regulation of A20 and other OTU deubiquitinases by reversible oxidation.
      ). Instead of a catalytic cysteine, metalloprotease DUBs catalyze isopeptide hydrolysis via a catalytic serine and a zinc ion cofactor (
      • Ambroggio X.I.
      • Rees D.C.
      • Deshaies R.J.
      JAMM: A metalloprotease-like zinc site in the proteasome and signalosome.
      ,
      • Shrestha R.K.
      • Ronau J.A.
      • Davies C.W.
      • Guenette R.G.
      • Strieter E.R.
      • Paul L.N.
      • Das C.
      Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.
      ). While the activity of DUBs from both families can be regulated, this difference in catalytic mechanism makes metalloproteases more resistant to oxidative stress (
      • Cotto-Rios X.M.
      • Békés M.
      • Chapman J.
      • Ueberheide B.
      • Huang T.T.
      Deubiquitinases as a signaling target of oxidative stress.
      ). The characteristics of both the cysteine protease and metalloprotease classes of DUBs are discussed further below.

      Cysteine proteases

      The cysteine protease class of DUBs is by far the most well studied of the two. This is due not only to their greater number, with over 80 known in humans, but also to them being the first DUBs to be structurally and mechanistically characterized, which led to the development of inhibitors designed to target the catalytic site (
      • Johnston S.C.
      • Larsen C.N.
      • Cook W.J.
      • Wilkinson K.D.
      • Hill C.P.
      Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.
      ,
      • Pickart C.M.
      • Rose I.A.
      Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin.
      ,
      • Schauer N.J.
      • Magin R.S.
      • Liu X.
      • Doherty L.M.
      • Buhrlage S.J.
      Advances in discovering deubiquitinating enzyme (DUB) inhibitors.
      ). Having inhibitors to this class of DUBs enabled faster identification and characterization of new members, thus resulting in better knowledge of their impact on cellular physiology and how they might be used in the manipulation of therapeutic systems (
      • Schauer N.J.
      • Magin R.S.
      • Liu X.
      • Doherty L.M.
      • Buhrlage S.J.
      Advances in discovering deubiquitinating enzyme (DUB) inhibitors.
      ). The six families of cysteine protease DUBs include the ubiquitin C-terminal hydrolase (UCH), ubiquitin-specific protease (USP), ovarian tumor (OTU), Machado–Josephin domain (MJD), K48 polyubiquitin-specific MINDY domain families, as well as the newest-discovered DUB family, zinc finger with UFM1-specific peptidase domain, named for its founding protein member (
      • Li Y.
      • Reverter D.
      Molecular mechanisms of DUBs regulation in signaling and disease.
      ).
      The UCH family of DUBs possesses four mammalian members (UCHL1, UCHL3, UCHL5, and BAP1) with UCHL3 being the first DUB to be structurally characterized (
      • Johnston S.C.
      • Larsen C.N.
      • Cook W.J.
      • Wilkinson K.D.
      • Hill C.P.
      Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.
      ). A primary feature of DUBs from the UCH family is a loop structure that covers the active site, limiting the size of substrate with which they can interact to small peptides, such as those that result from proteasomal or lysosomal degradation (
      • Johnston S.C.
      • Larsen C.N.
      • Cook W.J.
      • Wilkinson K.D.
      • Hill C.P.
      Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.
      ,
      • Johnston S.C.
      • Riddle S.M.
      • Cohen R.E.
      • Hill C.P.
      Structural basis for the specificity of ubiquitin C-terminal hydrolases.
      ). Some larger substrates, however, may be accommodated through unfolding (
      • Johnston S.C.
      • Riddle S.M.
      • Cohen R.E.
      • Hill C.P.
      Structural basis for the specificity of ubiquitin C-terminal hydrolases.
      ). One UCH DUB, BAP1, is involved in regulation of the cell cycle and DNA damage response (
      • Yu H.
      • Mashtalir N.
      • Daou S.
      • Hammond-Martel I.
      • Ross J.
      • Sui G.
      • Hart G.W.
      • Rauscher 3rd, F.J.
      • Drobetsky E.
      • Milot E.
      • Shi Y.
      • Affar El B.
      The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression.
      ). Germline mutations in BAP1, causing loss of activity, result in a predisposition to malignant tumors, such as malignant melanoma or renal cell carcinoma (
      • Masoomian B.
      • Shields J.A.
      • Shields C.L.
      Overview of BAP1 cancer predisposition syndrome and the relationship to uveal melanoma.
      ).
      The USP family of DUBs is the largest, comprised of 58 members in the human genome. Members of this family have a structure described as being in the form of a hand, with three subdomains: the thumb, palm, and fingers (Fig. 3) (
      • Hu M.
      • Li P.
      • Li M.
      • Li W.
      • Yao T.
      • Wu J.W.
      • Gu W.
      • Cohen R.E.
      • Shi Y.
      Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde.
      ,
      • Hu M.
      • Li P.
      • Song L.
      • Jeffrey P.D.
      • Chenova T.A.
      • Wilkinson K.D.
      • Cohen R.E.
      • Shi Y.
      Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14.
      ,
      • Renatus M.
      • Parrado S.G.
      • D'Arcy A.
      • Eidhoff U.
      • Gerhartz B.
      • Hassiepen U.
      • Pierrat B.
      • Riedl R.
      • Vinzenz D.
      • Worpenberg S.
      • Kroemer M.
      Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.
      ). The catalytic site sits between the thumb and palm subdomains, while the fingers are responsible for stabilizing the interaction with distal ubiquitin on substrates (
      • Hu M.
      • Li P.
      • Li M.
      • Li W.
      • Yao T.
      • Wu J.W.
      • Gu W.
      • Cohen R.E.
      • Shi Y.
      Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde.
      ).
      Figure thumbnail gr3
      Figure 3Conformations of cysteine proteases. Inactive (A, PDB: 1NB8) and active (B, PDB: 1NBF) conformations of the USP family cysteine protease USP7 are shown in cyan. Below are enlarged views of the catalytic triad positions. In the inactive state (A), the catalytic cysteine (Cys223) is positioned far from the other members of the catalytic triad (His464 and Asp481). This prevents the histidine from lowering the pKa to deprotonate the thiol of the cysteine and promote the active state. In the active state (B), USP7 is bound to a ubiquitin substrate, which induces a conformational shift that brings the catalytic cysteine closer to the other members of the catalytic triad, enabling its deprotonation into a reactive thiolate. In either case, the fingers domain (containing the ubiquitin interaction motif), as well as the palm and thumb domains (containing the catalytic center) are indicated.
      There are 17 members of the OTU family of DUBs, most of which display linkage specificity for ubiquitin substrates (
      • Mevissen T.E.
      • Hospenthal M.K.
      • Geurink P.P.
      • Elliott P.R.
      • Akutsu M.
      • Arnaudo N.
      • Ekkebus R.
      • Kulathu Y.
      • Wauer T.
      • El Oualid F.
      • Freund S.M.
      • Ovaa H.
      • Komander D.
      OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis.
      ). For example, ovarian tumor deubiquitinase, ubiquitin aldehyde binding (OTUB1) is specific for K48 chains, while Cezanne is specific for K11 chains, and ovarian tumor deubiquitinase (OTUD2) demonstrates activity for K11, K27, and K33 chains (
      • Mevissen T.E.
      • Hospenthal M.K.
      • Geurink P.P.
      • Elliott P.R.
      • Akutsu M.
      • Arnaudo N.
      • Ekkebus R.
      • Kulathu Y.
      • Wauer T.
      • El Oualid F.
      • Freund S.M.
      • Ovaa H.
      • Komander D.
      OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis.
      ,
      • Edelmann M.J.
      • Iphofer A.
      • Akutsu M.
      • Altun M.
      • di Gleria K.
      • Kramer H.B.
      • Fiebiger E.
      • Dhe-Paganon S.
      • Kessler B.M.
      Structural basis and specificity of human otubain 1-mediated deubiquitination.
      ,
      • Bremm A.
      • Freund S.M.
      • Komander D.
      Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne.
      ). This specificity is achieved through use of additional ubiquitin interaction sites that enable binding to longer chains of specific linkages, and it has led to OTU DUBs being known for regulating signaling pathways (
      • Mevissen T.E.
      • Hospenthal M.K.
      • Geurink P.P.
      • Elliott P.R.
      • Akutsu M.
      • Arnaudo N.
      • Ekkebus R.
      • Kulathu Y.
      • Wauer T.
      • El Oualid F.
      • Freund S.M.
      • Ovaa H.
      • Komander D.
      OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis.
      ). Another interesting aspect of the OTU family of DUBs is that some lack the asparagine or aspartate residue of the catalytic triad in DUBs (
      • Nijman S.M.
      • Luna-Vargas M.P.
      • Velds A.
      • Brummelkamp T.R.
      • Dirac A.M.
      • Sixma T.K.
      • Bernards R.
      A genomic and functional inventory of deubiquitinating enzymes.
      ). While some of these, like OTUB2, are predicted to be inactive, resulting from the absence of the negatively charged member of the catalytic triad to polarize the histidine, tumor necrosis factor alpha–induced protein 3 (A20) has been demonstrated to retain activity after an induced mutation of its catalytic aspartate residue (
      • Nanao M.H.
      • Tcherniuk S.O.
      • Chroboczek J.
      • Dideberg O.
      • Dessen A.
      • Balakirev M.Y.
      Crystal structure of human otubain 2.
      ,
      • Lin S.C.
      • Chung J.Y.
      • Lamothe B.
      • Rajashankar K.
      • Lu M.
      • Lo Y.C.
      • Lam A.Y.
      • Darnay B.G.
      • Wu H.
      Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20.
      ).
      The four members of the MJD family (ataxin 3 [ATXN3], ATXN3L, Josephin domain-containing 1, and Josephin domain-containing 2) all present a highly conserved catalytic Josephin domain, which contains two ubiquitin binding sites as well as two conserved histidines, along with the catalytic cysteine, that are necessary for catalysis (
      • Li Y.
      • Reverter D.
      Molecular mechanisms of DUBs regulation in signaling and disease.
      ,
      • Albrecht M.
      • Golatta M.
      • Wullner U.
      • Lengauer T.
      Structural and functional analysis of ataxin-2 and ataxin-3.
      ). This family is named for Machado–Josephin disease, a neurological disorder caused by an expansion of the CAG repeat motif in ATXN3, producing polyglutamine and causing protein misfolding and aggregation (
      • Kawaguchi Y.
      • Okamoto T.
      • Taniwaki M.
      • Aizawa M.
      • Inoue M.
      • Katayama S.
      • Kawakami H.
      • Nakamura S.
      • Nishimura M.
      • Akiguchi I.
      • Kimura J.
      • Narumiya S.
      • Kakizuka A.
      CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1.
      ). They are completely absent in yeast, presumably evolving to function in higher eukaryotes.
      The MINDY family of DUBs, which contains five members (MINDY1–4 and MINDY4B), has a unique catalytic triad of Cys, His, and Gln and is highly specific for K48 ubiquitin linkages (
      • Li Y.
      • Reverter D.
      Molecular mechanisms of DUBs regulation in signaling and disease.
      ,
      • Maurer T.
      • Wertz I.E.
      Length matters: MINDY is a new deubiquitinase family that preferentially cleaves long polyubiquitin chains.
      ). The members of this family have also been demonstrated to be autoinhibited prior to substrate binding, which causes a conformational shift to activate the enzyme (
      • Abdul Rehman S.A.
      • Kristariyanto Y.A.
      • Choi S.Y.
      • Nkosi P.J.
      • Weidlich S.
      • Labib K.
      • Hofmann K.
      • Kulathu Y.
      MINDY-1 is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes.
      ).
      Finally, the newest family, zinc finger with UFM1-specific peptidase domain, contains only one member of the same name, which has two ubiquitin-binding domains, both of which are required for its highly specific cleavage of K63 linkages (
      • Kwasna D.
      • Abdul Rehman S.A.
      • Natarajan J.
      • Matthews S.
      • Madden R.
      • De Cesare V.
      • Weidlich S.
      • Virdee S.
      • Ahel I.
      • Gibbs-Seymour I.
      • Kulathu Y.
      Discovery and characterization of ZUFSP/ZUP1, a distinct deubiquitinase class important for genome stability.
      ). Despite the variations in domain architectures and amino acid sequences, all of these families of DUBs have a catalytic core comprised of a cysteine and a histidine residue (
      • Li Y.
      • Reverter D.
      Molecular mechanisms of DUBs regulation in signaling and disease.
      ,
      • Suresh H.G.
      • Pascoe N.
      • Andrews B.
      The structure and function of deubiquitinases: Lessons from budding yeast.
      ).
      Mechanistically, cysteine protease enzymes rely on a reactive cysteine residue in the catalytic site (Fig. 3) (
      • Li Y.
      • Reverter D.
      Molecular mechanisms of DUBs regulation in signaling and disease.
      ,
      • Suresh H.G.
      • Pascoe N.
      • Andrews B.
      The structure and function of deubiquitinases: Lessons from budding yeast.
      ). This active/inactive state of cysteine protease DUBs is dependent on whether the cysteine contains an inactive thiol (-SH) or reactive thiolate (-S) group (
      • Ronau J.A.
      • Beckmann J.F.
      • Hochstrasser M.
      Substrate specificity of the ubiquitin and Ubl proteases.
      ). Transition between these states usually occurs through conformational shifts caused by substrate binding, affiliation with a protein complex, or PTM of the DUB itself (Fig. 3B) (
      • Komander D.
      • Clague M.J.
      • Urbe S.
      Breaking the chains: Structure and function of the deubiquitinases.
      ,
      • Hu M.
      • Li P.
      • Li M.
      • Li W.
      • Yao T.
      • Wu J.W.
      • Gu W.
      • Cohen R.E.
      • Shi Y.
      Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde.
      ,
      • Boudreaux D.A.
      • Maiti T.K.
      • Davies C.W.
      • Das C.
      Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation.
      ,
      • Maiti T.K.
      • Permaul M.
      • Boudreaux D.A.
      • Mahanic C.
      • Mauney S.
      • Das C.
      Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme.
      ). This conformational shift results in the polarization of the active site histidine, often achieved by the presence of an asparagine or aspartate residue (
      • Nijman S.M.
      • Luna-Vargas M.P.
      • Velds A.
      • Brummelkamp T.R.
      • Dirac A.M.
      • Sixma T.K.
      • Bernards R.
      A genomic and functional inventory of deubiquitinating enzymes.
      ,
      • Eletr Z.M.
      • Wilkinson K.D.
      Regulation of proteolysis by human deubiquitinating enzymes.
      ). This polarization lowers the pKa of the cysteine, causing deprotonation of the thiol and stabilization of the thiolate (Fig. 4) (
      • Komander D.
      Mechanism, specificity and structure of the deubiquitinases.
      ,
      • Suresh H.G.
      • Pascoe N.
      • Andrews B.
      The structure and function of deubiquitinases: Lessons from budding yeast.
      ). Once in the active thiolate state, the cysteine is able to undergo a nucleophilic attack on the isopeptide bond linking ubiquitin to its substrate or on the polyubiquitin chain, forming a thioester intermediate with the substrate, prior to release and reactivation of the DUB (Fig. 4) (
      • Komander D.
      Mechanism, specificity and structure of the deubiquitinases.
      ,
      • Zhang W.
      • Sulea T.
      • Tao L.
      • Cui Q.
      • Purisima E.O.
      • Vongsamphanh R.
      • Lachance P.
      • Lytvyn V.
      • Qi H.
      • Li Y.
      • Menard R.
      Contribution of active site residues to substrate hydrolysis by USP2: Insights into catalysis by ubiquitin specific proteases.
      ). Substrate release occurs when the DUB hydrolyzes the bonds of the ubiquitin/DUB intermediate, and the DUB resets back to a thiolate, ready to begin a new enzymatic cycle (
      • Komander D.
      Mechanism, specificity and structure of the deubiquitinases.
      ,
      • Zhang W.
      • Sulea T.
      • Tao L.
      • Cui Q.
      • Purisima E.O.
      • Vongsamphanh R.
      • Lachance P.
      • Lytvyn V.
      • Qi H.
      • Li Y.
      • Menard R.
      Contribution of active site residues to substrate hydrolysis by USP2: Insights into catalysis by ubiquitin specific proteases.
      ).
      Figure thumbnail gr4
      Figure 4DUB cysteine protease catalytic mechanism. A generalized mechanism of cysteine protease DUBs is shown, including the three most common members of the catalytic triad (Cys, His, and Asp) and the isopeptide bond of the ubiquitinated substrate. The general steps of the mechanism are as follows: 1, the histidine, depolarized by the aspartate, deprotonates the cysteine, converting its side chain from an inactive thiol to a reactive thiolate. 2, the thiolate of cysteine undergoes a nucleophilic attack on the acyl group of the ubiquitin isopeptide bond, forming a tetrameric intermediate. 3, the isopeptide bond is cleaved as the amide group of the isopeptide bond deprotonates the DUB histidine, freeing the substrate from ubiquitin, which is still bound as an intermediate with the DUB cysteine. 4, hydration of the DUB cysteine acyl intermediate utilizing a water molecule to convert the acyl intermediate to a carboxyl intermediate. 5, the intermediate bond between the ubiquitin and DUB cysteine is broken, reforming the ubiquitin monomer and thiolate. 6, the ubiquitin monomer and substrate are released from the DUB.
      One reason proposed for the inactive conformation of cysteine proteases is to protect the DUBs from oxidation by maintaining the cysteine in the inactive thiol form (
      • Lee J.G.
      • Baek K.
      • Soetandyo N.
      • Ye Y.
      Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells.
      ). This does not leave the cysteine immune to oxidation, but it does lessen its reactivity, and therefore may offer some amount of protection from oxidative stress. Because of the reactivity of cysteines with ROS, all cysteine proteases, including DUBs, are potentially susceptible to inhibition, due to covalent modification in the active site (
      • Turell L.
      • Zeida A.
      • Trujillo M.
      Mechanisms and consequences of protein cysteine oxidation: The role of the initial short-lived intermediates.
      ). This means that, during times of oxidative stress, a large number of DUBs may be rapidly inhibited through cysteine oxidation, increasing the amount of ubiquitin conjugation that occurs throughout the cell. During homeostasis, a number of substrates are regularly ubiquitinated and deubiquitinated (
      • Haglund K.
      • Dikic I.
      Ubiquitylation and cell signaling.
      ). In response to oxidative stress, accumulation of ubiquitin conjugates will derive from inhibition of DUBs, based on their varied ROS sensitivity, leading to modulation of pathways in accordance with cellular needs for oxidative stress response (
      • Lee J.G.
      • Baek K.
      • Soetandyo N.
      • Ye Y.
      Reversible inactivation of deubiquitinases by reactive oxygen species in vitro and in cells.
      ). In addition, other targets might accumulate due to proteasome inhibition (
      • Ding Q.
      • Keller J.N.
      Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins.
      ) or increased activity of E2s and E3s (
      • Obin M.
      • Shang F.
      • Gong X.
      • Handelman G.
      • Blumberg J.
      • Taylor A.
      Redox regulation of ubiquitin-conjugating enzymes: Mechanistic insights using the thiol-specific oxidant diamide.
      ). Although E2s and E3s are themselves enzymes containing a catalytic cysteine, only two yeast E2s, out of 12 known to exist, have been shown to be regulated under oxidative stress, adding to the balance of ubiquitin conjugates (
      • Simões V.
      • Harley L.
      • Cizubu B.K.
      • Zhou Y.
      • Pajak J.
      • Snyder N.A.
      • Bouvette J.
      • Borgnia M.J.
      • Arya G.
      • Bartesaghi A.
      • Silva G.M.
      Redox sensitive E2 Rad6 controls cellular response to oxidative stress via K63 ubiquitination of ribosomes.
      ,
      • Doris K.S.
      • Rumsby E.L.
      • Morgan B.A.
      Oxidative stress responses involve oxidation of a conserved ubiquitin pathway enzyme.
      ). However, the regulation of these enzymes is also only beginning to be elucidated.

      Metalloproteases

      The metalloprotease class of DUBs are comprised of zinc-dependent enzymes with JAB1/MPN/MOV34 (JAMM/MPN+) domains (
      • Ambroggio X.I.
      • Rees D.C.
      • Deshaies R.J.
      JAMM: A metalloprotease-like zinc site in the proteasome and signalosome.
      ,
      • Tran H.J.
      • Allen M.D.
      • Lowe J.
      • Bycroft M.
      Structure of the Jab1/MPN domain and its implications for proteasome function.
      ). The human genome encodes 14 genes with this domain, of which seven are capable of coordinating the catalytically required zinc ion and only six have been demonstrated to possess the ability to hydrolyze ubiquitin conjugates (AMSH, AMSH-LP, BRCA1/BRCA2-containing complex subunit 3, COP9 signalosome complex subunit 5, Myb-like, SWIRM and MPN domains 1, and regulatory particle non-ATPase 11 [RPN11]) (
      • Shrestha R.K.
      • Ronau J.A.
      • Davies C.W.
      • Guenette R.G.
      • Strieter E.R.
      • Paul L.N.
      • Das C.
      Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.
      ,
      • Nijman S.M.
      • Luna-Vargas M.P.
      • Velds A.
      • Brummelkamp T.R.
      • Dirac A.M.
      • Sixma T.K.
      • Bernards R.
      A genomic and functional inventory of deubiquitinating enzymes.
      ,
      • Davies C.W.
      • Paul L.N.
      • Kim M.I.
      • Das C.
      Structural and thermodynamic comparison of the catalytic domain of AMSH and AMSH-LP: Nearly identical fold but different stability.
      ).
      One of the earliest and best-studied examples of DUB metalloproteases is RPN11, one of three DUBs associated with the proteasome (the other two are the cysteine proteases USP14 and UCHL5) (
      • Verma R.
      • Aravind L.
      • Oania R.
      • McDonald W.H.
      • Yates 3rd, J.R.
      • Koonin E.V.
      • Deshaies R.J.
      Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome.
      ,
      • Lee B.H.
      • Lee M.J.
      • Park S.
      • Oh D.C.
      • Elsasser S.
      • Chen P.C.
      • Gartner C.
      • Dimova N.
      • Hanna J.
      • Gygi S.P.
      • Wilson S.M.
      • King R.W.
      • Finley D.
      Enhancement of proteasome activity by a small-molecule inhibitor of USP14.
      ,
      • Yao T.
      • Song L.
      • Xu W.
      • DeMartino G.N.
      • Florens L.
      • Swanson S.K.
      • Washburn M.P.
      • Conaway R.C.
      • Conaway J.W.
      • Cohen R.E.
      Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1.
      ). Used to destroy and recycle damaged or unwanted proteins from the cell, the proteasome, a 26S protein complex, is comprised of a barrel-shaped 20S catalytic core, which is capped on one or both ends by different regulatory particles (
      • Arendt C.S.
      • Hochstrasser M.
      Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly.
      ,
      • Tanaka K.
      The proteasome: Overview of structure and functions.
      ). While the 20S core particle is responsible for carrying out proteolysis of substrates, the 19S regulatory particle provides the high specificity for ubiquitinated substrates, through ubiquitin-binding receptors (
      • Tanaka K.
      The proteasome: Overview of structure and functions.
      ,
      • Livneh I.
      • Cohen-Kaplan V.
      • Cohen-Rosenzweig C.
      • Avni N.
      • Ciechanover A.
      The life cycle of the 26S proteasome: From birth, through regulation and function, and onto its death.
      ). Prior to substrate degradation, the polyubiquitin chain is removed and recycled to the pool of free ubiquitin (
      • Yao T.
      • Cohen R.E.
      A cryptic protease couples deubiquitination and degradation by the proteasome.
      ). RPN11 cleaves the link between the substrate and the proximal ubiquitin of the chain, thus removing the entire polyubiquitin chain at once (
      • Verma R.
      • Aravind L.
      • Oania R.
      • McDonald W.H.
      • Yates 3rd, J.R.
      • Koonin E.V.
      • Deshaies R.J.
      Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome.
      ,
      • Yao T.
      • Cohen R.E.
      A cryptic protease couples deubiquitination and degradation by the proteasome.
      ).
      The activity of RPN11 is dependent on its interaction with the proteasome as well as the proteasome ATPase activity responsible for unfolding of the substrate (
      • Worden E.J.
      • Dong K.C.
      • Martin A.
      An AAA motor-driven mechanical switch in Rpn11 controls deubiquitination at the 26S proteasome.
      ,
      • Finley D.
      Recognition and processing of ubiquitin-protein conjugates by the proteasome.
      ). This dependency on substrate unfolding delays the activity of RPN11 until the substrate is fully committed to degradation in the proteasome (
      • Worden E.J.
      • Dong K.C.
      • Martin A.
      An AAA motor-driven mechanical switch in Rpn11 controls deubiquitination at the 26S proteasome.
      ). Failure of RPN11 to remove the polyubiquitin chain results in steric hindrance of the substrate into the 20S core particle and thus prevents protein degradation, making RPN11 essential to the ubiquitin-proteasome system (UPS) (
      • Worden E.J.
      • Dong K.C.
      • Martin A.
      An AAA motor-driven mechanical switch in Rpn11 controls deubiquitination at the 26S proteasome.
      ). Meanwhile, the cysteine proteases USP14 and UCHL5, the other DUBs of the proteasome, instead serve a regulatory role, trimming the ubiquitin from substrates to limit their degradation (
      • Lee B.H.
      • Lee M.J.
      • Park S.
      • Oh D.C.
      • Elsasser S.
      • Chen P.C.
      • Gartner C.
      • Dimova N.
      • Hanna J.
      • Gygi S.P.
      • Wilson S.M.
      • King R.W.
      • Finley D.
      Enhancement of proteasome activity by a small-molecule inhibitor of USP14.
      ,
      • Lee M.J.
      • Lee B.H.
      • Hanna J.
      • King R.W.
      • Finley D.
      Trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes.
      ).
      Another metalloprotease DUB, AMSH, is involved in regulating endosomal membrane trafficking by removing K63-linked ubiquitin from cargoes (
      • McCullough J.
      • Clague M.J.
      • Urbe S.
      AMSH is an endosome-associated ubiquitin isopeptidase.
      ,
      • McCullough J.
      • Row P.E.
      • Lorenzo O.
      • Doherty M.
      • Beynon R.
      • Clague M.J.
      • Urbe S.
      Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery.
      ). The activity of AMSH determines whether substrate cargoes are ultimately degraded or recycled by the ESCRT pathway (
      • McCullough J.
      • Row P.E.
      • Lorenzo O.
      • Doherty M.
      • Beynon R.
      • Clague M.J.
      • Urbe S.
      Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery.
      ). This is a critical pathway for neuronal development, and mutations in AMSH have been linked to the developmental disorder microcephaly-capillary malformation (MIC-CAP) syndrome, making AMSH an important target for therapeutic treatment (
      • McDonell L.M.
      • Mirzaa G.M.
      • Alcantara D.
      • Schwartzentruber J.
      • Carter M.T.
      • Lee L.J.
      • Clericuzio C.L.
      • Graham Jr., J.M.
      • Morris-Rosendahl D.J.
      • Polster T.
      • Acsadi G.
      • Townshend S.
      • Williams S.
      • Halbert A.
      • Isidor B.
      • et al.
      Mutations in STAMBP, encoding a deubiquitinating enzyme, cause microcephaly-capillary malformation syndrome.
      ).
      The catalytic sites of the DUB metalloprotease class contain an aspartate, a serine, and two histidine residues (
      • Ambroggio X.I.
      • Rees D.C.
      • Deshaies R.J.
      JAMM: A metalloprotease-like zinc site in the proteasome and signalosome.
      ,
      • Shrestha R.K.
      • Ronau J.A.
      • Davies C.W.
      • Guenette R.G.
      • Strieter E.R.
      • Paul L.N.
      • Das C.
      Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.
      ). The catalytic site also requires a zinc ion, which is coordinated by the aspartate and histidine residues, as well as a water molecule. The mechanism of action by the DUBs in this class utilizes a zinc ion to generate a hydroxide ion from water to hydrolyze the isopeptide bond between ubiquitin and substrate (
      • Ambroggio X.I.
      • Rees D.C.
      • Deshaies R.J.
      JAMM: A metalloprotease-like zinc site in the proteasome and signalosome.
      ). This mechanism means that the DUBs in this class never form a covalent intermediate between the enzyme and substrate, making them resistant to classical DUB inhibitors as well as redox reactions, which typically covalently target the catalytic cysteine of the other DUB families to inhibit DUB activity (
      • Mevissen T.E.T.
      • Komander D.
      Mechanisms of deubiquitinase specificity and regulation.
      ).

      Cellular functions regulated by DUBs

      We have thus far presented a general overview of DUBs and the catalytic mechanisms through which they remove ubiquitin signals from substrates. However, DUBs do not only function to counteract ubiquitin signals. One of the most important functions of DUBs is to recycle and produce free ubiquitin (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ). This maintains a pool of ubiquitin from which the various ubiquitin enzymes may draw to target proteins for degradation or signaling (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ). The largest portion of this pool comes from both de novo synthesis of ubiquitin and from recycling of ubiquitin at the proteasome from proteins targeted for degradation (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ).
      In terms of protein synthesis, ubiquitin is unique in that it is not synthesized as a single protein (
      • Grou C.P.
      • Pinto M.P.
      • Mendes A.V.
      • Domingues P.
      • Azevedo J.E.
      The de novo synthesis of ubiquitin: Identification of deubiquitinases acting on ubiquitin precursors.
      ). Instead, it is encoded either with a C-terminally fused ribosomal protein, as is the case with UBA52 and UBA80 in mammals (Ubi1–3 in yeast), or as a ubiquitin polymer linked in a head-to-tail fashion followed by a variable length C-terminus (
      • Redman K.L.
      • Rechsteiner M.
      Identification of the long ubiquitin extension as ribosomal protein S27a.
      ,
      • Baker R.T.
      • Board P.G.
      The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.
      ,
      • Larsen C.N.
      • Krantz B.A.
      • Wilkinson K.D.
      Substrate specificity of deubiquitinating enzymes: Ubiquitin C-terminal hydrolases.
      ). UBA52 is a fusion of ubiquitin and the ribosomal protein eL40 while UBA80 is a fusion of ubiquitin and eS31 (
      • Baker R.T.
      • Board P.G.
      The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.
      ,
      • Kirschner L.S.
      • Stratakis C.A.
      Structure of the human ubiquitin fusion gene Uba80 (RPS27a) and one of its pseudogenes.
      ). For this reason, several DUBs, including UCHL3, USP9X, USP7, USP5, and Otulin, are required to hydrolyze these proteins releasing free ubiquitin monomers (
      • Grou C.P.
      • Pinto M.P.
      • Mendes A.V.
      • Domingues P.
      • Azevedo J.E.
      The de novo synthesis of ubiquitin: Identification of deubiquitinases acting on ubiquitin precursors.
      ). For the C-terminal fusion proteins, this occurs posttranslationally, while for ubiquitin polymers, it may occur either posttranslationally or cotranslationally (
      • Grou C.P.
      • Pinto M.P.
      • Mendes A.V.
      • Domingues P.
      • Azevedo J.E.
      The de novo synthesis of ubiquitin: Identification of deubiquitinases acting on ubiquitin precursors.
      ).
      Besides the production of free ubiquitin monomers from protein fusions and polymers, DUBs also recompose the pool of free ubiquitin by recycling it from substrates destined to degradation by the proteasome, as mentioned above (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ). Proteasome-associated DUBs, RPN11, USP14, and UCHL5, cleave the ubiquitin from the substrate, which is then unfolded by proteasomal ATPases and passed into the 20S core particle for degradation (
      • Tanaka K.
      The proteasome: Overview of structure and functions.
      ). USP14 (ubiquitin-binding protein [Ubp6] in yeast) and UCHL5 are cysteine proteases that bind to the regulatory particle of the proteasome and function to trim ubiquitin from substrates, potentially rescuing proteins from degradation (
      • de Poot S.A.H.
      • Tian G.
      • Finley D.
      Meddling with fate: The proteasomal deubiquitinating enzymes.
      ). Since RPN11 resides within the core particle in humans and yeast, it removes ubiquitin from substrates already committed to degradation (
      • Verma R.
      • Aravind L.
      • Oania R.
      • McDonald W.H.
      • Yates 3rd, J.R.
      • Koonin E.V.
      • Deshaies R.J.
      Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome.
      ). These DUBs thereby replenish the pool of free ubiquitin, rather than allow them to be degraded along with the substrate (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ). Since many DUBs are sensitive to oxidative stress, this can impact the DUB-dependent replenishment of the ubiquitin pool by simultaneously inhibiting DUBs that function at the synthesis and recycling steps (Fig. 1) (
      • Park C.W.
      • Ryu K.Y.
      Cellular ubiquitin pool dynamics and homeostasis.
      ). Without DUB-dependent replenishment of ubiquitin monomers, the amount of free ubiquitin would rapidly decrease as ubiquitin conjugation reactions occur, leading to limitation on the rapidity of ubiquitin signaling and widespread regulation of cellular pathways necessary to survival (
      • Haglund K.
      • Dikic I.
      Ubiquitylation and cell signaling.
      ).
      Another function of DUBs is to regulate the ubiquitin signaling network and thereby control a number of important cellular processes, including protein trafficking, DNA damage repair, and translation regulation (
      • Haglund K.
      • Dikic I.
      Ubiquitylation and cell signaling.
      ,
      • Millard S.M.
      • Wood S.A.
      Riding the DUBway: Regulation of protein trafficking by deubiquitylating enzymes.
      ,
      • Kee Y.
      • Huang T.T.
      Role of deubiquitinating enzymes in DNA repair.
      ,
      • Kapadia B.B.
      • Gartenhaus R.B.
      DUBbing down translation: The functional interaction of deubiquitinases with the translational machinery.
      ). These processes often require rapid and dynamic changes due to shifts in the cellular environment. Through modulation of DUB activity, proteins can be destabilized or stabilized rapidly through addition and removal of ubiquitin, respectively (
      • Komander D.
      Mechanism, specificity and structure of the deubiquitinases.
      ). This is particularly useful during cellular response to stress, where regulatory mechanisms occur, leading to activation or inhibition of DUBs required to stabilize stress factors or foster the degradation of unneeded proteins (
      • Mevissen T.E.T.
      • Komander D.
      Mechanisms of deubiquitinase specificity and regulation.
      ). Some highlights of the role of DUBs in these processes will be briefly discussed below.

      Protein trafficking

      Ubiquitin signaling plays a quite prominent role in trafficking (
      • Millard S.M.
      • Wood S.A.
      Riding the DUBway: Regulation of protein trafficking by deubiquitylating enzymes.
      ,
      • Clague M.J.
      • Urbe S.
      Endocytosis: The DUB version.
      ). Studies have shown that at the plasma membrane, ubiquitination occurs on adaptor proteins or even directly on cargoes to impact internalization (
      • Clague M.J.
      • Urbe S.
      Integration of cellular ubiquitin and membrane traffic systems: Focus on deubiquitylases.
      ,
      • Savio M.G.
      • Wollscheid N.
      • Cavallaro E.
      • Algisi V.
      • Di Fiore P.P.
      • Sigismund S.
      • Maspero E.
      • Polo S.
      USP9X controls EGFR fate by deubiquitinating the endocytic adaptor Eps15.
      ,
      • Foot N.
      • Henshall T.
      • Kumar S.
      Ubiquitination and the regulation of membrane proteins.
      ). Destabilization of adaptors by ubiquitin blocks internalization by preventing assembly of the core components of the endocytic machinery around the cargo (
      • Clague M.J.
      • Urbe S.
      Integration of cellular ubiquitin and membrane traffic systems: Focus on deubiquitylases.
      ). In contrast, ubiquitination of the cargoes induces internalization, targeting them to the endosome and, eventually, to destruction at the lysosome (
      • Piper R.C.
      • Dikic I.
      • Lukacs G.L.
      Ubiquitin-dependent sorting in endocytosis.
      ,
      • Cheng J.
      • Guggino W.
      Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.
      ,
      • Fujita H.
      • Iwabu Y.
      • Tokunaga K.
      • Tanaka Y.
      Membrane-associated RING-CH (MARCH) 8 mediates the ubiquitination and lysosomal degradation of the transferrin receptor.
      ,
      • Haglund K.
      • Sigismund S.
      • Polo S.
      • Szymkiewicz I.
      • Di Fiore P.P.
      • Dikic I.
      Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation.
      ). In this case, DUBs function to clear ubiquitin from the cargo, preventing its degradation and allowing it to undergo recycling to the surface (
      • Clague M.J.
      • Urbe S.
      Endocytosis: The DUB version.
      ,
      • Balut C.M.
      • Loch C.M.
      • Devor D.C.
      Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1.
      ). This is particularly important in cell surface receptor activation, as DUBs maintain the duration of signaling by preventing the degradation of cell surface receptor, as is the case with the DUB USP8 and epidermal growth factor receptor (EGFR) (
      • Berlin I.
      • Schwartz H.
      • Nash P.D.
      Regulation of epidermal growth factor receptor ubiquitination and trafficking by the USP8.STAM complex.
      ,
      • Hurley J.H.
      • Stenmark H.
      Molecular mechanisms of ubiquitin-dependent membrane traffic.
      ). DUBs, such as USP32 and USP7, can also influence intracellular membrane protein trafficking by similar methods (
      • Sapmaz A.
      • Berlin I.
      • Bos E.
      • Wijdeven R.H.
      • Janssen H.
      • Konietzny R.
      • Akkermans J.J.
      • Erson-Bensan A.E.
      • Koning R.I.
      • Kessler B.M.
      • Neefjes J.
      • Ovaa H.
      USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7.
      ,
      • Hao Y.H.
      • Fountain Jr., M.D.
      • Fon Tacer K.
      • Xia F.
      • Bi W.
      • Kang S.H.
      • Patel A.
      • Rosenfeld J.A.
      • Le Caignec C.
      • Isidor B.
      • Krantz I.D.
      • Noon S.E.
      • Pfotenhauer J.P.
      • Morgan T.M.
      • Moran R.
      • et al.
      USP7 acts as a molecular rheostat to promote WASH-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder.
      ).

      DNA damage repair

      DUBs function to regulate the DNA damage repair system, making it readily available during times of damage and stress (
      • Kee Y.
      • Huang T.T.
      Role of deubiquitinating enzymes in DNA repair.
      ,
      • He M.
      • Zhou Z.
      • Shah A.A.
      • Zou H.
      • Tao J.
      • Chen Q.
      • Wan Y.
      The emerging role of deubiquitinating enzymes in genomic integrity, diseases, and therapeutics.
      ). For example, under normal conditions, phosphorylated USP7 stabilizes the E3 ubiquitin ligase MDM2 (Mouse double minute 2), which ubiquitinates and targets p53 for degradation (
      • Sheng Y.
      • Saridakis V.
      • Sarkari F.
      • Duan S.
      • Wu T.
      • Arrowsmith C.H.
      • Frappier L.
      Molecular recognition of p53 and MDM2 by USP7/HAUSP.
      ,
      • Sarkari F.
      • La Delfa A.
      • Arrowsmith C.H.
      • Frappier L.
      • Sheng Y.
      • Saridakis V.
      Further insight into substrate recognition by USP7: Structural and biochemical analysis of the HdmX and Hdm2 interactions with USP7.
      ). Upon stress, USP7 is inactivated by dephosphorylation, which destabilizes MDM2, leading to increased levels of p53 (
      • Khoronenkova S.V.
      • Dianova I.I.
      • Ternette N.
      • Kessler B.M.
      • Parsons J.L.
      • Dianov G.L.
      ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.
      ). DUBs, like Ubp8 of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, also regulate the ubiquitination of histones and other DNA-binding proteins to increase or decrease access of stress/damage response elements to DNA (
      • Lee K.K.
      • Florens L.
      • Swanson S.K.
      • Washburn M.P.
      • Workman J.L.
      The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex.
      ). Finally, DUBs also play a role in stabilizing some of the response elements themselves, as is the case with USP47 and DNA polymerase β (Pol β), which is responsible for the DNA single-strand break and base excision repair mechanisms (
      • Parsons J.L.
      • Dianova I.I.
      • Khoronenkova S.V.
      • Edelmann M.J.
      • Kessler B.M.
      • Dianov G.L.
      USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase beta.
      ).

      Translation

      DUBs have also been found to influence translation by regulating ubiquitination of ribosomes themselves (
      • Kapadia B.B.
      • Gartenhaus R.B.
      DUBbing down translation: The functional interaction of deubiquitinases with the translational machinery.
      ). This places DUBs in an important position to regulate global protein synthesis. Recently, a new form of DUB regulation of ribosomes, termed RTU (redox control of translation by ubiquitin), has been discovered in yeast (
      • Dougherty S.E.
      • Maduka A.O.
      • Inada T.
      • Silva G.M.
      Expanding role of ubiquitin in translational control.
      ,
      • Silva G.M.
      • Finley D.
      • Vogel C.
      K63 polyubiquitination is a new modulator of the oxidative stress response.
      ). In the RTU, a DUB central to the pathway, Ubp2, becomes reversibly inhibited by ROS during oxidative stress (
      • Silva G.M.
      • Finley D.
      • Vogel C.
      K63 polyubiquitination is a new modulator of the oxidative stress response.
      ). This inhibition results in accumulation of K63-linked polyubiquitin chains on ribosomes mediated by the E2 Rad6 and E3 Bre1, leading to a buildup of polysomes, suggesting that this modification arrests translation elongation (
      • Simões V.
      • Harley L.
      • Cizubu B.K.
      • Zhou Y.
      • Pajak J.
      • Snyder N.A.
      • Bouvette J.
      • Borgnia M.J.
      • Arya G.
      • Bartesaghi A.
      • Silva G.M.
      Redox sensitive E2 Rad6 controls cellular response to oxidative stress via K63 ubiquitination of ribosomes.
      ,
      • Silva G.M.
      • Finley D.
      • Vogel C.
      K63 polyubiquitination is a new modulator of the oxidative stress response.
      ,
      • Zhou Y.
      • Kastritis P.L.
      • Dougherty S.E.
      • Bouvette J.
      • Hsu A.L.
      • Burbaum L.
      • Mosalaganti S.
      • Pfeffer S.
      • Hagen W.J.H.
      • Forster F.
      • Borgnia M.J.
      • Vogel C.
      • Beck M.
      • Bartesaghi A.
      • Silva G.M.
      Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.
      ,
      • Back S.
      • Gorman A.W.
      • Vogel C.
      • Silva G.M.
      Site-specific K63 ubiquitinomics provides insights into translation regulation under stress.
      ). Interestingly, deletion of UBP2 from yeast was found to increase the K63 ubiquitin signaling, even in the absence of oxidative stress (
      • Silva G.M.
      • Finley D.
      • Vogel C.
      K63 polyubiquitination is a new modulator of the oxidative stress response.
      ). This suggests that the K63 ubiquitination of the ribosome by Rad6/Bre1 may occur constitutively, but it is tightly regulated by Ubp2 (
      • Silva G.M.
      • Finley D.
      • Vogel C.
      K63 polyubiquitination is a new modulator of the oxidative stress response.
      ).
      K63-ubiquitinated ribosomes from cells undergoing oxidative stress are predominately isolated in a rotated pretranslocation state, suggesting that this stage of translation is lengthened or arrested (
      • Zhou Y.
      • Kastritis P.L.
      • Dougherty S.E.
      • Bouvette J.
      • Hsu A.L.
      • Burbaum L.
      • Mosalaganti S.
      • Pfeffer S.
      • Hagen W.J.H.
      • Forster F.
      • Borgnia M.J.
      • Vogel C.
      • Beck M.
      • Bartesaghi A.
      • Silva G.M.
      Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.
      ). As such, it is likely that ubiquitination of ribosomes occurs to reduce global translation during oxidative stress to reduce the synthesis of unneeded proteins and prevent accumulation of damaged ones. Other instances of DUBs influencing translation have been identified as well. USP21 and OTUD3 antagonize the ubiquitination of eS10 and uS10 that results from integrated stress response (ISR) and ribosome-associated quality control pathway (RQC) (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      ). These responses are induced by ribosome stalling, which occurs due to amino acid deprivation or defective mRNAs that lack 3′UTRs or stop codons (
      • Brandman O.
      • Stewart-Ornstein J.
      • Wong D.
      • Larson A.
      • Williams C.C.
      • Li G.W.
      • Zhou S.
      • King D.
      • Shen P.S.
      • Weibezahn J.
      • Dunn J.G.
      • Rouskin S.
      • Inada T.
      • Frost A.
      • Weissman J.S.
      A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.
      ). Ribosome ubiquitination by ISR and RQC results in ribosome dissociation and degradation of the associated mRNA and arrested peptide (
      • Brandman O.
      • Stewart-Ornstein J.
      • Wong D.
      • Larson A.
      • Williams C.C.
      • Li G.W.
      • Zhou S.
      • King D.
      • Shen P.S.
      • Weibezahn J.
      • Dunn J.G.
      • Rouskin S.
      • Inada T.
      • Frost A.
      • Weissman J.S.
      A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.
      ,
      • Pisareva V.P.
      • Skabkin M.A.
      • Hellen C.U.
      • Pestova T.V.
      • Pisarev A.V.
      Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes.
      ,
      • Shoemaker C.J.
      • Eyler D.E.
      • Green R.
      Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay.
      ).
      The small subunit of the ribosome, which is ubiquitinated during quality control processes, is rescued from degradation by the activity of the DUB USP10, which removes ubiquitin from the ribosomal proteins eS10, uS3, and uS5, allowing recycling of the small subunit for new translation reactions (
      • Meyer C.
      • Garzia A.
      • Morozov P.
      • Molina H.
      • Tuschl T.
      The G3BP1-family-USP10 deubiquitinase complex rescues ubiquitinated 40S subunits of ribosomes stalled in translation from lysosomal degradation.
      ). Other DUBs are also involved in this quality control pathway, including OTUB2, OTUD1, and UCHL1 although their roles are not yet fully understood (
      • Garshott D.M.
      • Sundaramoorthy E.
      • Leonard M.
      • Bennett E.J.
      Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized.
      ). Finally, Ubp3 and Otu2 in yeast are required for efficient translation (
      • Takehara Y.
      • Yashiroda H.
      • Matsuo Y.
      • Zhao X.
      • Kamigaki A.
      • Matsuzaki T.
      • Kosako H.
      • Inada T.
      • Murata S.
      The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation.
      ). Ubp3 is responsible for inhibiting polyubiquitination of eS7 by the E3 Hel2, which would trigger RQC and suppress translation (
      • Takehara Y.
      • Yashiroda H.
      • Matsuo Y.
      • Zhao X.
      • Kamigaki A.
      • Matsuzaki T.
      • Kosako H.
      • Inada T.
      • Murata S.
      The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation.
      ). The activity of Ubp3 leaves eS7 in the monoubiquitinated form (
      • Takehara Y.
      • Yashiroda H.
      • Matsuo Y.
      • Zhao X.
      • Kamigaki A.
      • Matsuzaki T.
      • Kosako H.
      • Inada T.
      • Murata S.
      The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation.
      ). Otu2 deubiquitinates eS7 in order to promote dissociation of the small ribosomal subunit from the mRNA upon completion of translation (
      • Takehara Y.
      • Yashiroda H.
      • Matsuo Y.
      • Zhao X.
      • Kamigaki A.
      • Matsuzaki T.
      • Kosako H.
      • Inada T.
      • Murata S.
      The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation.
      ). This cycling of ubiquitination/deubiquitination demonstrates the intricacy and importance of DUB regulation of fundamental cellular pathways.
      Not only do DUBs regulate translation elongation, but they also regulate translation initiation and ribophagy. USP9X and USP11 have been demonstrated to stabilize the initiation factor eIF4A and eIF4B, respectively (
      • Li Z.
      • Cheng Z.
      • Raghothama C.
      • Cui Z.
      • Liu K.
      • Li X.
      • Jiang C.
      • Jiang W.
      • Tan M.
      • Ni X.
      • Pandey A.
      • Liu J.O.
      • Dang Y.
      USP9X controls translation efficiency via deubiquitination of eukaryotic translation initiation factor 4A1.
      ,
      • Kapadia B.
      • Nanaji N.M.
      • Bhalla K.
      • Bhandary B.
      • Lapidus R.
      • Beheshti A.
      • Evens A.M.
      • Gartenhaus R.B.
      Fatty acid synthase induced S6Kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in DLBCL.