Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE

Wang,Jianmin; Liu,Yingli; Tang,Lijuan; Qi,Sufen; Mi,Yingjun; Liu,Dianwu; Tian,Qingbao

doi:10.3892/ijmm.2017.2984

Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE

Authors:
- Jianmin Wang
- Yingli Liu
- Lijuan Tang
- Sufen Qi
- Yingjun Mi
- Dianwu Liu
- Qingbao Tian
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Affiliations: Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China
Published online on: May 10, 2017 https://doi.org/10.3892/ijmm.2017.2984
Pages: 47-56
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The present study aimed to identify candidate substrates of ubiquitin-specific protease (USP)13 using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). USP13 is a well-characterized member of the USP family, which regulates diverse cellular functions by cleaving ubiquitin from ubiquitinated protein substrates. However, existing studies indicate that USP13 has no detectable hydrolytic activity in vitro. This finding implies that USP13 likely has different substrate specificity. In this study, a USP cleavage assay was performed using two different types of model substrates (glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase) to detect the deubiquitinating enzyme (DUB) activity of USP13. In addition, a proteomic approach was taken by using 2D-DIGE to detect cellular proteins whose expressoin is significantly altered in 293T cell lines following the overexpression of USP13 or its C345S mutant (the catalytically inactive form). The data indicated that USP13 still has no detectable DUB activity in vitro nor does C345S. The results of 2D-DIGE demonstrated that the expression of several proteins increased or decreased significantly in 293T cells following the overexpression of USP13. Mass spectroscopy analysis of gel spots identified 7 proteins, including 4 proteins with an increased expression, namely vinculin, thimet oligopeptidase, cleavage and polyadenylation specific factor 3, and methylosome protein 50, and 3 proteins with a decreased expression, namely adenylosuccinate synthetase, annexin and phosphoglycerate mutase. In addition, in the samples of 293T cell lines after the overexpression of USP13 and USP13 C345S, vinculin exhibited an increased expression, suggesting that it may be a candidate substrate of USP13. However, sufficient follow-up validation studies are required in order to determine whether vinculin protein directly interacts with USP13.

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1	Kim YK, Kim YS, Yoo KJ, Lee HJ, Lee DR, Yeo CY and Baek KH: The expression of Usp42 during embryogenesis and spermatogenesis in mouse. Gene Expr Patterns. 7:143–148. 2007. View Article : Google Scholar
2	Quesada V, Díaz-Perales A, Gutiérrez-Fernández A, Garabaya C, Cal S and López-Otín C: Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochem Biophys Res Commun. 314:54–62. 2004. View Article : Google Scholar : PubMed/NCBI
3	Baek SH, Park KC, Lee JI II, Kim KI II, Yoo YJ, Tanaka K, Baker RT and Chung CH: A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct N- and C-terminal extensions. Biochem J. 334:677–684. 1998. View Article : Google Scholar : PubMed/NCBI
4	D'Andrea A and Pellman D: Deubiquitinating enzymes: a new class of biological regulators. Crit Rev Biochem Mol Biol. 33:337–352. 1998. View Article : Google Scholar : PubMed/NCBI
5	Wing SS: Deubiquitinating enzymes - the importance of driving in reverse along the ubiquitin-proteasome pathway. Int J Biochem Cell Biol. 35:590–605. 2003. View Article : Google Scholar : PubMed/NCBI
6	Everett RD, Meredith M, Orr A, Cross A, Kathoria M and Parkinson J: A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J. 16:1519–1530. 1997. View Article : Google Scholar : PubMed/NCBI
7	Zhang W, Tian QB, Li QK, Wang JM, Wang CN, Liu T, Liu DW and Wang MW: Lysine 92 amino acid residue of USP46, a gene associated with 'behavioral despair' in mice, influences the deubiquitinating enzyme activity. PLoS One. 6:e262972011. View Article : Google Scholar : PubMed/NCBI
8	Dang LC, Melandri FD and Stein RL: Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry. 37:1868–1879. 1998. View Article : Google Scholar : PubMed/NCBI
9	Yin ST, Huang H, Zhang YH, Zhou ZR, Song AX, Hong FS and Hu HY: A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes. Biochem Biophys Res Commun. 416:76–79. 2011. View Article : Google Scholar : PubMed/NCBI
10	Timms KM, Ansari-Lari MA, Morris W, Brown SN and Gibbs RA: The genomic organization of isopeptidase T-3 (ISOT-3), a new member of the ubiquitin specific protease family (UBP). Gene. 217:101–106. 1998. View Article : Google Scholar : PubMed/NCBI
11	Stein RL, Chen Z and Melandri F: Kinetic studies of isopeptidase T: modulation of peptidase activity by ubiquitin. Biochemistry. 34:12616–12623. 1995. View Article : Google Scholar : PubMed/NCBI
12	Lacombe T and Gabriel JM: Further characterization of the putative human isopeptidase T catalytic site. FEBS Lett. 531:469–474. 2002. View Article : Google Scholar : PubMed/NCBI
13	Reyes-Turcu FE, Horton JR, Mullally JE, Heroux A, Cheng X and Wilkinson KD: The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell. 124:1197–1208. 2006. View Article : Google Scholar : PubMed/NCBI
14	Zhang YH, Zhou CJ, Zhou ZR, Song AX and Hu HY: Domain analysis reveals that a deubiquitinating enzyme USP13 performs non-activating catalysis for Lys63-linked polyubiquitin. PLoS One. 6:e293622011. View Article : Google Scholar
15	Bonnet J, Romier C, Tora L and Devys D: Zinc-finger UBPs: regulators of deubiquitylation. Trends Biochem Sci. 33:369–375. 2008. View Article : Google Scholar : PubMed/NCBI
16	Catic A, Fiebiger E, Korbel GA, Blom D, Galardy PJ and Ploegh HL: Screen for ISG15-crossreactive deubiquitinases. PLoS One. 2:e6792007. View Article : Google Scholar : PubMed/NCBI
17	Liu Y, Soetandyo N, Lee JG, Liu L, Xu Y, Clemons WM Jr and Ye Y: USP13 antagonizes gp78 to maintain functionality of a chaperone in ER-associated degradation. eLife. 3:e013692014. View Article : Google Scholar : PubMed/NCBI
18	Scortegagna M, Subtil T, Qi J, Kim H, Zhao W, Gu W, Kluger H and Ronai ZA: USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains. J Biol Chem. 286:27333–27341. 2011. View Article : Google Scholar : PubMed/NCBI
19	Yeh HM, Yu CY, Yang HC, Ko SH, Liao CL and Lin YL: Ubiquitin-specific protease 13 regulates IFN signaling by stabilizing STAT1. J Immunol. 191:3328–3336. 2013. View Article : Google Scholar : PubMed/NCBI
20	Chen M, Gutierrez GJ and Ronai ZA: Ubiquitin-recognition protein Ufd1 couples the endoplasmic reticulum (ER) stress response to cell cycle control. Proc Natl Acad Sci USA. 108:9119–9124. 2011. View Article : Google Scholar : PubMed/NCBI
21	Zhao X, Fiske B, Kawakami A, Li J and Fisher DE: Regulation of MITF stability by the USP13 deubiquitinase. Nat Commun. 2:4142011. View Article : Google Scholar : PubMed/NCBI
22	Liu J, Xia H, Kim M, Xu L, Li Y, Zhang L, Cai Y, Norberg HV, Zhang T, Furuya T, et al: Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell. 147:223–234. 2011. View Article : Google Scholar : PubMed/NCBI
23	Zhang J, Zhang P, Wei Y, Piao HL, Wang W, Maddika S, Wang M, Chen D, Sun Y, Hung MC, et al: Deubiquitylation and stabilization of PTEN by USP13. Nat Cell Biol. 15:1486–1494. 2013. View Article : Google Scholar : PubMed/NCBI
24	Geng J, Huang X, Li Y, Xu X, Li S, Jiang D, Liang J, Jiang D, Wang C and Dai H: Down-regulation of USP13 mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis. Respir Res. 16:1242015. View Article : Google Scholar : PubMed/NCBI
25	Liu YL, Zheng J, Tang LJ, Han W, Wang JM, Liu DW and Tian QB: The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth. Gene. 572:49–56. 2015. View Article : Google Scholar : PubMed/NCBI
26	Tian QB, Okano A, Nakayama K, Miyazawa S, Endo S and Suzuki T: A novel ubiquitin-specific protease, synUSP, is localized at the post-synaptic density and post-synaptic lipid raft. J Neurochem. 87:665–675. 2003. View Article : Google Scholar : PubMed/NCBI
27	Tang LJ, Li Y, Liu YL, Wang JM, Liu DW and Tian QB: USP12 regulates cell cycle progression by involving c-Myc, cyclin D2 and BMI-1. Gene. 578:92–99. 2016. View Article : Google Scholar
28	Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248–254. 1976. View Article : Google Scholar : PubMed/NCBI
29	Tang W: Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis. Methods Mol Biol. 876:67–82. 2012. View Article : Google Scholar : PubMed/NCBI
30	Shirano Y and Shibata D: Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level. FEBS Lett. 271:128–130. 1990. View Article : Google Scholar : PubMed/NCBI
31	Kataeva I, Chang J, Xu H, Luan CH, Zhou J, Uversky VN, Lin D, Horanyi P, Liu ZJ, Ljungdahl LG, et al: Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli. J Proteome Res. 4:1942–1951. 2005. View Article : Google Scholar : PubMed/NCBI
32	Volontè F, Marinelli F, Gastaldo L, Sacchi S, Pilone MS, Pollegioni L and Molla G: Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E coli. Protein Expr Purif. 61:131–137. 2008. View Article : Google Scholar
33	Chou CP: Engineering cell physiology to enhance recombinant protein production in Escherichia coli. Appl Microbiol Biotechnol. 76:521–532. 2007. View Article : Google Scholar : PubMed/NCBI
34	Larsen CN, Krantz BA and Wilkinson KD: Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. Biochemistry. 37:3358–3368. 1998. View Article : Google Scholar : PubMed/NCBI
35	Kolkman A, Dirksen EH, Slijper M and Heck AJ: Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling. Mol Cell Proteomics. 4:255–266. 2005. View Article : Google Scholar : PubMed/NCBI
36	Levy C, Khaled M and Fisher DE: MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med. 12:406–414. 2006. View Article : Google Scholar : PubMed/NCBI
37	Ziegler WH, Liddington RC and Critchley DR: The structure and regulation of vinculin. Trends Cell Biol. 16:453–460. 2006. View Article : Google Scholar : PubMed/NCBI
38	Das C, Hoang QQ, Kreinbring CA, Luchansky SJ, Meray RK, Ray SS, Lansbury PT, Ringe D and Petsko GA: Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1. Proc Natl Acad Sci USA. 103:4675–4680. 2006. View Article : Google Scholar : PubMed/NCBI
39	Frisan T, Coppotelli G, Dryselius R and Masucci MG: Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth. FASEB J. 26:5060–5070. 2012. View Article : Google Scholar : PubMed/NCBI