本文選題：E3泛素連接酶 + CHIP��； 參考：《浙江大學》2012年博士論文

【摘要】：泛素化修飾在機體的免疫系統(tǒng)中發(fā)揮著重要的調控作用,廣泛參與免疫系統(tǒng)的發(fā)育以及免疫應答反應的各個階段。關于E3泛素連接酶參與免疫應答反應調控,包括E3泛素連接酶新成員的發(fā)現(xiàn)或新調控機制的提出一直是免疫學研究領域的前沿熱點。因此,本實驗室進行了小鼠骨髓來源的樹突狀細胞(BMDC)和原代腹腔巨噬細胞全基因表達譜芯片的E3泛素連接酶表達譜分析。根據芯片數(shù)據和前期研究成果,我們選擇了E3泛素連接酶CHIP和Nrdp1開展了進一步的研究。CHIP(Carboxyl terminus of Hsc70-interacting protein,CHIP)是一個與分子伴侶相關的含有U-box功能域的E3泛素連接酶,目前關于CHIP在天然免疫中的調控作用以及其作用機制研究尚不清楚。在此,我們探討了CHIP在TLRs信號通路中的調控作用以及分子機制。在Raw264.7細胞、小鼠腹腔巨噬細胞、BMDC以及pDC細胞中,CHIP干擾后可以顯著抑制TLR2/4/7/9信號通路觸發(fā)的促炎性細胞因子和Ⅰ型干擾素(IL-6、TNF-α和IFN-β)的產生。在BMDC中,CHIP干擾后顯著抑制了LPS誘導的IL-12p70的產生以及BMDC的表型成熟。在APC中,CHIP干擾后能明顯抑制LPS/CpG誘導的NF-κB、IRF3和IRF7的活化以及核轉移,同時IL-6. IFN-β和CCL5的報告基因活化也顯著受到抑制；相應的,CHIP過表達則產生相反的結果。通過GST Pull-down和免疫共沉淀試驗,我們發(fā)現(xiàn)CHIP可以結合HSP70、TLR4/9以及Src和PKCζ,并通過共聚焦顯微鏡檢測進一步證實該相互作用。在穩(wěn)定表達CHIP-HA的Raw264.7細胞,CHIP能促進LPS/CpG誘導的Src和PKCζ的磷酸化；而CHIP被干擾后,Src和PKCζ的磷酸化受到明顯抑制。體外激酶活性實驗提示,在Raw264.7細胞中,CHIP干擾后能顯著抑制IRAK1和TBK1的激酶活性,而CHIP-HA過表達能明顯增強IRAK1和TBK1的激酶活性。體內和體外泛素化實驗提示CHIP能夠介導Src和PKCζ發(fā)生K63-linked的多聚泛素化并促使Src和PKCζ活化。通過以上的研究,我們對CHIP參與天然免疫調控得出以下結論：經TLRs配體如LPS/CpG刺激后,CHIP可以募集Src和PKCζ并介導其發(fā)生K63-linked的多聚泛素化而活化,進而通過NF-kB信號通路或TBK1和IRAK1信號通路活化IRF3和IRF7并最終促使促進炎性因子和干擾素的產生。本部分研究揭示了CHIP在病原體模式識別信號通路中發(fā)揮重要調節(jié)作用的一個新功能,較深入揭示了CHIP與Src/PKCζ復合體對TLR4/9的調節(jié)作用；該研究豐富了TLR信號傳導的分子機制,創(chuàng)新性地提出Src/PKCζ調節(jié)TBK1和IRAKI并進而活化IRF3/7的通路,為Ⅰ型干擾素的調節(jié)機制研究提出了新的角度。 我們前期工作研究了E3泛素連接酶Nrdp1參與天然免疫的作用及相關機制,但是對于Nrdp1在適應性免疫中的作用尚未有報導。我們發(fā)現(xiàn)Nrdp1優(yōu)勢表達于CD8+T細胞,顯示Nrdp1可能與CD8+T細胞的功能調節(jié)密切相關。同時發(fā)現(xiàn)Nrdp1E3泛素連接酶功能缺失(DN-Nrdp1)后能促進CD8+T細胞分泌IFN-γ、穿孔素和顆粒酶,并且抑制實驗性自身免疫性腦脊髓炎(EAE)的發(fā)展和促進EAE的緩解。為進一步驗證Nrdpl與EAE的關系,我們將DN-Nrdp1CD8+T細胞回輸,發(fā)現(xiàn)其可以抑制EAE的病程。這些結果提示Nrdp1可能參與調控CD8+T細胞的功能和T細胞介導的自身免疫性疾病的發(fā)生發(fā)展,但其機理又不同于目前所報道的其他E3泛素連接酶。有關Nrdp1參與CD8+T細胞功能調控的研究可能為E3泛素連接酶參與適應性免疫以及自身免疫性疾病提供新的機制解釋,并可能為自身免疫性疾病治療提出新的潛在靶點。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

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