E3泛素连接酶调控Toll信号介导的天然免疫反应分子机制的研究

Innate immunity is the first line of host defense against invading microorganisms, including bacteria, fungi and viruses. The Toll signaling pathway has been demonstrated to play important roles in mediating the innate immune response. Although the Toll-mediated innate immunity has been extensively studied, how Toll signaling is precisely controlled in balancing innate immune responses remains still poorly understood. Because excessive activation of the innate immunity signaling could be a damage signal to infected cells, and potentially causes the pathogenesis of autoimmunity and infectious diseases, it is important for cells to maintain a proper level of innate immune response. Thus, understanding these unknown mechanisms by which innate immune signaling is proper controlled is critical in immunology. In this proposal, we will use the Drosophila as a model and perform genetic screening and cell-based luciferase reporter assay screening to identify the ubiquitin E3 Ligases that are potentially involved in the regulation of Toll-mediated innate immune signaling pathway, and elucidate the molecular mechanism underlying the actions of these molecules in regulating innate immune responses. We will pay attention on a subtype of E3 ligases, which carry RING-figure domain. In our previous unpublished work, we have identified two E3 ubiquitin ligases (we referred it as Rbe1, RbE3-1, respectively). Our preliminary results have shown that both Rbe1 and RbE3-1 plays an important role in balancing Drosophila innate immune responses in a manner that depends on E3 activities. However, the molecular basis of how these two enzymes negatively regulate Drosophila innate immunity remains a mystery. In addition, we found that RbE3-1 is an evolutionarily conserved in both mouse (referred as mRbE3-1) and human (referred as hRbE3-1). Our preliminary results show both mRbE3-1 and hRbE3-1 have potential role in innate immunity, when cultured cells were challenged with LPS, which could activate Toll signaling in mammals. One of important aims of this proposal is to elucidate the molecular mechanism of how RbE3 proteins regulate innate immunity.

天然免疫反应是机体抵抗外界病原微生物侵扰的第一道防线，Toll信号通路是介导天然免疫反应的关键途径之一。尽管被广泛研究，但Toll信号通路在转导过程中信号活性的有效平衡和适时终止的调控机制仍不清楚。Toll信号转导活性的有效平衡和适时终止的调控对机体来说至关重要，否则可能导致细胞死亡或免疫相关疾病。本项目在前期工作基础上，以果蝇为先驱模型，通过细胞和遗传学筛选，挖掘调控果蝇天然免疫Toll途径的E3泛素连接酶，并试图阐明候选调控因子在果蝇Toll介导的天然免疫反应的分子调控机制。我们已有的实验发现果蝇存在两个E3连接酶（Rbe1和RbE3-1）在天然免疫反应的平衡和适时终止起非常重要的作用。我们将进一步探讨它们调控的分子机制和在高等动物中是否存在类似的调控机制。本项目的开展可能揭示天然免疫一个新调控机制，为新药研发和抗感染治疗提供理论基础。

天然免疫反应是机体抵抗外界病原微生物侵扰的第一道防线，Toll信号通路是介导天然免疫反应的关键途径之一。尽管被广泛研究，但Toll信号通路在转导过程中信号活性的有效平衡和适时终止的调控机制仍不清楚。我们研究结果表明分子伴侣蛋白Bam与Otu蛋白形成复合物，促进Otu聚集，增强其去泛素化酶活性，协助参与对免疫信号及寿命的调节，发现一定程度上的蛋白聚集能够在短时间的特定区域内提高蛋白酶活，调节生理状态以应对外界环境的变化，保持组织和个体的稳态，影响寿命及健康。此外，我们在研究泛素相关蛋白在机体抗病毒的免疫反应中的作用发现：1）USP27X通过去除RIG-I的K63位泛素来负调控RLRs介导的天然免疫信号；2）膜蛋白SDC4通过影响去泛素化酶CYLD和RIG-I的定位和相互作用调控RIG-I的泛素来参与调控RIG-I介导的抗病毒免疫反应；3）参与线粒体自噬的重要蛋白E3连接酶Parkin通过影响TRAF3蛋白的K48位的泛素调控其蛋白的稳定性，并进一步负调控抗病毒的天然免疫反应。这些发现将有助于我们进一步加深对天然免疫信号通路调控机制的理解。