【作者】 李志伟；

【导师】 吴琛； 游富平；

【作者基本信息】 河北大学 ， 细胞生物学， 2023， 硕士

【摘要】 天然免疫作为宿主抵抗外界病原入侵的第一道防线,通过一系列模式识别受体(Pattern recognition receptors,PRRs)感知来自病原体的病原相关分子模式(Pathogen associated molecular patterns,PAMPs)并激活下游信号通路,诱导干扰素(Interferon,IFN)和其他细胞因子的产生来发挥抵抗病原微生物的效应。IFN-β是天然免疫中最重要的效应分子之一,主要通过激活干扰素诱导基因(IFN-stimulated genes,ISGs)的表达发挥抗病毒作用。甲基转移酶样21A(Methyltransferase like 21A,METTL21A)是近年来新发现的蛋白质赖氨酸甲基转移酶,已有研究表明其可对HSP70(Heat shock protein of～70 k Da)蛋白家族进行三甲基化修饰,参与调控内质网应激。METTL蛋白家族其他成员如METTL3,METTL10,METTL14等在抗病毒天然免疫调控中发挥重要作用,但METTL21A是否参与天然免疫调控尚无报道。本研究首先利用CRISPR-Cas9技术构建了敲除METTL21A的HT29和Hela细胞系。水疱性口炎病毒(Vesicular Stomatitis Virus,VSV)感染HT29和Hela细胞后,与野生型细胞相比,METTL21A敲除细胞IFN-β表达水平显著升高,病毒载量显著降低,细胞抗病毒能力增强。在小鼠原代细胞中进行相同的实验,发现METTL21A敲除的小鼠腹腔巨噬细胞和骨髓来源巨噬细胞抗病毒能力同样增强。此外,转录组测序结果发现METTL21A敲除的细胞中XAF1、IFIT1等抗病毒基因表达上调。随后构建VSV感染的小鼠模型,与野生型小鼠相比,METTL21A敲除小鼠体重下降程度以及死亡率降低,小鼠血液中IFN-β以及抗病毒基因表达显著升高,病毒载量降低,小鼠的抗病毒能力增强。为了确定METTL21A调控抗病毒天然免疫的分子机制,通过免疫共沉淀富集FlagMETTL21A蛋白并进行蛋白质质谱鉴定,找到了与METTL21A相互作用的蛋白HSPA5。HSPA5能够控制IRE1、PERK以及ATF6信号通路的激活,研究发现VSV病毒感染的同时使用IRE1抑制剂处理细胞时,可消除METTL21A敲除细胞中IFN-β以及抗病毒基因的表达上调。而PERK和ATF6抑制剂并不会影响METTL21A敲除细胞中IFN-β以及抗病毒基因的表达。综上所述,本研究首次发现METTL21A参与抗病毒天然免疫调控。在病毒感染后,METTL21A与HSPA5相互作用调节IRE1信号通路的激活,进而影响IFN-β以及相关抗病毒基因的表达,调控细胞及小鼠的抗病毒能力。总之,研究METTL21A调控I型IFN抗病毒反应中的生物学功能有助于我们更全面地了解机体抵抗病毒感染的机制,将为相关免疫性疾病治疗提供一个新的潜在靶点。更多还原

【Abstract】 As the host’s first line of defense against external pathogenic invasion,innate immunity exerts its effects against pathogenic microorganisms through a series of pattern recognition receptors(PRRs)that sense pathogen associated molecular patterns(PAMPs)from the pathogen and activate downstream signaling pathways that induce the production of interferon(IFN)and other cytokines.IFN-β is one of the most important molecules in innate immunity,which plays an antiviral role mainly by activating the expression of IFN-induced genes(ISGs).Methyltransferase-like 21A(METTL21A)is a newly discovered protein lysine methyltransferase in recent years.Studies have shown that it can trimethylate HSP70(Heat shock protein of ～70k Da)protein family and participate in the regulation of endoplasmic reticulum stress.Other members of the METTL family,such as METTL3,METTL10,METTL14,play an important role in the regulation of antiviral innate immunity,but whether METTL21 A is involved in the regulation of innate immunity has not been reported.In this study,HT29 and Hela cell lines that knock out METTL21 A were first constructed using CRISPR-Cas9 technology.After HT29 and Hela cells were infected with vesicular stomatitis virus(VSV),the expression of IFN-β in METTL21 A knockout cells was significantly higher than that in wild type cells,while the viral expression was significantly decreased,and the antiviral ability of cells was enhanced.The same experiment was carried out in mouse primary cells,and it was found that the antiviral ability of METTL21 A knockout mouse peritoneal macrophages and bone marrow-derived macrophages were also enhanced.In addition,transcriptome sequencing showed that the expression of some antiviral genes in METTL21 A knockout cells was up-regulated.Then the model of VSV infected mice was constructed.Compared with wild type mice,the weight loss and mortality of METTL21 A knockout mice decreased,the expression of IFN-β and antiviral genes in blood significantly increased,the expression of virus decreased,and the antiviral ability of mice was enhanced.In order to determine the molecular mechanism of METTL21 A regulating antiviral innate immunity,Flag-METTL21 A protein was enriched by co-immunoprecipitation and identified by protein mass spectrometry,and the protein HSPA5 interacting with METTL21 A was found.HSPA5 controls the activation of IRE1,PERK and ATF6 signaling pathways.It was found that when cells were treated with IRE1 inhibitors instead of PERK and ATF6 inhibitors,the upregulation of IFN-β and antiviral genes in METTL21 A knockout cells were eliminated.In conclusion,this study revealed for the first time that METTL21 A is involved in the regulation of antiviral innate immunity.Upon viral infection,METTL21 A interacts with HSPA5 to regulate the activation of the IRE1 signaling pathway,which in turn affects the expression of IFN-β and related antiviral genes and regulates the antiviral ability of cells and mice.In short,studying the biological function of METTL21 A in regulating the antiviral response to type I IFN helps us to more comprehensively understand the mechanism by which the organization resists viral infection and will provide a new potential therapeutic target for related immune diseases.更多还原