细胞抑癌因子ARF与腺病毒E1A蛋白关系的研究

发布时间：2018-05-31 06:45

本文选题：ARF + 腺病毒E1A　；参考：《华东师范大学》2011年硕士论文

【摘要】：ARF是哺乳动物细胞内的一个重要抑癌因子(小鼠细胞中是P19ARF,人类细胞中是P14ARF)。一般生理条件下,ARF表达量保持着较低水平,但是当细胞受到一些致癌因素的刺激时,它会响应信号而表达量上升。ARF一般通过p53-依赖或p53-非依赖的途径实施其抑癌功效。E1A基因是腺病毒致病的关键基因,它主要是通过结合细胞内Rb蛋白和P300/CBP,然后启动自身病毒基因的转录及引发宿主细胞周期失控,最终可导致相关细胞的癌化。在本课题研究中,通过外源性的和内源性的免疫共沉淀首次发现了ARF与腺病毒E1A蛋白之间的相互作用,两者之间的结合并不依赖于p53的存在。在功能方面,发现P14ARF可以抑制E1A对于病毒基因组的转录激活与引起的细胞DNA复制。在探索其内在机制时,发现高表达P14ARF可以改变E1A原先的细胞核浆定位,E1A进入细胞核仁后加速了其被泛素化途径降解。而通过Knockdown方去下调内源性p14ARF,我们发现E1A的转录激活能力显著升高。分别构建了P14ARF和E1A的一系列蛋白片段,成功检测到两者相结合的区段。另外,还发现了E1A可以竞争性地阻断ARF-mdm2的结合。这些机制的发现,加上之前已有的文献报道E1A可以激活P14ARF的转录,本研究提出了一条存在于P14ARF与E1A之间的负反馈调控通路,这很有可能是宿主细胞针对腺病毒感染的一种防御机制。而对于病毒来说,这条通路的存在,使得病毒可以调控其感染途径与程度,以避免细胞引发对其有害的过度反应。
[Abstract]:ARF is an important tumor suppressor in mammalian cells (P19 ARF in mouse cells and P14 ARFN in human cells. The expression of ARF remains low under normal physiological conditions, but when the cells are stimulated by some carcinogenic factors, It responds to the signal and increases the expression of .ARF generally through a p53-dependent or p53-independent pathway to carry out its tumor suppressor effect. E1A gene is a key gene of adenovirus pathogenesis. It is mainly by binding the intracellular RB protein and P300% CBP, and then initiating the transcription of its own virus gene and causing the host cell cycle to lose control, which can eventually lead to the carcinogenesis of the associated cells. In this study, the interaction between ARF and adenovirus E1A protein was first discovered by exogenous and endogenous immunoprecipitation, and the binding between the two proteins was not dependent on the existence of p53. In terms of function, P14ARF can inhibit the transcriptional activation and DNA replication induced by E1A. In order to explore its intrinsic mechanism, it was found that overexpression of P14ARF could change the localization of E1A into nucleolus and accelerate its degradation by Ubiquitin pathway. By down-regulating endogenous p14 ARF by Knockdown, we found that the transcriptional activation ability of E1A was significantly increased. A series of protein fragments of P14ARF and E1A were constructed, and the combined regions were successfully detected. In addition, it was found that E 1A could competitively block the binding of ARF-mdm2. The discovery of these mechanisms, together with previous reports that E1A activates transcription of P14ARF, suggests a negative feedback regulatory pathway between P14ARF and E1A. This may be a defense mechanism against adenovirus infection in host cells. For viruses, the presence of this pathway allows the virus to regulate its infection pathway and degree to prevent cells from triggering harmful overreactions.
【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】：R346

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