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Molecular Cloning of Ubiquitin Specific Protease 46 and the Effect of Gene Mutation on Its Function
Zhang Jingkun1, Zhang Wei2*, Wei Qun3, Qi Sufen4, Mi Yingjun4, Jia Yuan5, Lü Hongzhi6, Zhao Jing7, Liu Shuxia2
1International Education College, Hebei Medical University, Shijiazhuang 050017, China; 2Department of Pathology, Hebei Medical University, Shijiazhuang 050017, China; 3Department of Nosocomial Infection Control, Hebei General Hospital, Shijiazhuang 050000, China; 4Department of Epidemiology and Statistics, Hebei Medical University, Shijiazhuang 050017, China; 5Department of Nosocomial Infection Control, the Second Hospital of Hebei Medical University, Shijiazhuang 050000, China; 6Orthopedic Research Institution of Hebei Province, the Third Hospital of Hebei Medical University, Shijiazhuang 050051, China; 7Department of Pharmacology, College of Chinese Integrative Medicine, Hebei Medical University, Shijiazhuang 050017, China
Abstract: This work was to investigate biological function and the effect of the missense mutation on the function of ubiquitin-specific protease 46 (usp46)， which is associated with ‘behavioral despair’ in mice. The total RNA was extracted from human esophageal cancer cell line TE-1. Reverse transcription polymerase chain reaction (RT-PCR) was applied to clone human usp46 gene. The relative expression levels of usp46 mRNA in mice were detected by quantitative Real-time PCR. The deubiquitinating enzyme activity assay was established to detect USP46 deubiquitinating enzymatic activity. Site-Directed Mutagenesis kit were used to generate usp46 (V89I) and detect its effect on deubiquitinating enzyme activity. The results suggested that the human usp46 gene had a 1 101 bp open reading frame， encoding 366 amino acids. The sequence alignment showed that usp46 was highly conservative in many species. While weakly expressed in skeletal muscle， usp46 mRNA strongly expressed in spleen of Mus musculus. The human USP46 protein had the deubiquitinating enzyme activity， and the activity of the V89I mutation was significantly decreased， which was only 37.67%±2.52% of the wild type (P<0.05). The results indicated that the expression levels of usp46 mRNA in different organs of Mus musculus were significantly different. Moreover， the activity of V89I missense mutation decreased significantly， which provided a new clue of the etiology of neuropsychiatric disorders such as depression.
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