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

Ubiquitination and deubiquitination are important processes for numerous intracellular mechanisms, and the imbalance of these two processes can cause severe diseases including cancer. Accordingly, deubiquitinating enzymes (DUBs) responsible for deubiquitination from their protein substrates become attractive targets for many studies. USP20, also known as VDU2, belongs to ubiquitin-specific protease (USP) subfamily of DUBs and has several important roles in cells as shown with other DUBs. USP20 stabilizes HIF-1α by abolishing von Hippel-Lindau protein (pVHL)-E3 ligase complex-mediated HIF-1α degradation. USP20 is also associated with β2 adrenergic receptor recycling. In addition, a previous study demonstrated that USP20 regulates Tax-induced NF-κB activation through its deubiquitinating activity. These studies provide a line of evidence that USP20 has critical roles in cellular functions. In this study, we generated and characterized a polyclonal and two monoclonal antibodies against USP20. It is feasible that USP20 antibodies can be useful to investigate USP20-related cellular mechanisms and to find novel substrates of USP20.