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.

14-3-3 is a family of highly conserved protein that is involved in a number of cellular processes. In this study, we identified that the high expression of 14-3-3γ in various cancer cell lines correlates with the invasiveness of the cancer cells. Overexpression of 14-3-3γ causes changes to the morphologic characteristics of cell transformation, and promotes cell migration and invasion. The cells overexpressed with 14-3-3γ have been shown to stimulate foci and tumor formation in SCID-NOD mice in concert with signaling components as reported with the 14-3-3β. In our previous study, we demonstrated that 14-3-3γ inhibits apoptotic cell death and mediates the promotion of cell proliferation in immune cell lines. Earlier, binding partners for 14-3-3γ were defined by screening. We found that USP37, one of deubiquitinating enzymes (DUBs), belongs to this binding partner group. Therefore, we investigated whether 14-3-3γ mediates proliferation in cancer cells, and 14-3-3γ by USP37 is responsible for promoting cell proliferation. Importantly, we found that USP37 regulates the stability of ubiquitin-conjugated 14-3-3γ through its catalytic activity. This result implies that the interactive behavior between USP37 and 14-3-3γ could be involved in the regulation of 14-3-3γ degradation. When all these findings are considered together, USP37 is shown to be a specific DUB that prevents 14-3-3γ degradation, which may contribute to malignant transformation via MAPK signaling pathway, possibly providing a new target for therapeutic objectives of cancer.

To increase the half-life of growth hormones, we proposed its long-lasting regulation through the ubiquitin-proteasome system (UPS). We identified lysine residues (K67, K141, and K166) that are involved in the ubiquitination of human growth hormone (hGH) using ubiquitination site prediction programs to validate the ubiquitination sites, and then substituted these lysine residues with arginine residues. We identified the most effective substituent (K141R) to prevent ubiquitination and named it AUT-hGH. hGH was expressed and purified in the form of hGH-His, and ubiquitination was first verified at sites containing K141 in the blood stream. Through the study, we propose that AUT-hGH with an increased half-life could be used as a long-lasting hGH in the blood stream.