【作者】 谢斌；

【导师】 王建莉； 曹雪涛；

【作者基本信息】 浙江大学 ， 免疫学， 2014， 博士

【摘要】 免疫应答是机体识别并清除“非我”的重要手段,当且仅当免疫应答强度控制在一定范围内才能有效清除病原微生物并维持自身稳态。本课题围绕着“表观分子HAT1的天然免疫功能研究”和"Siglec-G分子在病毒感染中负向调控I型干扰素产生的机制研究”两部分展开,以进一步加深我们对免疫调节与免疫交叉调控机制的认识,和对病毒感染性疾病和自身免疫性疾病的预防和诊疗提供新思路。第一部分B型乙酰转移酶HATl负向调节炎症反应及其机制的研究组蛋白的乙酰化-去乙酰化过程调节着众多生命活动,不同位点的乙酰化状态,不论单一还是混合的,将产生不同的生物学效应。新生组蛋白的乙酰化修饰H4K5Ac、H4K8Ac和H4K12Ac被认为与核小体的组装成熟密切相关,而染色质中H4K16、H3K56的乙酰化或去乙酰化则与基因的转录活化或沉默息息相关。组蛋白乙酰转移酶主要由A型乙酰化转移酶和B型乙酰化转移酶两类构成,前者包括GNAT家族、MYST家族、P300/CBP等,主要分布于细胞核内,后者主要包含组蛋白H4/H3乙酰转移酶1(Histone acetyltransferases, HAT1),其广泛分布于在胞浆,少量分布于核内；组蛋白去乙酰化酶可粗略分为两个大组分,HDAC1至HDAC11、SIRT1至SIRT7。然而,乙酰化-去乙酰化修饰在天然免疫应答过程中的功能尚不清楚。在小鼠原代腹腔巨噬细胞中通过干扰RNA技术筛选我们发现,HAT1能负向调控TLR4信号所介导的白介素6(interleukin6, IL-6)的产生。这完全出乎我们的意料,需要进一步验证上述筛选结果。HAT1干扰后的小鼠巨噬细胞,经各种病原体刺激后,包括PolyI:C、LPS、CpG-ODN刺激以及RNA病毒SeV和DNA病毒HSV-1感染,所诱导表达的炎症细胞因子IL-6和TNFα mRNA和蛋白水平均显著高于对照组,而I型干扰素表达相对不受影响。全细胞蛋白裂解液和胞浆胞核蛋白分离的免疫印迹实验提示,干扰后的腹腔巨噬细胞内经典的MAPK信号和NF-κB信号基本没有改变。究竟是什么原因导致了HAT1干扰后IL-6选择性上调呢?利用染色质免疫沉淀技术发现,HAT1干扰后小鼠巨噬细胞内IL-6启动子区组蛋白H3K4Me3和H3Ac水平明显升高。于是我们提出了可能的机制猜想：1、B型乙酰转移酶可能和A型乙酰转移酶存在相互竞争,包括组蛋白的相同或相邻位点的修饰；2、HAT1可能选择性靶向到某种免疫抑制分子的启动子区,即干扰后存在“负负得正”的现象；3、HAT1很可能存在与炎症密切相关的非组蛋白底物,直接参与该底物活性调节。后续将深入机制研究,并在动物模型上观察HAT1在天然免疫应答过程中的作用,以期为自身免疫性疾病的预防和诊疗提供理论保障和技术支持。第二部分Siglec-G负向调控RIG-Ⅰ介导IFNβ产生的分子机制研究RIG-Ⅰ分子被公认为胞浆RNA病毒识别的关键Sensor,由N端两个caspase活化与募集结构域(Caspase activation and recruitment domain, CARDs)、中间的含有DEAD基序的解旋酶/ATP酶结构域(DEAD Helicase/ATPase domain)和C端自我抑制结构域(C-terminal regulatory domain, CTD)共同构成,可识别短于lkb的双链RNA和5’三磷酸RNA。在静息时,RIG-Ⅰ处于闭合态,即CTD覆盖着CARDs,当外源性短链dsRNA进入到胞浆后,CTD打开并和解旋酶结构域一起识别、结合RNA,使得CARDs结构域暴露,亲和下游接头分子MAVS (Mitochondrial antiviral signaling)上CARDs发生寡聚化并形成朊蛋白样结构,进而活化Ⅰ型干扰素表达。为了进一步研究RIG-Ⅰ-MAVS-IFNβ信号通路的调控,尤其是RIG-Ⅰ翻译后修饰,我们研究团队前期进行了小鼠原代巨噬细胞RNA病毒VSV感染模型基因芯片测序分析,和HEK293细胞RIG-Ⅰ过表达后免疫沉淀-RIG-Ⅰ蛋白复合体-质谱分析,然后结合小鼠体内和体外实验,发现并证实唾液酸结合性免疫球蛋白样凝集素分子Siglec-G能负向调控IFNβ的产生；Siglec-G敲除的腹腔巨噬细胞经RNA病毒诱导后能产生更多的RIG-Ⅰ蛋白(RIG-Ⅰ半衰期被延长了),而接头分子MAVS表达相对恒定。即在RNA病毒感染下,Siglec-G能促进RIG-Ⅰ的降解。接下来,本课题对其中的分子机制进行了深入的探究。我们利用预实验结果"Siglec-G通过胞内段ITIM基序在病毒感染下能招募更多的SHP2分子”而“SHP2在一定条件下能和c-Cbl相互作用,并能降解部分受体酪氨酸激酶(RTKs)",推测c-Cbl可能是RIG-Ⅰ的E3连接酶。原代巨噬细胞中c-Cbl的干扰的确能增加IFNβ的表达；]HEK293T重建实验也证实c-Cbl能促进RIG-Ⅰ发生K48多聚泛素化而降解。SHP2敲除的巨噬细胞VSV感染中,干扰了Siglec-G不影响IFN β产生,但干扰了c-Cbl却能显著上调IFN β产生,从而明确了Siglec-G-SHP2-c-Cbl-RIG-I通路的上下游顺序。免疫共沉淀和免疫荧光共定位实验表明,Siglec-G-SHP2-c-Cbl-RIG-I四聚体客观存在于小鼠腹腔巨噬细胞、RAW264.7和HEK293T细胞中,其中RIG-I的CTD结构域为c-Cbl相互作用所必需。为了进一步明确c-Cbl靶向降解RIG-Ⅰ的位点,我们构建了一系列的RIG-Ⅰ点突变,当RIG-I第813位赖氨酸突变为精氨酸后,则具备了抵抗c-Cbl K48位泛素化降解的功能。将RIG-Ⅰ K813R和WT质粒分别转染入HEK293T细胞中,荧光报告基因结果显示,K813R更能诱发IFNβ活化,接着将上述质粒分别电转染入RIG-Ⅰ敲除的腹腔巨噬细胞,结果发现813位点点突变后IFNβ mRNA产生水平相对更高。综上所述,RNA病毒能诱导上调Siglec-G分子招募接头分子SHP2和E3连接酶c-Cbl,靶向RIG-Ⅰ赖氨酸813位发生K48泛素化修饰将其降解,减少了IFN-β产生从而实现RNA病毒自身的逃逸。更多还原

【Abstract】 Part I. B type acetyltransferase HAT1negatively regulates TLR-triggered inflammatory innate response and the underlying mechanism.Histone acetylation-deacetylation regulates many biological processes, and specific acetylation marks, either singly or in combination, produce distinct outcomes. For example, the acetylation pattern on newly synthesized histones H4K5Ac, H4K8Ac and H4K12Ac is important for their assembly into nucleosomes by histone chaperones; deacetylation of H4K16and H3K56is important for spreading of heterochromatin components, whereas acetylation of these sites serves as a marker of transcript activation. HATs are classified into two groups, HAT A and HAT B, depending on the mechanism of catalysis and on cellular localization. Type A HATs are found in the nucleus, where they transfer the acetyl group from Acetyl-CoA to an ε-NH2group of histone N-tails after the assembly into nucleosomes, including GNAT family, MYST family, P300/CBP. Conversely, Type B HATs act mostly in the cytoplasm and transfer the acetyl group from Acetyl-CoA to an ε-NH2group of free histones prior to their deposition on the DNA, maybe only incuding HAT1(Histone acetyltransferases-1, HAT1). Histone deacetylase can be roughly divided into two major components, HDAC1to HDAC11, and SIRT1to SIRT7. However, acetylation-deacetylation function in inflammatory innate immunity remained to be fully investigated.By RNA interference screening technique, we found that HAT1can negatively regulate TLR4-triggered interleukin6production in primary peritoneal macrophages. It seems not concordance with our conventional ideas "acetylation almost activates transcription". So we have to make sure of the above screening results. Q-PCR and ELISA results demonstrated that there was more increased mRNA and protein expression of IL-6and TNFa in HAT1-silenced peritoneal macrophages in response to stimuli, including challenge with TLR ligands PolyI:C, LPS, CpG-ODN, or infected with RNA virus SeV and DNA virus HSV-1, while the expression of type I interferons was almost unaffected. Immunoblotting experiments suggested that classic MAPK signaling and NF-κB signal remained unchanged in those HAT1-silenced macrophages. How does HAT1selectively target proinflammatory cytokines to regulate inflammatory innate response? Using chromatin immunoprecipitation technique (Chip), we discoveried that the H3K4Me3and H3Ac level at IL-6promoter was significantly increased once HAT1was silenced. Thus, we supposed that, B-type and A-type acetyltransferase type may compete for each other, including modifying the same or adjacent sites of histone, or HAT1may selectively target certain immunosuppressive molecule promoter region, or HAT1may acetylate non-histone substrates for controlling inflammatory transcription activity. The underlying mechanism of HAT1-mediated negative regulation of TLR-trioggered inflammatory innate immunity needs further study, for shedding light on prevention, diagnosis and therapy of autoimmune diseases. Part Ⅱ. Siglec-G negatively regulates RNA virus-mediated IFN β production by promoting c-Cbl-mediated RIG-I degradationRIG-I is the most important sensor for recognizing cytoplasmic viral RNA, which composed of two N-terminal caspase recruitment domains (CARDs), a central DEAD box helicase/ATPase domain, and a C-terminal regulatory domain (CTD). RIG-I recognizes relatively short dsRNA (up to1kb), and the presence of a5’triphosphate end greatly enhances its type Ⅰ IFN-inducing activity. In the resting state, CTD of RIG-I covers over its CARDs and the whole molecular comformation is closed. When virus RNAs invade into the cytoplasm, CTD is opened and recognizes dsRNA with Helicase domain, reseasing its own CARDs and associating with CARDs of MAVS, eventually more and more MAVS in mitochondrion outermembrane oligomerize to form a prion-like structure, and convert signal to activate IFNβ transcription. To invstigate the precise regulation of RIG-I-MASV-IFN pathway, especially RIG-I itself modification, we used two methods, Genearray analysis of gene expression profile in mouse primary peritoneal macrophages upon VSV infection, and LC-MS/MS analysis of RIG-I-Flag-Immunoprecipitation from HEK293T cells, then combined in vitro and in vivo experiments to confirm that sialic acid binding immunoglobulin-like lectin molecule Siglec-G can negatively regulate IFNβ production in response to RNA virus infection.In this subject, we went further to investigate the molecular mechanism by which Siglec-G controls the IFN β production. Followed by ImmunoBlot, Siglec-G knockout peritoneal macrophages generated more RIG-I protein after VSV infection, but the adapter MAVS relatively remained unchanged. By using the protein synthesis inhibitor cycloheximide to treatment the macrophages before VSV infection, we found that the half-life of RIG-I is extended in Siglec-G-deficient macrophages, indicating that Siglec-G could promote RIG-I degradation by an unkown mechanism. Using Siglec-G-Flag-Immunoprecipitation in RAW264.7and following LC-MS/MS, along with the proved idea "Siglec-G through its ITIM motif to recruit more SHP2molecule under VSV infection" and "c-Cbl can associate with SHP2upon SDF-1stimulation", we found c-Cbl might be a E3ligase for RIG-I. Overexpression experiemnts in HEK293T cells suggested that RIG-I could be greatly degradated by c-Cbl. In SHP2-knockout macrophages,VSV-induced IFN β production could be negatively regulated by c-Cbl siRNA but not Siglec-G siRNA, which stuggested Siglec-G was the upstream, then SHP2, and latter c-Cbl. Co-immunoprecipitation and Immunofluorescence colocalization experiments confirmed that Siglec-G-SHP2-c-Cbl-RIG-I tetramer exists objectively in mouse peritoneal macrophages, RAW264.7and HEK293cells, whereas the CTD domain of RIG-I is essential for its interaction with c-Cbl. In order to identify which amino acid sites of RIG-I was targeted by c-Cbl, we constructed a series of RIG-I point mutations. The K813R mutation caused near-complete loss of K48ubiquitination and degradation of RIG-I. Transfection of RIG-I-K813R induced IFN-β transcription more markedly in HEK293T cells, RAW264.7and RIG-I-deficient macrophages. In summary, RNA viruses can up regulate Siglec-G to recruit adaptor molecule SHP2and E3ligase c-Cbl, targeting RIG-I lysine813for degradation to inhibit anti-viral innate immunity.更多还原