β-arrestin亚细胞定位以及对TLR-IL-1R通路的调控

发布时间：2018-06-20 01:45

本文选题：β-arrestin-TRAF6相互作用 + TLR-IL-1R信号通路　；参考：《中国科学院研究生院（上海生命科学研究院）》2007年硕士论文

【摘要】： Toll样受体-白介素1受体(TLR-IL-1R)信号转导在宿主的免疫应答和抵抗病原入侵方面发挥着极为重要的作用。TLR-IL-1R信号的过度激活会导致机体产生一些自身免疫性疾病。因此TLR-IL-1R信号必须被精细调控,研究机体对该信号的调控机制有助于我们更好地认识人体的免疫调控,从而有效防治自身免疫疾病等伤害。 β抑制蛋白(β-arrestin)是一类多功能的蛋白,其经典功能是作为接头蛋白介导G蛋白偶联受体(GPCR)的脱敏和内吞。近年来的研究显示,β-arrestin还参与GPCR和其他信号通路间的通讯或直接调控其他信号的转导,如β-arrestin介导β2AR信号对免疫系统的调节作用。但β-arrestin与TLR-IL-1R信号间的关系则未见报道。我们的研究发现,β-arrestin可以直接结合TLR-IL-1R下游关键分子TRAF6,抑制NF-κB的激活,从而抑制TLR-IL-1R下游多种细胞因子的转录和表达。在脂多糖(LPS)刺激下,与对照野生型小鼠成纤维细胞(MEF)比较,β-arrestin1/β-arrestin2双敲除小鼠成纤维细胞中Ccl2、TNFα和IκBδ等细胞因子的转录水平明显升高。将β-arrestin1或β-arrestin2重新转回双敲除的MEF细胞时,LPS刺激导致Ccl2、TNFα和IκBδ转录水平的升高则受到明显抑制。此外,在Hela细胞中过表达β-arrestin1或β-arrestin2也都能抑制IL-1β刺激下Ccl2、IL-6和IL-8转录水平的升高。进一步检测蛋白表达水平发现,在THP-1细胞中用RNAi的方法降低内源β-arrestin1的表达水平可以增强THP-1细胞在LPS刺激下的细胞因子表达。这些研究从细胞水平上证明β-arrestin结合TRAF6可以负调控TLR-IL-1R信号通路,抑制TLR-IL-1R激活导致的下游细胞因子表达。接下来,我们通过腹腔注射LPS建立内毒素刺激小鼠模型的研究发现,注射LPS情况下,β-arrestin2基因敲除小鼠比对照野生型小鼠产生更多的Ccl2, TNFα, IL-1β, IL-6和IκBδ等,进一步从动物水平证实了β-arrestin是TLR-IL-1R信号的一个负调控因子的结论。 Arrestin基因家族在进化上具有高度的保守性,分为有器官特异性分布的Visual arrestin基因家族和普遍分布的β-arrestin基因家族。对于β-arrestin基因家族的研究长期集中在哺乳动物中。最近,有文献报道了斑马鱼β-arrestin2基因在胚胎发育过程中参与了smoothen信号通路。但通过文献检索和序列查找,我们至今没有发现低等非哺乳类脊椎动物中关于β-arrestin1基因的报道。本研究通过Genebank中一小段高度相似序列,使用RACE方法第一次从斑马鱼中克隆获得了β-arrestin1基因,并通过多序列比对和进化分析,定位了该基因上具有的β-arrestin家族保守的Clathrin结合域,AP-2和MAPK结合位点。激光共聚交荧光显微镜的结果显示,外源表达斑马鱼GFP-β-arrestin1蛋白在细胞质和细胞核内均有分布,这和已知的β-arrestin1蛋白亚细胞定位一致。进一步的β2AR受体内吞实验则在功能上验证了新克隆的基因能够参与GPCR信号转导,具有β-arrestin家族保守的功能。此外,实时定量RT-PCR的结果显示,斑马鱼β-arrestin1和β-arrestin2基因在发育过程中都存在时序表达的现象。这提示了斑马鱼β-arrestin可能在胚胎发育过程中具有重要的生物学功能。因此,本文报道了第一个被克隆的非哺乳类脊椎动物中的β-arrestin1基因,并从序列、蛋白亚细胞定位以及功能上验证了该基因的进化地位,提示了该基因潜在的生物学功能。
[Abstract]:The Toll like receptor - IL-1 receptor (TLR-IL-1R) signal plays an extremely important role in the host's immune response and resistance to pathogenic invasion. The overactivation of.TLR-IL-1R signals will lead to some autoimmune diseases. Therefore, the TLR-IL-1R signal must be carefully regulated, and the mechanism of the body's regulation of the signal is studied. It helps us to better understand the immune regulation of human body, so as to effectively prevent and control autoimmune diseases.
Beta suppressor (beta -arrestin) is a kind of multifunctional protein whose classic function is to mediate the desensitization and endocytosis of G protein coupled receptor (GPCR) as a joint protein. Recent studies have shown that beta -arrestin also participates in the communication between GPCR and other signaling pathways or directly regulates the transduction of his signal, such as beta -arrestin mediated beta 2AR signal to immunity The regulatory role of the epidemic system. However, the relationship between beta -arrestin and TLR-IL-1R signal has not been reported.
Our study found that beta -arrestin can directly bind to the key molecule TRAF6 of downstream TLR-IL-1R, inhibit the activation of NF- kappa B, and inhibit the transcription and expression of a variety of cytokines in the downstream of TLR-IL-1R. Under the stimulation of lipopolysaccharide (LPS), the beta -arrestin1/ beta -arrestin2 double knockout in mice is compared with that of the control wild type mouse fibroblasts (MEF). The transcriptional level of cell factors such as Ccl2, TNF A and I kappa B Delta in the cells was significantly increased. When the beta or beta -arrestin2 was retransferred to the double knockout MEF cells, LPS stimulation led to Ccl2, TNF A and I kappa B delta transcriptional levels were significantly inhibited. Further detection of Ccl2, IL-6 and IL-8 transcriptional levels. Further detection of protein expression levels found that the expression level of endogenous beta -arrestin1 decreased by RNAi in THP-1 cells can enhance the expression of cytokines in THP-1 cells under LPS stimulation. These studies show that beta -arrestin binding TRAF6 can negatively regulate TLR-IL-1R TLR-IL-1R on the cell level. The signal pathway inhibited the expression of downstream cytokine induced by TLR-IL-1R activation. Next, we found that the mice injected with LPS by intraperitoneal injection of endotoxin stimulated mice model. In the case of LPS injection, the beta -arrestin2 knockout mice produced more Ccl2, TNF a, IL-1 beta, IL-6 and I kappa B Delta, and further from the animals. The level confirms that beta -arrestin is a negative regulator of TLR-IL-1R signal.
The Arrestin gene family is highly conserved in evolution, divided into the Visual arrestin gene family with an organ specific distribution and the widely distributed beta -arrestin family. The study of the beta -arrestin gene family has been concentrated in mammals for a long time. Recently, there have been reports of the development of the zebrafish beta -arrestin2 gene in embryo development. Smoothen signaling pathway is involved in the process, but we have not found a report on the gene of beta -arrestin1 in lower non mammalian vertebrates through literature retrieval and sequence search.
In this study, the beta -arrestin1 gene was obtained from zebrafish for the first time by RACE method, and the conservative Clathrin binding domain of the beta -arrestin family, the binding site of AP-2 and MAPK in the gene, and the laser copolymerization fluorescence microscope were obtained by using the method of multiple sequence alignment and evolutionary analysis in Genebank. The results showed that the GFP- beta -arrestin1 protein was distributed in both cytoplasm and nucleus of zebrafish, which was in accordance with the known subcellular localization of beta -arrestin1 protein. Further beta 2AR receptor endocytosis test demonstrated that the newly cloned gene could be involved in GPCR signal transduction and had the conserved function of the beta -arrestin family. In addition, the results of real-time quantitative RT-PCR show that the gene of zebrafish beta -arrestin1 and beta -arrestin2 have time series expression during development, which suggests that zebrafish beta -arrestin may have important biological functions during the development of embryo. Therefore, this paper reports the beta -a of the first cloned non mammalian vertebrate. The rrestin1 gene was verified by sequence, protein subcellular location and function, indicating the potential biological function of the gene.
【学位授予单位】：中国科学院研究生院（上海生命科学研究院）
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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