USP2a通过去泛素化TRAF6负调控NF-κB的激活

发布时间：2018-08-07 13:46

【摘要】：脊椎动物常受到外界微生物的入侵,因此物种自身形成了一套清除感染病原体的机制——免疫系统。免疫系统主要由两部分构成：天然免疫和获得性免疫。天然免疫是机体防御外来病原体入侵的第一道防线,它包含了巨噬细胞和树突状细胞。获得性免疫主要在免疫后期阶段参与清除病原体的活动并产生免疫记忆。免疫系统主要依赖抗原识别受体(pattern recognition receptor,简称PRR)识别病原相关分子模式(pathogen-associated molecular pattern,简称PAMP)。这些识别受体主要被分为TLR、 RLR、 NLR以及一些DNA受体等。机体的受体在识别了病原体后,能够通过招募下游的接头蛋白进行免疫信号转导,从而激活免疫信号通路。在免疫信号通路中能够激活一些转录因子,包括IRF3, IRF7, AP-1, NF-κB等,这些转录因子能够促进其下游的I型干扰素或者炎症因子的转录与表达,从而激活免疫反应。转录因子NF-κB在包括免疫反应在内的众多生命活动中发挥了重要的功能。因此,激活NF-κB的信号反应途径受到严格的调控,以免产生对机体有害的后果。在我们的研究中发现,一个名叫USP2a (ubiquitin-specific protease2isoform a)的蛋白能够抑制TLR/IL-1β和仙台病毒诱导激活的NF-κB。过量表达USP2a能够抑制IL-1β和仙台病毒诱导的NF-κB的激活以及下游炎症因子的产生。我们也构建了敲除USP2基因的HCT116细胞,并用这些细胞进行了实验。结果表明敲除了USP2a能够增强TAK1和IKK复合物的磷酸化和降解,IKBα的磷酸化及降解,NF-κB的激活以及下游炎症因子的转录。由此我们判断USP2a是一个抑制NF-κB激活的蛋白。我们也寻找了USP2a作用的分子,并发现USP2a能够持续性的和TRAF6(tumor necrosis factor receptor-associated factor6)发生相互作用,并且这种作用是不受刺激例如IL-1β的影响。由于USP2a是一个去泛素化酶家族蛋白,我们也验证了其去泛素化活性。我们发现USP2a能够去除TRAF6上被IL-1p和仙台病毒诱导产生的K63位连接的泛素化链。而且缺失了USP2a能够促进TRAF6上这种泛素化链的生成。我们也发现了对USP2a去泛素化功能而言至关重要的氨基酸残基。我们的实验结果提供了一个相对广谱的负调控NF-κB激活的机制。泛素化是免疫信号转导中一个重要的信号传导机制,同时去泛素化对信号转导的调控也至关重要。目前人们了解到的去泛素化酶还不多,还有近百种去泛素化酶其功能还不被人们知晓,因此对去泛素化的研究还有待进一步深入。而对于USP2a这个蛋白来说,其功能的了解我们做得也不够完整。从它同家族的蛋白中我们可以了解去泛素化酶的作用位点很多,USP2a可能不只作用于TRAF6一个位点,而这个蛋白还有一些其他的间接形式,对于这些间接形式的研究进行的也不是很充分。而USP2a是通过怎样的机制去除TRAF6上的泛素化,我们目前也不是很清楚,这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 伪 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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1

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