泛素特异性蛋白酶46的分子克隆及基因突变对其功能的影响

Molecular Cloning of Ubiquitin Specific Protease 46 and the Effect of Gene Mutation on Its Function

该文探讨了行为绝望相关基因—泛素特异性蛋白酶46(usp46)基因的生物学功能及编码区错义突变对其功能的影响。从人类食管癌细胞株TE-1中提取总RNA,RT-PCR扩增获得人类usp46基因,q RT-PCR检测小鼠各组织中该基因的m RNA相对表达水平;运用去泛素化酶活性检测体系,将USP46与模型底物表达质粒共转化,研究USP46的活性;采用定点突变法构建错义突变V89I,用上述方法检测突变对酶活性的影响。结果发现,人类usp46基因具有1 101 bp的开放阅读框,编码366个氨基酸;序列比对显示,其在各个物种中高度保守;在小鼠脾脏中m RNA表达量较高,而骨骼肌中表达较少;人类的USP46蛋白具有去泛素化酶活性,V89I突变后,其活性显著下降,仅为野生型的37.67%±2.52%(P<0.05)。结果表明,usp46基因在小鼠各组织中的m RNA表达水平存在差异;人类USP46蛋白具有去泛素化酶活性,而且其编码区V89I错义突变后,其活性显著下降,为抑郁症等神经精神性疾病的病因学研究提供了新的线索。

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 m RNA 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（V89 I） 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 m RNA strongly expressed in spleen of Mus musculus. The human USP46 protein had the deubiquitinating enzyme activity, and the activity of the V89 I mutation was significantly decreased, which was only 37.67%±2.52% of the wild type（P0.05）. The results indicated that the expression levels of usp46 m RNA in different organs of Mus musculus were significantly different. Moreover, the activity of V89 I missense mutation decreased significantly, which provided a new clue of the etiology of neuropsychiatric disorders such as depression.