本文選題：Tip60　切入點(diǎn)：輻射敏感性　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年博士論文　論文類型：學(xué)位論文

【摘要】：Tip60(Tat interaction protein,60kD)最初是作為HIV病毒蛋白Tat相互作用的細(xì)胞內(nèi)乙酰轉(zhuǎn)移酶蛋白而被發(fā)現(xiàn)的。它是MYST家族成員之一,具有乙酰轉(zhuǎn)移酶活性,可以將乙酰輔酶A的乙酰基團(tuán)轉(zhuǎn)移到組蛋白的賴氨酸殘基上,中和了組蛋白尾的陽性電荷,削弱了組蛋白-DNA和組蛋白-組蛋白之間的相互接觸。通過這種方式不僅直接改變?nèi)旧|(zhì)的結(jié)構(gòu),也調(diào)整特定蛋白與染色質(zhì)之間的相互作用。這種乙�；�(lián)合其它轉(zhuǎn)錄后修飾方式一起,創(chuàng)造一種多位點(diǎn)修飾模式,使包括許多轉(zhuǎn)錄因子在內(nèi)的蛋白易于識(shí)別和結(jié)合,促進(jìn)了基因表達(dá)調(diào)控、DNA損傷修復(fù)、細(xì)胞周期進(jìn)程等過程的進(jìn)行。 對(duì)Tip60的研究發(fā)現(xiàn),它在細(xì)胞內(nèi)信號(hào)傳遞、DNA損傷修復(fù)、細(xì)胞周期和檢查點(diǎn)控制、細(xì)胞凋亡等多個(gè)方面發(fā)揮作用。Tip60在轉(zhuǎn)錄調(diào)控方面具有雙重作用,可以通過其乙酰轉(zhuǎn)移酶的活性對(duì)目的基因起到轉(zhuǎn)錄激活,或者通過募集組蛋白去乙酰化酶到目的基因的啟動(dòng)子上實(shí)現(xiàn)轉(zhuǎn)錄抑制作用。有研究報(bào)道,Tip60通過MYST結(jié)構(gòu)域與TEL相互作用,作為TEL的轉(zhuǎn)錄共抑制子發(fā)揮作用;還可以乙酰化AR并加強(qiáng)其反式活化功能。 特別值得一提的是,Tip60對(duì)DNA損傷修復(fù)的調(diào)節(jié)也是它不容忽視的一個(gè)重要功能。有文獻(xiàn)報(bào)道,當(dāng)Tip60與乙酰輔酶A的結(jié)合位點(diǎn)Q377、G380突變后,細(xì)胞修復(fù)DNA損傷能力下降;Tip60引起γH2AX的乙酰化并參與UBC13對(duì)γH2AX的泛素化和與H2AX的交換有關(guān);Tip60的活性通過與H3K9me3的結(jié)合而被活化;Tip60是p53發(fā)揮正確功能所必需的,直接乙�；痯53,調(diào)控p53下游靶基因的轉(zhuǎn)錄。目前認(rèn)為Tip60參與DNA損傷修復(fù)主要通過兩條途徑:一方面,是對(duì)PI3K家族蛋白尤其是ATM的乙�；瘑�(dòng)ATM自磷酸化并活化,進(jìn)而磷酸化其下游包括p53、BRCA1、γH2AX等多個(gè)底物分子,參與損傷修復(fù);另一方面,則是以NuA4-Tip60復(fù)合物的形式介導(dǎo)染色質(zhì)重構(gòu),通過乙酰化DNA損傷位點(diǎn)附近的H4和H2AX,使核小體間相互作用減弱,結(jié)構(gòu)松弛,染色質(zhì)結(jié)構(gòu)得以打開。 種種證據(jù)表明,Tip60是DNA損傷后細(xì)胞修復(fù)不可或缺的關(guān)鍵分子。本研究利用Tip60基因沉默和轉(zhuǎn)染Tip60基因的骨肉瘤細(xì)胞模型探討了Tip60對(duì)電離輻射誘發(fā)的DNA損傷修復(fù)及細(xì)胞周期阻滯的影響以及相關(guān)機(jī)制,并探討了其大規(guī)模染色質(zhì)松弛的功能,獲得如下主要結(jié)果: 1、利用轉(zhuǎn)染Tip60基因的細(xì)胞模型,發(fā)現(xiàn)正常情況下,過表達(dá)Tip60對(duì)骨肉瘤細(xì)胞的生長(zhǎng)發(fā)揮一定的負(fù)調(diào)控作用; 2、利用Tip60基因沉默的細(xì)胞模型,發(fā)現(xiàn)Tip60的缺失導(dǎo)致細(xì)胞輻射敏感性明顯增加,單細(xì)胞凝膠電泳、脈沖場(chǎng)凝膠電泳以及γH2AX foci及免疫印跡等多個(gè)實(shí)驗(yàn)指標(biāo)觀察都證實(shí)Tip60的抑制導(dǎo)致細(xì)胞對(duì)DNA損傷的修復(fù)能力降低; 3、利用Tip60穩(wěn)定表達(dá)的細(xì)胞模型,與對(duì)照細(xì)胞比較,通過單細(xì)胞凝膠電泳、脈沖場(chǎng)凝膠電泳以及γH2AX foci及免疫印跡等實(shí)驗(yàn)發(fā)現(xiàn)Tip60的過表達(dá)引起細(xì)胞DNA損傷識(shí)別感應(yīng)增強(qiáng),提高細(xì)胞應(yīng)對(duì)DNA損傷修復(fù)的能力; 4、證實(shí)Tip60在DNA損傷刺激的情況下與DNA-PKcs有相互作用,但與DNA-PK復(fù)合物的另一個(gè)亞基Ku70沒有相互作用,并且Tip60的高表達(dá)對(duì)電離輻射刺激下Ku70、Ku80蛋白的表達(dá)也沒有明顯作用; 5、證實(shí)Tip60的乙酰轉(zhuǎn)移酶活性抑制劑漆樹酸可以抑制電離輻射后DNA-PKcs pT2609的磷酸化; 6、利用Tip60穩(wěn)定表達(dá)的細(xì)胞模型,通過流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)Tip60的過表達(dá)引起電離輻射誘發(fā)G2/M期阻滯較對(duì)照細(xì)胞延長(zhǎng),同時(shí)發(fā)現(xiàn),這種延長(zhǎng)G2/M期阻滯的作用可能與Cyclin B和Cdk 1的表達(dá)下調(diào)有關(guān); 7、利用本實(shí)驗(yàn)室建立的染色質(zhì)大規(guī)模松弛觀察技術(shù),證實(shí)Tip60在電離輻射后,對(duì)染色質(zhì)大規(guī)模松弛的作用明顯增強(qiáng);同時(shí)發(fā)現(xiàn)Tip60 Ser86、Ser90以及乙酰輔酶A結(jié)合位點(diǎn)Gln377、Gly380的分別突變不會(huì)破壞Tip60參與大規(guī)模染色質(zhì)重構(gòu)的功能,但Ser90的模擬磷酸化突變卻降低了Tip60參與大規(guī)模染色質(zhì)重構(gòu)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 孫薏;黃越承;徐勤枝;王會(huì)平;隋建麗;周平坤;;HIV-1 Tat蛋白對(duì)細(xì)胞周期相關(guān)基因表達(dá)及輻射細(xì)胞周期阻滯的影響[J];軍事醫(yī)學(xué)科學(xué)院院刊;2006年02期

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本文編號(hào)：1614417