核仁蛋白Def与半胱氨酸蛋白酶CAPN3的互作研究及其在核仁中的靶蛋白鉴定

发布时间：2018-01-09 20:22

本文关键词：核仁蛋白Def与半胱氨酸蛋白酶CAPN3的互作研究及其在核仁中的靶蛋白鉴定　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：核仁不仅是核糖体合成与加工的场所,也与细胞周期调控、DNA损伤修复、压力信号应答等生化过程有关。核仁作为细胞核内致密的非膜细胞器,富集了超过4,500种不同蛋白质。然而,人们对于这些蛋白质在核仁中的功能以及这些蛋白在核仁中是如何被调控的所知甚少。本实验室之前的研究发现核仁蛋白Def(Digestive-organexpansion factor)能够介导半胱氨酸蛋白酶 CAPN3(Calpain3)降解p53。但是我们并不清楚Def如何帮助CAPN3降解p53,也不知Def-CAPN3在核仁内是否还存在其他靶蛋白。本研究首先发现人CAPN3能够降解p53A138V、p53M2371、p53R248W和p53R273P,而不能降解p53R175H。p53R175H作为癌症样品中出现频率最高的p53点突变,提示了 CAPN3在肿瘤形成中的意义。通过免疫共沉淀实验,我们证明Def与人CAPN3能够直接互作,并在核仁内形成蛋白复合体。我们发现这种互作在斑马鱼上也具有保守性,斑马鱼Def能够与Capn3b(人CAPN3在斑马鱼上的同源蛋白)在核仁内形成蛋白复合体。进一步的研究发现,Def能够通过磷酸化修饰调控其与CAPN3的结合能力,招募CAPN3进入核仁降解p53。此外,Def-CAPN3对细胞周期及斑马鱼器官发育的调控也是部分通过p53实现的。在两个不同的Caapn3b基因敲除品系斑马鱼的肝脏细胞中,p53都大量积累于核仁。虽然该斑马鱼纯合突变体可正常繁育,但是成年鱼表现出体长变短、躯体易侧弯的症状,与capn3基因突变引起的先天性肢带型肌肉营养不良(LGMD2A)症状类似。为了寻找Def-CAPN3在核仁内的其他靶蛋白,我们通过蛋白质组学技术分析了capn3b基因敲除斑马鱼与野生型斑马鱼肝脏细胞核仁中差异表达的蛋白,发现了 119个在capn3b突变鱼中上调(超过1.45倍)的核蛋白,其中核仁蛋白33个。对其中8个候选蛋白进行体内、体外的验证实验,证明其中7个(LmnA、Nkap、Ddx18、Cdk9、Hmgb1、Tra2a、Bbofl)蛋白能够被CAPN3降解,而且能够被Def诱导降解。此外,通过对蛋白质组学数据的深入分析,我们发现Capn3b还参与了免疫系统的调节,这解释了为何部分LGMD2A患者在发病早期出现炎症反应。综上所述,我们的研究首次阐明了 Def-CAPN3降解途径能够在核仁内通过对靶蛋白的降解,调控细胞周期进程及器官发育。作为首个斑马鱼核仁蛋白质组学研究,我们的工作不仅为斑马鱼核仁蛋白功能的研究提供了帮助,也通过这种方法鉴定出了 7个Def-CAPN3的新底物,为Def-CAPN3的功能研究提供了新的基础。此外,我们所构建的capn3b基因敲除斑马鱼能够作为LGMD2A疾病研究模型,将来可用于药物筛选。
[Abstract]:Nucleoli are not only the sites for ribosome synthesis and processing, but also related to the biochemical processes such as cell cycle regulation, DNA damage repair, pressure signal response, etc. Nucleoli act as dense non-membranous organelles in the nucleus. Enriched more than 4,500 different proteins. Little is known about the function of these proteins in nucleoli and how they are regulated in nucleoli. Digestive-organexpansion factor can mediate cysteine protease CAPN3 and Calpain3). However, we do not know how Def can help CAPN3 to degrade p53. It is also unknown whether there are other target proteins in the nucleoli of Def-CAPN3. In this study, we first found that human CAPN3 can degrade p53A138VP53M2371. P53R248W and p53R273Pcould not degrade p53R175H.p53R175H as the most frequent point mutation of p53 in cancer samples. By immunoprecipitation, we have proved that Def and human CAPN3 can interact directly with each other. We found that this interaction is also conserved in zebrafish. Zebrafish Def can form protein complex in nucleoli with Capn3b (the homologous protein of human CAPN3 on zebrafish). Def can regulate its binding ability to CAPN3 by phosphorylation and enlist CAPN3 into nucleolus to degrade p53. in addition. The regulation of Def-CAPN3 on cell cycle and organ development of zebrafish is also partly mediated by p53. In two different Caapn3b gene knockout strains of zebrafish liver cells. Although the homozygous mutant of zebrafish could be bred normally, adult fish showed the symptoms of shorter body length and easy lateral curvature. Similar to the capn3 gene mutation caused by congenital limb-band muscular dystrophy (LGMD2A). In order to search for other target proteins of Def-CAPN3 in nucleoli. We analyzed the differentially expressed proteins in liver nuclei of capn3b knockout zebrafish and wild type zebrafish by proteomics. One hundred and nineteen nucleoproteins were identified in capn3b mutant fish, of which 33 were nucleolus proteins. Eight candidate proteins were tested in vitro and in vivo. It was proved that seven of them, Ddx18Cdk9, Hmgb1Tra2aBboflon, could be degraded by CAPN3. In addition, through the in-depth analysis of proteomics data, we found that Capn3b is also involved in the regulation of the immune system. This explains why some patients with LGMD2A develop inflammation at the early stage of the disease. Our study demonstrated for the first time that Def-CAPN3 degradation pathway can degrade target proteins in nucleoli. As the first zebrafish nucleolus proteomics research, our work not only provides help for the study of zebrafish nucleolar protein function. Seven new substrates of Def-CAPN3 were also identified by this method, which provided a new basis for the functional study of Def-CAPN3. Our constructed capn3b knockout zebrafish can be used as a LGMD2A disease research model and can be used for drug screening in the future.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q75

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