【作者】 李强；

【导师】 邹声泉；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 腺病毒(Ad)已成为探索真核细胞转录、RNA转录后加工及细胞周期调控基本原理的重要工具。研究证明通过持续表达Ad E1B和E1A蛋白,能使哺乳动物细胞转化为恶性肿瘤。Ad12 E1B N端能特异性与Sin3结合并相互作用,尽管Sin3A在胞核的功能作为一个转录辅阻遏物,是组蛋白去乙酰化酶和其它转录抑制酶蛋白复合物中的主要组成成分,但它在细胞核内的的作用仍然不清楚。P53-E1B的相互作用导致P53的转导激活功能丧失以及促进Ad介导的细胞异常转化[1,2]。研究证明,E1B与p53的TAD区域结合,可能阻断p53与转录物的相互作用,从而抑制p53转录活性[3,4]。然而,E1B 55-kDa通过结合一个p53抑制区域单独是否能抑制作为一个转录激活因子的p53功能,是否除E1B 55-kDa外可能还存在其它的机制使p53功能失活仍不清楚。本研究通过实验证实Ad12 E1B 55kDa不能单独抑制p53功能,而是需要联合其它的辅阻遏蛋白阻抑p53的功能,如Ad12 E1A。因此,对Ad E1B 55-kDa深入的研究能告诉我们很多有关恶性肿瘤细胞生物学特性的基本过程。E1B 55-kDa也是病毒泛素化连接酶的一个底物结合亚单元,作用于p53和MRN复合物的一个或多个亚单元促使蛋白降解。E1B 55-kDa抑制区域的功能需要一个迄今未证实的细胞内辅阻遏物。E1B 55-kDa/E4orf6泛素连接酶也是许多细胞内病毒后期蛋白合成效率所需的,但机制并不清楚。这些问题告诉我们,虽然我们对Ad研究了解的已经很多,但是还有更多的问题需要我们去研究了解,才能更好的利用Ad为人类的健康作出贡献。人类腺病毒有将近50种不同的血清分型,它们虽然有着相似的蛋白质样外壳形态和线性双链DNA基因组,然而它们之间却有着很重要功能的差别。例如,静脉注射Ad5病毒颗粒导致明显的肝脏局灶性坏死,以及最近报道的Ad5经凝血因子X(FX)转导肝细胞,而许多其它的Ad血清型却不具有这个特性,这表明Ad5外壳蛋白六位体(hexon)介导hexon-FX相互作用,许多其它的Ad蛋白也存在超变区,它们也被赋予不同的功能。在目前已研发的三代腺病毒载体中,大多以5型(Ad5)、2型(Ad2)为基础,但即使是第三代腺病毒载体也必须有辅助病毒存在才能在特定的细胞系中发挥作用。研究证明,Ad12的DNA在病毒感染早期就能紧密结合在哺乳动物细胞的染色质[5],Ad12的DNA也能够选择性的稳定结合至染色质的特定位点[6],且Ad12DNA的末端共价结合蛋白能更有效率的被哺乳动物细胞摄取[7]。因此研究理解腺病毒12型与其它不同血清型的差异,是为设计出更好的基因转移载体,以达到提高治疗效率和降低毒性的目的。我们课题组在邹声泉教授带领下,已对恶性肿瘤的生物学特性进行了一系列的研究,特别是在早期诊断,早期治疗方面取得了一些阶段性成果,为进一步研究细胞恶性转化的发生学机制及靶向治疗肿瘤的合适的腺病毒基因载体,由国家留学基金委资助联合培养博士研究生,公派至美国佛罗里达大学医学院Shands癌症中心Daiqing Liao副教授的实验室,进行与细胞恶性转化密切相关的腺病毒癌蛋白E1B、E1A的生物学功能的相关研究。本研究旨在前期研究结果的基础上,对腺病毒癌蛋白E1B和E1A蛋白与被感染细胞内的辅阻抑蛋白相互作用的研究,并观察这些蛋白发挥的生物学功能作用及翻译后修饰对其功能的影响,探索肿瘤发生的机制及设计更好的腺病毒基因载体,为肿瘤的早期诊断和治疗提供可靠的实验依据和理论基础。1.腺病毒12型E1B 55-kDa与宿主细胞内Sin3蛋白相互作用的研究目的研究腺病毒12型E1B 55-kDa (Ad12 E1B 55-kDa)蛋白在宿主细胞内与辅阻遏蛋白Sin3A结合的方式特点,及对E1B-Sin3A在细胞内定位的影响。方法采用分子生物学方法构建包含PAH1而缺失NLS的Sin3A突变体,以及构建包含NLS而缺失C端序列的Sin3A突变体,突变E1B S476及S477位点,构建多种不同Sin3A的序列；以及去除一段E1B序列突变体,用免疫荧光方法观察其在Saos2细胞中的定位。结果1. Sin3A的aa439-443 (KKKPK)为其定位于细胞核的核定位信号(NLS)序列元件,不包含此序列元件的Sin3A突变体与E1B结合后共定位于细胞浆,而包含NLS序列元件的Sin3A突变体能与E1B结合后共定位于细胞核。2. Ad12 E1B 55-kDa的C端S476/477的磷酸化修饰能与不包含NLS序列的Sin3A突变体结合后共定位于细胞核,而未磷酸化修饰的E1B在细胞浆E1B-Sin3A结合。3. Ad12 E1B 55-kDa的N端序列调节其定位于细胞核内,而缺失N端82aa的E1B则定位于核孔复合体(NPCs)。结论1. Sin3A的NLS序列元件调节其与E1B结合及细胞内共定位表达。2. Ad12 E1B的C端磷酸化修饰调节E1B-Sin3A结合及在宿主细胞核内的定位表达。3. Ad12 E1B的N端具有调节其进出宿主细胞核的作用。2.腺病毒12型E1B 55-kDa的SUMO化修饰在宿主细胞内作用的研究目的研究Ad12 E1B 55-kDa的SUMO化修饰对其在宿主细胞内定位及其与细胞内辅阻遏蛋白Sin3A、Sin3B相互作用的影响。方法用分子生物学方法构建及突变E1B的多种序列,并设计构建Rapmycin可诱导下的HA-SUMO1-FRB融合突变体及2×FKB-E1B融合突变体,在Saos2细胞内表达,用免疫荧光方法(IF)检测其蛋白复合体在细胞内的定位。用抗-FLAG(?)M2-免疫磁珠共沉淀(Flag M2-IP)及蛋白免疫印记法(WB)分析E1B在细胞内是否与SUMO蛋白结合。结果1. Ad12 E1B的Lys88和N末端区域的其它赖氨酸是SUMO1的修饰位点；而Ad12E1B不能被SUMO3修饰。2.未被SUMO1修饰的Ad12 E1B定位于细胞核,而SUMO1修饰后的Ad12 E1B定位在细胞浆。3. SUMO1修饰的Ad12 E1B阻止其与辅阻遏蛋白Sin3A和Sin3B的结合。结论1. Ad12 E1B 55-kDa能被SUMO1修饰,且修饰后调节其定位于细胞浆内及NPCs。2. Ad12 E1B 55-kDa的SUMO1修饰不再与Sin3B在细胞内的结合。3.腺病毒12型E1B 55-kDa、E1A对病毒自身DNA合成的调节作用目的研究腺病毒12型E1B 55-kDa、E1A及SUMO1修饰E1B 55-kDa的对腺病毒自我复制的调节作用。方法采用分子生物学方法构建E1A266, wtE1B, SUMO1-E1B 55-kDa片段,单独或与E1A共表达于LN-229细胞株中,采用缺失表达E1A E3区域的Ad3875感染细胞株。并使用病毒DNA量分析法分析各组中病毒DNA表达的量。结果1. Ad12 E1B 55-kDa以及E1A蛋白单独分别能刺激病毒DNA的合成。2. Ad12 E1B 55-kDa以及E1A蛋白共同能显著刺激大量病毒DNA的合成。3. SUMO1修饰的Ad12 E1B 55-kDa蛋白虽定位于胞浆,仍能联合E1A适度刺激病毒DNA的合成。结论1. Ad12 E1B 55-kDa及E1A均有刺激腺病毒自身DNA合成作用。2. SUMO1修饰的Ad12 E1B 55-kDa虽表达在宿主细胞浆内,但被仍能联合E1A显著促进病毒DNA合成。4.腺病毒12型E1B 55-kDa、E1A调节p53介导的转录研究目的研究Ad12 E1B 55-kDa和E1A对p53介导的转录活性的调节作用。方法采用肾肿瘤细胞株(G401)和稳定表达Ad12 E1B 55-kDa的G401-CC3,及染色质免疫共沉淀(ChIP)方法分析p53靶基因p21和Mdm2启动子上Ad12E1B 55-kDa是否在染色质水平上与p53结合；采用WB及实时定量PCR方法分析p53靶基因p21、Mdm2的蛋白及p21核苷酸表达量；采用双荧光素酶报告基因方法分析表达目的基因质粒质粒(单独或联合转染质粒：p53,E1A,E1B; E1A, E1B, p53, p300)的缺失p53的结肠癌细胞株(HCT116)中Mdm2启动子活性；采用WB分析表达E1B或者E1B+E1A质粒的G401-CC3细胞株中p53靶基因的表达量;采用分子生物学方法突变或者剪切掉E1A的CR1、CR2或者CR3功能区,检测其协同E1B抑制p53功能是否有改变。结果1. Ad12 E1B 55-kDa在染色质水平上与P21启动子上的p53 BS2及Mdm2启动子上P53RE2区域结合。2. Ad12 E1B 55-kDa能稳定激活p53的表达。3. Ad12 E1B 55-kDa或E1A单独不能抑制p53介导的mdm2启动子转录活性及p21蛋白的表达,但共同能抑制p53介导的mdm2启动子转录活性及p21蛋白的表达。4. Ad12 E1B 55-kDa和E1A能显著减弱组蛋白乙酰化酶p300的共激活p53介导的转录作用。5.E1A的p300,Rb及CtBP结合位点可能不是联合E1B 55-kDa抑制p53介导的转录活性的关键性序列。结论1. Ad12 E1B 55-kDa在染色质水平上与p53结合后能持续稳定的激活p53介导的转录。2. Ad12 E1B 55-kDa单独不能抑制p53介导的转录,但能联合E1A共同抑制p53介导的转录。3.E1A的p300,Rb和CtBP结合位点均不是联合E1B调节p53介导的转录作用的序列元件。更多还原

【Abstract】 Adenovirus is an important tool for detecting fundamental theory of cell biology. Cells can be transformed to neplastic cancer cell by continued expression of the ad E1B and E1A proteins. Nevertheless as we know that Sin3A as a transcriptional corepressor locates in the nucleus, it’s unclear if Sin3A express in the cytoplasm. It has been documented that E1B-p53 interaction results in the inactivation of p53 mediated transactivation function and facilitates Ad-mediated cell transformation [1,2], Our data demonstrate Ad 12 E1B 55-kDa cann’t suppresses p53 mediated transcription, and it can suppress p53 mediated transcription with corepressor Ad12 E1A. Abstractly, adenovirus leaves a lot to tell us about basic cell active.E1B 55-kDa is a substrate of the ubiqutin ligase; it conjugates p53 or other subunits of the MRN complex for target proteosomal degradation. Although E1B-55kDa can surpresses p53 gene mediated function as a transcriptional repressor by conjugating a corepressor to it. So EIB 55-kDa requires an unclear cellular corepressor for completed it. These issues need us that in spite of Ads have taught us too much, but they still have left a lot to tell us.There are nearly 50 distinct serotypes of the human adenoviruses. They share similar proteinaceous capsid morphology and linear double-stranded DNA genomes. However, important functional differences have been observed among the Ad serotypes. For examples, intravenous injection of Ad5 viral particles results in significant liver sequestration of the virus. It was reported recently that Ad5 transduces liver cells via the coagulation factor X (FX), whereas many other Ad serotypes do not share this property. It was shown that the hypervariable regions of the Ad5 coat protein hexon mediate hexon-FX interaction. Hypervariable regions also exist in many other Ad proteins. They conceivably also confer distinct functions. From now on, there are three generations of adenovirus vector, and most of them made from Ad2 or Ad5. Though the third generation of Ads transfer vector still require helper virus for its function in the cells. Ad 12 DNA conjugates with cell chromosomes after virus infection[5], Ad12 DNA is firmly at selective chromosomal sites[6]. Ad12 DNA with the terminal protein still covalently attached (Ad12 DNA-TP) is more efficiently taken up by mammalian cells[7]. Understanding differences among these Ad serotypes may allow for the design of better gene transfer vectors for therapeutic purposes with improved efficacy and reduced toxicity.Under the leading and direction of Professor Shengquan Zou, we have studied on the biological of malignant tumor, especially on its early diagnosis, early treatment. I have been assined to Dr. Daiqing Liao’s lab, Shands cancer center, University od Florida, to further study the mechanism of cell malignant transformation and properly adenoviral genetic vector for treating cancer as a joint Doctor training candidate sponsored by China Scholarship Council. We have studied on the biological function of p53, especially on its interaction with adenorirus oncopreins E1B and E1A in the cancer cells. The present study is based on the previous results, concentrating on the mechanism of adenovirus oncopreins E1B and E1A interaction with infected host celluar corepressor, trying to provide an experimental basis for further study and excpted to refer some new approachs for cancer early diagnosis and treatment. Objects:To investigate adenovirus type 12 E1B 55-kDa protein in regulating its interactions with host proteins Sin3 and intracellular location.Methods:By using molecular biology method to construct various constructs spanning different regions of Sin3A and the modification of E1B, then transfected them into Saos2 cells and determined their subcellular localization using IF.Results:1. The aa439-443 (KKKPK) of Sin3A is critcal for its location in nuclear. Sin3A constructs containing PAH1 in the absence of the NLS was found together with E1B in cytoplasmic bodies but they failed to colocalize in the nucleus. when the NLS is included, constructs lacking C-terminal sequence localized exclusively in the nucleus and colocalized with E1B in nuclear dots.2. Phosphorylation at Ser476 and Ser477 of Ad12 E1B also appears to have an important role in regulating its interaction with Sin3A.3. N terminal of Adl2 E1B 55-kDa stalled is important for its entrance into nuclearConclusions:1. The NLS of Sin3A regulates its interaction of E1B-Sin3A and its intracellular location.2. Phosphorylation of C terminal of Ad12 E1B regulates its intracellular location.3. N terminal of Ad 12 E1B 55-kDa regulates its traffic from nuclear. Objects:To investigate the SUMO modification of Ad12 E1B 55-kDa in regulating its intracullar location and its interaction with the host proteins Sin3A and Sin3B.Methods:By using molecular biology method to construct and mutate various E1B and design a system that allowed for inducible attachment of SUMO1 to E1B is that two copies of rapamycin-binding domain FKBP were fused to the N-terminus of Ad12 E1B to make 2xFKBP-E1B and the rapamycin-binding domain of the mTOR kinase, FRB, was fused to the C-terminus of SUMO1 to produce HA SUMO1-FRB, then expression in Saos2 cells. By using immunfluoresence and western blotting to detect the location and associated proteins of E1B complex.Results:1. Lys88 and perhaps an additional lysine within the N terminal domain of Ad 12 E1B are not sites of SUM03 modification but SUMO1.2. Exclusion of SUMO1 modified Ad12 E1B from the nucleus to cytoplasm.3. SUMO1-modification of Ad12 E1B prevents its interaction with Sin3 corepressor proteins.Conclusions:1. SUMO1 modification of Ad12 E1B 55-kDa sequesters into cytoplasm.2. SUMO1 modification of Ad12 E1B 55-kDa block its interaction with Sin3A and Sin3B. Objects:To investigate Ad12 E1B, E1A and SUMO1 modification of adenovirus type 12 E1B 55-kDa proteins in regulating viral DNA synthesis.Methods:The expression of constructs E1A266, wtE1B and SUMO1-E1B 55-kDa in LN229 cells by Ad3875 virus infection. We detect the amount of virus DNA replication by viral replication assay.Results:1. Ad12 E1B 55-kDa or Ad12 E1A is able to stimulate viral DNA replication.2. Ad E1B 55-kDa is significant able to stimulate viral DNA replication in cooperation with E1A.3. SUMO1-E1B, despite exclusive localization in the cytoplasm, can still support viral DNA replication in cooperation with Ad12 E1A.Conclusions:1. The critical roles of Ad12 E1B 55-kDa and E1A in regulating viral replication.2. The important roles of SUMO modification of Ad12 E1B 55-kDa in regulating viral replication with Ad 12 E1A. Objects:To investigate the mechanism in Ad12 E1B 55-kDa and E1A represses p53-mediated transcription.Methods:We used anti-p53 and anti-ElB 55-kDa antibodies for ChIP in G401 and G401-CC3 cell lines, then PCR amplified two regions of the p21 and Mdm2 promoter to detect whether E1B only associates with the p53-binding sites of the p21 and Mdm2 promoter; p53 and its target geneorteins were analyzed in Western blotting analysis and quantitative real-time PCR analysis of p21 mRNA levels in G401 and G401-CC3 cells, the mRNA levels were normalized against that of beta-actin; HCT116 cells were transfected with firefly luciferase reporter under the control of the Mdm2 promoter and the control Renilla luciferase reporter alone (Reporter) or together with other indicated expression plasmids (P53, E1A, E1B, E1A, E1B, P53, P300), Firefly luciferase activities were normalized against the Renilla luciferase activities, Shown are average values of two independent experiments with standard deviations. G401 cells were stably transduced with lentiviral vector expressing GFP, GFP-E1B, GFP-E1B and E1A, or E1A and treated with TSA (1μM) or etoposide (50μM) for 24h for Western blotting assays; we also have made a number of E1A mutant constructs to test whether these mutants could cooperate with E1B to repress the Mdm2 promoter in reporter gene assays.Results:1. Ad12 E1B 55-kDa associates with p53-binding sites of the p21 and mdm2 promoter in cells.2. Ad12 E1B 55-kDa alone could stable the expression of p53. 3. E1A can cooperate with the E1B 55-kDa for suppressing p53-mediated transcription.4. Coexpression of E1A and E1B 55-kDa completely reversed p300-mediated coactivation.5. The CBP/p300, Rb and CtBP-binding motif are not required for cooperation with E1B 55-kDa in repressing p53.Conclusions:1. Ad12 E1B 55-kDa binds to p53 in chromatin in cells and active the expression of p53.2. Ad12 E1B 55-kDa or E1A alone could not suppress p53-mediated transcription, but coexpression of E1A along with E1B 55-kDa resulted in repression of p53 target genes.3. The binding sites for p300, Rb and CtBP in E1A may not be necessary for cooperation with E1B 55-kDa.更多还原