肿瘤抑制因子PTEN的抗病毒功能及其机制研究

发布时间：2018-10-23 18:31

【摘要】：识别病原微生物是激活天然免疫反应的第一步,该过程是通过一系列模式识别受体(pattern recognition receptors, PRRs)来识别病原相关的分子模式(pathogen-associated molecular patterns, MAMPs)来实现的。例如,在大多数细胞中,RIG-I样受体(retinoic acid-inducible gene I (RIG-I)-like receptors, RLRs)识别病毒感染产生的双链RNA (dsRNA)或者单链RNA (ssRNA);在一些免疫细胞中,Toll样受体(toll-like receptors, TLRs)识别经过内吞作用进入内体的病毒dsRNA和ssRNA。模式识别受体一旦激活后,会激起下游的信号级联反应,导致Ⅰ型干扰素和促炎症因子的大量产生。干扰素调控因子3 (interferon-regulatory factor 3, IRF3)作为IFN-β诱导通路的关键转录因子,在天然免疫反应过程中起着不可或缺的作用。细胞识别病原体后,IRF3会在几个磷酸化接受体簇上磷酸化,继而发生构象变化,进行同源二聚化、入核并结合到目的基因的启动子序列干扰素刺激反应原件(interferon-stimulated response elments, ISRE)上。IRF3在IFN-β诱导通路中必不可少的角色决定了它必须被精细的调控,从而使宿主对病毒感染做出准确适应的免疫反应。IRF3上游的激酶TBK1和IKKε早已被报道,但是负责IRF3去磷酸化过程的磷酸酶仍然未知。肿瘤抑制因子PTEN (Phosphatase and tensin homolog deleted on chromosome ten)是突变率最高的几个抑癌因子之一。PTEN通过其脂质磷酸酶活性来拮抗PI3K-Akt信号通路,从而控制一系列细胞生理过程,在肿瘤抑制过程中起着核心的作用。尽管PTEN的肿瘤抑制功能研究得非常透彻,但关于PTEN在抗病毒免疫方面的功能还未见报道。本研究中,我们报道PTEN在抗病毒天然免疫中的新功能,我们发现PTEN能够促进病毒诱导的Ⅰ型干扰素的表达,从而在抗病毒天然免疫反应中起着关键的作用。我们的研究发现,PTEN在Ⅰ型干扰素诱导通路中的作用依赖于其磷酸酶活性,但不依赖于PI3K-Akt通路。IRF3是I型干扰素诱导通路的关键转录因子,而PTEN通过促进IRF3的入核转运从而促进病毒诱导的I型干扰素的表达。使用多种病毒以及核酸类似物诱导干扰素产生的实验证明,PTEN 参与了所有IRF3介导的干扰素反应。进一步研究表明,IRF3除已知的磷酸化激活位点之外,还存在磷酸化失活位点,本研究鉴定出三个磷酸化负调控位点,包括Ser82、Ser97和Ser339,其中Ser97位点的磷酸化导致IRF3不能入核,而PTEN则通过其磷酸酶活性对Ser97磷酸化位点行使去磷酸化功能,从而促进IRF3入核,激活I型干扰素的表达。病毒感染实验表明,PTEN缺失的各类细胞(包括肿瘤细胞)抗病毒干扰素反应显著降低,进而病毒复制效率上升,宿主细胞凋亡增加。本研究建立了PTEN条件性敲除小鼠实验体系,相对于野生型小鼠,PTEN条件缺失型小鼠干扰素反应明显降低,对水泡性口炎病毒(VSV)高度易感,因此在小鼠模型中证实了上述发现的生理功能。总之,我们的研究发现PTEN除抗肿瘤功能之外,还在抗病毒天然免疫中发挥关键作用；揭示了IRF3磷酸化负调控的新机制和PTEN磷酸酶对IRF3负调控位点的去磷酸化功能。这些工作表明了PTEN和IRF3在肿瘤抑制和抗病毒天然免疫之间的交互作用,也部分解释了为什么肿瘤细胞一般对病毒感染更加敏感,因此该研究为研发新的抗病毒策略提供了新知识,对于筛选与应用溶瘤病毒治疗PTEN缺失型癌症具有重要的临床意义。
[Abstract]:Identifying pathogenic microorganisms is the first step in activating a natural immune response, which is achieved by identifying the pathogen-related molecular patterns (MAMPs) through a series of pattern recognition receptors (PRRs). For example, in most cells, RIG-I-like receptors (RIG-I)-like rectins, RLRs) recognize double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) generated by viral infections; in some immune cells, Toll-like receptors (TLRs) identify viral RNA and ssRNA that have been internalized into the endosomes. Once activated, the pattern recognition receptors cause downstream signal cascade reactions leading to a large number of type I interferon and pro-inflammatory factors. interferon regulatory factor 3 (iRF3) plays an indispensable role in the process of innate immune response. After cell recognition of a pathogen, IRF3 is phosphorylated on several phosphorylated receptor clusters, followed by conformational changes, homodimerizing, nucleus-entering, and binding to the promoter sequence interferon-stimulated response (IRE) of the target gene. The essential role of IR3 in the IFN-induced pathway determines that it must be finely regulated so as to allow the host to respond accurately to viral infections. The kinases TBK1 and IKK upstream of IRF3 have long been reported, but phosphatases responsible for the IRF3 dephosphorylation process are still unknown. Tumor suppressor gene PTEN is one of several tumor suppressor factors with the highest mutation rate. PTEN antagonizes the T-Akt signaling pathway through its lipid phosphatase activity, thereby controlling a series of cellular physiological processes, which play a central role in tumor suppression. Although the tumor suppressor function of PTEN is very thorough, the function of PTEN in anti-viral immunity has not been reported. In this study, we report the new function of PTEN in innate anti-virus innate immunity, and we have found that PTEN can promote the expression of viral-induced type I interferon and play a key role in anti-viral innate immune response. Our findings suggest that the role of PTEN in the type I interferon inducible pathway depends on its phosphatase activity, but does not depend on the p38-Akt pathway. IRF3 is a key transcription factor for the I-type interferon inducible pathway, while PTEN promotes the expression of viral-induced I-type interferon by promoting the migration of IRF3 into the nucleus. Experiments using a variety of viruses and nucleic acid analogs to induce interferon production demonstrate that PTEN is involved in all IRF3-mediated interferon reactions. Further studies have shown that, in addition to the known phosphorylation activation sites, IRF3 also has phosphorylation sites, which identify three phosphorylated negative regulatory sites, including Ser82, Ser97 and Ser339, where phosphorylation of Ser97 sites results in IRRF3 not entering the nucleus, PTEN confirmed the phosphorylation of Ser97 phosphorylation site by its phosphatase activity, thus promoting the expression of IRF3 in nuclear and I-type interferon. The virus infection experiment shows that the anti-virus interferon response of all kinds of cells (including tumor cells) deleted by PTEN is remarkably reduced, and the replication efficiency of the virus is increased, and the apoptosis of the host cell is increased. In this study, PTEN conditioned knock-out mouse experimental system was established. Compared with wild-type mice, there was a significant decrease in interferon response in PTEN-deficient mice, and it was highly susceptible to blister-inflammatory virus (VSV). Therefore, the physiological function of PTEN was confirmed in mouse model. In conclusion, we have found that PTEN plays a key role in anti-virus innate immunity in addition to anti-tumor function, and reveals the new mechanism of phosphorylation negative regulation of IRF3 and the dephosphorylation function of PTEN phosphatase to the negative regulatory site of IR3. These work demonstrate the interaction between PTEN and IRF3 between tumor suppression and anti-viral innate immunity, and partly explain why tumor cells are generally more sensitive to viral infections, thus providing new knowledge for the development of new antiviral strategies, It has important clinical significance for screening and applying soluble tumor virus in the treatment of PTEN-deficient cancer.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

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