Abnormal regulation of the ubiquitin-proteasome system (UPS) has been known to be involved in the pathogenesis of a variety of human diseases. A number of studies have focused on the identification of small modifiers for the UPS. Even though the proteasome inhibitor Bortezomib (Velcade®) has been approved for the therapy of multiple myeloma and mantle cell lymphoma, there are still no DUB inhibitors endorsed for clinical usage. Since deubiquitinating enzymes (DUBs) are becoming as a new class of modifiers in the UPS, potential drugs that target specific DUBs have been investigated with the development of experimental technologies for screening small inhibitor molecules. However, the molecular mechanisms of these molecules are poorly understood. In order to design and develop specific small inhibitor molecules for specific DUBs, identification of specific substrates and molecular structures for each DUB is required. Here, we review structures, substrates, and small inhibitor molecules of DUBs identified up to date, providing a clear rationale for the development of novel small inhibitor molecules of DUBs for cancer.

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All organs consisting of single cells are consistently maintaining homeostasis in response to stimuli such as free oxygen, DNA damage, inflammation, and microorganisms. The cell cycle of all mammalian cells is regulated by protein expression in the right phase to respond to proliferation and apoptosis signals. Post-translational modifications (PTMs) of proteins by several protein-editing enzymes are associated with cell cycle regulation by their enzymatic functions. Ubiquitination, one of the PTMs, is also strongly related to cell cycle regulation by protein degradation or signal transduction. The importance of deubiquitinating enzymes (DUBs), which have a reversible function for ubiquitination, has recently suggested that the function of DUBs is also important for determining the fate of proteins during cell cycle processing. This article reviews and summarizes the diverse roles of DUBs, including DNA damage, cell cycle processing, and regulation of histone proteins, and also suggests the possibility for therapeutic targets.

The Lethal giant larvae (Lgl) gene encodes a cortical cytoskeleton protein, Lgl, and is involved in maintaining cell polarity and epithelial integrity. Previously, we observed that Mgl-1, a mammalian homologue of the Drosophila tumor suppressor protein Lgl, is subjected to degradation via ubiquitin-proteasome pathway, and scaffolding protein RanBPM prevents the turnover of the Mgl-1 protein. Consequently, overexpression of RanBPM enhances Mgl-1-mediated cell proliferation and migration. Here, we analyzed the ability of ubiquitin-specific protease 11 (USP11) as a novel regulator of Mgl-1 and it requires RanBPM to regulate proteasomal degradation of Mgl-1. USP11 showed deubiquitinating activity and stabilized Mgl-1 protein. However, USP11-mediated Mgl-1 stabilization was inhibited in RanBPM-knockdown cells. Furthermore, in the cancer cell migration, the regulation of Mgl-1 by USP11 required RanBPM expression. In addition, an in vivo study revealed that depletion of USP11 leads to tumor formation. Taken together, the results indicated that USP11 functions as a tumor suppressor through the regulation of Mgl-1 protein degradation via RanBPM.

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HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N-and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSPp53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage.Posttranslational modification of numerous proteins in eukaryotic cells relies on the counterbalancing effect of ubiquitination and deubiquitination. Most proteins contain at least one or more lysine specific ubiquitination sites, and the ubiquitination process is catalyzed by the sequential actions of E1 ubiquitin-activating, E2 ubiquitin-conjugating, and E3 ubiquitin ligase enzymes, followed by protein transfer to the 26S proteasome. This process is referred as the ubiquitin proteasome pathway (UPP) 1 . In addition to the monoubiquitin chain, free ubiquitins can be conjugated to ubiquitin molecules attached to target proteins to link polyubiquitin chains. Structural and functional analyses of polyubiquitin chains indicate that polyubiquitin chains can make diverse conformation depending on ubiquitination of its lysine residues at Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63, and these are involved in the regulation of intracellular signaling 2 . The ubiquitination process is reversible and mono-or poly-ubiquitin chains can be removed by various deubiquitinating enzyme (DUBs) 3 . Approximately, ~100 DUBs are encoded in human genome that can be classified into at least six families; ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Josephin domains (MJDs), JAB1/MPN/MOV34 (JAMMs), and monocyte chemotactic protein-induced proteases (MCPIPs) 4 . The USP, UCH, OTU and MJD are known as cysteine proteases, and JAMM is known as metalloproteases 5,6 . The USPs specifically detach covalently bound ubiquitins from lysine sites to regulate substrate stabilization and intracellular localization 7 . Recent studies have shown that the USP family

USP20, one of deubiquitinating enzymes (DUBs) belonging to the subfamily of ubiquitin-specific protease (USP), regulates ubiquitin-mediated protein degradation. So far, USP20 has been identified as a binding protein and a regulator of hypoxia-inducible factor (HIF)-1α, β-adrenergic receptor, and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). In order to investigate other biological functions of USP20 with its novel substrates, we searched for putative substrates through two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. We found several putative substrates, some of which are related to cancer metabolism or neural disorders. Among these, the pyruvate kinase isoenzyme M2 (PKM2) had a high identity score. Most cancer cells contain a specific metabolic pathway, referred to as the Warburg effect. One well-known function of PKM2 is a main regulator in cancer metabolic pathways, and PKM2 promotes the Warburg effect and tumor growth. In addition, both PKM2 and HIF-1α upregulate the expression of target genes. From this evidence, it is expected that USP20 would be associated with the metabolic pathway through the regulation of PKM2 ubiquitination. Despite various roles of DUBs, the biological functions of USP20 in cellular mechanisms are poorly understood. Herein, we investigated the inter-action between PKM2 and USP20. Our results suggest a new molecular pathway in cancer metabolism through the regulation of PKM2.

SOD2 is a key mitochondrial antioxidant enzyme and its perturbation leads to oxidative cell death, which results in various disorders. In this study, we identified a deubiquitinating enzyme USP36 that regulates the protein stability of SOD2. The regulatory effect of USP36 on SOD2 was initially identified by 2-DE and MALDI-TOF/MS analyses. In addition, endogenous USP36 and SOD2 were shown to interact in an immunoprecipitation assay, which was verified using the yeast two-hybrid system. Furthermore, we demonstrated that SOD2 binds with ubiquitin molecules to form polyubiquitination chains and undergoes degradation through the ubiquitin-proteasomal pathway. Finally, USP36 was shown to be a specific deubiquitinating enzyme that reduces the ubiquitination level of SOD2 and was involved in SOD2 protein stability by extending its half-life.