本文關(guān)鍵詞：裂殖酵母中核仁蛋白Dnt1與腫瘤抑制因子Chfr的同源蛋白Dma1相互作用的研究　出處：《廈門大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】： 在裂殖酵母S.pombe中,人類腫瘤抑制因子Chfr的同源蛋白Dma1是紡錘體檢驗(yàn)點(diǎn)蛋白和胞質(zhì)分裂的抑制因子,它通過抑制SIN(septation initiation network)信號(hào)途徑來阻止胞質(zhì)分裂的起始;在有絲分裂后期Dma1同時(shí)定位在紡錘體極體(spindle pole body,簡稱SPB;相當(dāng)于哺乳動(dòng)物的中心體)和有絲分裂環(huán)上。而核仁蛋白Dnt1與出芽酵母中的Net1/Cfi1具有同源序列,是一個(gè)新發(fā)現(xiàn)的有絲分裂早期SIN的抑制因子;整個(gè)細(xì)胞周期Dnt1都在核仁內(nèi)有定位,在有絲分裂后期還會(huì)定位在紡錘體及SPB上�；贒ma1和Dnt1都有在SPB上定位,并且均可調(diào)控胞質(zhì)分裂過程,因此本論文主要研究Dma1和Dnt1兩種蛋白的相互作用關(guān)系。 我們將Dma1和Dnt1分別在細(xì)菌和dnt1△的酵母細(xì)胞中表達(dá)后,進(jìn)行pull-down實(shí)驗(yàn),純化后的結(jié)果顯示Dma1與Dnt1在體外有著直接的相互作用;隨后我們?cè)诩?xì)菌內(nèi)表達(dá)Dma1的全長和FHA區(qū)域缺失突變體,讓其分別與酵母細(xì)胞內(nèi)表達(dá)的Dnt1-13myc進(jìn)行體外結(jié)合實(shí)驗(yàn),Western blot檢測結(jié)果發(fā)現(xiàn)兩種蛋白之間的相互作用是通過Dma1的FHA區(qū)域介導(dǎo)的,而FHA區(qū)域,被認(rèn)為是一個(gè)可以識(shí)別并結(jié)合到被磷酸化的肽鏈或蛋白上的區(qū)域。本論文中分別用CIAP和λ-PPase對(duì)蛋白進(jìn)行去磷酸化處理,結(jié)果發(fā)現(xiàn)Dnt1蛋白磷酸化后才可以與Dma1相結(jié)合。 因?yàn)镈ma1與磷酸化Dnt1在有絲分裂中期的相互作用可以達(dá)到最強(qiáng),而CDK1和Plo1是調(diào)節(jié)酵母細(xì)胞周期中的兩個(gè)關(guān)鍵激酶,其活性都是在有絲分裂中期達(dá)到最高;我們分別利用CDK1位點(diǎn)突變的dnt1(7A)、dnt1(11A)和Plo1突變體plo1-24c、plo1-25以及與Plo激酶活性有關(guān)的突變體cut12.1檢測這兩種激酶對(duì)Dnt1磷酸化的影響,結(jié)果顯示CDK1和Plo1可能并不影響Dnt1的磷酸化進(jìn)而影響Dnt1與Dma1的相互作用。 本論文初步闡明了Dma1與Dnt1蛋白的相互作用機(jī)制,在正常的有絲分裂中期被磷酸化后的Dnt1可以很強(qiáng)地結(jié)合到Dma1上,以達(dá)到在這一時(shí)期抑制Dma1的E3泛素連接酶活性的目的,保護(hù)Dma1的底物不會(huì)在這一時(shí)期過早被降解。鑒于裂殖酵母中的Dma1是哺乳動(dòng)物中Chfr的同源蛋白,對(duì)于Dma1的深入研究將加深我們對(duì)于Chfr的功能的認(rèn)識(shí)。
[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.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

1 王小韻;李小毛;;CHFR基因在腫瘤中的研究現(xiàn)狀及意義[J];廣東醫(yī)學(xué);2012年09期

2 苗W歐，

本文編號(hào)：1414757