本文選題：斑馬魚 + 核仁�。� 參考：《浙江大學(xué)》2015年博士論文

【摘要】：核仁因子Def在真核生物中保守,是核糖體小亞基加工復(fù)合體的組分,與U3snoRNA及核仁蛋白Mpp10和Sas10互作,參與核糖體RNA的加工；另外,在人和斑馬魚中,Def與分別與半胱氨酸蛋白酶CAPN3和Capn3b互作,介導(dǎo)p53蛋白在核仁中降解。Def作為一個(gè)如此重要的多功能蛋白,蛋白的活性需要受到精細(xì)的調(diào)控,但目前卻知之甚少。本課題主要著眼于Def的翻譯后修飾研究,利用遺傳學(xué),細(xì)胞生物學(xué),生物化學(xué)和分子生物學(xué)的研究方法,旨在尋找調(diào)控Def-Capn3b降解途徑的翻譯后修飾位點(diǎn)。本研究首先通過(guò)堿性磷酸酶處理蛋白樣品,結(jié)合蛋白免疫印跡的方法,發(fā)現(xiàn)Def蛋白存在磷酸化修飾,而且其中一部分磷酸化修飾,發(fā)生在具有介導(dǎo)p53降解功能的Def肽段上。接著,我們利用定點(diǎn)突變的方法,在這一重要的肽段上,找到了5個(gè)磷酸化修飾位點(diǎn),分別為S50, S58, S62, S87和S92。之后通過(guò)構(gòu)建并研究磷酸化位點(diǎn)突變的轉(zhuǎn)基因斑馬魚,發(fā)現(xiàn)磷酸化修飾通過(guò)調(diào)節(jié)細(xì)胞周期從G1到S以及G2到M的進(jìn)程,調(diào)節(jié)斑馬魚肝臟發(fā)育。更為重要的是,研究也發(fā)現(xiàn)磷酸化修飾是Def在核仁中介導(dǎo)p53降解所必需。本研究的成果,為Def-Capn3蛋白降解途徑的信號(hào)通路研究,奠定了堅(jiān)實(shí)的基礎(chǔ)。此外,本研究還發(fā)現(xiàn)DefN端具有的酸性氨基酸(E和D)富集的特性,導(dǎo)致其在SDS-PAGE上的分子量,比其理論分子量大13 kDa。通過(guò)分析Def來(lái)源的13個(gè)蛋白片段遷移速率與不同類型氨基酸比例之間的關(guān)系,我們發(fā)現(xiàn)蛋白分子量的差異與酸性氨基酸的比例,呈線性正相關(guān)：y=276.5x-31.33(x代表酸性氨基酸的比例,y代表每個(gè)氨基酸的平均△MW)。并且該方程成功地預(yù)測(cè)了其它13個(gè)酸性蛋白在SDS-PAGE上的分子量。該方程的建立,成功地解決了酸性氨基酸影響蛋白遷移速率這一問(wèn)題。綜上所述,論文以解析Def-Capn3b蛋白降解途徑為研究目的,著重開展Def蛋白的生化功能研究,發(fā)現(xiàn)Def磷酸化修飾調(diào)控細(xì)胞周期和p53降解,以及酸性氨基酸影響蛋白遷移速率的特性并建立了計(jì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q51

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Liver development in zebrafish (Danio rerio)[J];遺傳學(xué)報(bào);2009年06期

2 ;Ribosome Biogenesis Factor Bmsl-like Is Essential for Liver Development in Zebrafish[J];遺傳學(xué)報(bào);2012年09期

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本文編號(hào)：2024802