【摘要】：轉化生長因子β(Transforming Growth Factor β,TGFβ)是一種多功能的蛋白因子,在促進腫瘤的轉移中具有十分重要的作用。TGFβ信號的起始是由細胞膜上的受體與配體結合,隨后受體通過其蛋白激酶活性對R-Smad進行磷酸化,并促使其能夠與Smad4結合,最終將信號傳遞到細胞核內。研究發(fā)現(xiàn),細胞核蛋白質SNIP1(Smad Nuclear Interacting Protein 1)能夠直接與Smad4結合,并能夠通過破壞Smad4與其共激活子p300的結合,抑制TGFβ下游基因的激活。SNIP1在調控TGFβ信號通路中具有重要的作用,但是對于其本身是否會受到翻譯后修飾的調控還研究得較少。本實驗室前期工作顯示,SNIP1能夠發(fā)生SUMO化修飾,并且SUMO化修飾主要發(fā)生在第5、30、108位點的賴氨酸殘基上。SUMO是一種小的類泛素樣蛋白修飾物,能通過影響基因組的完整性,蛋白在細胞核內的定位以及轉錄活性等,改變蛋白的功能。本論文通過一系列分子、生化和細胞生物學實驗對SNIP1的SUMO修飾及其對TGFβ信號通路的影響進行了鑒定,發(fā)現(xiàn)SUMO修飾能夠使SNIP1失去對TGFβ信號通路的抑制作用。從報告基因、mRNA水平以及蛋白水平分析,SUMO修飾的SNIP1不再抑制TGFβ下游基因如PAI-1和MMP-2的表達。針對這些結果的分子機理研究顯示,SNIP1能夠通過抑制TGFβ誘導的Smad復合物的形成以及Smad/p300的相互作用來抑制TGFβ信號通路的激活。然而SUMO化修飾使得SNIP1喪失抑制Smad復合物的形成以及Smad/p300相互作用的能力,從而影響SNIP1在TGFβ信號通路中的活性。進一步的細胞功能研究顯示,SNIP1能夠抑制TGFβ誘導的A549細胞的遷移與侵襲,然而SUMO修飾的SNIP1失去了抑制效應。同時SNIP1能夠通過抑制RhoA的激活,來影響應力纖維的形成,然而SUMO修飾的SNIP1失去了這個功能。綜上所述,我們認為SUMO可以使得SNIP1喪失抑制Smad復合物以及Smad/p300形成的能力,從而影響SNIP1在TGFβ信號通路中的活性,最終為TGFβ誘導的腫瘤細胞遷移與侵襲提供了有利的條件。
[Abstract]:Transforming growth factor 尾 (Transforming Growth Factor 尾 (TGF 尾) is a multifunctional protein factor, which plays an important role in promoting tumor metastasis. The initiation of TGF 尾 signal is the binding of receptor on cell membrane to ligand. The receptor then phosphorylates R-Smad through its protein kinase activity and enables it to bind to Smad4 and eventually transmit the signal to the nucleus. It has been found that nuclear protein SNIP1 (Smad Nuclear Interacting Protein 1 can directly bind to Smad4 and inhibit the activation of downstream TGF 尾 gene by destroying the binding of Smad4 to its co-activator p300. SNIP1 plays an important role in the regulation of TGF 尾 signaling pathway. However, little research has been done on whether or not it will be regulated by post-translational modification. Our previous work has shown that SNIP1 can undergo SUMO modification, and SUMO modification mainly occurs on the lysine residue at site 5: 30108. SUMO is a small ubiquitin like protein modifier that can affect the integrity of genome. The localization and transcriptional activity of proteins in the nucleus change the function of proteins. In this paper, a series of molecular, biochemical and cellular biological experiments were carried out to identify the SUMO modification of SNIP1 and its effect on TGF 尾 signaling pathway. It was found that SUMO modification could make SNIP1 lose its inhibitory effect on TGF 尾 signaling pathway. From the analysis of reporter gene, mRNA level and protein level, SUMO modified SNIP1 no longer inhibited the expression of TGF 尾 downstream genes such as PAI-1 and MMP-2. The molecular mechanism of these results shows that SNIP1 can inhibit the activation of TGF 尾 signaling pathway by inhibiting the formation of Smad complexes induced by TGF 尾 and the interaction of Smad/p300. However, the modification of SUMO makes SNIP1 lose the ability to inhibit the formation of Smad complex and the interaction of Smad/p300, thus affecting the activity of SNIP1 in TGF 尾 signaling pathway. Further studies on cell function showed that SNIP1 could inhibit the migration and invasion of A549 cells induced by TGF 尾, but SUMO modified SNIP1 had no inhibitory effect. At the same time, SNIP1 can affect the formation of stress fibers by inhibiting the activation of RhoA, but SUMO modified SNIP1 loses this function. In conclusion, we suggest that SUMO can make SNIP1 lose the ability to inhibit the formation of Smad complex and Smad/p300, thus affect the activity of SNIP1 in TGF 尾 signaling pathway, and finally provide favorable conditions for TGF 尾 -induced tumor cell migration and invasion.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R73-3

【參考文獻】

相關期刊論文 前3條

1 馬艷;劉虹;張浩;邵榮光;;TGF-β信號通路調控乳腺癌上皮-間質轉化的研究進展[J];藥學學報;2015年04期

2 李玲;文娟;庹勤慧;廖端芳;;SUMO化對DAXX亞細胞定位與功能的影響[J];生理學報;2013年01期

3 林俠;陳曄光;孟安明;馮新華;;利用功能基因組學方法研究SMAD去磷酸化-TGF-β超家族信號轉導的終止機制(英文)[J];遺傳學報;2007年01期

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