International Journal of Molecular Sciences Review Molecular Mechanisms of DUBs Regulation in Signaling and Disease Ying Li and David Reverter * Departament de Bioquimica i Biologia Molecular, Institut de Biotecnologia i de Biomedicina, Universitat Autonòma de Barcelona, 08193 Bellaterra, Spain; liyingbaobei123@gmail.com * Correspondence: david.reverter@uab.cat Abstract: The large family of deubiquitinating enzymes (DUBs) are involved in the regulation of a plethora of processes carried out inside the cell by protein ubiquitination. Ubiquitination is a basic pathway responsible for the correct protein homeostasis in the cell, which could regulate the fate of proteins through the ubiquitin–proteasome system (UPS). In this review we will focus on recent advances on the molecular mechanisms and specificities found for some types of DUBs enzymes, highlighting illustrative examples in which the regulatory mechanism for DUBs has been understood in depth at the molecular level by structural biology. DUB proteases are responsible for cleavage and regulation of the multiple types of ubiquitin linkages that can be synthesized inside the cell, known as the ubiquitin-code, which are tightly connected to specific substrate functions. We will display some strategies carried out by members of different DUB families to provide specificity on the cleavage of particular ubiquitin linkages. Finally, we will also discuss recent progress made for the development of drug compounds targeting DUB proteases, which are usually correlated to the progress of many pathologies such as cancer and neurodegenerative diseases.  Keywords: ubiquitin; deubiquitinating enzymes; UPS; proteasome; protein degradation; DUBs;  USPs; ubiquitin-code; structural analysis Citation: Li, Y.; Reverter, D. Molecular Mechanisms of DUBs Regulation in Signaling and Disease. Int. J. Mol. Sci. 2021, 22, 986. https:// 1. Introduction doi.org/10.3390/ijms22030986 The ubiquitin–proteasome System (UPS) is the major pathway for the degradation of more than 80% of intracellular proteins. Ubiquitination is the major signal to label proteins Academic Editor: for degradation, it controls their fates and contributes to the correct cellular homeostasis Suresh Ramakrishna during all cell cycle phases [1,2]. After a particular protein receives a signal for degradation, Received: 30 December 2020 Accepted: 18 January 2021 such as phosphorylation, DNA damage, protein misfolding, or others, a cascade of ubiqui- Published: 20 January 2021 tinating enzymes, namely the ubiquitin-activating enzymes E1, the ubiquitin conjugation enzymes E2, and the ubiquitin ligases E3, labels the protein target with a ubiquitin chain Publisher’s Note: MDPI stays neutral to be carried to the 26S-proteasome, which degrades ubiquitinated-target proteins and with regard to jurisdictional claims in recycles ubiquitin for reuse [3]. Several E1, tens of E2 and hundreds of E3 enzymes have published maps and institutional affil- been found in human, and they are responsible or the formation of the isopeptide bond iations. between a lysine residue in the target and the C-terminus of ubiquitin. Cells degrade unnecessary proteins in this highly specific manner to prevent cell damage induced by incorrect interaction with other proteins or abnormal protein aggregation. In addition to label proteins for degradation by the UPS pathway, different types of ubiquitin tags can be Copyright: © 2021 by the authors. specifically used by proteins in signaling pathways other than degradation [1,4]. Licensee MDPI, Basel, Switzerland. Deubiquitination refers to the process by which ubiquitin molecules are removed This article is an open access article from ubiquitin-labeled proteins under the proteolytic action of deubiquitinating enzymes, distributed under the terms and which cleaves off the isopeptide bond between the C-terminal tail of ubiquitin and the conditions of the Creative Commons target lysine, thus changing the fate of ubiquitinated proteins and in some instances by Attribution (CC BY) license (https:// preventing them from degradation [5,6]. Deubiquitination has an opposite function to creativecommons.org/licenses/by/ the action of ubiquitin E3 ligases and their balance inside the cell is essential for protein 4.0/). homeostasis and the correct function of the cell. The human genome encodes about Int. J. Mol. Sci. 2021, 22, 986. https://doi.org/10.3390/ijms22030986 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 986 2 of 24 100 deubiquitinase (DUB) genes [7], in contrast to more than 800 genes encoding ubiquitin E3 ligases. These numbers highlight the major role of the ubiquitin pathway in the cell, which must provide a high selectivity grade in the regulation of protein targets. Ubiquitin can form different types of chain linkages, depending on the lysine residue used in ubiquitin as a target to build poly-ubiquitin chains. Additionally, four differ- ent types of protein ubiquitination in substrates enrich the diversity in ubiquitination, namely monoubiquitination, multi-monoubiquitination, homotypic polyubiquitination, and heterotypic polyubiquitination [8,9]. In homotypic polyubiquitination, multiple ubiq- uitins are linked to each other to form a chain, and are connected to a certain lysine residue (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) or to the amino-terminal methionine. Also, protein ubiquitination could be accompanied by some other ubiquitin-like modifiers (UBL), such as SUMO, NEDD8, and ISG15. Heterotypic chains include mixed ubiquitin chains and branched ubiquitin chains. In heterotypic polyubiquitination, by generating different connection groups on different ubiquitin molecules, a mixed ubiquitination se- quence can be formed. Several modifications can also occur on each ubiquitin, resulting in branched polymers. Due to the diversity of ubiquitinated chain linkage styles, which has been named as “ubiquitin code”, particular DUBs have evolved to have specific preferences for the different types of ubiquitin chains [6,7,10,11]. According to sequence and structure similarity, deubiquitylating enzymes can be divided into seven families, each presenting a unique structural fold: Ubiquitin-specific proteases (USPs), Ubiquitin C-terminal hydrolases (UCHs), Ovarian tumor proteases (OTUs), Machado–Joseph Disease protease family (MJDs), MINDY protease family (MIU- containing DUB family), JAMM family (Jad1/Pad/MPN domain-containing metalloen- zymes), and the newly discovered ZUFSP/Mug105 family (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1) [12,13] (Table 1). Amid them, the JAMM family are zinc-dependent metalloproteinases, while the other six families are cysteine proteases, displaying a characteristic cysteine residue in the context of the active site catalytic triad. Except for MJDs, the other families are highly conserved in yeast and humans (Table 1). Among the 99 DUBs members, 11 are considered pseudo-enzymes due to the lack of key residues for deubiquitinating activity, nevertheless they still can allosterically activate other active deubiquitinating enzymes and other types of active enzymes, thereby executing a critical biological role [14]. Table 1. The seven classes of deubiquitinating enzymes (DUBs). Species/DUBs USP OTU JAMM MJD UCH MINDY ZUP1 Homo sapiens 56 17 12 4 4 5 1 Danio rerio 44 11 12 3 4 5 1 Drosophila melanogaster 23 5 10 1 3 1 0 Schizosaccharomyces pombe 11 1 4 0 2 1 1 Saccharomyces cerevisiae 11 3 7 0 1 1 0 UBD (ubiquitin-binding domain) is a type of modular domain that can non-covalently bind to ubiquitin or ubiquitinated substrates, which is widely present in deubiquitinating enzymes. Almost 20 different UBDs families have been discovered so far. According to the structure of UBDs, they are divided into 5 categories: α helix, Zinc finger, PH domain, Ubc-like, and others. The most common in DUBs are UBA (ubiquitin-associated domain), UBL (ubiquitin-like modifiers), UIM (ubiquitin-interacting motif), ZnF-UBP (zinc finger ubiquitin-specific protease domain), and ZnF-A20s (A20-type zinc fingers) [7,15,16]. Re- cent research has discovered a new UBD named CoCUN [17]. In the process of DUBs recognition and hydrolysis of ubiquitin chains, UBD plays an indispensable role. For ex- ample, OTUD1 can specifically hydrolyze the Lys63 ubiquitin chain, but the lack of the Int. J. Mol. Sci. 2021, 22, 986 3 of 24 UIM domain decreases its specificity [18]. Also, the adjacent UBL domains of USP7 are indispensable to achieve complete deubiquitinating activity [19]. The mechanism by which deubiquitinating enzymes exert their function in the cell can be complex, in some cases it is not only deubiquitination, but it can also promote ubiq- uitination by recycling ubiquitin molecules, proofreading of the ubiquitination process, and decomposing ubiquitination inhibitors [20]. In this review, we will focus attention on advances in explaining molecular mechanisms and specificities found in some DUBs enzymes, highlighting illustrative examples in which the regulatory mechanism for DUBs has been understood in depth at the molecular level. We will also discuss on DUB specificity and terminate by indicating new advances encouraging their development as therapeutic targets. 2. DUBs in Diseases The large number of deubiquitinating enzymes are not only playing a role in the normal physiological activities of cells, but can also be relevant for the occurrence and development of tumorigenesis and other pathologies [21–23]. Target proteins for deubiqui- tinating enzymes include enzymes, transcription factors, signal transduction molecules, immune response proteins, viral proteins, epigenetic factors, and many other regulators of cell homeostasis, also including products of known oncogenes or tumor suppressor genes [24–31]. Therefore, disease treatment strategies with DUBs as molecular targets have a broad development value and promising clinical application prospects [32]. Many stud- ies have shown DUBs involvement in the regulation of Wnt/β-catenin signaling, TGF-β (transforming growth factor-β), Akt (Protein Kinase B), NF-κB (nuclear factor kappa-light- chain-enhancer of activated B cells) and other cancer-related pathways [33–36]. Several DUBs are involved in the regulation of the Wnt/β-catenin signaling. For ex- ample, overexpression of USP5 will cause an increase in the amount of transcription factor FoxM1, which will increase the content of β-catenin, resulting in faster cell proliferation and carcinogenesis in many tumors [37]. UCH37 can specifically bind and deubiquitinate transcription factor 7 (Tcf7) to activate Wnt signaling in human liver cancer cells [38]. In pediatric high-grade glioma, abnormal expression of UCH-L1 can promote tumor formation [39]. Abnormal activation of USP7 can lead to colorectal cancer [40]. USP14 over- expression is closely related to hepatocellular carcinoma [41]. USP4 is related to colorectal cancer since the overexpression of USP4 exists in many types of tumors, thus it is consid- ered to be a potential oncogene [42–45]. Most DUBs will promote the occurrence of tumors, but a small number of DUBs play the opposite role. For example, the tumor suppressor cylindromatosis (CYLD), which is expressed in tumor cells, inhibits the proliferation and spread of tumor cells. CYLD is not expressed in multiple myeloma, and inhibits the growth of multiple myeloma by acting on the Dvl substrate [46,47]. Most DUBs reduce the degradation of protein targets by protecting them from the proteasome, thereby a high concentration of TGF-β, promoted by specific DUBs, might lead to tumor occurrence and metastasis. Deubiquitinating enzyme USP10 promotes the metas- tasis of hepatocellular carcinoma metastasis by deubiquitinating and stabilizing Smad4 [48]. But there are special cases, CYLD can inhibit oral squamous cell in the metastasis of carci- noma, CYLD controls the downstream TGF-β pathway by regulating Smad7, thereby in- hibiting the occurrence and development of cancer [49,50]. DUBs also play an important role in regulating the NF-κB pathway. OTULIN/CYLD can interact with LUBAC (linear ubiquitin chain assembly complex) through the PUB (PN- Gase/UBA or UBX) domain of HOIP (a catalytic subunit of LUBAC), thereby inhibiting the NF-κB pathway and exerting anti-tumor effects [51]. Studies have shown that alterations in the CYLD gene are closely related to HPV-associated cancers, it activates NF-κB and is implicated in invasion and metastasis [52–54]. USP15 potentiates NF-κB activation by differentially stabilizing TAB2 and TAB3 [55]. The Akt pathway promotes the survival and proliferation of cells in the organ- ism, and its expression is increased in cancer cells [56]. The increased expression of Int. J. Mol. Sci. 2021, 22, 986 4 of 24 USP4 will reduce the ubiquitination process of downstream PRL-3 and increase the content of PRL-3, thereby activating the PI3K/Akt pathway. The PI3K/Akt pathway is over- activated, causing tumorigenesis and other diseases [43]. In osteosarcoma cells, over- expression of USP22 leads to increased activity of the PI3K/Akt pathway and causes cancer [57]. USP22 plays a vital role in the development of chemoresistant hepatocellular carcinoma cells [58]. 3. DUBs Families and Linkage Specificity 3.1. Ovarian Tumor-Related Proteases, OTUs The OTU domain was first discovered in the ovarian tumor gene of Drosophila melanogaster by bioinformatics methods [59]. After structural analysis, it was found that although the structure is different from any other known DUB enzyme, it belongs to the cysteine protease class and contained the characteristic catalytic triad. There are 17 OTUs in humans, and members of the OTU family have been revealed to possess obvious recogni- tion specificity for different types of polyubiquitin chain connections, thus disclosing many principles of DUBs chain linkage specificity. For instance, OTULIN cleaves linear ubiqui- tin [60]. OTUB2 prefers K63 di-ubiquitin substrate [18,61]. OTUB1 only cleaves K48-linked chains [62]. The specificity of OTU family members to recognize polyubiquitin chains of different connection types is likely to be the basis for their recognition of target substrates. 3.2. Ubiquitin-Specific Processing Enzymes, USPs/Ubiquitin-Specific Proteases, UBPs Within the deubiquitinating enzymes, the ubiquitin-specific proteases family (USPs) contains the major number of members (more than 50) and its large structural diversity relies on the presence of multiple domains. All members are cysteine proteases and contain the characteristic catalytic triad in the active site of the catalytic USP domain. The conserved USP domain consists of three subdomains that were initially described as thumb, palm, and fingers of a human right hand in the USP7/HAUSP structure [63]. The catalytic triad of the active site is located between the palm and thumb subdomains, and the finger subdomain is responsible for the interaction with the ubiquitin substrate [64]. In addition to the catalytic domain, other domains of USPs contribute to a large diversity of functions, such as zinc finger domains, ubiquitin-like domains, ubiquitin-associated domain (UBA), ubiquitin-interacting motif (UIM). Some of them are associated with the specific recognition of substrates and poly-ubiquitin chains. Most USP members have no specific linkage-type preference in vitro [65], but in vivo there are some exceptions, for example, CYLD shows K63-linked specificity [66]; USP30 preferentially processes K6-linked chains [67]. Also, some of them show preference for different types of UBL such as USPL1 and USP18 for SUMO and ISG15, respectively [68,69]. 3.3. Jad1/Pad/MPN Domain-Containing Metalloenzymes, JAMM Family/MPN The JAMMs is the only family of deubiquitinating enzymes belonging to the met- alloprotease family, which contain a Zn atom in the active site. Members of this family are occasionally subunits of large protein complexes, such as COP9 signalosome complex (CSN5 and CSN6 subunits), the 26S-proteasome (PSMD14/Rpn11 and PSMD7/Rpn8 sub- units), and the Eukaryotic translation initiation factor 3 (EIF3) (EIF3H and EIF3F sub- units) [70–76]. In this family, AMSH, AMSH-LP, and BRCC36 have been shown to have specificity for K63-linked ubiquitin chains [77,78], and CNS5/COPS5 has activity against NEDD8, another type of ubiquitin-like modifier [79]. 3.4. Machado–Joseph Disease Proteases, MJDs/Josephin Currently, four members of the MJDs/Josephin family are found in humans, namely ATXN3/ataxin3, ATXN-3L, JOSD-1 and JOSD-2. All members have a highly conserved catalytic triad formed by one cysteine and two histidine residues. In addition to the Josephin domain, ATXN3 and ATXN3L also have ubiquitin interaction motifs (UIMs), which are likely to mediate the interaction between protein and ubiquitin in protein multimers [80]. Int. J. Mol. Sci. 2021, 22, 986 5 of 24 ATXN3 can bind both K48 and K63-linked ubiquitin chains, but preferentially shows activity for K63 linkages [81]. ATXN3L binds to K48 and K63-linked ubiquitin chains in vitro [82]. JOSD1 only has activity for monoubiquitin and JOSD2 cleaves K48 and K63-linked ubiquitin chains in vitro [83]. 3.5. Ubiquitin C-Terminal Hydrolases, UCHs UCHs are also cysteine proteases, including UCHL1, UCHL3, UCHL5/UCH37, and BAP1 (BRCA 1 associated protein-1). Substrates are usually small proteins like short polypeptides or small protein domains. The narrow pocket on the active site of UCHs and the limitation of the diameter of the loop precludes binding and catalysis of large ubiquitinated proteins, to a certain extent [84–86]. UCH-L1 can cleave short peptides from the C-terminus of ubiquitin in vitro [87]. UCH-L3 cleaves the C-terminus of NEDD8 or ubiquitin [88]. UCH-L5 participates in the formation of 19S regulatory particles, it can hydrolyze ubiquitin chains [89]. BAP1 can also regulate the polyubiquitination of the transcriptional cofactor HCF-1 (host cell factor 1) [90]. 3.6. MIU-Containing Novel DUB Family, MINDY MINDY family members, include MINDY1, -2, -3, -4, and -4B in humans, are specific for K48-linked ubiquitin chains [91]. The crystal structure of MINDY1 exhibited a novel protein fold without homology to any other type of DUB [91]. MINDY-1 prefers to cleave long polyubiquitin chains and cleaves the distal ubiquitin component [7,91,92]. In the absence of the substrate, the active site in MINDY-1 has a non-productive conformation, which is remodeled and the catalytic triad rearranged upon substrate binding [91]. 3.7. Zinc Finger with UFM1-Specific Peptidase Domain Protein/C6orf113/ZUP1 ZUP1/ZUFSP in humans and Mug105 in fission yeast are representative members of a seventh DUB family. The ZUFSP family seems equidistant to the UFSP and ATG4 families, and the catalytic domain is related to the UFM1-protease UFSP2, but ZUFSP is not a member of UFSP [93]. Whereas the UFM1-specific protease UFSP2 has no detectable DUB activity, ZUFSP has linkage-selective DUB activity [12]. Human ZUFSP selectively interacts and cleaves long K63-linked ubiquitin chains using tandem ubiquitin-binding domains, but it displays poor activity toward mono- or di-Ub substrates. Mug105 prefers K48-linked chains [12,94]. The discovery of other members of the family will probably assist the analysis of the linkage specificity, as well as to shed light on structural and functional properties. 4. Regulatory Mechanisms of DUB Activity and Specificity Research on deubiquitinating enzymes shows that although deubiquitinating enzymes are classified into several distinct families, the structural differences between members have caused their functional diversity. Here, we have selected a few classical, special, or interesting examples published in recent years to explain the molecular mechanism of DUB regulation at the molecular level, based on their crystal structures. These in-depth examples of DUB regulation may inspire researchers to shed light on deubiquitinating enzymes with still unknown mechanisms. 4.1. USP7, a Conformational Rearrangement of the Catalytic Triad USP7/HAUSP has been deeply studied from the structure/function standpoint and its molecular signaling pathway is well established and considered a potential thera- peutic target for cancer [95]. The human USP7 gene is located on chromosome 16, con- tains 1102 amino acid residues and is mainly located in the nucleus, but also slightly distributed in the cytoplasm [95]. USP7 is highly conserved in mammals, with a 98.6% sequence identity between human, mouse and rat [96]. USP7 is a multidomain protein with seven domains: In addition to the “canonical” USP catalytic domain (aa208-560), it contains one N-terminal TRAF-like domain (Tumor necrosis factor Receptor-Associated Factor), target for cancer [95]. The human USP7 gene is located on chromosome 16, contains 1102 amino acid residues and is mainly located in the nucleus, but also slightly distributed in the cytoplasm [95]. USP7 is highly conserved in mammals, with a 98.6% sequence identity between human, mouse and rat [96]. USP7 is a multidomain protein with seven domains: Int. J. Mol. Sci. 2021, 22, 986 6 of 24 In addition to the “canonical” USP catalytic domain (aa208-560), it contains one N-termi- nal TRAF-like domain (Tumor necrosis factor Receptor-Associated Factor), and five con- secutive C-terminal UBL domains (Ubiquitin-like domains) [97] (Figure 1A,B). UBL do- mains canconsecutive and five be also found in other C-terminal UBLUSPs and are domains known to play (Ubiquitin-like a role[97] domains) in the regulation (Figure 1A,B). of UBL the domains activity enzymatic can be also [98].found USP7incan other USPs cleave and K6, areK33, K11, known to and K48, playK63-linked a role in theubiquitin regu- lation of the chains [63,99].enzymatic activity [98]. USP7 can cleave K6, K11, K33, K48, and K63-linked ubiquitin chains [63,99]. Figure 1. Active site rearrangements in USP7. (A) Schematic representation of the USP7 domain organization. (B) Model Figure 1.activation for the Active siteofrearrangements in and USP7 by ubiquitin USP7. (A) Schematic ubiquitin-like representation modifiers of the(different (UBL) domains USP7 domain organization. colors). (B) Model (C) Crystal structure forofthe activation of USP7 by ubiquitin and ubiquitin-like modifiers (UBL) domains (different colors). (C) Crystal the dimer of the USP7CD-UBL45-ubiquitin complex. Ubiquitin (yellow), the C-terminal peptide after UBL5 (orange). struc- ture of the dimer of the USP7CD-UBL45-ubiquitin complex. Ubiquitin (yellow), the C-terminal peptide after UBL5 (or- (D) Comparison of the active sites between USP7CD-UBL45-Ub and USP7CD. Active USP catalytic domain (blue), non-active ange). (D) Comparison of the active sites between USP7CD-UBL45-Ub and USP7CD. Active USP catalytic domain (blue), USP catalytic domain (gray). Dotted lines are distances between active site Cys and His residues (PDB: 5JTV, 5FWI). non-active USP catalytic domain (gray). Dotted lines are distances between active site Cys and His residues (PDB: 5JTV, 5FWI). The initial crystal structure of the catalytic domain of USP7 defined the characteristic right hand-like structural fold of the USP family, containing the active site catalytic The triad: initial His464, Cys223, crystal structure and Asp481.of the catalytic domain Remarkably, of USP7 in contrast defined to other USPs,the characteristic the cysteine right and histidine of the USP7 catalytic domain are in a non-productive configuration triad: hand-like structural fold of the USP family, containing the active site catalytic in Cys223, His464, and the apoenzyme and Asp481. Remarkably, need to be activated byinstructural contrast torearrangement other USPs, the cysteine upon and his- ubiquitin- tidine bindingof the USP7 [63,98] catalytic (Figure domain 1B,C). are in a non-productive The N-terminal TRAF-like domainconfiguration in the apoen- and the C-terminal zyme and need UBL domains areto be activated important bythestructural sites for binding ofrearrangement upon ubiquitin-binding USP7 to other proteins [63]. USP7 can interact [63,98] with p53, (Figure HDM2, 1B,C). HDMX, MCM-BP, The N-terminal TRAF-likeUbE2E1, vIRF1, domain andvIRF4, and EBNA1 the C-terminal UBLthrough domains theimportant are TRAF-likesites domainfor [100–106]. the binding of USP7 to other proteins [63]. USP7 can interact with Interestingly, the active site of USP7 shows an inactive conformation in its apo form, but in the presence of ubiquitin, the active site is remodeled and the catalytic triad aligned [64] (Figure 1D). UBL domains also play a role in the activation of the catalytic domain [107]. Deletion of the USP7 C-terminal UBL domains have an impact on the USP7 activity and then USP7 cannot bind ubiquitin chains [19,97]. According to the struc- ture of the USP7CD-UBL4-5-Ubiquitin complex (Figure 1C), the C-terminal extension after UBL-5 domain binds a cavity next to the active site and promotes its activation. The C- terminal peptide seems to stabilize the active conformation instead of significantly change Int. J. Mol. Sci. 2021, 22, 986 7 of 24 the structure of the catalytic domain. It is accommodated in a hydrophobic cavity by several contacts, such as Ile1098 and Ile1100, and can also bind the C-terminal Arg74 of ubiqui- tin [97]. This regulatory C-terminus extension of USP7 is essential for the enhancement of the USP7 catalytic activity [97,108]. Among all C-terminal UBL domains, the presence of the UBL4-5 domain is enough to reconstitute the USP7 activation in vitro [19]. USP7 activation can be triggered by interaction with other substrates to the C-terminal UBL domains, such as binding to GMPS (guanosine 50 -monophosphate synthetase) [19]. Other USP7 substrates also bind to the UBL domains, such as DNMT1, ICPO, RNF169, and UHRF1 [109–113]. Therefore, the C- terminal UBL domains have a role in the regulation of USP7 by inhibiting or enhancing enzyme activity [104]. In the cell, different substrates compete to bind to the same surface on USP7 [100]. USP7 has been related to many diseases, such as lung cancer, cervical cancer, her- pes simplex virus, and nasopharyngeal carcinoma by Epstein–Bar virus (EBV) [114–117]. USP7 plays an important role in tumorigenesis and progression by deubiquitinating a variety of tumor regulatory molecules [95] and is a promising target for the development of lead compounds. 4.2. Ubp8, Needs Partner Proteins for Activity Ubp8, the yeast protein homolog of human USP22, is a subunit of the SAGA complex, which is a multifunctional complex protein involved in many biological processes such as transcription activation and mRNA transport [118]. The center module for catalyzing deubiquitination is called the DUB region and contains four subunits: Ubp8, Sgf11, Sus1, and Sgf73 [119–121] (Figure 2A,B). Ubp8 deubiquitinates histone H2B [122]. Ubp8 can sufficiently perform its deubiquitinating activity with the assistance of the other partners of the complex: Sgf11, Sus1, and Sgf73 [123]. The active site residues of Ubp8 are catalytically well-arranged in the absence of ubiquitin [119,121,124]. However, the ZnF-UBP (zinc finger-ubiquitin binding) domain of Ubp8 lacks vital residues for ubiquitin-binding, since the ubiquitin-binding area is at the interface with the Sgf11 and Sus1 subunits, it needs other proteins of the complex to help its interaction with ubiquitin [98,124,125] (Figure 2C). SAGA promotes deubiquitination through the specific binding of the zinc finger domain of Sgf11 to the acidic pocket of ubiquitinated nucleosomes [124]. Moreover, Sgf11 plays a regulatory role in stabilizing the active conformation of the catalytic active site of Ubp8 [121]. The C-terminal zinc finger domain of Sgf11 (Sgf11-ZnF) binds to the Ubp8 USP domain next to the catalytic site, making extensive contacts with the loop containing the active site residues (called loop L2) (Figure 2D). The N-terminal 100 residues of Sgf73 connects the DUB domain with other parts of the SAGA complex and regulates the spatial structure of the complex [121,126]. The zinc fingers of Sgf11 and Sgf73 are essential for the activity of the DUB module: Deleting Sgf11-ZnF will greatly reduce the activity of the enzyme, while the absence of the zinc finger of Sgf73 will cause Sgf73 to dissociate from the DUB module, thereby abolishing the deubiquitinase activity [124,127]. It has been proposed a role for Sgf73, Sgf11, and Sus1 in stabilizing a conformation of Ubp8 that can easily interact with ubiquitin [119]. Overexpression USP22 is a marker of aggressive cancer phenotypes like metastasis, and therapy resistance studies have shown that catalyzing SAGA subunit USP22 to control CCND1 ubiquitination is important for the progression of cancer cells through the G1 cell cycle [128]. USP22 promotes the advance of gastric cancer by stabilizing BMI1 protein [129]. Also, USP22 plays a carcinogenic effect in prostate cancer by regulating cell proliferation and DNA repair [130]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 26 Int. J. Mol. Sci. 2021, 22, 986 8 of 24 Thestructure Figure2.2.The Figure structureofofUbp8 Ubp8ininthe theSAGA SAGAdeubiquitinating deubiquitinatingmodule. module.(A)(A) Crystal Crystal structure structure ofof the the SAGA SAGA DUB DUB module module incomplex complexwith withmonoubiquitinated monoubiquitinatednucleosome nucleosome (PDB: 4ZUX). ◦ in (PDB: 4ZUX). (B)(B) View View of the of the complex complex rotated rotated 180 180°. (C). Structure (C) Structure of of Ubp8/Sgf11/Sus1/Sgf73 Ubp8/Sgf11/Sus1/Sgf73 bound bound toto ubiquitinaldehyde ubiquitin aldehyde(PDB: (PDB:3MHS). 3MHS).(D) (D)Zoom-up Zoom-upand andsuperposition superposition ofof the active site with or without with the Sgf11-ZnF or without domain. the Sgf11-ZnF Ubp8 domain. (blue), Ubp8 Sus1Sus1 (blue), (orange), Sgf73 (orange), (pink), Sgf73 sgf11 (pink), (wheat), sgf11 ubiquitin (wheat), (yellow), ubiquitin zinc (yellow), atom zinc atom (purple) (purple) (PDB:(PDB: 4FJC 4FJC and 3MHS). and 3MHS). 4.3. The active USP25, an site residues of Ubp8 Auto-Inhibitory are catalytically well-arranged in the absence of ubiq- Homotetramer uitin [119,121,124]. However, the ZnF-UBP (zinc finger-ubiquitin binding) domain of USP25 is a special member of the USP family due to its auto-inhibitory mechanism. Ubp8 lacks vital residues for ubiquitin-binding, since the ubiquitin-binding area is at the The 180 amino acids insertion in the middle of the catalytic domain of USP25 plays a crucial role interface with the in the catalytic Sgf11There activity. and Sus1 subunits, are two it needsofother conformations proteins USP25: of and Tetramer the complex dimer. Thetodimer help its interaction with ubiquitin [98,124,125] (Figure 2C). SAGA promotes deubiquitination USP25 is active, while the tetramer USP25 has a low activity [131] (Figure 3A–C). According to through structuralthe specific biology binding analysis of the zinc [131–133], finger domain an inhibitory of Sgf11 loop binds to the acidic pocket the ubiquitin-binding of surface ubiquitinated nucleosomes [124]. Moreover, Sgf11 plays a regulatory role in of the USP domain in the tetramer conformation, resulting in a failure of USP25 to cleave stabilizing the active conformation ubiquitin of thecan substrates. USP25 catalytic switchactive in the site ofbetween cell Ubp8 [121]. The(active) dimer C-terminal and zinc fin- tetramer ger domain of Sgf11 (Sgf11-ZnF) binds to the Ubp8 USP domain next to the catalytic (inactive) [133]. This special feature is unique to USP25, and it has not been found in any site, making other USPextensive member contacts to date.with the loop containing the active site residues (called loop cial role in the catalytic activity. There are two conformations of USP25: Tetramer an dimer. The dimer USP25 is active, while the tetramer USP25 has a low activity [131] (Fi ure 3A–C). According to structural biology analysis [131–133], an inhibitory loop bind the ubiquitin-binding surface of the USP domain in the tetramer conformation, resultin in a failure of USP25 to cleave ubiquitin substrates. USP25 can switch in the cell betwee Int. J. Mol. Sci. 2021, 22, 986 9 of 24 dimer (active) and tetramer (inactive) [133]. This special feature is unique to USP25, an it has not been found in any other USP member to date. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 26 Figure 3. The structure of USP25. (A) Schematic cartoon representation of the USP25 tetramer and dimer assemblies. Figure 3. The structure Each monomer of USP25. is colored. (A) Schematic (B) Crystal structure ofcartoon representation the USP25 of the5O71 tetramer (PDB: USP25 andtetramer and dimer 6HEL). Each dimer assemblies. Each is colored blue or monomer light pink.is(C) colored. Crystal(B) Crystalofstructure structure the USP25of dimer. the USP25 tetramer IL-loop, (PDB:insertion coiled-coil 5O71 and 6HEL). and Each dimer protease ubiquitin-specific is colored blue or (USP)-like light pink. domain are(C) Crystal(D) marked. structure Bindingofinterface the USP25 dimer. of the IL-loop, IL-loop coiled-coil with the insertion and ubiquitin-binding siteubiquitin-specific of USP25. IL-loopprotease (USP)- (red), active site like domain are marked. (D) Binding interface of the IL-loop with the ubiquitin-binding site of USP25. (green), binding surface (yellow), the interface contacts of IL-loop are labelled. (E) View of D rotated 90 . IL-loop ◦ (red), active site (green), binding surface (yellow), the interface contacts of IL-loop are labelled. (E) View of D rotated 90°. USP28 has 57% amino acid sequence identity with USP25, but USP28 is not regulated USP28 by this has 57% amino autoinhibitory acid sequence mechanism. USP28 identity with USP25, has always but USP28 a dimeric is not regulated conformation, never a by this autoinhibitory mechanism. USP28 has always a dimeric conformation, never tetramer [133]. The unique coiled-coil helical insertion domain in the middle of the catalytic a te- tramer [133]. The unique coiled-coil helical insertion domain in the middle of domains of USP25 and USP28 is essential for the formation of dimers [132] (Figure 3D,E). the catalytic domains Probablyof USP25 due to theand USP28 slightly is essential different for theofformation orientation the dimer,ofUSP28 dimers [132]form cannot (Figure 3D,E). a tetramer Probably due to the slightly different orientation of the dimer, USP28 cannot form a te- tramer structure. Therefore, despite USP25 and USP28 possess high sequence and struc- tural similarities, they display different regulatory mechanisms. Other USP members, such as USP4, USP7, and USP14, also have a certain degree of auto-inhibition, but not through tetrameric inhibitory conformations like USP25. Studies Int. J. Mol. Sci. 2021, 22, 986 10 of 24 structure. Therefore, despite USP25 and USP28 possess high sequence and structural similarities, they display different regulatory mechanisms. Other USP members, such as USP4, USP7, and USP14, also have a certain degree of auto-inhibition, but not through tetrameric inhibitory conformations like USP25. Stud- ies have shown that the activity of USP14 is significantly improved after binding the proteasome [134,135]. The DUSP-UBL domain at the N-terminus of USP4 plays an indis- pensable role in the catalytic activity of USP4, since the dissociation rate of ubiquitin will affect the catalytic activity of USP4, the DUSP domain can promote the dissociation of ubiquitin, thereby improving the catalytic activity of USP4 [136]. USP25 might be an oncogene, it participates in the stabilization of tankyrases that can regulate oncogenic pathways and have an impact on the invasion and migration of cancer cells [137,138]. A latest research shows that USP25 is a new target to induce degradation of oncogenic BCR-ABL protein in Ph-positive leukemia cells [139]. 4.4. OTUB1, a Non-Canonical Inhibition of the E2-Conjugating Enzyme OTUB1 (OTU domain-containing ubiquitin aldehyde binding protein 1) is an exten- sively studied member of the OTU deubiquitinating family. The human OTUB1 gene is located in chromosome 11 and encodes an enzyme of 271 residues [140]. Its subcellular location is regulated by casein kinase 2 (CK2), which phosphorylates Ser16 of OTUB1 mov- ing it from the cytoplasm to the nucleus [141]. OTUB1 can participate in the regulation of p53 stability, immune response, signal transduction, DNA damage repair, and other biological processes [142–146]. OTUB1 is a K48 specific deubiquitinating enzyme, and in addition to the DUB activity, OTUB1 has also emerged as a special DUB that can suppress the activity of E2 conjugating enzymes, inhibiting thus the ubiquitination process [143]. Researches have convincingly illustrated a unique mechanism for OTUB1 performing different biological functions through the interaction with E2-conjugating enzymes (such as UBC13 and UBCH5) [147,148]. OTUB1 contains two different specific binding sites for ubiquitin: The proximal ubiquitin-binding site binds the donor ubiquitin, and the distal ubiquitin-binding site binds free ubiquitin [149,150] (Figure 4A,B). E2 binding to OTUB1 stabilizes an N-terminal disordered helix that interacts with the proximal ubiquitin and enhances the deubiquitinating activity of OTUB1 for K48-linked chains (Figure 4A–C). The affinity of OTUB1 for the charged E2-Ub is increased when a free ubiquitin monomer binds to the distal site of OTUB1 [147–149]. However, when E2 is charged with the ubiquitin thioester linkage, it locates its donor ubiquitin in the proximal ubiquitin-binding site of OTUB1, and in this scenario, the deubiquitinating activity of OTUB1 for K48-linked chains is inhibited and the charged E2 cannot deliver ubiquitin to substrate and the ubiquitination conjugation activity of E2 is inhibited. Also, free ubiquitin binding to the distal site of OTUB1 has been shown to compete with the binding of K48-linked chains and inhibits the deubiquitinating activity of OTUB1 [148]. Likewise, binding of uncharged E2s to OTUB1 stimulates OTUB1 cleavage of K48-linked substrates [150]. Thereby, the relative proportion of E2 versus E2~Ub and the local concentration of K48-linked polyubiquitin chains as well as free ubiquitin decide if OTUB1 plays a role either as DUB or as an inhibitor of ubiquitination [150]. Consequently, in addition to its DUB activity, OTUB1 also has the special func- tion to bind different E2 ubiquitin conjugation enzymes, causing inhibition of ubiqui- tin transfer and regulation ubiquitination in a non-catalytically manner. Recently, an- other role for OTUB1 has been found by the binding of the E2-conjugating enzyme UBE2E1 (UBCH6) [151]. UBE2E1 is ubiquitinated in vivo and can be targeted to the proteasome for degradation. In vitro, OTUB1 can non-catalytically inhibit the autoubiquitination activity of UBE2E1, thus preventing it from degradation [149]. Probably, more mechanisms for the regulation of other E2 conjugation activities by OTUB1 will be disclosed in the future. to the distal site of OTUB1 has been shown to compete with the binding of K48-linked chains and inhibits the deubiquitinating activity of OTUB1 [148]. Likewise, binding of un- charged E2s to OTUB1 stimulates OTUB1 cleavage of K48-linked substrates [150]. Thereby, the relative proportion of E2 versus E2~Ub and the local concentration of K48- Int. J. Mol. Sci. 2021, 22, 986 linked polyubiquitin chains as well as free ubiquitin decide if OTUB1 plays a role 11 of 24 either as DUB or as an inhibitor of ubiquitination [150]. Figure 4. The structure of OTUB1 and E2. (A) Crystal structure of OTUB1. (B) Crystal structure of OTUB1-ubiquitin Figure 4. The structure of OTUB1 and E2. (A) Crystal structure of OTUB1. (B) Crystal structure of OTUB1-ubiquitin alde- aldehyde-UBC13~Ub. hyde-UBC13~Ub. (C) Superposition (C) Superposition of OTUB1 of OTUB1 structures structures in in (A,B). (A,B). Ubiquitin Ubiquitin (yellow),UBC13 (yellow), UBC13(green), (green),OTUB1 OTUB1 without without ubiquitin (white-blue), OTUB1 with ubiquitin (blue), N-terminal ubiquitin-binding helix (blue) is disordered in theinapo- ubiquitin (white-blue), OTUB1 with ubiquitin (blue), N-terminal ubiquitin-binding helix (blue) is disordered the apoenzyme. (PDB: 4DHZ and enzyme. (PDB: 4DHZ and 2ZFY). 2ZFY). OTUB1 is involved in many cancers [142]. For example, OTUB1 regulates epithelial- mesenchymal cell transformation through the PI3K-AKT-GSK3β signaling pathway in colorectal cancer [141]. OTUB1 activates RhoA to promote prostate cancer cell invasion and promote tumorigenesis in prostate cancer [152]. OTUB1 represses the ubiquitination and degradation of FOXM1 in breast cancer [146]. The isoform OTUB1 promotes G1-S phase transition, inhibits cell apoptosis, and promotes tumor cell proliferation as well as invasion in gastric cancer [153]. But so far, there are no specific inhibitors for OTUB1. 4.5. OTULIN, with a Little Help of Ubiquitin for Activation OTULIN (OTU domain DUB with Linear Linkage specificity), also known as Gumby/ FAM105b, is a member of the ovarian tumor-associated protease family (OTUs) and it is a deubiquitinating enzyme that specifically hydrolyzes Met1-Ub linear linkages [154]. The human OTULIN gene is located in chromosome 5 and encodes a protein of 352 amino acids [18,36]. OTULIN can actively cleave the connection point between Met1 and Ub in the linear chain, but it has a very weak activity in the connection point between ubiquitin and the substrate lysine. The distal and proximal Met1-Ub linear linkages can respectively interact with the ubiquitin-binding sites S1 and S1’ (Figure 5A). Point mutations in S1 or S1’ will reduce the affinity of OTULIN for Met1-Ub, thereby reducing the DUB activity of OTULIN [154]. In the absence of substrate, the histidine and cysteine residues of the catalytic triad of OTULIN (Cys129, His339, and Asn341) display two alternating conformations [155], with occupancy rates of 28% and 36% for cysteine and histidine, respectively (Figure 5B), exhibiting an auto-inhibited active site conformation. Interestingly, the interaction of the linear ubiquitin linkage substrate to the active site of OTULIN is essential for activation. Indeed, the proximal ubiquitin directly participates in the activity of the catalytic center. Particularly, the carbonyl group of Met1 interferes with the non-productive conformation of His339, thereby changing it to an active conformation (Figure 5C,D). Ubiquitin Glu16, at the binding site of Met1-Ub and OTULIN, has a decisive effect on catalytic activity. The Glu16 side chain of the proximal ubiquitin is inserted into the active site, replacing the position of Asp336, and further restricting the His339 confor- mation [154]. Glu16 and Asp336 prompts His339 into an active conformation to further deprotonates Cys129 [156]. At the same time, Glu16 cooperates with Asn341 to further increase the catalytic activity of the catalytic center. Mutation of Glu16 to Ala reduces the catalytic efficiency of OTULIN by 240-fold [154,155]. The distal and proximal Met1-Ub linear linkages can respectively interact with the ubiquitin-binding sites S1 and S1’ (Figure 5A). Point mutations in S1 or S1’ will reduce the affinity of OTULIN for Met1-Ub, thereby reducing the DUB activity of OTULIN [154]. In the absence of substrate, the histidine and cysteine residues of the catalytic triad of OTU- LIN (Cys129, His339, and Asn341) display two alternating conformations [155], with oc- Int. J. Mol. Sci. 2021, 22, 986 12 of 24 cupancy rates of 28% and 36% for cysteine and histidine, respectively (Figure 5B), exhib- iting an auto-inhibited active site conformation. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 26 Figure 5. The structure of OTULIN. (A) Structure of OTULIN OTU domain in complex with Met1-di-ubiquitin. Figure 5.(blue), OTULIN The structure ubiquitinof(yellow). OTULIN.(B) (A)Rearrangement Structure of OTULIN OTU domain of the active in complex site residues in the with Met1-di-ubiquitin. absence (white blue) of OTULIN bound (blue), ubiquitin (yellow). (B) Rearrangement of the active site residues in the absence (white blue) Met1-di-ubiquitin. (C) Rearrangement of active site residues in the presence (blue) of bound Met1-di-ubiquitin. of bound Met1-di- (D) ubiquitin. (C)ofRearrangement Superposition (B,C). (PDB: 3ZNZof active site residues in the presence (blue) of bound Met1-di-ubiquitin. (D) Superposition and 3ZNV). of (B,C). (PDB: 3ZNZ and 3ZNV). OTULIN regulates immune receptor signaling by interacting with LUBAC. OTULIN de- ficiencyInterestingly, results in severethe interaction of the linear auto-inflammatory ubiquitin disease linkage in patients substrate [157]. OTULIN to the canactive site protect of OTULIN is essential for activation. Indeed, the proximal ubiquitin the liver against cell death and cancer, OTULIN deficiency in non-hematopoietic cells directly participates in the activity causing of the catalytic liver pathology [156,158].center. OTULINParticularly, the carbonyl inhibits liver inflammationgroup of hepatocellular and Met1 interferes with the non-productive carcinoma conformation via restraining FADD- and RIPK1of His339, thereby changing kinase-mediated it to apoptosis hepatocyte an active confor- [159]. mation Recent (Figure studies 5C,D). have shown Ubiquitin Glu16, at that OTULIN may therelated binding to site HIV-1of Met1-Ub activationand OTULIN, has [160]. a decisive effect on catalytic activity. The Glu16 side chain of the proximal ubiquitin is 4.6. RPN11, inserted Cleaves into the Ubiquitin the active Chain inthe site, replacing the position 26S Proteasome of Asp336, and further restricting the His339 There conformation [154]. Glu16 are three important and Asp336enzymes deubiquitinating prompts in His339 human into an activeUSP14, proteasomes: confor- mation and UCH37, to further Rpn11deprotonates [2]. Rpn11 is a Zn2+ dependent Cys129 [156]. At the sameof protein time, Glu16 cooperates the JAMM/MPN with protein Asn341 family to further [161], whichincrease is located thestraight catalytic activity over of the the gate catalytic channel center. of the 20SMutation proteasome of Glu16 cen- to[162]. ter Ala reduces The 26Sthe catalytic efficiency proteasome of OTULIN mainly recognizes by 240-foldtarget ubiquitinated [154,155]. proteins through the OTULIN regulates immune receptor signaling by two intrinsic ubiquitin receptors Rpn10 and Rpn13, and cleaves the 48-linkedinteracting with LUBAC. OTULIN polyubiquitin deficiency chain by theresults in severe auto-inflammatory deubiquitinating activity of Rpn11,disease whichinassists patients the[157]. OTULIN cancore 20S proteasome pro- tect thetoliver particle against degrade cell death proteins and cancer, [163,164]. Rpn11 OTULIN is activated deficiency in non-hematopoietic by proteasome association as wellcells ascausing by ATPliverhydrolysis pathology [165]. [156,158]. OTULIN inhibits liver inflammation and hepatocellular Rpn11 is carcinoma viahard to purify restraining alone,and FADD- butRIPK1 in complex with Rpn8hepatocyte kinase-mediated forms a stable and func- apoptosis [159]. tionally Recent DUB studies heterodimer have shown[166,167] (Figure that OTULIN may 6A). Rpn8toand related Rpn11 HIV-1 are canonical activation [160]. MPN domains, but Rpn8 is catalytically inactive and does not contain the JAMM Zn-binding motif and it has 4.6. RPN11, mainly Cleaves a supporting the Ubiquitin Chainrole in thefor26SRpn11 [166]. Also, Rpn11 does not show Proteasome complete features of a fully active DUB structure, There are three important deubiquitinating enzymes it lacks conserved in humanresidues responsible proteasomes: USP14, for ubiquitin-binding and stabilization of the isopeptide bond UCH37, and Rpn11 [2]. Rpn11 is a Zn2+ dependent protein of the JAMM/MPN protein for cleavage [166,167]. family [161], which is located straight over the gate channel of the 20S proteasome center [162]. The 26S proteasome mainly recognizes ubiquitinated target proteins through the two intrinsic ubiquitin receptors Rpn10 and Rpn13, and cleaves the 48-linked polyubiqui- tin chain by the deubiquitinating activity of Rpn11, which assists the 20S proteasome core particle to degrade proteins [163,164]. Rpn11 is activated by proteasome association as well as by ATP hydrolysis [165]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 26 Int. J. Mol. Sci. 2021, 22, 986 13 of 24 Figure6.6. Structure Figure Structure of Ubiquitin-Bound Rpn11-Rpn8. of Ubiquitin-Bound Rpn11-Rpn8.(A) (A)Structure StructureofofRpn11-Rpn8 Rpn11-Rpn8 with with ubiquitin. ubiquitin. Ubiquitin Ubiquitin (yellow), (yellow), Rpn11 (blue), Rpn8 Rpn11(blue), Rpn8 (gray), (gray),Zinc Zinc(purple) (purple)(B) (B)Comparison Comparison of of conserved conserved binding binding sites sites of Rpn11 of Rpn11 andand AMSH-LP AMSH-LP for the for the Ile44-ubiquitin-binding Ile44-ubiquitin-bindingsurface. surface.Rpn11 Rpn11(blue), (blue),AMSH-LP AMSH-LP(gray).(gray).Val83 Val83andandPhe87 Phe87are arefrom fromRpn11, Rpn11,Val328 Val328and andPhe332 Phe332are are from from AMSH-LP. AMSH-LP. (C) Comparison (C) Comparison of theofactive the active sites sites of of Rpn11 Rpn11 and Rpn11-Ub. and Rpn11-Ub. (PDB:(PDB: 5U4P,5U4P, 5W83,5W83, 4O8X4O8X and 2ZNV). and 2ZNV). Rpn11 Rpn11and andAMSH-LP AMSH-LP bind ubiquitin bind ubiquitinin ainsimilar orientation, a similar but their orientation, but interaction with their interaction the withIle44 thehydrophobic patch ispatch Ile44 hydrophobic quiteisdifferent. It is known quite different. that Val328 It is known thatand Phe332 Val328 andof AMSH- Phe332 of LP interact interact AMSH-LP with ubiquitin Ile44, but with ubiquitin structural Ile44, comparison but structural showsshows comparison that the thatequivalent the equiv- positions in Rpn11 alent positions (Val83 and in Rpn11 Phe87) (Val83 and are oriented Phe87) in an opposite are oriented directiondirection in an opposite from AMSH- from LP (Figure 6B). Phe87 of Rpn11 does not bind ubiquitin but participates AMSH-LP (Figure 6B). Phe87 of Rpn11 does not bind ubiquitin but participates in stabi- in stabilizing the heterodimer interface of Rpn11 and Rpn8, which may help to place Rpn11 lizing the heterodimer interface of Rpn11 and Rpn8, which may help to place Rpn11 above above the central pore of the proteasome the central and allow translocation-coupled pore of the proteasome substrate deubiquitination and allow translocation-coupled [167]. substrate deubiquiti- nationUbiquitin [167]. binding to Rpn11 does not require substrate interaction with the proteasome RP (regulatory Ubiquitin particle) binding [168]. The MPN to Rpn11 does domain not require of Rpn11 has three substrate essential interaction regulatory with the pro- regions: Insertion 1 (Ins-1), Insertion 2 (Ins-2) and the catalytic loop. teasome RP (regulatory particle) [168]. The MPN domain of Rpn11 has three essential Ins-1 and Ins-2 are nec- reg- essary for activity in many MPN domain [77]. Ins-2 region determines the DUB ulatory regions: Insertion 1 (Ins-1), Insertion 2 (Ins-2) and the catalytic loop. Ins-1 and Ins- specificity for a specific 2 are necessaryubiquitin connection for activity in manytypeMPN bydomain interaction [77].with Ins-2the proximal region ubiquitin determines [77]. the DUB However, Ins-2 of Rpn11 is not ordered and does not interact with the proximal ubiquitin (Figure 6A) [167]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 15 of 26 Int. J. Mol. Sci. 2021, 22, 986 specificity for a specific ubiquitin connection type by interaction with the proximal ubiq- 14 of 24 uitin [77]. However, Ins-2 of Rpn11 is not ordered and does not interact with the proximal ubiquitin (Figure 6A) [167]. In other JAMM members, the Ins-1 loop has a permanently active β-hairpin structure, In other such JAMM or as in AMSH members, AMSH-LP, thebut Ins-1 theloop Ins-1has a permanently loop of Rpn11 is active closedβ-hairpin and inactive.structure, Ac- such cording to the crystal structure of the Rpn11-Rpn8 heterodimer bound to ubiquitin, theto as in AMSH or AMSH-LP, but the Ins-1 loop of Rpn11 is closed and inactive. According theIns-1 crystal loopstructure of Rpn11ofchanges the Rpn11-Rpn8 heterodimer its conformation bound to with different ubiquitin, states the Ins-1 loop of the proteasome, be-of Rpn11 changes its conformation with different states of the proteasome, fore and after ubiquitin-binding, from the closed state to the β-hairpin active state [169].before and after ubiquitin-binding, from the closed state to the β-hairpin active state [169]. The Ins-1 loop is required for DUB activity [76,169]. The newly formed β-hairpin and res- The Ins-1 loop is required idues Arg74for DUB activity and Gly75 from[76,169]. ubiquitinThe newly formed constitute and residues β-hairpintriple-strand an anti-parallel Arg74 and β structure, Gly75 fromplacing thereby ubiquitintheconstitute an anti-parallel scissile isopeptide bond attriple-strand β structure, the active site for cleavagethereby [169] placing (Figure the 6 scissile A,C).isopeptide bond at the active site for cleavage [169] (Figure 6A,C). Rpn11 Rpn11 is is ananimportant important potential potential target targetforfordrug drugtherapy therapy[161]. [161].RPN11 RPN11may mayfunction functionas anasoncogene an oncogenethat that promotes proliferation promotes proliferationandandmigration migrationof of breast cancer breast cancercells cells[170]. [170]. 4.7.4.7. USP12-UAF1-WDR20, USP12-UAF1-WDR20,Help HelpofofPartners Partners for for Activation Activation Prostate Prostate cancer cancer(PC)(PC)isisa acommon commonmale male malignant malignant tumor [171]. Androgen tumor [171]. Androgenreceptor receptor (AR) (AR)plays playsa key role a key in regulating role in regulating thethe gene transcription gene transcription program program ofof PC [172]. PC [172].USP12 USP12isisa aco- activator of androgen co-activator receptor of androgen involved receptor in PC involved through in PC through deubiquitination deubiquitination andandstabilization stabiliza- of tion AR [24,173]. USP12 of AR [24,173]. (370(370 USP12 amino aminoacids) shows acids) showsa high a highdegree degree ofof homology homology (88%) (88%)withwith Usp46 Usp46 (366(366 amino amino acids). acids). USP1-associated USP1-associated factor1 1(UAF1) factor (UAF1)isisa a80KDa 80KDamultidomain multidomainprotein pro- tein formed formed by anby an N-terminal N-terminal WD40 WD40 repeat repeat domain, domain, a aCentral CentralAuxiliary Auxiliary domain domainand anda C- a C- terminal terminal SUMO-likedomain SUMO-like domain[174,175] [174,175] (Figure (Figure 7A). UAF1 UAF1 isis aa USP USPmodulator modulatorthat thatcan can bind to and activate bind to and activate three human deubiquitinases: USP1, USP12, and USP46 human deubiquitinases: USP1, USP12, and USP46 [176,177]. [176,177]. The Theactive activesite of of site free USP12 free USP12hashas a catalytically active a catalytically conformation, active but itsbut conformation, activity is low.isThe its activity low. The narrow catalytic cleft of free USP12 needs to undergo a conformationaltochange narrow catalytic cleft of free USP12 needs to undergo a conformational change accom-to modate ubiquitin accommodate [174].[174]. ubiquitin Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 26 Figure 7. Structure of USP12-UAF1-WDR20. (A) Structure of with USP12-UAF1. (B) Structure of with USP12-UAF1(9-580)- Figure 7. Structure of USP12-UAF1-WDR20. (A) Structure of with USP12-UAF1. (B) Structure of with USP12-UAF1(9- Ub.580)-Ub. (C) Active site of USP12. (D) Differences of Fingers domain in the absence and presence of UAF1. (E) Structure of (C) Active site of USP12. (D) Differences of Fingers domain in the absence and presence of UAF1. (E) Structure USP12-UAF1(9-580)-WDR20. of USP12-UAF1(9-580)-WDR20. FreeFree USP12 (pink), USP12 UAF1(deep (pink), UAF1(deepblue), WDR20 blue), WDR20(green), ubiquitin (green), ubiquitin(yellow). (yellow).(PDB: (PDB:5K16, 5K16, 518W, 5K1B, and5K1B, 518W, 5K1C).and 5K1C). UAF1 interacts with the distal end of the USP12 Finger domain, causing a series of structural changes in USP12, which are then transmitted to the essential ubiquitin contact loop adjacent to the catalytic cleft, thereby enhancing the activity of USP12 (Figure 7B,C) [174,177]. The interaction of UAF1 can stabilize the outer edge of the Fingers domain, Int. J. Mol. Sci. 2021, 22, 986 15 of 24 UAF1 interacts with the distal end of the USP12 Finger domain, causing a series of structural changes in USP12, which are then transmitted to the essential ubiquitin contact loop adjacent to the catalytic cleft, thereby enhancing the activity of USP12 (Figure 7B,C) [174,177]. The interaction of UAF1 can stabilize the outer edge of the Fingers domain, transforming it from an unstable state into a regular β chain, completing the four-stranded β structure of the Fingers domain [174]. Mutation analysis confirmed the important role of the Fingers domain in USP12 for its activation by UAF1 binding [178] (Figure 7D). WDR20 (WD repeat-containing protein 20) is a single domain protein containing WD40-repeat motifs [174]. USP12 can be further activated by WDR20 [179,180]. WDR20 in- teracts with the USP12 Palm domain, and it is paralleled with the WD40 repeat domain of UAF1 (Figure 7E). The binding sites of UAF1 and WDR20 are located on an opposite surface to the catalytic triad of USP12, but they can allosterically activate USP12 through different structural rearrangements. UAF1 activation of USP12 requires structural fine-tuning at the ubiquitin-binding site, while WDR20 directly regulates the catalytic center of the enzyme remotely, transforming USP12 into a compact structure similar to USP46-Ub [174]. Interestingly, it has been shown that USP12-Ub and UAF1 have two possible binding sites: The Finger domain and the back of the ubiquitin-binding cleft [178]. The second UAF1 binding site has low affinity and partially overlaps the WDR20-USP12 binding inter- face, thus the binding of WDR20 to USP12/UAF1 may prevent the second UAF1 molecule from binding to USP12, so it may have a regulatory effect [178]. Also, UAF1 can interact with USP1 and USP46 in addition to USP12 [177]. USP1 is considered an important drug target [181]. USP1 was the first deubiquitinating enzyme found to enhance its activity through UAF1 interaction, displaying an increase up to 35- fold [176]. The USP1-UAF1 protein complex is an important regulator in DNA damage response pathways [182]. In addition to PC, USP12 is involved in other diseases. USP12 is a potential target for the treatment of Hepatocellular Carcinoma (HCC), which can prevent G2/M and trigger apoptosis [183]. Also, USP12 causes autophagy and confer neuroprotection in Huntington’s disease (HD) [184]. 5. Conclusions In this review, we have introduced and described the mechanisms for the catalytic regulation in different types of DUBs. Most of the discovered DUBs have a similar basic mechanism of deubiquitinating activity for cleavage of the isopeptide bond, but with the presence of regulatory domains adjacent to the catalytic domain or by the formation of tetrameric complex assemblies, DUBs have developed specific strategies to regulate the interaction and cleavage of the different types of linkages of the ubiquitin-code, which will probably reveal layers of complexity that are still not completely understood. Deubiquitinating enzymes play an essential role in the process of life, and the occur- rence and development of many diseases are directly related to them [21–23]. The in-depth study of the molecular mechanism of deubiquitination will help us to understand and treat the physiological functions and diseases. Therefore, research on deubiquitinating enzymes has always attracted much attention to the pharmaceutical industry. Research in recent decades has shown that deubiquitinating enzymes can regulate multiple signaling pathways [33–36], suggesting to researchers the development of new medicines based on the pathogenesis of different diseases that can target deubiquitinating enzymes. For ex- ample, the development of deubiquitinating enzyme inhibitors [185], especially selective inhibitors, may have a good therapeutic effect [186,187]. As one of the most well-studied DUBs, USP7 has many small molecule inhibitors. USP7 is currently an important targeted protein for tumor therapy. Studies have found that USP7 inhibitors P5091 and Parthenolide inhibits Wnt signaling and colorectal tumor growth; specific inhibitor HBX 19818 can selectively reduce the activity of USP7; USP7 small-molecule inhibitors P5091 and P22077 can induce tumor cell apoptosis by enhancing intracellular oxidative stress response and endoplasmic reticulum stress response [40,188–191]. Struc- Int. J. Mol. Sci. 2021, 22, 986 16 of 24 tural biology helped to reveal some inhibitory mechanisms of small molecule inhibitors on DUBs. As ubiquitin-competitive small molecules, FT671 and FT827 have high affinity and specificity for inhibiting USP7, they target the dynamic pockets nearby the catalytic center of the apo-USP7 [192]. FT671 extends into the Fingers subdomain and catalytic center of USP7, reducing the stability of the USP7 substrate, leading to P53 target gene transcription and tumor growth inhibition in mice. FT827 extends to the catalytic cen- ter and forms a covalent bond with the catalytic Cys223 and thus inhibiting the activity of USP7 [192]. XL188 is another selective inhibitor of USP7, which occupies some sub- sites in the substrate-binding cleft, resulting in the increase of tumor suppressor proteins P53 and P21 [193]. Other DUBs-related inhibitors have also been discovered, for example, capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11, which can stabilize proteasome substrates and block the proliferation of cancer cells [194]. A specific inhibitor of USP14, b-AP15, can significantly increase the apoptosis of leukemia cells [195]. IU1-47 is a specific inhibitor of USP14, which inhibits tumors by reducing the expression of tau protein in cells [196]. There are also some newly discovered USP14 candidate in- hibitors [197,198]. ML323 is a highly effective inhibitor of USP1-UAF1 deubiquitinase complex, blocking the allosteric activation pathway may be another strategy to study and discover USP inhibitors [178,199,200]. It is worth noting that there are still many unsolved problems in the research of deu- biquitinating enzymes, and it is likely that novel regulatory mechanisms, similar to the examples depicted in this review, will be uncovered for other DUBs in the future. The de- velopment of inhibitory DUB compounds, to treat tumor growth and other pathologies, are based on a better understanding of the structure and regulatory mechanism in the context of each particular DUB. Research on DUB inhibitor drugs has made some progress, but more efforts will be needed to overcome all steps required for clinical application and drug development. 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