Tip60蛋白对细胞DNA损伤反应的调节作用及机制研究

发布时间：2018-03-15 04:22

  本文选题：Tip60　切入点：辐射敏感性　出处：《中国人民解放军军事医学科学院》2011年博士论文　论文类型：学位论文

【摘要】：Tip60(Tat interaction protein,60kD)最初是作为HIV病毒蛋白Tat相互作用的细胞内乙酰转移酶蛋白而被发现的。它是MYST家族成员之一,具有乙酰转移酶活性,可以将乙酰辅酶A的乙酰基团转移到组蛋白的赖氨酸残基上,中和了组蛋白尾的阳性电荷,削弱了组蛋白-DNA和组蛋白-组蛋白之间的相互接触。通过这种方式不仅直接改变染色质的结构,也调整特定蛋白与染色质之间的相互作用。这种乙酰化联合其它转录后修饰方式一起,创造一种多位点修饰模式,使包括许多转录因子在内的蛋白易于识别和结合,促进了基因表达调控、DNA损伤修复、细胞周期进程等过程的进行。 对Tip60的研究发现,它在细胞内信号传递、DNA损伤修复、细胞周期和检查点控制、细胞凋亡等多个方面发挥作用。Tip60在转录调控方面具有双重作用,可以通过其乙酰转移酶的活性对目的基因起到转录激活,或者通过募集组蛋白去乙酰化酶到目的基因的启动子上实现转录抑制作用。有研究报道,Tip60通过MYST结构域与TEL相互作用,作为TEL的转录共抑制子发挥作用;还可以乙酰化AR并加强其反式活化功能。 特别值得一提的是,Tip60对DNA损伤修复的调节也是它不容忽视的一个重要功能。有文献报道,当Tip60与乙酰辅酶A的结合位点Q377、G380突变后,细胞修复DNA损伤能力下降;Tip60引起γH2AX的乙酰化并参与UBC13对γH2AX的泛素化和与H2AX的交换有关;Tip60的活性通过与H3K9me3的结合而被活化;Tip60是p53发挥正确功能所必需的,直接乙酰化p53,调控p53下游靶基因的转录。目前认为Tip60参与DNA损伤修复主要通过两条途径:一方面,是对PI3K家族蛋白尤其是ATM的乙酰化启动ATM自磷酸化并活化,进而磷酸化其下游包括p53、BRCA1、γH2AX等多个底物分子,参与损伤修复;另一方面,则是以NuA4-Tip60复合物的形式介导染色质重构,通过乙酰化DNA损伤位点附近的H4和H2AX,使核小体间相互作用减弱,结构松弛,染色质结构得以打开。 种种证据表明,Tip60是DNA损伤后细胞修复不可或缺的关键分子。本研究利用Tip60基因沉默和转染Tip60基因的骨肉瘤细胞模型探讨了Tip60对电离辐射诱发的DNA损伤修复及细胞周期阻滞的影响以及相关机制,并探讨了其大规模染色质松弛的功能,获得如下主要结果: 1、利用转染Tip60基因的细胞模型,发现正常情况下,过表达Tip60对骨肉瘤细胞的生长发挥一定的负调控作用; 2、利用Tip60基因沉默的细胞模型,发现Tip60的缺失导致细胞辐射敏感性明显增加,单细胞凝胶电泳、脉冲场凝胶电泳以及γH2AX foci及免疫印迹等多个实验指标观察都证实Tip60的抑制导致细胞对DNA损伤的修复能力降低; 3、利用Tip60稳定表达的细胞模型,与对照细胞比较,通过单细胞凝胶电泳、脉冲场凝胶电泳以及γH2AX foci及免疫印迹等实验发现Tip60的过表达引起细胞DNA损伤识别感应增强,提高细胞应对DNA损伤修复的能力; 4、证实Tip60在DNA损伤刺激的情况下与DNA-PKcs有相互作用,但与DNA-PK复合物的另一个亚基Ku70没有相互作用,并且Tip60的高表达对电离辐射刺激下Ku70、Ku80蛋白的表达也没有明显作用; 5、证实Tip60的乙酰转移酶活性抑制剂漆树酸可以抑制电离辐射后DNA-PKcs pT2609的磷酸化; 6、利用Tip60稳定表达的细胞模型,通过流式细胞术检测发现Tip60的过表达引起电离辐射诱发G2/M期阻滞较对照细胞延长,同时发现,这种延长G2/M期阻滞的作用可能与Cyclin B和Cdk 1的表达下调有关; 7、利用本实验室建立的染色质大规模松弛观察技术,证实Tip60在电离辐射后,对染色质大规模松弛的作用明显增强;同时发现Tip60 Ser86、Ser90以及乙酰辅酶A结合位点Gln377、Gly380的分别突变不会破坏Tip60参与大规模染色质重构的功能,但Ser90的模拟磷酸化突变却降低了Tip60参与大规模染色质重构的功能。
[Abstract]:Tip60 (Tat interaction protein, 60kD) initially as HIV virus protein Tat interactions of intracellular acetyl transferase protein was found. It is a member of the MYST family, has acetyltransferase activity and lysine residues can be acetyl groups of acetyl coenzyme A is transferred to the group of proteins, and the the positive charge of the histone tail, weakened the interaction between histone -DNA and histone histone. In this way not only directly alter the structure of chromatin, but also adjust the specific interactions between proteins and chromatin. The acetylation and other post-translational modification together, creating a multilocus modified model so, including many transcription factors, protein is easy to recognize and bind, promote gene expression regulation, DNA repair, cell cycle process and other processes.
In the Tip60 study, it in cell signaling, DNA repair, cell cycle checkpoint and control many aspects of cell apoptosis play a role in.Tip60 has a dual role in transcriptional regulation, can play on gene transcriptional activation through its acetyltransferase activity, inhibition of transcription through recruitment or implementation histone deacetylase to target gene promoter. Studies have reported that the Tip60 MYST domain and TEL structure interaction, TEL as a transcriptional co repressor can also play a role; acetylation of AR and enhance its trans activation function.
It is particularly worth mentioning is that an important function of Tip60 on DNA damage and repair regulation is also can not be ignored. It is reported, when the binding site of Q377 Tip60 and acetyl coenzyme A, G380 mutation, decreased ability of cells to repair DNA damage caused by Tip60 gamma; acetylation of H2AX and UBC13 on H2AX the ubiquitination and exchange with H2AX; the activity of Tip60 by combination of H3K9me3 and Tip60 is p53 and is activated; the proper function required by the direct acetylation of p53, transcription of downstream target genes of p53. It is believed that Tip60 involved in DNA damage repair mainly through two ways: on the one hand, is to PI3K protein family especially acetylation of ATM promoter ATM autophosphorylation and activation and phosphorylation of downstream including p53, BRCA1, H2AX and many other gamma substrate molecules involved in injury and repair; on the other hand, is in the form of chromatin mediated NuA4-Tip60 complex Guide Reconfiguration, by acetylation of H4 and H2AX near the DNA damage site, the interaction between the nucleosomes is weakened, the structure is relaxed, and the chromatin structure is opened.
The evidence suggests that Tip60 is a key molecule essential DNA cell injury after repair. By using the model of osteosarcoma cells Tip60 gene silencing and transfected with Tip60 gene to investigate the effect of DNA damage repair and cell cycle arrest of Tip60 induced by ionizing radiation and related mechanism, and discusses the function of large-scale mass relaxation staining. The main results are as follows:
1, using the cell model transfected with Tip60 gene, it was found that under normal conditions, overexpression of Tip60 could play a negative regulatory role in the growth of osteosarcoma cells.
2, the use of cell model for silencing Tip60 gene, and found that loss of Tip60 leads to cell radiation sensitivity increased significantly, reduce the single cell gel electrophoresis, pulsed field gel electrophoresis and gamma H2AX foci and Western blotting. Many experiments have confirmed that the observed inhibition of Tip60 resulted in cells to DNA damage repair ability;
3, using Tip60 stable expression cell model, compared with the controls, by single cell gel electrophoresis, pulsed field gel electrophoresis and gamma H2AX foci and Western blotting assays showed that overexpression of DNA induced cell damage identification induced enhancement of Tip60, improve the ability of cells to respond to DNA damage and repair;
4, it was confirmed that Tip60 interacted with DNA-PKcs in the presence of DNA damage, but it had no interaction with the other subunit Ku70 of DNA-PK complex, and the high expression of Tip60 had no obvious effect on Ku70 and Ku80 protein expression stimulated by ionizing radiation.
5, it was proved that Tip60's acetyltransferase activity inhibitor lacquer acid could inhibit the phosphorylation of DNA-PKcs pT2609 after ionizing radiation.
6, by using Tip60 cell model stably expressed and detected by flow cytometry, it was found that the over expression of Tip60 caused the G2/M phase arrest induced by ionizing radiation prolonged compared with the control cells. Meanwhile, it was found that the prolongation of G2/M phase arrest might be related to the downregulation of Cyclin B and Cdk 1.
7, established by our laboratory large-scale chromatin relaxation observation technique, Tip60 confirmed that after ionizing radiation, the chromatin mass relaxation effect is obviously enhanced; at the same time that Tip60 Ser86, Ser90 and acetyl coenzyme A binding sites of Gln377, Gly380 were broken Tip60 mutations do not function of participation in large-scale chromatin remodeling, but the simulation of phosphoric acid the Ser90 mutation was reduced by Tip60 in large-scale chromatin remodeling.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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