G3BP1通过cGAS调控抗病毒天然免疫的作用机制研究

发布时间：2019-05-28 17:15

【摘要】：天然免疫系统是机体防御病原微生物入侵的第一道防线。当微生物侵入机体时,天然免疫细胞能够迅速识别并激活相应的免疫反应,促进免疫系统杀灭并清除入侵的病原微生物。这一识别过程主要通过位于细胞膜或者细胞内的模式识别受体(Pattern recognition receptors,PRRs)来实现的,它可以识别某些病原微生物共有的高度保守的分子结构,如蛋白质、核酸、脂蛋白、脂多糖等等,称为病原相关分子模式(Pathogen-associated molecular patterns,PAMPs)。目前已知的PRRs主要包括TLRs(Toll-like receptors)家族、NLRs(NOD-like Receptors)家族、CLRs(C-type lectin receptors)家族、RLRs(RIG-I-like receptors)家族和一些胞浆DNA感受器(DNA sensors),其中DNA感受器是识别胞浆DNA的一类重要PRRs,在抗病毒免疫反应中具有重要作用。正常情况下DNA只存在于真核细胞的细胞核和线粒体中。在病毒感染或细胞损伤时出现在胞浆中的游离DNA是一种重要PAMP。这些DNA被胞内DNA感受器识别并激活干扰素反应,在机体的抗病毒免疫中发挥重要作用。cGAS是2013年被鉴定到的一个胞内DNA感受器,被认为是识别胞浆DNA的最主要受体,在胞浆DNA诱导的干扰素通路中具有重要作用,同时cGAS介导的信号通路过度激活与多种自身免疫疾病密切相关。目前对于cGAS信号通路的调控知之甚少,寻找该信号通路新的关键调控因子,有助于我们深入认识机体抗病毒天然免疫反应的调控机理,也将为治疗自身免疫性疾病提供新的靶点及策略。本课题通过质谱鉴定到cGAS的相互作用蛋白质G3BP1(GTPase-activating protein-(SH3domain)-binding protein 1)。G3BP1具有核酸结合能力。已知G3BP1能够启动组装一个由多种蛋白和mRNA组成的复合体-应激颗粒(Stress Granules,SGs),从而在细胞抗病毒免疫特别是抗RNA病毒免疫中具有重要作用。然而,G3BP1是否参与胞浆DNA诱导的免疫反应尚未见报道。为了研究G3BP1在胞浆DNA诱导干扰素通路中的作用,我们首先利用CRISPR/Cas9技术构建了G3bp1基因敲除细胞系。在多种不同来源的胞浆DNA刺激下,敲除G3bp1都能明显抑制胞浆DNA诱导的信号通路激活和I型干扰素产生,表明G3BP1在DNA诱导的I型干扰素通路中具有重要的正向调控作用。cGAS作为一种核苷转移酶,在识别并结合胞浆DNA后会发生构象改变,并迅速催化ATP和GTP形成小分子第二信使cGAMP(Cyclic GMP-AMP),进而激活接头蛋白质STING及下游信号通路,引起转录因子IRF3(Interferon-regulatory factor 3)活化,启动I型干扰素表达。利用cGAMP刺激细胞,我们发现G3BP1缺失并不影响cGAMP诱导的信号通路激活,提示G3BP1对DNA诱导I型干扰素通路的调控作用可能是通过调节cGAS功能实现的。cGAMP作为cGAS的产物,其合成量直接指示了cGAS酶活性。我们用LC-MS/MRM(Liquid chromatography-mass spectrometry with multiple-reaction monitoring)技术直接检测胞浆DNA诱导cGAMP的量,结果发现G3BP1缺失显著影响cGAMP合成,证明G3BP1对cGAS的调控作用是通过影响cGAS功能实现的。已知G3BP1具有解旋酶活性,能够部分解离双链DNA和双链RNA,所以我们推测G3BP1可能参与cGAS识别DNA的过程。我们构建了DNA-binding实验体系,发现G3BP1缺失明显降低了cGAS对DNA的结合能力。由此证明G3BP1是通过影响cGAS的DNA结合能力调控DNA诱导的I型干扰素产生。DNA病毒感染细胞时,会将自身DNA注入细胞进行复制和蛋白质表达。这些DNA也会被胞浆DNA感受器识别,激活干扰素通路,引发抗病毒效应。为了研究G3BP1在抗DNA病毒免疫反应中的作用,我们利用DNA病毒HSV1(Herpes simplex virus 1)感染细胞并检测I型干扰素产生以及病毒复制情况,发现G3BP1缺失明显抑制DNA病毒诱导的I型干扰素产生,与此同时,对HSV1 mRNA和病毒滴度的检测显示HSV1复制能力在G3BP1缺失细胞中明显增强,说明G3BP1在抗DNA病毒的免疫反应中具有重要作用。综上,在本项研究中我们鉴定到一个胞浆DNA识别信号通路新的功能调控分子G3BP1,并阐释了G3BP1通过调控cGAS的DNA结合能力影响其功能的分子机制,揭示了G3BP1在抗DNA病毒感染中的重要作用。已知G3BP1通过SGs在抗病毒特别是RNA病毒的免疫反应中具有重要作用。我们的研究发现G3BP1通过调节cGAS的功能影响I型干扰素产生,在DNA病毒诱导的免疫反应中也发挥关键作用。这一结果为深入认识cGAS的功能调控机制和G3BP1在抗病毒免疫中的功能提供了新的实验依据。我们的实验结果提示G3BP1有可能作为抗病毒免疫和自身免疫性疾病治疗的新靶点。
[Abstract]:The natural immune system is the first line of defense against the invasion of pathogenic microorganisms. When the microorganisms invade the body, the natural immune cells can quickly identify and activate the corresponding immune response, promote the immune system to kill and clear the invading pathogenic microorganisms. This recognition process is achieved primarily by pattern recognition receptors (PRRs) located within a cell membrane or cell, which can identify highly conserved molecular structures common to certain pathogenic microorganisms, such as proteins, nucleic acids, lipoproteins, lipopolysaccharides, and the like, Referred to as the pathogen-related molecular pattern (PMPs). The PRRs currently known mainly include the TLRs (Toll-like receptors) family, the NLRs (NOD-like receptors) family, the CLRs (C-type lectins) family, the RLRs (RIG-I-like receptors) family and some of the cytoplasmic DNA receptors, wherein the DNA receptors are a class of important PRRs that recognize the cytoplasmic DNA and play an important role in the anti-viral immune response. Normally, the DNA is present only in the nucleus and mitochondria of the eukaryotic cells. The presence of free DNA in the cytoplasm at the time of viral infection or cell damage is an important PAMP. These DNA are recognized and activated by intracellular DNA receptors and play an important role in the anti-viral immunity of the body. CGAS is an intracellular DNA receptor that was identified in 2013 and is believed to be the most important receptor for identifying the cytoplasmic DNA, which plays an important role in the cytoplasmic DNA-induced interferon pathway, and the overexpression of cGAS-mediated signaling pathways is closely related to a variety of autoimmune diseases. At present, little is known about the regulation and control of the signal path of the cGAS, and a new key control factor for the signal path is found, so that the regulation mechanism of the anti-viral natural immune response of the organism can be deeply recognized, and a new target point and a strategy are also provided for the treatment of an autoimmune disease. The interaction protein G3BP1 (GTPase-activating protein-(SH3domain)-binding protein 1) was identified by mass spectrometry. G3BP1 has a nucleic acid binding capacity. G3BP1 is known to be able to initiate the assembly of a complex-stress particle (SGs) consisting of a variety of proteins and mRNAs, thereby having an important role in cell antiviral immunity, in particular in anti-RNA viral immunity. However, whether G3BP1 is involved in the cellular DNA-induced immune response has not been reported. In order to study the role of G3BP1 in the cytoplasmic DNA-induced interferon pathway, the G3bp1 knockout cell line was first constructed using the CRISPR/ Cas9 technique. The knockout of G3bp1 could significantly inhibit the activation of the signaling pathway and the production of type I interferon, which indicated that G3BP1 had an important positive control in the DNA-induced type I interferon pathway. cGAS, as a nuclear transfer enzyme, can change in conformation after identification and binding to the cytoplasm DNA, and rapidly catalyze ATP and GTP to form the second messenger cGGAMP (Cyclic GMP-AMP) of the small molecule, thereby activating the terminal protein STRING and the downstream signal path, causing the transcription factor IRF3 (Interferon-region factor 3) to be activated, Start the expression of type I interferon. Using cGAMP to stimulate the cells, we found that the absence of G3BP1 did not affect the activation of the signal pathway induced by cGAMP, suggesting that the regulatory effect of G3BP1 on the DNA-induced type I interferon pathway may be achieved by modulating the cGAAS function. CGAMP is the product of cGAS, and its synthetic quantity directly indicates the activity of cGAS. We use the LC-MS/ MRM (Liquid chromatography-mass spectrometry with multiple-action monitoring) to directly detect the amount of cGAMP induced by the cytoplasmic DNA. The results show that the G3BP1 deletion significantly affects the synthesis of cGAMP, and it is proved that the regulation of G3BP1 on cGAS is realized by the effect of the function of cGAS. It is known that G3BP1 is capable of partially dissociating double-stranded DNA and double-stranded RNA, so that G3BP1 may be involved in the process of recognizing DNA by cGAS. We constructed a DNA-binding experiment system, and found that the deletion of G3BP1 significantly reduced the binding capacity of cGAS to DNA. This demonstrates that G3BP1 is produced by the regulation of DNA-induced type I interferon by influencing the DNA binding ability of cGAS. In the case of a DNA-infected cell, the self-DNA is injected into the cell for replication and protein expression. These DNA will also be identified by the cytoplasmic DNA receptors, activate the interferon pathway, and initiate antiviral effects. In order to study the role of G3BP1 in the anti-DNA virus immune response, we used the DNA virus HSV1 (Hades simplex virus 1) to infect the cells and detect the production of type I interferon and the replication of the virus, and it was found that the deletion of G3BP1 clearly inhibited the production of type I interferon induced by the DNA virus, at the same time, The detection of HSV1 mRNA and virus titer showed that the replication ability of HSV1 was significantly enhanced in the G3BP1-deficient cells, suggesting that G3BP1 had an important role in the immune response of the anti-DNA virus. In this study, we identified a new functional regulatory molecule G3BP1 in a cytoplasmic DNA recognition signal pathway and explained the important role of G3BP1 in the anti-DNA virus infection by regulating the molecular mechanism of the DNA binding ability of cGAS to affect its function. G3BP1 is known to play an important role in the immune response of the virus, in particular the RNA virus, by SGs. Our study found that G3BP1 plays a key role in the immune response induced by DNA virus by regulating the function of cGAS to affect the production of type I interferon. The results provide a new experimental basis for the in-depth understanding of the function and regulation mechanism of cGAS and the function of G3BP1 in the anti-viral immunity. Our results suggest that G3BP1 may be a new target for the treatment of anti-viral and autoimmune diseases.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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