G3BP1通過cGAS調(diào)控抗病毒天然免疫的作用機制研究
發(fā)布時間：2019-05-28 17:15
【摘要】：天然免疫系統(tǒng)是機體防御病原微生物入侵的第一道防線。當微生物侵入機體時,天然免疫細胞能夠迅速識別并激活相應的免疫反應,促進免疫系統(tǒng)殺滅并清除入侵的病原微生物。這一識別過程主要通過位于細胞膜或者細胞內(nèi)的模式識別受體(Pattern recognition receptors,PRRs)來實現(xiàn)的,它可以識別某些病原微生物共有的高度保守的分子結構,如蛋白質、核酸、脂蛋白、脂多糖等等,稱為病原相關分子模式(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)家族和一些胞漿DNA感受器(DNA sensors),其中DNA感受器是識別胞漿DNA的一類重要PRRs,在抗病毒免疫反應中具有重要作用。正常情況下DNA只存在于真核細胞的細胞核和線粒體中。在病毒感染或細胞損傷時出現(xiàn)在胞漿中的游離DNA是一種重要PAMP。這些DNA被胞內(nèi)DNA感受器識別并激活干擾素反應,在機體的抗病毒免疫中發(fā)揮重要作用。cGAS是2013年被鑒定到的一個胞內(nèi)DNA感受器,被認為是識別胞漿DNA的最主要受體,在胞漿DNA誘導的干擾素通路中具有重要作用,同時cGAS介導的信號通路過度激活與多種自身免疫疾病密切相關。目前對于cGAS信號通路的調(diào)控知之甚少,尋找該信號通路新的關鍵調(diào)控因子,有助于我們深入認識機體抗病毒天然免疫反應的調(diào)控機理,也將為治療自身免疫性疾病提供新的靶點及策略。本課題通過質譜鑒定到cGAS的相互作用蛋白質G3BP1(GTPase-activating protein-(SH3domain)-binding protein 1)。G3BP1具有核酸結合能力。已知G3BP1能夠啟動組裝一個由多種蛋白和mRNA組成的復合體-應激顆粒(Stress Granules,SGs),從而在細胞抗病毒免疫特別是抗RNA病毒免疫中具有重要作用。然而,G3BP1是否參與胞漿DNA誘導的免疫反應尚未見報道。為了研究G3BP1在胞漿DNA誘導干擾素通路中的作用,我們首先利用CRISPR/Cas9技術構建了G3bp1基因敲除細胞系。在多種不同來源的胞漿DNA刺激下,敲除G3bp1都能明顯抑制胞漿DNA誘導的信號通路激活和I型干擾素產(chǎn)生,表明G3BP1在DNA誘導的I型干擾素通路中具有重要的正向調(diào)控作用。cGAS作為一種核苷轉移酶,在識別并結合胞漿DNA后會發(fā)生構象改變,并迅速催化ATP和GTP形成小分子第二信使cGAMP(Cyclic GMP-AMP),進而激活接頭蛋白質STING及下游信號通路,引起轉錄因子IRF3(Interferon-regulatory factor 3)活化,啟動I型干擾素表達。利用cGAMP刺激細胞,我們發(fā)現(xiàn)G3BP1缺失并不影響cGAMP誘導的信號通路激活,提示G3BP1對DNA誘導I型干擾素通路的調(diào)控作用可能是通過調(diào)節(jié)cGAS功能實現(xiàn)的。cGAMP作為cGAS的產(chǎn)物,其合成量直接指示了cGAS酶活性。我們用LC-MS/MRM(Liquid chromatography-mass spectrometry with multiple-reaction monitoring)技術直接檢測胞漿DNA誘導cGAMP的量,結果發(fā)現(xiàn)G3BP1缺失顯著影響cGAMP合成,證明G3BP1對cGAS的調(diào)控作用是通過影響cGAS功能實現(xiàn)的。已知G3BP1具有解旋酶活性,能夠部分解離雙鏈DNA和雙鏈RNA,所以我們推測G3BP1可能參與cGAS識別DNA的過程。我們構建了DNA-binding實驗體系,發(fā)現(xiàn)G3BP1缺失明顯降低了cGAS對DNA的結合能力。由此證明G3BP1是通過影響cGAS的DNA結合能力調(diào)控DNA誘導的I型干擾素產(chǎn)生。DNA病毒感染細胞時,會將自身DNA注入細胞進行復制和蛋白質表達。這些DNA也會被胞漿DNA感受器識別,激活干擾素通路,引發(fā)抗病毒效應。為了研究G3BP1在抗DNA病毒免疫反應中的作用,我們利用DNA病毒HSV1(Herpes simplex virus 1)感染細胞并檢測I型干擾素產(chǎn)生以及病毒復制情況,發(fā)現(xiàn)G3BP1缺失明顯抑制DNA病毒誘導的I型干擾素產(chǎn)生,與此同時,對HSV1 mRNA和病毒滴度的檢測顯示HSV1復制能力在G3BP1缺失細胞中明顯增強,說明G3BP1在抗DNA病毒的免疫反應中具有重要作用。綜上,在本項研究中我們鑒定到一個胞漿DNA識別信號通路新的功能調(diào)控分子G3BP1,并闡釋了G3BP1通過調(diào)控cGAS的DNA結合能力影響其功能的分子機制,揭示了G3BP1在抗DNA病毒感染中的重要作用。已知G3BP1通過SGs在抗病毒特別是RNA病毒的免疫反應中具有重要作用。我們的研究發(fā)現(xiàn)G3BP1通過調(diào)節(jié)cGAS的功能影響I型干擾素產(chǎn)生,在DNA病毒誘導的免疫反應中也發(fā)揮關鍵作用。這一結果為深入認識cGAS的功能調(diào)控機制和G3BP1在抗病毒免疫中的功能提供了新的實驗依據(jù)。我們的實驗結果提示G3BP1有可能作為抗病毒免疫和自身免疫性疾病治療的新靶點。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
本文編號：2487227
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
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