Molecular mechanisms for antiviral signaling and regulation by MDA5 and TRIM65

MDA5 和 TRIM65 抗病毒信号传导和调节的分子机制

基本信息

批准号：
10206037
负责人：
Sun Hur
金额：
$ 44.25万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10206037
关键词：
Address Affinity Agonist Antiviral Agents Attention Autoimmune Binding Biochemical Cells Collaborations Complex DNA Methyltransferase Inhibitor Data Discrimination Disease Double-Stranded RNA Event Filament Goals Grant HIV Immune response Immune signaling Immunologic Receptors Infection Inflammation Inflammatory Influenza A Virus, H1N1 Subtype Ionizing radiation Ligands Link Mediating Methods Modeling Molecular Molecular Conformation Pathogenesis Physiological Play Process Public Health RNA Regulation Repression Research Role Severe Acute Respiratory Syndrome Signal Pathway Signal Transduction Sterility Structure Therapeutic Time Viral Virus Virus Diseases Work base cancer immunotherapy cancer therapy global health human disease immune function influenzavirus innate immune pathways novel novel therapeutic intervention pandemic disease receptor therapeutic target ubiquitin-protein ligase viral RNA

项目摘要

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.

概括 MDA5 是一种保守的先天免疫受体，可在感染过程中检测病毒 RNA 并激活最近的研究表明，MDA5 不仅可以在感染期间被激活，而且在没有感染的各种生理条件下，这种“无菌”也可以发生炎症。引起炎症性疾病的发病机制，但同时也可能具有治疗益处，例如以癌症免疫治疗为例，在过去的几年里，我的实验室已经定义了分子框架。为了了解 MDA5 如何识别病毒 dsRNA 并激活下游信号传导，我们发现了这一点。 MDA5 在与 dsRNA 结合后组装成丝，并且丝的形成是有效的 dsRNA 结合和下游信号激活尽管取得了进展，但我们的研究仍存在关键差距。了解 MDA5 是如何被激活的以及其活性是如何被调节的，也就是说，MDA5 的确切身份是什么。在病毒感染和无菌炎症条件下刺激 MDA5 的 dsRNA 是什么？ MDA5 丝形成后的分子事件导致抗病毒信号激活。建议通过关注 TRIM65（一个泛素 (Ub) E3 连接酶对于 MDA5 信号传导至关重要。我们和其他人之前的研究表明，K63 连接的多聚泛素链 (K63-Ubn) 在 TRIM65 被推测是负责 MDA5 介导的抗病毒信号传导的 E3 连接酶。 MDA5 的 K63-Ubn 缀合然而，事实是否如此，如果是，具体是如何以及何时缀合的。 TRIM65 对 MDA5 的作用尚不清楚，在我们的初步分析中，我们发现 TRIM65 直接结合。 MDA5，并且这种结合严格依赖于 MDA5 丝的形成。这一观察表明。 TRIM65 作为配体辨别和信号激活的检查点发挥着核心作用。发现 TRIM65 pull-down 可用于特异性分离组装在激动剂上的 MDA5 丝 dsRNA，远离与丰富的 ssRNA 结合的 MDA5 的非活性复合物。这一发现有望带来新的发现。鉴定 MDA5 配体的方法，这是该领域长期以来寻求的里程碑。进展，我们在这里建议解决MDA5功能的两个核心问题，即信号机制（目标1）和RNA配体选择性（目标2），从TRIM65的新角度更具体地说，我们将。确定 TRIM65 激活和调节 MDA5 的结构和生化机制（目标 1）并开发一种新的 TRIM65 Pull-down 策略来识别 MDA5 的 RNA 配体。我们相信，所提出的工作将展示 E3 连接酶如何直接参与自我连接与非自我歧视和免疫信号过程，并将提供一个研究其他模型 E3 连接酶在免疫功能及其他方面的作用。此外，我们的研究还可能指导新的治疗方法针对 MDA5 功能及其抗病毒信号通路的策略。