裂殖酵母中核仁蛋白Dnt1与肿瘤抑制因子Chfr的同源蛋白Dma1相互作用的研究

发布时间：2018-01-12 15:06

  本文关键词：裂殖酵母中核仁蛋白Dnt1与肿瘤抑制因子Chfr的同源蛋白Dma1相互作用的研究　出处：《厦门大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： Dma1 Dnt1 SIN信号途径

【摘要】： 在裂殖酵母S.pombe中,人类肿瘤抑制因子Chfr的同源蛋白Dma1是纺锤体检验点蛋白和胞质分裂的抑制因子,它通过抑制SIN(septation initiation network)信号途径来阻止胞质分裂的起始;在有丝分裂后期Dma1同时定位在纺锤体极体(spindle pole body,简称SPB;相当于哺乳动物的中心体)和有丝分裂环上。而核仁蛋白Dnt1与出芽酵母中的Net1/Cfi1具有同源序列,是一个新发现的有丝分裂早期SIN的抑制因子;整个细胞周期Dnt1都在核仁内有定位,在有丝分裂后期还会定位在纺锤体及SPB上。基于Dma1和Dnt1都有在SPB上定位,并且均可调控胞质分裂过程,因此本论文主要研究Dma1和Dnt1两种蛋白的相互作用关系。 我们将Dma1和Dnt1分别在细菌和dnt1△的酵母细胞中表达后,进行pull-down实验,纯化后的结果显示Dma1与Dnt1在体外有着直接的相互作用;随后我们在细菌内表达Dma1的全长和FHA区域缺失突变体,让其分别与酵母细胞内表达的Dnt1-13myc进行体外结合实验,Western blot检测结果发现两种蛋白之间的相互作用是通过Dma1的FHA区域介导的,而FHA区域,被认为是一个可以识别并结合到被磷酸化的肽链或蛋白上的区域。本论文中分别用CIAP和λ-PPase对蛋白进行去磷酸化处理,结果发现Dnt1蛋白磷酸化后才可以与Dma1相结合。 因为Dma1与磷酸化Dnt1在有丝分裂中期的相互作用可以达到最强,而CDK1和Plo1是调节酵母细胞周期中的两个关键激酶,其活性都是在有丝分裂中期达到最高;我们分别利用CDK1位点突变的dnt1(7A)、dnt1(11A)和Plo1突变体plo1-24c、plo1-25以及与Plo激酶活性有关的突变体cut12.1检测这两种激酶对Dnt1磷酸化的影响,结果显示CDK1和Plo1可能并不影响Dnt1的磷酸化进而影响Dnt1与Dma1的相互作用。 本论文初步阐明了Dma1与Dnt1蛋白的相互作用机制,在正常的有丝分裂中期被磷酸化后的Dnt1可以很强地结合到Dma1上,以达到在这一时期抑制Dma1的E3泛素连接酶活性的目的,保护Dma1的底物不会在这一时期过早被降解。鉴于裂殖酵母中的Dma1是哺乳动物中Chfr的同源蛋白,对于Dma1的深入研究将加深我们对于Chfr的功能的认识。
[Abstract]:In S. pombe, the homologous protein Dma1 of human tumor suppressor Chfr is the inhibitory factor of spindle checkpoint protein and cytokinesis. It inhibits the initiation of cytokinesis by inhibiting the SIN(septation initiation network signaling pathway. At the late mitotic stage, Dma1 was located in the spindle pole body of the spindles. Nucleolar protein Dnt1 is homologous to Net1/Cfi1 in budding yeast. It is a newly discovered inhibitor of SIN in the early stage of mitosis. The whole cell cycle Dnt1 was located in nucleolus and fusiform and SPB in late mitosis, and SPB based on Dma1 and Dnt1. Therefore, the interaction between Dma1 and Dnt1 proteins was studied in this paper. After Dma1 and Dnt1 were expressed in yeast cells of bacteria and dnt1, the pull-down experiment was carried out. The purified results showed that Dma1 and Dnt1 had direct interaction in vitro. Then we expressed the full-length and FHA region deletion mutants of Dma1 in the bacteria and let them bind to the Dnt1-13myc expressed in yeast cells in vitro. The results of Western blot analysis showed that the interaction between the two proteins was mediated by the FHA region of Dma1, while the FHA region. It is considered to be a region that can be recognized and bound to the phosphorylated peptide chain or protein. In this paper, CIAP and 位 -PPase were used to dephosphorize the protein, respectively. The results showed that Dnt1 protein was phosphorylated before it could bind to Dma1. Because the interaction between Dma1 and phosphorylated Dnt1 is strongest in the middle of mitosis, CDK1 and Plo1 are two key kinases in the regulation of yeast cell cycle. Their activity reached the highest in the middle stage of mitosis. We used the CDK1 site-mutant dnt1- 7 Agnont 11A) and the Plo1 mutant plo1-24c, respectively. Plo1-25 and cut12.1, a mutant associated with the activity of Plo kinase, were used to detect the effect of these two kinases on the phosphorylation of Dnt1. The results showed that CDK1 and Plo1 may not affect the phosphorylation of Dnt1 and then affect the interaction between Dnt1 and Dma1. In this paper, the mechanism of interaction between Dma1 and Dnt1 protein was elucidated. Dnt1, which was phosphorylated in normal mitotic metaphase, could be strongly bound to Dma1. In order to achieve the purpose of inhibiting the E3 ubiquitin ligase activity of Dma1 in this period. The substrate that protects Dma1 will not be degraded prematurely during this period, since Dma1 in merozoite yeast is the homologous protein of Chfr in mammals. The in-depth study of Dma1 will deepen our understanding of the function of Chfr.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

【共引文献】

相关期刊论文 前3条

1 王小韵;李小毛;;CHFR基因在肿瘤中的研究现状及意义[J];广东医学;2012年09期

2 苗W歐，

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