Protein Stability of Mitochondrial Superoxide Dismutase SOD2 is Regulated by USP36 Myung-Sun Kim, Suresh Ramakrishna, Key-Hwan Lim, Jun-Hyun Kim, and Kwang-Hyun Baek * Department of Biomedical Science, CHA General Hospital, CHA University, Seoul 135-081, Republic of Korea ABSTRACT 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. J. Cell. Biochem. 112: 498–508, 2011. ß 2010 Wiley-Liss, Inc. KEY WORDS: DEUBIQUITINATION; OXIDATIVE STRESS; ROS U biquitination and deubiquitination play a key role in maintaining cell homeostasis and are involved in the regulation of a number of biological processes such as cell cycle control, transcriptional regulation, immune response, apoptosis, oncogenesis, pre-implantation, and intracellular signaling path- ways [Ciechanover, 1998; Hershko and Ciechanover, 1998; Amerik and Hochstrasser, 2004; Baek, 2006; Ramakrishna et al., in press]. Protein ubiquitination is a critical event that is mediated by sequential enzymatic actions via ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). A novel ubiquitination factor (E4) required for efficient poly- ubiquitination has been identified in yeast [Ciechanover, 1998; Koegl et al., 1999]. These polyubiquitinated proteins are then directed towards the 26S proteasome for ubiquitin dependent protein degradation [Pickart, 2001]. Deubiquitination, which is a process counteracting protein ubiquitination, is catalyzed by deubiquitinating (DUB) enzymes that are involved in the removal of ubiquitin by hydrolyzing the Ub-substrate isopeptide bond. DUB enzymes are primarily classified into 5 different classes: the ubiquitin-specific protease (USP), the ubiquitin C-terminal hydrolase (UCH), the ovarian tumor (OTU), the Josephin domain are papain-like cysteine proteases and the Jab1/MPN domain-associated metalloisopeptidase (JAMM) [Baek, 2002; Amerik and Hochstrasser, 2004]. USP36, an USP, is primarily localized to the nucleoli and is required to maintain normal nucleolar structure [Endo et al., 2009b]. USP36 was found to interact and stabilize nucleolar proteins nucleophosmin/B23 and fibrillarin by reducing its ubiquitination level and preventing it from protein degradation. The deubiquiti- nating activity of USP36 controls transcriptional regulation and ribosome biogenesis in response to the changes in environmental conditions. In addition, the siRNA of USP36-transfected cells showed slow a proliferation rate, indicating that this protein is involved in regulating normal cellular proliferation [Endo et al., 2009b]. Recently, it has been reported that nucleophosmin/B23 is responsible for recruiting USP36 in the nucleoli and regulating its deubiquitinating function on nucleolar proteins [Endo et al., 2009a]. Previously, we reported that HeLa DUB-1 was a C-terminal truncated form of USP36 and was shown to have PEST motifs and was polyubiquitinated [Kim et al., 2004, 2005]. Elevated expression Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 112:498–508 (2011) 498 Abbreviations used: CBB, Coomassie Brilliant Blue; CHX, cyclohexamide; DUB enzyme, deubiquitinating enzyme; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; ROS, reactive oxygen species; SOD, superoxide dismutase; Ub, ubiquitin; UPP, ubiquitin-proteasomal pathway; USP, ubiquitin-specific protease. Grant sponsor: Korea Health 21 R&D Project, Ministry for Health, Welfare and Family affairs, Republic of Korea; Grant number: 01-PJ10-PG6-01GN13-0002; Grant sponsor: Biomedical Poteome Research Center; Grant number: A03000. *Correspondence to: Prof. Kwang-Hyun Baek, Department of Biomedical Science, CHA General Hospital, CHA University, 606-16 Yeoksam 1-Dong, Gangnam-Gu, Seoul 135-081, Republic of Korea. E-mail: baek@cha.ac.kr Received 7 April 2010; Accepted 25 October 2010  DOI 10.1002/jcb.22940  ß 2010 Wiley-Liss, Inc. Published online 15 November 2010 in Wiley Online Library (wileyonlinelibrary.com).