p53调控IFITM3棕榈酰化修饰介导的抗日本脑炎病毒作用研究

发布时间：2018-04-27 09:42

  本文选题：p53 + IFITM3　； 参考：《中国农业科学院》2015年博士论文

【摘要】：日本脑炎病毒(Japanese encephalitis virus,JEV)是一种经蚊虫传播的黄病毒,能引起中枢神经系统损伤和猪的繁殖障碍,具有重要的公共卫生学意义。肿瘤抑制因子p53具有参与细胞周期捕获、细胞代谢、诱导细胞凋亡和先天性免疫的作用。与野生型小鼠相比,JEV感染p53缺失小鼠能引起较高病毒血症、死亡率,脑组织出现更为严重的炎症反应和病理变化。表明p53在宿主抗JEV感染中具有重要作用。除了已报道的p53增强I-IFN介导的抗病毒先天性免疫和诱导细胞凋亡外,是否还存在其他抗病毒先天性免疫调控机制?为了进一步阐明p53抗JEV的分子机制及深入理解p53介导的细胞与JEV相互作用,遂进行了本课题研究。JEV感染小鼠脑组织表达谱芯片发现,JEV感染的小鼠脑组织中IFITM3 mRNA水平显著上调,p53WT小鼠IFITM3 mRNA水平高于p53KO小鼠。这种变化与p53转录调控的靶基因IRF9、TLR3和GBP1等具有相似性,提示两者间存在某种联系。p53生物学功能的发挥多依赖于其转录活性,然而,在分析p53不同状态下,IFITM3 mRNA水平变化时,发现IFITM3的转录并不受p53的影响,说明两者并没有直接关系。但当对IFITM3蛋白表达变化进行检测时,发现IFITM3的蛋白丰度随p53信号通路的激活而明显上升,表明p53间接上调IFITM3的蛋白表达。考虑到蛋白棕榈酰化修饰对蛋白定位、稳定性、生物学功能的重要作用,对p53上调IFITM3蛋白丰度与蛋白棕榈酰化修饰的关系进行了评估,发现棕榈酰化抑制剂2-BP处理废除了p53与IFITM3间的调控关系。IFITM3棕榈酰化修饰的抑制后其蛋白表达明显下降,而转录水平未受影响。进一步探究证明2-BP处理后,IFITM3蛋白稳定性明显下降,半衰期由5h衰减为2h,溶酶体途径抑制剂Leupeptin处理能明显恢复IFITM3蛋白含量,并延长其半衰期至10h,说明IFITM3降解的主要途径为溶酶体途径。IFIITM3棕榈酰化修饰能增强IFITM3蛋白稳定性,抑制其在溶酶体中降解,也证实了p53可以间接调控IFITM3棕榈酰化修饰并增强其蛋白稳定性。蛋白棕榈酰化修饰受DHHC蛋白和PPT的可逆调节,因此,我们假设p53直接转录调控负责IFITM3棕榈酰化修饰的某些酶,如正向调控具有蛋白乙酰转移酶活性的DHHC家族成员(负责蛋白棕榈酰化修饰),或负调控乙酰蛋白硫酯酶APTs和蛋白棕榈酸硫酯酶PPTs(负责蛋白去棕榈酰化修饰),从而间接调节IFITM3的蛋白稳定性。随后对p53转录调控ZDHHC和PPT家族成员的能力进行了分析,发现在不同细胞系中ZDHHC1 mRNA水平均随p53状态而改变。对ZDHHC1蛋白水平检测也证实p53同样可以上调IFITM3的蛋白水平,从而说明了p53转录调控IFITM3表达。IFITM3蛋白棕榈酰化修饰利于其蛋白稳定性,ZDHHC1若修饰IFITM3,其蛋白表达和稳定性也会增加。ZDHHC1过表达上调IFITM3蛋白表达,并不影响其转录水平;ZDHHC1knockdown降低了IFITM3的蛋白丰度,说明了p53调控的中间分子ZDHHC1的确利于IFITM3蛋白表达与稳定性。进一步试验确认了ZDHHC1和IFITM3的表达变化与p53的蛋白表达变化存在一致性,证实了p53-ZDHHC1-IFITM3调控通路的存在。为阐明p53-ZDHHC1-IFITM3通路在JEV复制过程中的作用,分别对各个分子单独或协同作用于JEV复制的影响进行了分析。证实了p53通过转录调控ZDHHC1表达间接增强IFITM3蛋白稳定性,从而抑制JEV复制的作用。另外,JEV也存在拮抗p53抗病毒先天性免疫的机制,NS4A蛋白可将p53扣留在细胞浆中,抑制p53入核和转录活性的发挥,并利于JEV的复制。本研究证实了p53-ZDHHC1-IFITM3调控通路的存在,并阐明了抑制JEV复制的生物学作用,揭示了p53抗JEV复制的新的分子机制,也说明了p53参与抗病毒先天性免疫的多样性与灵活性。p53调控ZDHHC1的表达,将p53与蛋白棕榈酰化修饰联系起来,拓展了p53的生物学作用。p53-ZDHHC1-IFITM3通路的发现为进一步研究p53抗病毒的分子机制及开发抗病毒药物提供了一定参考依据。
[Abstract]:Japanese encephalitis virus (JEV) is a kind of yellow virus transmitted by mosquitoes, which can cause the damage of the central nervous system and the reproductive barrier of pigs. It has important public health significance. The tumor suppressor factor p53 has the role of participating in cell cycle capture, cell metabolism, inducing cell apoptosis and innate immunity. JEV infected mice with p53 deficiency can cause hyperviremia, mortality, and more serious inflammatory and pathological changes in the brain tissue. It shows that p53 plays an important role in the host anti JEV infection. It is still existing in addition to the reported p53 enhanced I-IFN mediated antiviral congenital immunity and induction of apoptosis. In order to further elucidate the molecular mechanism of p53 anti JEV and to understand the interaction of p53 mediated cells and JEV, this subject studied the brain tissue expression profile of.JEV infected mice, and the level of IFITM3 mRNA in the brain tissues of JEV infected mice was significantly up-regulated, and IFITM3 mRNA in p53WT mice. It is higher than p53KO mice. This change is similar to the target gene IRF9, TLR3 and GBP1 of p53 transcriptional regulation, suggesting that there is a certain link between the biological function of.P53 and its transcriptional activity. However, when the mRNA level of IFITM3 is changed in different states of p53, the transcription of IFITM3 is not affected by p53, indicating two But when the expression of IFITM3 protein was detected, it was found that the protein abundance of IFITM3 increased obviously with the activation of p53 signaling pathway, indicating that p53 up-regulated the protein expression of IFITM3. Considering the role of protein palmitic modification on protein localization, stability, and biological function, p53 up regulation of IFITM3 eggs The relationship between white abundance and protein palmiylation modification was evaluated. Palmitoylation inhibitor 2-BP treatment abolished the regulatory relationship between p53 and IFITM3. The protein expression decreased markedly after the inhibition of.IFITM3 palmitic modification, while the transcriptional level was not affected. Further exploration proved that after 2-BP treatment, the stability of IFITM3 protein decreased significantly. The half-life decreased from 5h to 2h, and the lysosome pathway inhibitor Leupeptin treatment could obviously restore the IFITM3 protein content and prolong its half-life to 10h, indicating that the main pathway for the degradation of IFITM3 is that the lysosome pathway.IFIITM3 palmitic modification can enhance the stability of IFITM3 protein and inhibit its degradation in the lysosome, which also confirms that p53 can be indirectly regulated. IFITM3 palmiylation modifies and enhances its protein stability. Protein palmitylation modification is reversible by DHHC protein and PPT, so we assume that p53 direct transcriptional regulation is responsible for some enzymes of IFITM3 palmiylation modification, such as the positive regulation of DHHC family members with protein acetyltransferase activity (responsible for protein palmitic modification), or The negative regulation of acetyl protein thioesterase APTs and protein palmitate thioesterase PPTs (responsible for protein depalmiylation modification) indirectly regulates the protein stability of IFITM3. Subsequently, the ability of p53 transcriptional regulation of ZDHHC and PPT family members is analyzed. It is found that the level of ZDHHC1 mRNA in different cell lines is changed with p53 state. To ZDHHC1 eggs. The white level test also confirmed that p53 can also increase the protein level of IFITM3, thus indicating that p53 transcriptional regulation IFITM3 expression of.IFITM3 protein palmiylation is beneficial to its protein stability. If ZDHHC1 modifies IFITM3, its protein expression and stability also increase the over expression of.ZDHHC1 to up regulate the expression of IFITM3 protein, and does not affect its transcriptional level; ZDHH; ZDHH. C1knockdown reduced the protein abundance of IFITM3, indicating that the intermediate molecule ZDHHC1 regulated by p53 was indeed beneficial to the expression and stability of IFITM3 protein. Further experiments confirmed that the changes in the expression of ZDHHC1 and IFITM3 were consistent with the changes in the protein expression of p53, and confirmed the existence of p53-ZDHHC1-IFITM3 modulation pathway. In the process of JEV replication, the effect of individual or synergistic action on JEV replication is analyzed. It is confirmed that p53 can indirectly enhance the stability of IFITM3 protein by transcriptional regulation of ZDHHC1, thus inhibiting the role of JEV replication. In addition, JEV also has a mechanism to antagonize the innate immunity of p53, and NS4A protein can be used for P. 53 detain in the cytoplasm, inhibit the play of p53 nucleation and transcriptional activity, and facilitate the replication of JEV. This study confirmed the existence of p53-ZDHHC1-IFITM3 regulation pathway, clarified the biological function of inhibiting the replication of JEV, revealed the new molecular mechanism of p53 anti JEV replication, and also explained the diversity and spirit of p53 to participate in the antiviral innate immunity. The activity of.P53 regulates the expression of ZDHHC1, linking p53 with protein palmitic modification, expanding the discovery of the biological role of p53 and the discovery of the.P53-ZDHHC1-IFITM3 pathway, which provides a reference for further research on the molecular mechanism of p53 and the development of antiviral drugs.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

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