【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(gòu)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(chǎn)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(guān)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(zhuǎn)導(dǎo),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(zhuǎn)錄因子,包括IRF3, IRF7, AP-1, NF-κB等,這些轉(zhuǎn)錄因子能夠促進其下游的I型干擾素或者炎癥因子的轉(zhuǎn)錄與表達,從而激活免疫反應(yīng)。 轉(zhuǎn)錄因子NF-κB在包括免疫反應(yīng)在內(nèi)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(yīng)途徑受到嚴格的調(diào)控,以免產(chǎn)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(dǎo)激活的NF-κB。過量表達USP2a能夠抑制IL-1β和仙臺病毒誘導(dǎo)的NF-κB的激活以及下游炎癥因子的產(chǎn)生。我們也構(gòu)建了敲除USP2基因的HCT116細胞,并用這些細胞進行了實驗。結(jié)果表明敲除了USP2a能夠增強TAK1和IKK復(fù)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(zhuǎn)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(dǎo)產(chǎn)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(guān)重要的氨基酸殘基。我們的實驗結(jié)果提供了一個相對廣譜的負調(diào)控NF-κB激活的機制。 泛素化是免疫信號轉(zhuǎn)導(dǎo)中一個重要的信號傳導(dǎo)機制,同時去泛素化對信號轉(zhuǎn)導(dǎo)的調(diào)控也至關(guān)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(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392.1

【相似文獻】

相關(guān)期刊論文 前10條

1 Amrie C. Grammer;Peter E. Lipsky;;Competition between TRAF2 and TRAF6 Regulates NF-κB Activation in Human B Lymphocytes[J];Chinese Medical Sciences Journal;2010年01期

2 陳鋒;何生松;邱榮元;龐然;許娟娟;董繼華;;Influence of Silencing TRAF6 with shRNA on LPS/TLR4 Signaling in vitro[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2010年03期

3 辛利軍;TRAF:一種重要的接頭分子[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2002年03期

4 李珊;楊桂文;安利國;;TRAF1以及它的生物學(xué)功能[J];科技信息(學(xué)術(shù)研究);2008年25期

5 ;TRAF-mediated regulation of immune and inflammatory responses[J];Science China(Life Sciences);2010年02期

6 王新禹;黃懿;李禮;魏群;;腫瘤壞死因子受體作用因子3(TRAF3)結(jié)構(gòu)及生物學(xué)功能[J];中國生物化學(xué)與分子生物學(xué)報;2012年06期

7 孫利;王曉輝;關(guān)薇;唐劉君;王建;楊曉明;汪思應(yīng);;TRAF6-GST融合蛋白在大腸桿菌中的表達及純化[J];生物技術(shù)通訊;2011年05期

8 張波;周芳亮;盧芳國;嚴杰;王成;周建林;張健;;過表達TRAF2降解外源性綠色熒光蛋白[J];中國生物化學(xué)與分子生物學(xué)報;2012年08期

9 王沂;張秀軍;劉青;余源;慈雅麗;王洋;;人TRAF3IP3基因的克隆及真核表達[J];細胞與分子免疫學(xué)雜志;2013年05期

10 曾凡龍;衛(wèi)榮華;朱敬;沈君豪;;以TRAF2為樣本檢驗證實的一種通過模式關(guān)鍵位點研究未知蛋白質(zhì)功能的方法[J];湖北醫(yī)藥學(xué)院學(xué)報;2013年05期

相關(guān)會議論文 前10條

1 ;Interaction with TRAF6 increases the stability of CRF2-12[A];河南省細胞生物學(xué)學(xué)會第二屆會員代表大會暨學(xué)術(shù)研討會論文摘要集[C];2009年

2 ;HSP27 regulates IL-1 stimulated IKK activation through interacting with TRAF6 and affecting its ubiquitination[A];中國病理生理學(xué)會受體和信號轉(zhuǎn)導(dǎo)專業(yè)委員會暨消化專業(yè)委員會聯(lián)合學(xué)術(shù)會議論文匯編[C];2008年

3 王新禹;Yi Huang;Li Li;魏群;;Calmodulin fusion system facilitates TNF receptor-associated factor 3's TRAF domain to highly express in soluble form[A];第十屆全國酶學(xué)學(xué)術(shù)討論會論文集[C];2011年

4 Ying LU;Bao-Chun JIANG;Xin ZHANG;Zhi-Jun ZHANG;De-Li CAO;Yong-Jing GAO;;MicroRNA-146a infusion ameliorates neuropathic pain by reducing the expression of its target gene TRAF6 in spinal cord[A];中國神經(jīng)科學(xué)學(xué)會第十屆全國學(xué)術(shù)會議論文摘要集[C];2013年

5 彭炎炎;Ruidan Xu;Xiaofeng Zheng;;HSCARG Negatively Regulates the Cellular Antiviral RLR Pathwayby regulating TRAF3 Ubiquitination[A];中國生物化學(xué)與分子生物學(xué)會第十一次會員代表大會暨2014年全國學(xué)術(shù)會議論文集——專題報告三[C];2014年

6 ;The IRAK-1-BCL10-MALT1-TRAF6-TAK1 Cascade Mediates Signaling to NF- k. B from Toll-like Receptor 4[A];2006年度學(xué)術(shù)研討會論文摘要匯編[C];2006年

7 ;β-Arrestins negatively regulate Toll-like/IL-1-receptor signalling through their functional interaction with TRAF6[A];第五屆海峽兩岸細胞生物學(xué)學(xué)術(shù)研討會論文摘要集[C];2005年

8 張文;張p，

本文編號：2170209