核仁因子Def磷酸化修饰调控细胞周期和p53降解的研究

发布时间：2018-06-16 02:25

本文选题：斑马鱼 + 核仁　；参考：《浙江大学》2015年博士论文

【摘要】：核仁因子Def在真核生物中保守,是核糖体小亚基加工复合体的组分,与U3snoRNA及核仁蛋白Mpp10和Sas10互作,参与核糖体RNA的加工；另外,在人和斑马鱼中,Def与分别与半胱氨酸蛋白酶CAPN3和Capn3b互作,介导p53蛋白在核仁中降解。Def作为一个如此重要的多功能蛋白,蛋白的活性需要受到精细的调控,但目前却知之甚少。本课题主要着眼于Def的翻译后修饰研究,利用遗传学,细胞生物学,生物化学和分子生物学的研究方法,旨在寻找调控Def-Capn3b降解途径的翻译后修饰位点。本研究首先通过碱性磷酸酶处理蛋白样品,结合蛋白免疫印迹的方法,发现Def蛋白存在磷酸化修饰,而且其中一部分磷酸化修饰,发生在具有介导p53降解功能的Def肽段上。接着,我们利用定点突变的方法,在这一重要的肽段上,找到了5个磷酸化修饰位点,分别为S50, S58, S62, S87和S92。之后通过构建并研究磷酸化位点突变的转基因斑马鱼,发现磷酸化修饰通过调节细胞周期从G1到S以及G2到M的进程,调节斑马鱼肝脏发育。更为重要的是,研究也发现磷酸化修饰是Def在核仁中介导p53降解所必需。本研究的成果,为Def-Capn3蛋白降解途径的信号通路研究,奠定了坚实的基础。此外,本研究还发现DefN端具有的酸性氨基酸(E和D)富集的特性,导致其在SDS-PAGE上的分子量,比其理论分子量大13 kDa。通过分析Def来源的13个蛋白片段迁移速率与不同类型氨基酸比例之间的关系,我们发现蛋白分子量的差异与酸性氨基酸的比例,呈线性正相关：y=276.5x-31.33(x代表酸性氨基酸的比例,y代表每个氨基酸的平均△MW)。并且该方程成功地预测了其它13个酸性蛋白在SDS-PAGE上的分子量。该方程的建立,成功地解决了酸性氨基酸影响蛋白迁移速率这一问题。综上所述,论文以解析Def-Capn3b蛋白降解途径为研究目的,着重开展Def蛋白的生化功能研究,发现Def磷酸化修饰调控细胞周期和p53降解,以及酸性氨基酸影响蛋白迁移速率的特性并建立了计算方程。
[Abstract]:Nucleolar factor Def, conserved in eukaryotes, is a component of ribosomal small subunit processing complex that interacts with U3snoRNA and nucleolar proteins Mpp10 and Sas10 to process ribosomal RNA. In human and zebrafish, the interaction of Def with cysteine protease CAPN3 and Capn3b mediates the degradation of p53 protein in nucleoli as such an important multifunctional protein, the activity of which needs to be carefully regulated, but little is known. This study focuses on post-translational modification of Def, and uses genetic, cytobiological, biochemical and molecular biological methods to find posttranslational modification sites that regulate the degradation pathway of Def-Capn3b. In this study, the protein sample was treated with alkaline phosphatase and the protein was imprinted by Western blot. It was found that there was phosphorylation modification in Def protein, and some of the phosphorylation modification occurred on the Def peptide which mediated the degradation of p53. Then, using site-directed mutagenesis, we found five phosphorylation modification sites in this important peptide domain, namely S50, S58, S62, S87 and S92. By constructing and studying transgenic zebrafish with phosphorylation site mutation, it was found that phosphorylation could regulate the liver development of zebrafish by regulating the process of cell cycle from G1 to S and G2 to M. More importantly, phosphorylation modification was also found to be essential for def mediated p53 degradation. The results of this study laid a solid foundation for the study of signal pathway of Def-Capn3 protein degradation pathway. In addition, we also found that the def N terminal has the characteristics of acidic amino acid E and D) enrichment, resulting in its molecular weight on SDS-PAGE, higher than its theoretical molecular weight of 13 kDa. By analyzing the relationship between the migration rate of 13 protein fragments from Def and the proportion of different amino acids, we found that the difference of molecular weight of protein and the proportion of acidic amino acids, A linear positive correlation was found that the proportion of the acidic amino acids represented by the ratio of 276.5x-31.33nx represented the average MWH of each amino acid. The equation successfully predicted the molecular weight of 13 other acidic proteins on SDS-PAGE. The equation has successfully solved the problem that acid amino acids affect protein migration rate. In conclusion, the purpose of this study was to analyze the degradation pathway of Def-Capn3b protein, and to study the biochemical function of Def protein. It was found that Def phosphorylation could regulate cell cycle and p53 degradation. And the characteristics of acidic amino acids affecting the protein migration rate were established and the calculation equation was established.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q51

【参考文献】