Molecular mechanisms for antiviral signaling and regulation by MDA5 and TRIM65

总结 MDA 5是一种保守的先天免疫受体，在感染期间检测病毒RNA并激活 抗病毒免疫反应最近的研究表明，MDA 5不仅在感染过程中被激活， 而且在没有感染的各种生理条件下也是如此。这种“无菌”炎症可以 导致炎症性疾病的发病机制，但同时，对于 例如癌症免疫治疗。在过去的几年里，我的实验室已经定义了 了解MDA 5如何识别病毒dsRNA并激活下游信号传导。我们发现 MDA 5在与dsRNA结合后组装成细丝，并且细丝的形成是有效的细胞增殖所必需的。 dsRNA结合和下游信号激活。然而，尽管取得了进展， 了解MDA 5是如何激活的，以及它的活性是如何调节的。也就是说， 在病毒感染和无菌炎症条件下刺激MDA 5的dsRNA是什么， MDA 5丝形成后的分子事件导致抗病毒信号激活。这个目标 一个建议是通过关注TRIM 65来解决MDA 5功能的这两个知之甚少的方面， 泛素（Ub）E3连接酶，对MDA 5信号传导至关重要。 我们和其他人以前的研究表明，K63-连接的polyUb链（K63-Ubn）在细胞内起着重要的作用。 在MDA 5介导的抗病毒信号传导中的作用。TRIM 65被推测为负责E3连接酶。 MDA 5的K63-Ubn缀合。然而，事实上是否如此，如果是的话，究竟如何以及何时 TRIM 65对MDA 5的作用尚不清楚。在我们的初步分析中，我们发现TRIM 65直接结合 这种结合严格依赖于MDA 5丝的形成。这一观察表明， TRIM 65作为配体识别和信号激活的检查点起着核心作用。而且我们 发现TRIM 65 pull-down可用于特异性分离组装在激动剂上的MDA 5细丝 dsRNA，远离与丰富的ssRNA结合的MDA 5的无活性复合物。这一发现预示着一部小说 鉴定MDA 5配体的方法，这是该领域长期寻求的里程碑。在此基础上， 进展，我们在这里提出解决两个核心问题的MDA 5功能，即信号传导机制 (Aim 1）和RNA配体选择性（目标2），从TRIM 65的新视角。具体来说，我们将 确定TRIM 65激活和调节MDA 5的结构和生化机制（目的1） 并开发了一种新的TRIM 65下拉策略来识别MDA 5的RNA配体。 我们相信，这项工作将证明E3连接酶如何直接参与自我调节。 vs.非自我歧视和免疫信号传导过程，并将提供一个模型，研究其他 E3连接酶在免疫功能及其他方面的作用。此外，我们的研究还可以指导新的治疗方法， 靶向MDA 5功能及其抗病毒信号通路的策略。

SUMMARY MDA5 is a conserved innate immune receptor that detects viral RNAs during infection and activates antiviral immune response. Recent studies have shown that MDA5 can be activated not only during infection, but also under various physiological conditions in the absence of infection. Such “sterile” inflammation can cause pathogenesis of inflammatory disorders, but at the same time, can be therapeutically beneficial, for example during cancer immunotherapies. Over the last few years, my lab has defined the molecular framework for understanding how MDA5 recognizes viral dsRNA and activates downstream signaling. We discovered that MDA5 assembles into filaments upon binding to dsRNA and that the filament formation is required for efficient dsRNA binding and downstream signal activation. Despite the progress, however, there are key gaps in our understanding of how MDA5 is activated and how its activity is regulated. That is, what is the exact identity of dsRNA that stimulates MDA5 both in the virus-infected and sterile inflammatory conditions, and what are the molecular events following MDA5 filament formation leading up to antiviral signal activation. The goal of this proposal is to address these two poorly understood aspects of MDA5 function by focusing on TRIM65, a ubiquitin (Ub) E3 ligase essential for MDA5 signaling. Previous studies from us and others showed that K63-linked polyUb chains (K63-Ubn) plays an important role in MDA5-mediated antiviral signaling. TRIM65 has been speculated to be the E3 ligase responsible for the K63-Ubn conjugation of MDA5. However, whether this is in fact the case, and if so, exactly how and when TRIM65 acts on MDA5 have been unclear. In our preliminary analysis, we found that TRIM65 directly binds MDA5, and this binding is strictly dependent on MDA5 filament formation. This observation suggests that TRIM65 plays a central role as a check-point for ligand discrimination and signal activation. Furthermore, we found that TRIM65 pull-down can be used for specific isolation of MDA5 filament assembled on agonist dsRNA, away from the inactive complexes of MDA5 bound to abundant ssRNAs. This finding promises a novel method for identifying MDA5 ligands, the long-sought-after milestone in the field. Building upon these progresses, we here propose to address two central questions on MDA5 functions, i.e. signaling mechanism (Aim 1) and RNA ligand selectivity (Aim 2), from the new perspective of TRIM65. More specifically, we will determine the structural and biochemical mechanisms by which TRIM65 activates and regulates MDA5 (Aim 1) and develop a novel TRIM65 pull-down strategy to identify the RNA ligands for MDA5. We believe that the proposed work would demonstrate how an E3 ligase can directly participate in the self vs. non-self discrimination and immune signaling processes, and would provide a model for investigating other E3 ligases in immune functions and beyond. Furthermore, our research may also guide new therapeutic strategies to target MDA5 functions and its antiviral signaling pathway.