【摘要】：脊椎動物常受到外界微生物的入侵,因此物種自身形成了一套清除感染病原體的機制——免疫系統(tǒng)。免疫系統(tǒng)主要由兩部分構成：天然免疫和獲得性免疫。天然免疫是機體防御外來病原體入侵的第一道防線,它包含了巨噬細胞和樹突狀細胞。獲得性免疫主要在免疫后期階段參與清除病原體的活動并產生免疫記憶。免疫系統(tǒng)主要依賴抗原識別受體(pattern recognition receptor,簡稱PRR)識別病原相關分子模式(pathogen-associated molecular pattern,簡稱PAMP)。這些識別受體主要被分為TLR、 RLR、 NLR以及一些DNA受體等。機體的受體在識別了病原體后,能夠通過招募下游的接頭蛋白進行免疫信號轉導,從而激活免疫信號通路。在免疫信號通路中能夠激活一些轉錄因子,包括IRF3, IRF7, AP-1, NF-κB等,這些轉錄因子能夠促進其下游的I型干擾素或者炎癥因子的轉錄與表達,從而激活免疫反應。 轉錄因子NF-κB在包括免疫反應在內的眾多生命活動中發(fā)揮了重要的功能。因此,激活NF-κB的信號反應途徑受到嚴格的調控,以免產生對機體有害的后果。在我們的研究中發(fā)現(xiàn),一個名叫USP2a (ubiquitin-specific protease2isoform a)的蛋白能夠抑制TLR/IL-1β和仙臺病毒誘導激活的NF-κB。過量表達USP2a能夠抑制IL-1β和仙臺病毒誘導的NF-κB的激活以及下游炎癥因子的產生。我們也構建了敲除USP2基因的HCT116細胞,并用這些細胞進行了實驗。結果表明敲除了USP2a能夠增強TAK1和IKK復合物的磷酸化和降解,IKBα的磷酸化及降解,NF-κB的激活以及下游炎癥因子的轉錄。由此我們判斷USP2a是一個抑制NF-κB激活的蛋白。我們也尋找了USP2a作用的分子,并發(fā)現(xiàn)USP2a能夠持續(xù)性的和TRAF6(tumor necrosis factor receptor-associated factor6)發(fā)生相互作用,并且這種作用是不受刺激例如IL-1β的影響。由于USP2a是一個去泛素化酶家族蛋白,我們也驗證了其去泛素化活性。我們發(fā)現(xiàn)USP2a能夠去除TRAF6上被IL-1p和仙臺病毒誘導產生的K63位連接的泛素化鏈。而且缺失了USP2a能夠促進TRAF6上這種泛素化鏈的生成。我們也發(fā)現(xiàn)了對USP2a去泛素化功能而言至關重要的氨基酸殘基。我們的實驗結果提供了一個相對廣譜的負調控NF-κB激活的機制。 泛素化是免疫信號轉導中一個重要的信號傳導機制,同時去泛素化對信號轉導的調控也至關重要。目前人們了解到的去泛素化酶還不多,還有近百種去泛素化酶其功能還不被人們知曉,因此對去泛素化的研究還有待進一步深入。而對于USP2a這個蛋白來說,其功能的了解我們做得也不夠完整。從它同家族的蛋白中我們可以了解去泛素化酶的作用位點很多,USP2a可能不只作用于TRAF6一個位點,而這個蛋白還有一些其他的間接形式,對于這些間接形式的研究進行的也不是很充分。而USP2a是通過怎樣的機制去除TRAF6上的泛素化,我們目前也不是很清楚,這都有待于下一步的研究。
[Abstract]:Vertebrates are often invaded by external microbes, so the species itself has formed a set of mechanisms-immune system-to remove infectious pathogens. The immune system consists of two parts: innate immunity and acquired immunity. Innate immunity is the first line of defense against foreign pathogens. It contains macrophages and dendritic cells. Acquired immunity is mainly involved in the clearance of pathogens and immune memory in the late stage of immunity. The immune system mainly depends on the antigen recognition receptor (pattern recognition receptor, (PRR) to recognize the pathogen-associated molecular pattern (abbreviated as PAMP). These receptors are mainly divided into TLR, RLR, NLR and some DNA receptors. After recognizing the pathogen, the receptor can activate the immune signal pathway by recruiting downstream junction protein for immune signal transduction. Some transcription factors, including IRF3, IRF7, AP-1, NF- 魏 B, can be activated in the immune signaling pathway. These transcription factors can promote the transcription and expression of interferon type I or inflammatory factors downstream, thus activating the immune response. NF- 魏 B plays an important role in many life activities, including immune response. Therefore, the signaling pathway that activates NF- 魏 B is strictly regulated to avoid harmful consequences. In our study, we found that a protein called USP2a (ubiquitin-specific protease2isoform a) could inhibit TLR/IL-1 尾 and Sendai virus induced activation of NF- 魏 B. Overexpression of USP2a could inhibit the activation of NF- 魏 B induced by IL-1 尾 and Sendai virus and the production of downstream inflammatory factors. We also constructed HCT116 cells knockout USP2 gene, and used these cells to carry out experiments. The results showed that knockout USP2a could enhance phosphorylation and degradation of IKB 偽 and activation of NF- 魏 B and transcription of downstream inflammatory factors in TAK1 and IKK complexes. We conclude that USP2a is a protein that inhibits NF- 魏 B activation. We also looked for molecules of USP2a interaction and found that USP2a can interact sustainably with TRAF6 (tumor necrosis factor receptor-associated factor6) and that this action is not affected by stimuli such as IL-1 尾. Since USP2a is a family of diubiquitin proteins, we have also verified its deubiquitin activity. We found that USP2a can remove the Ubiquitin chain of K63 site connection induced by IL-1p and Sendai virus on TRAF6. Moreover, the absence of USP2a can promote the formation of the ubiquitin chain on TRAF6. We have also identified amino acid residues that are critical to the USP2a's deubiquification function. Our results provide a relatively broad spectrum mechanism for negative regulation of NF- 魏 B activation. Ubiquitin is an important signal transduction mechanism in immune signal transduction. At present, there are still about 100 kinds of diubiquitin enzymes whose functions are not known, so the research on deubiquification still needs to be further studied. For USP2a, the protein, we don't know enough about its function. From its family of proteins, we can see that there are many sites at which the deubiquitin enzyme acts, and that the USP2a may act on more than one site of TRAF6, and that there are some other indirect forms of the protein. Nor is the study of these indirect forms sufficient. However, the mechanism by which USP2a removes ubiquification from TRAF6 is not clear, which remains to be studied in the next step.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392.1

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