【作者】 刘金凤；

【导师】 孙志贤； 宋宜；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 基因组的完整性是一切生命遗传和代谢的基础。DNA损伤反应(DNA damage response)机制在维持基因组稳定性中起着决定性的作用。DNA损伤反应是生物在长期进化中发展的一整套防御系统,它包括损伤监视、转录调控、周期调控、损伤修复、凋亡诱导等细胞生物学事件,是保证机体自稳平衡(homeostasis)的重要机制,是遗传性疾病、衰老、肿瘤等医学研究热点领域的核心内容之一。与核基质相连的,以早幼粒白血病突变蛋白(the promyeocytic leukemia protein, PML)为核心分子组装而成的多蛋白复合物PML核体(PML nuclear bodies,PML NBs),能作为DNA损伤的动态感受器(dynamic sensors),通过其组份的组装(assemble)和去组装(disassemble)协同调控DNA损伤修复,细胞周期阻滞和细胞凋亡等信号途径,是维持基因组稳定性的关键多蛋白复合物之一,对其功能及调控机制的研究不仅能够推进对DNA损伤反应信号转导网络的认识,更可为以PML途径相关信号分子为靶的抗肿瘤治疗策略提供理论指导。正是鉴于PML和PML NBs在电离辐射诱发的DNA损伤反应中的重要作用及其组份高度动态变化的特点,我们采用免疫共沉淀的方法比较了未照射和10Gy照射后6小时的乳腺癌肿瘤细胞系MCF-7细胞中PML结合蛋白的变化,而后选取差异条带进行质谱鉴定,以寻找能定位到PML NBs中并可能在IR诱发的DNA损伤反应中具有重要功能作用的分子。最终我们鉴定得到了124个潜在的PML NBs定位蛋白质,并验证得到4个之前未有报道的,能在电离辐射前后差异定位到PML NBs的分子(NPM1、MVP、G3BP1、DHX9)。在以上研究的基础上选取与基因组稳定性密切相关的核磷蛋白(Nucleophosmin,NPM1/B23)分子进行进一步研究,以探讨NPM1-PML相互作用及其在辐射诱导的DNA损伤反应中的调控机制和功能意义。主要研究进展如下:一、PML NBs的功能蛋白质组学分析1.建立辐射激活PML NBs的细胞模型。检测了多个肿瘤细胞中PML的表达,选择PML表达量高的MCF-7细胞作为本研究的细胞模型;采用免疫荧光的方法实时观测了辐照后不同时间点MCF-7细胞中PML NBs的动态变化,选择PML NBs反应活跃的照后6h和未照射的MCF-7细胞作为筛选辐照相关PML NBs定位分子的细胞模型2.采用免疫沉淀联合质谱的方法分离鉴定了IR致DNA损伤反应中PML的相互作用候选分子,并建立了一套相对标准化的数据评价体系,分别从免疫沉淀的特异性、候选分子生物学功能以及相关数据库检索分析三个方面对候选分子与靶蛋白之间的相互作用进行可靠性评价。3.选出NPM1、G3BP1、SND1、DHX9、及MVP等5个可信度高的候选分子,采用Co-IP和免疫荧光的实验方法验证它们与PML的相互作用,其中NPM1、G3BP1、DHX9及MVP 4个为阳性,进一步表明所获数据具有较高的可靠性。数据的系统分析为揭示PML NBs的生物学功能及作用机制提供了有价值的线索。二、PML与NPM1相互作用在DNA损伤反应中的功能及调控机制研究1.研究NPM1在DSBs(Double stand breaks)损伤反应中的功能意义。在γ射线诱发的DSBs损伤反应中,NPM1蛋白在辐照后24 h表达增高明显,其定位出现从核仁到核浆的扩散,与此相对应,MCF-7细胞在辐照后24h呈现G2/M期细胞显著增多;提示,NPM1的蛋白表达及定位改变可能参与细胞G2/M阻滞(arrest)调控。2. NPM1敲低增高MCF-7细胞的辐射敏感性,NPM1在IR诱导的DNA损伤反应中发挥着促进DSBs损伤修复,促进G2/M阻滞和抗辐射诱导的细胞凋亡的作用。γ-H2AX免疫荧光检测发现,敲低NPM1表达后能够明显延缓电离辐射所致的双链断裂损伤修复;细胞周期检测发现,NPM1敲低后G2/M阻滞显著降低,文献报道,在UV诱发的SSBs(Single strand breaks)损伤反应中,NPM1敲低导致G2/M阻滞升高;提示,NPM1可能通过不同的分子机制调控DSBs、SSBs损伤反应;细胞凋亡检测发现,NPM1敲低后,细胞在辐照后24h开始凋亡显著增加。3.研究发现PML参与介导辐射反应中NPM1调控细胞G2/M期阻滞及凋亡的信号转导。敲低NPM1导致PML的表达升高,caspase 3激活,凋亡增加;同时p21下调,辐射诱导的G2/M阻滞降低;双敲低NPM1和PML则可部分地恢复敲低NPM1诱发的细胞G2/M期阻滞减少和凋亡增加,同时,caspase 3激活受到抑制,但p21的表达依然下调。结果表明,辐射诱导的DNA损伤反应中,NPM1的上调不仅能通过抑制PML(PML-IV)- - -caspase 3信号途径抑制细胞凋亡,还同时能调控PML(PML-IV)- - -caspase 3信号途径以外的其他凋亡诱导途径;对于辐射诱导的细胞G2/M阻滞,敲低PML表达对NPM1敲低造成的G2/M期阻滞减少的逆转并非通过PML调控p53下游分子p21的途径实现,PML下调可通过p21非依赖途径激活G2/M阻滞。4.免疫荧光共聚焦显微镜结果显示在照射后6小时,NPM1和PML存在明显的共定位。交互的CoIP实验证实NPM1和PML在细胞内存在相互作用,GST-pull down实验研究发现,体外PML和NPM1也存在直接的相互作用,NPM1与PML相互作用的区段位于NPM1的N端1-117aa,而PML与NPM1相互作用的区段位于PML的1-360aa的RBCC结构和394-433aa。5. NPM1通过促进PML的泛素-蛋白酶体途径降解负调控PML表达。NPM1过表达能够明显抑制PML的表达,同样抑制NPM1的表达能够增加PML的表达,表明NPM1可以负调控PML的表达水平;泛素-蛋白酶体抑制剂MG132能够抑制NPM1对PML蛋白表达的下调,表明NPM1能通过促进PML的泛素-蛋白酶体途径降解负调控PML表达。综上,本研究通过蛋白质组学技术筛选获得一系列γ射线致DNA损伤反应中PML的相互作用候选分子,是迄今为止检索到的最大规模的对PML NBs的蛋白质组学分析,该数据对推进PML NBs在DNA损伤反应中的功能机制研究具有重要意义。同时,首次系统研究了新鉴定的PML相互作用分子NPM1在DSBs反应中的功能作用及其调节PML表达的分子机制。本研究不仅深化了对PML NBs参与DNA损伤反应的功能机制的认识,更揭示了NPM1调控PML参与细胞周期及凋亡调控的新信号途径,预示其具有更为重要的生物学意义。更多还原

【Abstract】 The integrity of the genome is fundamental to the propagation and metabolism of all living beings. To maintain the integrity of the genome, cells have evolved the ability to detect and propagate an initial DNA damage signal to elicit cellular responses that include cell cycle arrest, DNA repair, and apoptosis, which collectively have been termed the DNA damage response (DDR). DNA damage response machinery, as an important mechanism to keep cell homeostasis, is one of the core contents in research area of genetic disease, ageing, and cancer. Recent evidence suggests that PML NBs, a multi-protein complex associated with nuclear matrix, play crucial roles in maintaining genomic stability, and are dynamic sensors of DNA damage. After irradiation, PML NBs dynamically recruit or release important proteins involved in cell cycle regulation, DNA repair, and apoptosis.Although it is reported that PML NBs play an important role in the DNA damage responses of mammalian cells, the molecular mechanisms underlying the biological processes occurring within this nuclear domain in DNA damage responses are not yet fully elucidated. Therefore, to understand the function of PML in the DDR, it is essential to characterize its dynamic interactions during specific stresses. As such, in this current study, using a combination of immunoprecipitation and mass spectrometry, we analyzed the composition of PML NBs in irradiation-treated MCF-7 cells. We identified a total of 124 proteins, which may localize to PML NBs after irradiation. Four proteins, NPM1, MVP, G3BP1, and DHX9, were verified to co-localize with PML differentially before and after IR treatment. Based on these results, NPM1, identified as a novel PML-associating protein, was selected for further investigation. The primary results of this study are as follows:I. Functional proteomics analysis of PML NBs1. We first surveyed the expression of PML in 13 cell lines and chose MCF-7 cells with higher PML expression for further study. To obtain results that could reveal the dynamic components in PML NBs in irradiation responses, we monitored the number and subcellular distribution of PML NBs before and after irradiation by immunofluorescence. Finally, un-irradiated cells and cells irradiated for 6 h were collected for co-immunoprecipitation and mass spectrometry (MS).2. A proteomic approach, using CoIP, mono-dimensional electrophoresis, and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation, of which 15 were previously reported PML-NB proteins and their homologues. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell cycle regulation, cell death regulation, and response to stress, confirming the plurifunctional nature of PML NBs.3. Four proteins, NPM1, MVP, G3BP1, and DHX9, were verified to co-localize with PML differentially before and after IR treatment. We also visualized the colocalization of MVP, G3BP1, and NPM1 with PML after irradiation in MCF-7 cells using fluorescence microscopy. The proteome of PML NBs presented in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.Based on above results, NPM1, identified as a novel PML-associating protein, was selected for further investigation.II. The function and molecular mechanisms that the interaction of NPM1 and PML had in DNA damage response.1. Function of NPM1 in DSBs damage response. The protein level of NPM1 in MCF-7 cells increased afterγirradiation. We observed the translocation of NPM1 from nucleolus to nucleus 24h afterγirradiation. Correspondingly, cells in G2/M phase increased significantly. Collectively, these data implied a role for NPM1 in G2/M arrest.2. The knockdown of NPM1 expression delayed the repair of DSBs along with slower disappearance ofγ-H2AX focies. Decrease in G2/M arrest and increase in apoptosis were noted in NPM1 siRNA-transfected MCF-7 cells compared with negative control siRNA-transfected cells after irradiation induction. These data indicated that transfection with NPM1 siRNA made MCF-7 cells more sensitive to irradiation-induced cell-killing. In irradiation-induced DDR, NPM1 played a role of promoting the repair of DNA damage and G2/M arrest, inhibiting apoptosis.3. Double-knockdown of NPM1 and PML confirmed that NPM1 regulated G2/M arrest and apoptosis through PML protein. On one hand, knockdown of NPM1 induced increased PML expression, Caspase 3 activation, and apoptosis increase; double-knockdown of NPM1 and PML partly inhibited apoptosis with unactivated Caspase 3; these data indicated that down-regulation of PML inhibited the knockdown of NPM1 induced increase of apoptosis through activating Caspase 3. On the other hand, knockdown of NPM1 induced increased PML expression, decreased p21 expression, and G2/M arrest; double-knockdown of NPM1 and PML partly recovered G2/M arrest with samely downregulated p21 expression; these data indicated that down-regulation of PML recovered the knockdown of NPM1 induced decrease of G2/M arrest through p21-independent pathways.4. The immunofluorescence staining and confocal observations were performed on MCF-7 cells transfected with GFP-NPM1 using antibodies against PML, and demonstrated a co-localization of PML and NPM1 in the nucleolus. We conformed the interaction of PML and NPM1 in vivo and in vitro using co-immunoprecipatition and GST-pull down. Binding sites of NPM1 was mapped within the domain of 1-117 in its N terminus. Binding sites of PML was mapped within the domain of 1-360 amino acids and 394-433 amino acids.5. Over-expression of NPM1 inhibited the expression of PML significantly, likewise, the knockdown of NPM1 expression up-regulated the expression of PML, indicating NPM1 could negatively regulate the expression of PML. Further research revealed that NPM1 regulated PML protein level through ubiqutin/protesome parthway.Taken together, the proteome of PML NBs presented here will help to draw a more detailed picture about the role of PML NBs in DNA damage response regulation and tumor suppression. The identification of new NPM1-PML pathway has further provided the mechanistic explanation for PML NBs-regulated cell cycle and apoptosis responses. Keywords: DNA damage response; proteome; cell cycle; apoptosis; PML NBs; PML; NPM1更多还原