SNIP1的SUMO修饰对其在TGFβ信号通路中的影响及分子机制的研究

发布时间：2018-11-25 13:30

【摘要】：转化生长因子β(Transforming Growth Factor β,TGFβ)是一种多功能的蛋白因子,在促进肿瘤的转移中具有十分重要的作用。TGFβ信号的起始是由细胞膜上的受体与配体结合,随后受体通过其蛋白激酶活性对R-Smad进行磷酸化,并促使其能够与Smad4结合,最终将信号传递到细胞核内。研究发现,细胞核蛋白质SNIP1(Smad Nuclear Interacting Protein 1)能够直接与Smad4结合,并能够通过破坏Smad4与其共激活子p300的结合,抑制TGFβ下游基因的激活。SNIP1在调控TGFβ信号通路中具有重要的作用,但是对于其本身是否会受到翻译后修饰的调控还研究得较少。本实验室前期工作显示,SNIP1能够发生SUMO化修饰,并且SUMO化修饰主要发生在第5、30、108位点的赖氨酸残基上。SUMO是一种小的类泛素样蛋白修饰物,能通过影响基因组的完整性,蛋白在细胞核内的定位以及转录活性等,改变蛋白的功能。本论文通过一系列分子、生化和细胞生物学实验对SNIP1的SUMO修饰及其对TGFβ信号通路的影响进行了鉴定,发现SUMO修饰能够使SNIP1失去对TGFβ信号通路的抑制作用。从报告基因、mRNA水平以及蛋白水平分析,SUMO修饰的SNIP1不再抑制TGFβ下游基因如PAI-1和MMP-2的表达。针对这些结果的分子机理研究显示,SNIP1能够通过抑制TGFβ诱导的Smad复合物的形成以及Smad/p300的相互作用来抑制TGFβ信号通路的激活。然而SUMO化修饰使得SNIP1丧失抑制Smad复合物的形成以及Smad/p300相互作用的能力,从而影响SNIP1在TGFβ信号通路中的活性。进一步的细胞功能研究显示,SNIP1能够抑制TGFβ诱导的A549细胞的迁移与侵袭,然而SUMO修饰的SNIP1失去了抑制效应。同时SNIP1能够通过抑制RhoA的激活,来影响应力纤维的形成,然而SUMO修饰的SNIP1失去了这个功能。综上所述,我们认为SUMO可以使得SNIP1丧失抑制Smad复合物以及Smad/p300形成的能力,从而影响SNIP1在TGFβ信号通路中的活性,最终为TGFβ诱导的肿瘤细胞迁移与侵袭提供了有利的条件。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-3

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