【作者】 王居平；

【导师】 邵吉民； 秦樾；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 Toll样受体4(Toll Like Receptor4,TLR4)在巨噬细胞的抗感染(特别是抗病毒)过程中发挥重要作用。TLR4可与配体革兰阴性菌的脂多糖(Lipopolysaccharide,LPS)结合,并与MD2、CD14两个分子形成MD2/TLR4/CD14复合体而活化。活化的TLR4激活两条经典的信号转导通路：一条是MyD88依赖性信号转导路径,主要接头蛋白为MAL(TIRAP)和MyD88, TLR4通过招募Mal(TIRAP)/Myd88,激活一系列蛋白酸磷酸化激酶(如TAK1、IKKγ, IKKαA, IKKβ)、导致转录因子NF-κB激活并入核而启动炎症因子基因表达。这些炎症因子如白介素6(Inter leukin 6, IL6)、IL12. IL17.肿瘤坏死因子α(Tumor necrosis factorα, TNFα)的表达释放参与巨噬细胞的炎症免疫反应。另一条是MyD88非依赖性信号转导途径,其主要接头蛋白为TRAM和TRIF。TLR4通过招募TRAM/TRIF,激活另一些蛋白酸磷化激酶(如TBK1、IKKs/IKKi)、导致转录因子干扰素调节因子3(IFN-regulated factor3, IRF3)激活并入核,来诱导包括干扰素β(Interferon beta,IFNβ)的转录,最终引起免疫反应。丝氨酸磷酸化被认为在TLR4的信号通路(尤其在两条通路的中下游)的激活,起到了至关重要的作用。然而至今为止,TLR4和TLR4的接头蛋白是如何被激活的,却毫无所知。最近,本实验室率先报道的蛋白乙酰化参与I型干扰素受体信号转导(Ref)为TLR4上游信号转导通路得激活提供了极为重要的线索。本论文研究证实了乙酰化和甲基化修饰对LPS引起的TLR4信号通路的激活,细胞因子合成释放以及巨噬细胞的炎症免疫调控功能具有重大的生物学意义。(一)TLR4乙酰化修饰在LPS活化的炎症信号转导中的作用1、LPS刺激巨噬细胞系Raw264.7细胞后,利用泛乙酰化抗体进行免疫印迹检测发现TLR4可发生乙酰化修饰。免疫荧光法和免疫共沉淀法证实LPS刺激可促进CBP出核并与TLR4相互作用；抑制内源CBP表达后,TLR4乙酰化显著降低,表明CBP介导TLR4的乙酰化修饰。为探索TLR4乙酰化修饰位点,我们构建了两个突变体即第732位和第813赖氨酸(K)分别突变为精氨酸(R)(K732R,K813R),并与CBP共表达后检测乙酰化程度。结果表明,与野生型TLR4相比,两个突变体的乙酰化程度显著降低。质谱法检测到TLR4第813位K发生乙酰化修饰。此外,我们分别制备了K732和K813乙酰化修饰的特异性抗体,发现LPS刺激后TLR4的K732和K813乙酰化显著升高。以上结果表明,LPS刺激可促进CBP介导的TLR4第732位和第813位赖氨酸的乙酰化修饰。2、应用免疫共沉淀法分析TLR4及其两个突变体K732R和K813R与TRAM、TRIF和MyD88三个接头蛋白的相互作用,结果表明,K732R突变阻断了TLR4与TRIF和MyD88的相互作用,而Κ813R突变阻断了TLR4与TRAM的相互作用。报告基因技术分析显示CBP过表达后,TLR4乙酰化程度增强,导致NF-κB和IRF3反应元件活性增加。相反,Κ732R和K813R突变均可降低IRF3反应元件的活性,而Κ732R突变则还可以使NF-κB反应元件活性降低。在TLR4-/-小鼠骨髓来源的巨噬细胞(Bone Marrow-Derived Macrophage, BMDM)中,应用半定量RT-PCR的方法分析了乙酰化对TLR4两条不同通路终产物的影响,结果显示,Κ732R和K813R突变可分别抑制IFNβ的合成释放,而Κ732R突变还可抑制IL6合成释放。以上结果表明TLR4第732位和第813位赖氨酸的乙酰化分别调控下游不同的信号通路。3、LPS刺激巨噬细胞系Raw264.7细胞,利用泛乙酰化抗体进行免疫印迹检测发现IRF3可发生乙酰化修饰。抑制内源CBP表达后,IRF3乙酰化显著降低,表明CBP介导IRF3的乙酰化修饰。为探索IRF3乙酰化修饰位点,我们用质谱法检测到IRF3第77位的Κ发生乙酰化修饰。在此基础上,我们构建了突变体Κ77R,并与CBP共表达后检测乙酰化程度。结果表明,与野生型IRF3相比,突变体的乙酰化程度显著降低。免疫印记法分析表明,加入去乙酰化酶6(Histone Deacetylase 6, HDAC 6)后,CBP/P300介导的IRF3的乙酰化程度显著下降,表明HDAC6介导IRF3的去乙酰化。免疫共沉淀法结果显示K77R突变阻断了IRF3二聚体的形成,而去乙酰化酶的抑制剂曲古抑菌素(Trichostatin A, TSA)和烟酰胺(Nicotinamide,NAM)则增强IRF3二聚体间的相互作用。免疫荧光法证实抑制内源CBP和K77R突变均能阻止IRF3入核。以上结果表明LPS诱导的乙酰化促进IRF3形成二聚体然后进入细胞核。主要结论：(1)LPS通过CBP介导TLR4第732位和第813位赖氨酸发生乙酰化修饰；(2)TLR4第732位赖氨酸的乙酰化参与了MyD88和TRAM/TRIF介导的信号通路的活化,而第813位赖氨酸的乙酰化则仅仅参与了TRAM/TRIF介导的信号通路的活化；(3)LPS诱导第77位赖氨酸的乙酰化促进IRF3形成二聚体进入细胞核。(二)TLR4甲基化修饰在LPS活化的炎症信号转导中的作用1、免疫共沉淀法分析表明TLR4可以与IRF3直接相互作用。LPS刺激巨噬细胞系Raw264.7细胞,利用泛甲基抗体进行免疫印迹检测发现TLR4可发生甲基化修饰。为探索TLR4甲基化修饰位点,我们构建两个突变体R731K,R812K,并与甲基化转移酶PRMT2共表达后检测甲基化程度。结果表明,与野生型TLR4相比,两个突变体的甲基化显著降低。以上结果表明,LPS刺激可促进PRMT2介导的TLR4第731位和第812位精氨酸的甲基化修饰2、应用免疫共沉淀法分析TLR4及其两个突变体R731K和R812K与IRF3的相互作用,结果表明,R812K突变阻断了TLR4与IRF3的相互作用。IRF3 R285K的突变也阻断TLR4与IRF3的相互作用。报告基因技术分析表明PRMT2过表达后,TLR4甲基化程度增强,导致IRF3反应元件活性增加。相反,R812K突变则降低IRF3反应元件的活性。在BMDM细胞中,用定量RT-PCR的方法检测了甲基化对TLR4-IRF3信号通路终产物的影响,结果显示,TLR4 R812K突变抑制IFNβ的合成释放。同样的结论在在293T细胞中也获得。以上结果表明,TLR4第812位精氨酸甲基化参与了IRF3的活化及其与TLR4的相互作用。3、LPS刺激巨噬细胞系Raw264.7细胞,利用泛甲基抗体进行免疫印迹检测发现IRF3可发生甲基化修饰。为探索IRF3甲基化修饰位点,我们构建了突变体R285K,并与PRMT2共表达后检测甲基化程度。结果表明,与野生型IRF3相比,突变体的甲基化程度显著降低。免疫共沉淀法和免疫荧光法证实R285K突变阻断了IRF3二聚体的形成并且阻止其进入细胞核。以上结果表明,LPS诱导的甲基化促进IRF3形成二聚体进入细胞核。主要结论：(1)LPS通过PRMT2介导TLR4第731位和第812位精氨酸发生甲基化修饰；(2)TLR4第812位精氨酸的甲基化参与了其与IRF3的直接相互作用以及IRF3的活化；(3)LPS诱导第285位精氨酸的甲基化促进IRF3形成二聚体进入细胞核。更多还原

【Abstract】 Toll like receptor 4 (TLR4) plays a crucial role in anti-infection, especially antiviral responses. Upon LPS binding, TLR4 is activated, forms a complex with CD 14, MD2, and then triggers downstream signal pathways. One of them is dependent on myeloid differentiation factor 88(MyD88). Through binding to TIR domain containing adaptor protein(TIRAP) and MyD88, TLR4 activates the nuclear factor kappa B(NF-κB), which translocates to the nucleus and induces the expressions of a number of anti-inflammatory genes such as interleukin 6 (IL6), IL12, IL17, and tumor necrosis factor a(TNF-a). The other pathway is MyD88-independent. Through binding to TIR domain containing adaptor protein inducing IFNβ(TRIF) and TRIF related adaptor molecule(TRAM), TLR4 activates IFN-regulated factor-3(IRF3) and IRF7, which induce the expressions of a series of antiviral genes including interferon beta (IFN(3).Posttranslational modifications, including acetylation and methylation, are crucial for gene transcription and signal transduction. However, the posttranslational modifications of TLR4 are largely unknown. Here we explored TLR4 acetylation and methylation in TLR4 activation.I. The role of TLR4 acetylation in inflammatory signal transduction activated by LPS1. Western blot analysis using a pan-acetylation antibody showed that TLR4 was acetylated after LPS stimulation in Raw264.7 cells. Immunofluorescence (IF) analysis showed that creb-binding protein (CBP) translocated to the cytoplasm upon LPS stimulation. Co-immunoprecipitation (Co-IP) experiments showed that LPS stimulation induced the binding of CBP to TLR4. TLR4 acetylation was reduced when CBP expression was down-regulated, indicating that CBP mediated TLR4 acetylation. The acetylation of TLR4 significantly decreased when lysine 732 (K732) or lysine 813 (K813) was mutated to arginine (R). Mass spectrometry analysis recovered K813 as the acetylation site. In addition, LPS-stimulated TLR4 acetylation was detected by specific antibodies targeting acetyl-K732 and acetyl-K813. Altogether, our data strongly indicated that LPS induced TLR4 K813 and K732 acetylation which was mediated by CBP.2. K732R mutation interrupted the interaction between TLR4 with TRIF and MyD88, while K813R mutation interrupted the interaction between TLR4 and TRAM. Luciferase analysis demonstrated that enhancement of TLR4 acetylation by overexpression of CBP increased the activities of NF-κB-and IRF3-responsive elements. Both K732R and K813R mutations decreased the IRF3-responsive element activity, besides K732R mutation decreased the NF-KB-responsive element activity. Reverse transcription PCR analysis revealed that both K732R and K813R mutations inhibited the production of IFN(3 in bone marrow-derived macrophage (BMDM) from TLR4-/-mouse, while only K732R mutation inhibited the production of IL6. The above results indicated that the acetylation of lysine 732 and 813 differentially regulated downstream signaling pathways.3. Western blot analysis using a pan-acetylation antibody showed that IRF3 was acetylated after LPS stimulation in Raw264.7 cells. IRF3 acetylation was reduced when CBP expression was down-regulated, indicating that CBP mediated IRF3 acetylation. Mass spectrometry analysis recovered K77 as the acetylation site. The acetylation of IRF3 significantly decreased when lysine 77 was mutated to arginine. IRF3 acetylation was significantly reduced when histone deacetylase 6 (HDAC6) was overexpressed, indicating that HDAC6 mediated IRF3 deacetylation. Co-IP analysis showed that K77R mutation interrupted IRF3 dimerization. The inhibitors of deacetylase trichostatin A (TSA) and nicotinamide (NAM) increased IRF3 dimerization. Both down-regulating CBP expression and K77R mutation prevented IRF3 from translocating to the nucleus. The above results indicated that acetylation at K77 induced by LPS promoted IRF3 dimerization and transloction to the nucleus. Main conclusions:(1)LPS induced TLR4 K732 and K813 acetylation mediated by CBP.(2) Acetylation at K732 is essential for both MyD88-and TRAM/TRIF-mediated signaling pathways activation, while acetylation at K813 is essential for TRAM/TRIF-mediated signaling activation. (3)Acetylation at K77 induced by LPS promoted IRF3 dimerization and transloction to the nucleus.Ⅱ. The role of TLR4 methylation in inflammatory signal transduction activated by LPS1. Co-IP analysis showed that TLR4 bound to IRF3 directly. Western blot analysis showed that TLR4 was methylated after LPS stimulation. Overexpression of PRMT2 increased TLR4 methylation. The methylation significantly decreased when R731 or R812 was mutated to K, indicating that LPS induced TLR4 methylation at R731 and R812.2. Co-IP analysis showed that R812K mutation interrupted the interaction between TLR4 and IRF3. Luciferase analysis showed that enhancement of TLR4 methylation by overexpression of PRMT2 increased the activities of IRF3-responsive elements, while R812K mutation decreased it. RT-PCR analysis revealed that TLR4 R812K mutation inhibited the production of IFNβin BMDM from TLR4-/-mouse. These data indicated that R812 methylation is essential for TLR4 binding to IRF3 and IRF3 activation.3. Western blot analysis also showed that IRF3 was methylated under LPS stimulation. The methylation of IRF3 significantly decreased when R285 was mutated to K. Co-IP analysis showed R285K mutation in IRF3 interrupted its dimerization and prevented IRF3 from translocating to the nucleus. The above results indicated that methylation induced by LPS promoted IRF3 dimerization and transloction to nucleus.Main conclusions:(1)LPS induced TLR4 R731 and R812 methylation mediated by PRMT2. (2)TLR4 R812 methylation mediated the direct interaction between TLR4 and IRF3 and IRF3 activation. (3)Methylation at R285 induced by LPS promoted IRF3 dimerization and transloction to the nucleus.更多还原