title: The catalytic triad of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation.
abstract: Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro.
authors: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China; Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China. Electronic address: songzb484@nenu.edu.cn.
authors: Zhen Bo
authors: ZB
authors: Song
authors: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China; Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China; Department of Handsurgery, China-Japan Union Hospital, Jilin University, Changchun 130033, China. Electronic address: liubiao20112013@163.com.
authors: Biao
authors: B
authors: Liu
authors: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China; Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China. Electronic address: liyuyin20112013@163.com.
authors: Yu Yin
authors: YY
authors: Li
authors: Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China. Electronic address: wuping20112013@163.com.
authors: Ping
authors: P
authors: Wu
authors: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China. Electronic address: baoyl800@nenu.edu.cn.
authors: Yong Li
authors: YL
authors: Bao
authors: Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China; Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China. Electronic address: huangyx20112013@163.com.
authors: Yan Xin
authors: YX
authors: Huang
authors: Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China. Electronic address: wuy705@nenu.edu.cn.
authors: Yin
authors: Y
authors: Wu
authors: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China; Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China. Electronic address: sunlg20112013@163.com.
authors: Lu Guo
authors: LG
authors: Sun
authors: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China; Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China. Electronic address: yucl885@nenu.edu.cn.
authors: Chun Lei
authors: CL
authors: Yu
authors: Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China. Electronic address: suny040@nenu.edu.cn.
authors: Ying
authors: Y
authors: Sun
authors: Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China. Electronic address: zhenglh015@nenu.edu.cn.
authors: Li Hua
authors: LH
authors: Zheng
authors: Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China; Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China. Electronic address: liyx486@nenu.edu.cn.
authors: Yu Xin
authors: YX
authors: Li
mesh_terms: D000818:Animals; D016466:CHO Cells; D063646:Carcinogenesis; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D000072000:NF-KappaB Inhibitor alpha; D012697:Serine Endopeptidases; C446379:testis-specific protease 50
keywords: Catalytic triad; Cell proliferation; Mechanism; Mutants; Serine protease; TSP50