蛋白磷酸酶PPM1A对KAP1和SIRT1的调节作用

发布时间：2018-07-07 19:54

本文选题：ppM1A + KAp1　；参考：《华东师范大学》2011年硕士论文

【摘要】：KAP1(又称TIF1β,TRIM28等)是一种转录中介因子,能介导多种转录调控过程。大量研究表明,KAP1在大多数情况下以共抑制因子的形式参与转录调控复合体的形成,其在精细胞发育、胚胎早期发育等生理过程中发挥了重要的调控作用。但是,调节KAP1自身活性的机制尚不明确。人类SIRT1基因是依赖于NAD+的组蛋白去乙酰化酶,也是一种新的抗肿瘤药物靶点。SIRT1的同源基因Sir2能够延长酵母、线虫、果蝇的寿命,在基因组稳定性、细胞代谢调节和衰老上起着不可缺少的作用,现已发现SIRT1能结合p53、E2F1、FOXO、NF-κB等非组蛋白,有抵抗氧化逆境、抑制肿瘤细胞凋亡和提高肿瘤细胞耐药性的作用。研究表明,SIRT1有着丰富的磷酸化修饰,明确磷酸化对SIRT1生物学功能的影响,对以SIRT1为靶点的药物开发具有重要意义。结合分子克隆、病毒包装、稳定单克隆细胞筛选、RNA干扰、免疫荧光、免疫共沉淀、Western Blot和荧光定量PCR等技术进行研究后,本文发现,丝/苏蛋白磷酸酶PPM1A是KAP1和SIRT1的特异位点去磷酸化酶,且其对后两者特异位点的去磷酸化能影响后两者的功能。过表达PPM1A能使KAP1 Ser473去磷酸化,而当PPM1A的表达被抑制后,UV诱导的KAP1磷酸化响应衰减变慢,KAP1的下游抑制基因表达增强。PPM1A和SIRT1两者结合,过表达PPM1A能使SIRT1 Thr 719去磷酸化,且使SIRT1对组蛋白H3的Lys18去乙酰化作用减弱。本文发现了PPM1A的两个新的底物,同时找到了调控KAP1和SIRT1功能的一个新的蛋白,完善了和PPM1A、KAP1、SIRT1三者有关的信号通路,为研究这三个蛋白提供了新的思路。
[Abstract]:KAP1 (TIF1 尾 trim28 et al.) is a transcriptional mediator that mediates many transcriptional processes. A large number of studies have shown that KAP1 is involved in the formation of transcriptional regulatory complex in the form of co-inhibitory factor in most cases, and it plays an important role in the development of spermatocytes and early embryonic development and other physiological processes. However, the mechanism of regulating the activity of KAP1 is unclear. Human SIRT1 gene is a NAD-dependent histone deacetylase. It is also a new antitumor drug target. SIRT1 homologous gene Sir2 can prolong the lifespan of yeast, nematode and Drosophila. Cell metabolism regulation and senescence play an indispensable role. It has been found that SIRT1 can bind to non-histone proteins such as p53 E2F1F1 FOXON- 魏 B, which can resist oxidative stress, inhibit apoptosis of tumor cells and increase drug resistance of tumor cells. The results show that SIRT1 has rich phosphorylation modification, and it is of great significance to study the effects of phosphorylation on the biological function of SIRT1 and to develop drugs targeting SIRT1. Combined with molecular cloning, virus packaging, stable monoclonal cell screening, RNA interference, immunofluorescence, immunoprecipitation Western Blot and fluorescent quantitative PCR, it was found that, PPM1A is a specific site dephosphorylase of KAP1 and SIRT1, and its dephosphorylation of the latter two specific sites can affect the function of the latter two. Overexpression of PPM1A resulted in dephosphorylation of KAP1 Ser473. However, when the expression of PPM1A was inhibited, the UV-induced phosphorylation response of KAP1 decreased and the downstream inhibitory gene expression of KAP1 increased. PPM1A overexpression combined with SIRT1 Thr719, and PPM1A overexpressed PPM1A could dephosphorize SIRT1 Thr719. SIRT1 reduced the Lys18 deacetylation of histone H 3. In this paper, two new substrates of PPM1A were found, a new protein regulating the function of KAP1 and SIRT1 was found, and the signal pathway related to PPM1A, KAP1 and SIRT1 was improved, which provided a new idea for the study of these three proteins.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341;Q55

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