E3泛素连接酶CHIP和Nrdp1在免疫应答反应中的调控作用及其机制研究

发布时间：2018-04-19 06:34

本文选题：E3泛素连接酶 + CHIP　；参考：《浙江大学》2012年博士论文

【摘要】：泛素化修饰在机体的免疫系统中发挥着重要的调控作用,广泛参与免疫系统的发育以及免疫应答反应的各个阶段。关于E3泛素连接酶参与免疫应答反应调控,包括E3泛素连接酶新成员的发现或新调控机制的提出一直是免疫学研究领域的前沿热点。因此,本实验室进行了小鼠骨髓来源的树突状细胞(BMDC)和原代腹腔巨噬细胞全基因表达谱芯片的E3泛素连接酶表达谱分析。根据芯片数据和前期研究成果,我们选择了E3泛素连接酶CHIP和Nrdp1开展了进一步的研究。CHIP(Carboxyl terminus of Hsc70-interacting protein,CHIP)是一个与分子伴侣相关的含有U-box功能域的E3泛素连接酶,目前关于CHIP在天然免疫中的调控作用以及其作用机制研究尚不清楚。在此,我们探讨了CHIP在TLRs信号通路中的调控作用以及分子机制。在Raw264.7细胞、小鼠腹腔巨噬细胞、BMDC以及pDC细胞中,CHIP干扰后可以显著抑制TLR2/4/7/9信号通路触发的促炎性细胞因子和Ⅰ型干扰素(IL-6、TNF-α和IFN-β)的产生。在BMDC中,CHIP干扰后显著抑制了LPS诱导的IL-12p70的产生以及BMDC的表型成熟。在APC中,CHIP干扰后能明显抑制LPS/CpG诱导的NF-κB、IRF3和IRF7的活化以及核转移,同时IL-6. IFN-β和CCL5的报告基因活化也显著受到抑制；相应的,CHIP过表达则产生相反的结果。通过GST Pull-down和免疫共沉淀试验,我们发现CHIP可以结合HSP70、TLR4/9以及Src和PKCζ,并通过共聚焦显微镜检测进一步证实该相互作用。在稳定表达CHIP-HA的Raw264.7细胞,CHIP能促进LPS/CpG诱导的Src和PKCζ的磷酸化；而CHIP被干扰后,Src和PKCζ的磷酸化受到明显抑制。体外激酶活性实验提示,在Raw264.7细胞中,CHIP干扰后能显著抑制IRAK1和TBK1的激酶活性,而CHIP-HA过表达能明显增强IRAK1和TBK1的激酶活性。体内和体外泛素化实验提示CHIP能够介导Src和PKCζ发生K63-linked的多聚泛素化并促使Src和PKCζ活化。通过以上的研究,我们对CHIP参与天然免疫调控得出以下结论：经TLRs配体如LPS/CpG刺激后,CHIP可以募集Src和PKCζ并介导其发生K63-linked的多聚泛素化而活化,进而通过NF-kB信号通路或TBK1和IRAK1信号通路活化IRF3和IRF7并最终促使促进炎性因子和干扰素的产生。本部分研究揭示了CHIP在病原体模式识别信号通路中发挥重要调节作用的一个新功能,较深入揭示了CHIP与Src/PKCζ复合体对TLR4/9的调节作用；该研究丰富了TLR信号传导的分子机制,创新性地提出Src/PKCζ调节TBK1和IRAKI并进而活化IRF3/7的通路,为Ⅰ型干扰素的调节机制研究提出了新的角度。我们前期工作研究了E3泛素连接酶Nrdp1参与天然免疫的作用及相关机制,但是对于Nrdp1在适应性免疫中的作用尚未有报导。我们发现Nrdp1优势表达于CD8+T细胞,显示Nrdp1可能与CD8+T细胞的功能调节密切相关。同时发现Nrdp1E3泛素连接酶功能缺失(DN-Nrdp1)后能促进CD8+T细胞分泌IFN-γ、穿孔素和颗粒酶,并且抑制实验性自身免疫性脑脊髓炎(EAE)的发展和促进EAE的缓解。为进一步验证Nrdpl与EAE的关系,我们将DN-Nrdp1CD8+T细胞回输,发现其可以抑制EAE的病程。这些结果提示Nrdp1可能参与调控CD8+T细胞的功能和T细胞介导的自身免疫性疾病的发生发展,但其机理又不同于目前所报道的其他E3泛素连接酶。有关Nrdp1参与CD8+T细胞功能调控的研究可能为E3泛素连接酶参与适应性免疫以及自身免疫性疾病提供新的机制解释,并可能为自身免疫性疾病治疗提出新的潜在靶点。
[Abstract]:Ubiquitination plays an important role in the regulation of the immune system in each stage of broad participation in the development of the immune system and the immune response. On the E3 ubiquitin ligase involved in immune response regulation, including new members of the E3 ubiquitin ligase discovery or a new control mechanism has been put forward is a key issue in immunology research field. Therefore, the laboratory of murine bone marrow derived dendritic cells (BMDC) and primary peritoneal macrophage gene expression microarray of E3 ubiquitin ligase expression analysis. According to the microarray data and previous research results, we selected the E3 ubiquitin ligase CHIP and Nrdp1 on.CHIP (Carboxyl terminus of Hsc70-interacting to protein CHIP.) is U-box containing a domain related with the molecular chaperone E3 ubiquitin ligase, at about CHIP in innate immunity in The regulation and its mechanism is not clear. In this study, we investigate the role of CHIP in the TLRs signaling pathway and the molecular mechanism in mouse peritoneal macrophages, Raw264.7 cells, BMDC and pDC cells, CHIP interference can inhibit TLR2/4/7/9 signaling pathway triggered by proinflammatory cytokines and type I interferon (IL-6, TNF- alpha and IFN- beta) production. In BMDC, CHIP interference significantly inhibited the LPS induced IL-12p70 and BMDC phenotype of mature. In APC, after CHIP interference can inhibit LPS/CpG induced NF- kappa B, IRF3 and IRF7 activation, nuclear transfer, and IL-6. IFN- beta and CCL5 reporter gene activation also significantly inhibited; accordingly, overexpression of CHIP on the contrary results. By GST Pull-down and immunoprecipitation experiments, we found that CHIP can be combined with HSP70, TLR4/9, Src and PKC, And through confocal microscopy further confirmed the interaction. The stable expression of CHIP-HA Raw264.7 cells, CHIP can promote LPS/CpG induced Src and PKC phosphorylation and zeta; CHIP interference, Src and PKC zeta phosphorylation was inhibited. Suggesting that in vitro kinase activity experiments in Raw264.7 cells, CHIP interference after IRAK1 and TBK1 can significantly inhibit the kinase activity, while overexpression of CHIP-HA significantly increased IRAK1 and TBK1 kinase activity in vitro and in vivo ubiquitination experiments suggest that CHIP mediated Src and PKC zeta K63-linked polyubiquitination and led Src and PKC zeta activation. Through the above research, we participate in the CHIP draw the following conclusions: innate immune regulation by TLRs ligands such as LPS/CpG stimulation, CHIP can raise Src and PKC and the K63-linked zeta mediated polyubiquitination and activation, and then through the NF-kB pathway or TBK1 And the activation of IRAK1 pathway in IRF3 and IRF7 and ultimately promote the inflammatory factor and interferon production. This study reveals a new function of CHIP play an important role in pathogen pattern recognition in the signal path, deeply reveals the CHIP and Src/PKC complexes of TLR4/ 9 zeta effect; the study enriched molecular the mechanism of TLR signal transduction pathway, proposes Src/PKC zeta regulation of TBK1 and IRAKI and then the activation of IRF3/7, put forward a new perspective for the research on the adjustment mechanism of type I interferon.
Our previous work on the E3 Nrdp1 ubiquitin ligase involved in innate immune function and related mechanism, but for Nrdp1 in adaptive immunity function has not yet been reported. We found that Nrdp1 expression in CD8+T cells, and CD8+T cells showed that Nrdp1 may be closely related to the regulation function. At the same time that the Nrdp1E3 ubiquitin ligase (DN-Nrdp1) after missing to promote the IFN- secretion of CD8+T cells, perforin and granzyme, and inhibition of experimental autoimmune encephalomyelitis (EAE) in the development and promotion of EAE. In order to further ease the relationship between the Nrdpl and EAE verification, we will DN-Nrdp1CD8+T cell transfusion, found that it can inhibit the course of EAE. These results suggest that the occurrence and development of the function of Nrdp1 may be involved in the regulation of CD8+T cell and T cell mediated autoimmune disease, other ubiquitin E3 but its mechanism is different from the reported connection enzyme. The study of Nrdp1 involved in the regulation of CD8+T cell function may provide new mechanisms for E3 ubiquitin ligase to participate in adaptive immunity and autoimmune diseases, and may provide new potential targets for the treatment of autoimmune diseases.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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