三角帆蚌 β-arrestin 在TLR 介导的免疫中的调控作用及分子机制

Toll-like receptors (TLR) are key elements which recognize pathogen associated molecular patterns, start the body's natural immune response and determine the type and strength of the acquired immune response. However, the excessive activation of TLR can lead to the occurrence and development of a variety of diseases such as autoimmune. Compare with mammals, having more diverse types of TLR indicates shellfish has strong TLR signaling network. β-arrestin are negative regulators TLR/ NF-κB signaling pathway in mammals. We found that LPS stimulation could significantly up-regulate of TLR and down-regulate the mRNA expression of β arrestin in Hyriopsis cumingii. This project intends to clone TLR receptors and the downstream signaling molecules of Hyriopsis cumingii, to search the specificity of TLR receptor ligands and clarify the types of shellfish TLR, distribution, signal transduction and the rule of the response, to further observe regulating effect of key genes of the TLR signal transduction pathways and some inflammatory factors after the β-arrestin expression change using gene transfection, RNA interference and co-immunoprecipitation, to analysis protein interactions of β-arrestin and key components of TLR signal transduction pathways, and clarify the molecular adjustment mechanism of TLR mediated innate immune by β-arrestin.

Toll样受体是机体识别病原相关分子模式、启动固有免疫反应、决定获得性免疫反应的类型及强度的关键性分子。然而TLR的过度活化可导致自身免疫等多种疾病的发生。贝类TLR类型较哺乳动物具有多样性，表明贝类具有强大的TLR信号网络。哺乳动物中β-arrestin 可负调控TLR信号通路。我们发现LPS 刺激三角帆蚌可导致TLR表达上调而β-arrestin 表达下调。因此本项目拟克隆三角帆蚌TLR受体及其信号传递的下游分子，通过试验手段寻找三角帆蚌TLR受体的特异性配体，阐明贝类TLR的类型，分布及信号传导和应答规律。利用基因转染、RNA干扰，免疫共沉淀技术等方法观察β-arrestin 表达上调及下调对TLR信号转导途径中关键基因及部分炎症因子的调节作用，分析β-arrestin与TLR信号转导途径中关键组分的相互作用，阐明β-arrestin调节贝类TLR介导的先天性免疫的分子机制。

Toll 样受体（TLRs）可识别不同的病原体相关分子模式，在先天性免疫应答中扮演着不可或缺的角色。Toll信号通路的过度活化可导致多种自身免疫性疾病。β-arrestin是抑制蛋白 (arrestins) 家族的重要成员，可以通过多种途径调节机体的免疫反应。本课题首先克隆出了三角帆蚌3种TLR受体、转录因子NF-κB、TRAF家族以及β-arrestin基因，荧光定量和原位杂交方法检测了PAMPs刺激后这些基因的表达变化，结果表明无论是在三角帆蚌鳃还是肝胰腺中，这些基因及下游靶基因在刺激后均显著升高，表明它们均参与贝类免疫应答；进一步结合启动子活性分析、RNA干扰等方法阐明 β-arrestin可负向调节TLR信号通路；表达了TLR、NF-κB、全长及截短型的TRAF6及其它TRAF蛋白，通过重组蛋白与病原菌的互作阐明了三种TLR配体特异性；GST-Pull实验表明β-arrestin只能与TRAF6重组蛋白结合，缺少环指结构和MATH结构都不会影响HcTRAF6与β-arrestin的体外互作，推测 β-arrestin对TLR信号通路的调控是通过与TRAF6的结合来实现的。这些结果基本阐明 β-arrestin调节贝类TLR介导的先天性免疫的分子机制。