AP-2β和p53相互作用并调控CRYAB基因的转录

发布时间：2018-06-03 05:13

  本文选题：CRYAB + AP-2β　； 参考：《湖南师范大学》2011年硕士论文

【摘要】：晶状体蛋白CRYAB是小热激蛋白家族的一员,许多胁迫或病理条件可以诱导CRYAB基因的表达。在胁迫条件下CRYAB作为分子伴侣起作用,可阻止变性蛋白聚集和细胞凋亡,促进细胞存活。在许多神经性疾病和恶性肿瘤中CRYAB基因过量表达。因此,研究CRYAB基因的转录调控机制具有重要意义。 为了鉴定调控CRYAB基因转录的调节因子,我们通过生物信息学软件查找CRYAB基因启动子上潜在的转录因子结合位点。除了以前报道的转录因子p53结合位点,我们还发现了转录因子AP-2的四个结合位点。随后,我们将包含有四个AP-2结合位点及一个p53结合位点的CRYAB启动子序列克隆到荧光素酶报告基因载体pGL3-basic中,并将这个质粒分别与AP-2α、AP-2β共转入含野生型p53的细胞系HeLa中,通过荧光素酶分析实验证明过表达AP-2a或AP-2β都能增强CRYAB启动子的转录活性,且AP-2p的作用更强。但在无p53的细胞系H358中,单独表达AP-2p对CRYAB启动子活性没有影响,而共转入AP-2p和p53时CRYAB启动子活性高于单独转入p53时的活性。这些结果暗示AP-2β通过p53增强CRYAB启动子活性。为了进一步验证这个结果,我们利用了一个只含有p53结合位点而没有AP-2结合位点的荧光素酶报告基因载体pp53-TA-Luc去检测p53转录激活活性。将pp53-TA-Luc与AP-2p、p53共转入H358细胞中,发现单独转染AP-2β不影响pp53-TA-Luc荧光素酶活性,而AP-2p和p53共转染时,pp53-TA-Luc活性比单独转染p53时提高60.4%,说明AP-2β能增强p53的转录激活活性。 为了探讨AP-2p增强p53的转录激活活性的分子机制,我们通过免疫共沉淀实验证实AP-2p蛋白能和p53蛋白相互作用。并且,我们还发现AP-2β和p53共表达时,AP-2β能增强p53蛋白的稳定性。由此我们得到结论,AP-2β与p53结合并加强p53的稳定性,从而增强由p53介导的CRYAB基因的转录。
[Abstract]:Crystallin CRYAB is a member of small heat shock protein family. Many stress or pathological conditions can induce the expression of CRYAB gene. Under stress, CRYAB, acting as a molecular chaperone, can prevent denatured protein aggregation and cell apoptosis and promote cell survival. CRYAB gene is overexpressed in many neurological diseases and malignant tumors. Therefore, it is of great significance to study the transcriptional regulation mechanism of CRYAB gene. In order to identify the regulatory factors that regulate the transcription of CRYAB gene, we searched the potential transcription factor binding sites on the promoter of CRYAB gene by bioinformatics software. In addition to previously reported transcription factor p53 binding sites, we also found four binding sites of transcription factor AP-2. Subsequently, we cloned the CRYAB promoter sequence containing four AP-2 binding sites and one p53 binding site into the luciferase reporter gene vector pGL3-basic, and cotransfected the plasmid with AP-2 伪 AP-2 尾 into the wild type p53 cell line HeLa. The results of luciferase analysis showed that overexpression of AP-2a or AP-2 尾 could enhance the transcriptional activity of CRYAB promoter, and AP-2p played a more important role. However, in the cell line H358 without p53, the expression of AP-2p alone had no effect on the activity of CRYAB promoter, but the activity of CRYAB promoter was higher in co-transfer of AP-2p and p53 than that in p53 alone. These results suggest that AP-2 尾 enhances the activity of CRYAB promoter through p53. To further verify this result, we used pp53-TA-Luc, a luciferase reporter gene vector containing only p53 binding sites but no AP-2 binding sites, to detect p53 transcriptional activation. Pp53-TA-Luc and AP-2ptp53 were co-transfected into H358 cells. It was found that transfection of AP-2 尾 alone did not affect the activity of pp53-TA-Luc luciferase, while the activity of pp53-TA-Luc luciferase in AP-2p and p53 co-transfection was 60.4% higher than that in p53 alone, indicating that AP-2 尾 could enhance the transcriptional activation of p53. In order to investigate the molecular mechanism of AP-2p enhancing p53 transcriptional activation, we demonstrated that AP-2p protein can interact with p53 protein by immunoprecipitation. Moreover, we also found that AP-2 尾 can enhance the stability of p53 protein when AP-2 尾 and p53 co-express. It is concluded that AP-2 尾 binds to p53 and enhances the stability of p53, thus enhancing the transcription of CRYAB gene mediated by p53.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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