中文摘要：

Artemis是1个具有多种生物学功能的磷酸化蛋白,它在基因毒性应激引发的细胞周期检测点调控中起重要作用,但其调控机制知之甚少.为了探讨UVC等DNA复制阻滞应激引发的Artemis磷酸化及蛋白表达水平对细胞周期蛋白E的调控作用和调控机制.首先以Western印迹方法检测Artemis S516-645A突变细胞和Artemis表达降低细胞的细胞周期蛋白E的表达水平,发现ArtemisS516-645A突变细胞和多种Artemis siRNA转染细胞的细胞周期蛋白E表达水平均高于对照细胞.在此基础上,为分析细胞周期蛋白E表达受调控的分子机制,在稳定表达各种磷酸化状态Artemis的HEK-293细胞中导入外源性启动子转录驱动的细胞周期蛋白E表达质粒,发现表达Artemis S516-645A突变体的细胞中外源性的细胞周期蛋白E蛋白表达水平也高于野生型细胞.进一步的研究发现在Artemis蛋白表达降低的细胞中与泛素结合的细胞周期蛋白E减少而蛋白稳定性增加.本研究还发现Artemis蛋白对细胞周期蛋白E的调控过程是不依赖于p53和p21表达的.这些结果表明,Artemis S516-645A突变和Artemis表达降低都可以引起细胞周期蛋白E蛋白水平升高,该调控作用是在转录后水平发生的,可能是干扰了细胞周期蛋白E的泛素化介导的蛋白降解过程,并且该调控作用是独立于p53-p21信号通路的.

英文摘要：

Artemis is a phosphorylated protein with multiple functions.Artemis plays an important role in the checkpoint regulation induced by genotoxic stress.However,little is known about the regulating mechanism.To investigate the role of Artemis phosphorylation state as well as Artemis protein level on the regulation of cyclin E and further explore the regulating mechanism,cyclin E protein levels in the Artemis S516-645A mutant and in the Artemis knockdown cells were analyzed by Western blotting.Cyclin E protein expression level was higher in the Artemis S516-645A mutant or multiple Artemis siRNA transfected cell than that in the control treated cells,respectively.Plasmid encoding cyclin E which was driven by exogenous promoter was introduced into the cells expressing different phosphorylation state of Artemis to examine the molecular mechanism of this regulation.Exogenous promoter driven cyclin E expression was also higher in Artemis S516-645A mutant than that in wild type cells.The in vivo ubiquitination assay was further employed to examine the ubiquitin mediated degradation process and found that less cyclin E was ubiquitinated in Artemis depleted cells.Additional study performed in p53 or p21 deficient HCT cells verified this regulation on cyclin Ewas independent of p53 and p21.Taken together,Artemis S516-645A mutation or Artemis knockdown caused increased cyclin E protein expression and the regulation was carried out at post-transcriptional level.The ubiquitin mediated cyclin E proteolysis was impaired in Artemis depleted cells,and the regulation was independent of p53-p21 pathway.