蛋白磷酸酶PP1和RAF激酶抑制蛋白RKIP在模式识别受体信号中的调控作用及分子机制

发布时间：2018-01-03 13:40

本文关键词：蛋白磷酸酶PP1和RAF激酶抑制蛋白RKIP在模式识别受体信号中的调控作用及分子机制　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：Toll样受体(TLRs)是一类重要的模式识别受体,主要表达于巨噬细胞和树突状细胞表面,特异性识别病原体中特定的分子结构,激活下游的信号通路,最终激活天然免疫细胞产生炎性因子和Ⅰ型干扰素,构成机体免疫系统抵御病原体入侵的第一道屏障。TLRs不仅启动天然免疫应答,控制炎症反应的性质、强度和持续时间,还可以调节获得性免疫应答的强度和类型。所以,TLRs信号过度活化或者活化不足会导致机体免疫功能异常和疾病的发生。到目前为止,对TLRs信号通路调控的分子机制还没有完全研究清楚,因此,对TLRs信号转导调控机制的深入研究具有重要的理论意义和应用价值。蛋白磷酸酶在免疫应答中的调控作用受到越来越多的重视。蛋白磷酸酶1(PP1)在细胞中广泛表达,在细胞存活和分化等方面具有重要的调控作用,但是到目前为止PP1在TLRs信号活化诱导的天然免疫反应中的作用尚不是十分清楚。我们研究发现,在小鼠巨噬细胞中,PP1以磷酸酶依赖的方式显著抑制由TLRs3、TLRs4和TLRs9活化引起的MAPK以及NF-κB信号通路的活化,进一步影响了下游TNF-α和IL-6这些炎性细胞因子的产生。进一步研究发现PP1通过作用于TLRs/IL-1R通路中关键分子TGF-β活化激酶1(TAK1)丝氨酸412位点的磷酸化负向调控TLRs信号转导过程。在这个过程中PP1调节亚基DNA损伤诱导蛋白34(GADD34)介导了PP1特异性与TAK1相互作用,将其丝氨酸412位点去磷酸化。GADD34的表达抑制会显著影响PP1与TAK1的结合,从而影响下游信号通路活化以及炎性因子的产生。综上,我们的研究发现了PP1负向调控巨噬细胞中TLRs触发的炎性细胞因子产生,证明了PP1是通过其调节亚基GADD34特异性作用于TAK1分子丝氨酸412位点的去磷酸化,防止TLRs触发的免疫应答反应过度的持续活化,保护机体在炎症反应过程中维持稳态。我们的研究结果为进一步了解TLRs通路的精细调控提供了新的思路。病毒感染是威胁人类健康的重要危险因素之一。在抗病毒天然免疫反应中,模式识别受体维甲酸诱导的基因Ⅰ样受体(RLRs),包括RIG-I和MDA5可以识别胞浆中的病毒RNA。常用于活化RIG-I信号的RNA病毒有水泡口炎病毒(VSV)、仙台病毒(SeV)等。而DNA病毒被相应的DNA识别受体如DAI,AIM2等识别。病毒RNA识别受体和DNA识别受体活化后通过招募接头分子MAVS或者STING活化下游TBKl等分子激活下游信号,引起IRF3的核转位,最终引起Ⅰ型干扰素和炎性因子的产生,激发机体抗病毒免疫反应。在本研究中,我们发现RAF激酶抑制蛋白RKIP正向调控抗病毒天然免疫反应。干扰或者敲除小鼠巨噬细胞中RKIP的表达,显著抑制了病毒感染引起的I型干扰素的产生。小鼠巨噬细胞株中过表达RKIP则促进了病毒感染诱导的Ⅰ型干扰素的产生。在RKIP基因缺陷的小鼠巨噬细胞中VSV的滴度显著升高,RKIP基因敲除小鼠的肝脏、脾脏和肺脏组织中VSV的复制率和滴度较对照组小鼠显著增加,血清中的Ⅰ型干扰素的产生明显降低。机制研究发现在病毒感染的情况下,RKIP分子通过靶向作用于TBK1分子,促进TBKl的自身磷酸化,进而促进下游Ⅰ型干扰素的产生,发挥抗病毒功能。进一步研究发现RKIP促进TBKl活化和抗病毒的功能依赖于其丝氨酸109的磷酸化。有趣的是,TBKl分子磷酸化RKIP丝氨酸109位点,当RKIP的丝氨酸109位点磷酸化后显著促进RKIP与TBK1的相互作用,进一步促进了TBK1的活化,诱导Ⅰ型干扰的产生,发挥抗病毒功能。我们的研究结果首次揭示了TBK1与RKIP之间存在的正反馈调节效应,为深入了解抗病毒反应的分子机制提供了新的思路。此外,我们的研究发现了RKIP分子正向调控机体抗病毒天然免疫反应的新功能,提出了一种新的机体抵抗病毒感染,防止病毒免疫逃逸的新机制。
[Abstract]:Toll - like receptor ( TLRs ) is a kind of important pattern recognition receptor , which is mainly expressed on the surface of macrophages and dendritic cells , specifically recognizes the specific molecular structure in the pathogen , activates the downstream signaling pathway , and finally activates the innate immune cells to produce inflammatory factors and type I interferon . The TLRs not only initiates the innate immune response , but also controls the nature , intensity and duration of inflammatory responses . In this process , we found that pp1 inhibited the activation of the MAPK and NF - 魏B signal pathway induced by TLRs3 , TLRs4 and TLRs9 activation and further affected downstream TNF - 伪 and IL - 6 phosphorylation . In conclusion , we have found that pp1 negatively regulates the production of inflammatory cytokines triggered by TLRs in macrophages . It is shown that pp1 is the dephosphorylation of serine 412 locus in TAK1 molecule by its regulatory subunit GAD34 , which prevents excessive activation of immune response reaction triggered by TLRs , and protects the organism in the course of inflammatory reaction . Our findings provide a new idea for further understanding the fine regulation of TLRs pathway . In the anti - viral innate immune response , the pattern recognition receptor retinoic acid - induced gene I - like receptor ( RLRs ) , which includes the identification of RNA in the cytoplasm of DNA viruses , is recognized by the corresponding DNA recognition receptors , such as DAI , AIM 2 , etc . The DNA viruses are recognized by corresponding DNA recognition receptors such as DAI , and the DNA virus is activated by the corresponding DNA recognition receptors such as DAI , and so on . The nuclear translocation of IRF3 is caused , which ultimately leads to the generation of type I interferon and inflammatory factor , and the antiviral immune response of the organism is excited . In the present study , we found that the inhibition of anti - viral innate immune response in macrophages of mice induced by viral infection was significantly inhibited by interfering or knocking out the expression of rKIP in the macrophages of mice . In the presence of viral infection , the replication rate and titer in the liver , spleen , and lung tissues of mice were significantly increased . The mechanism of the study found that the activation of TBKl and the interaction of TBK1 with TBK1 were promoted by targeting the TBK1 molecule . The results show that the positive feedback regulation effect between TBK1 and rKIP provides a new idea for further understanding the molecular mechanism of anti - viral response . In addition , our research has found that the new mechanism of anti - viral innate immune response of the organism against virus infection is proposed , and a new mechanism for preventing virus immune escape is proposed .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

【共引文献】