Molecular mechanism of RIG-I and RIPLET in antiviral signaling

RIG-I和RIPLET抗病毒信号传导的分子机制

基本信息

批准号：
10254221
负责人：
Sun Hur
金额：
$ 53.1万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-17 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10254221
关键词：
Address Antiviral Agents Autoimmune Binding Biochemical Biochemistry Biological Assay C-terminal Cell physiology Cellular biology Coiled-Coil Domain Collaborations Crystallization Crystallography Data Discrimination Disease Dissection Double-Stranded RNA Ensure Enzymes Filament Goals Grant HIV Immune Immunologic Receptors In Vitro Infection Inflammatory Influenza A Virus, H1N1 Subtype Investigation Lead Length Ligands Link Molecular N-terminal Nature Pathogen detection Pathogenesis Pathogenicity Play Polyubiquitin Protein Engineering Proteins Public Health RNA RNA Binding RNA Recognition Motif Repression Role Series Severe Acute Respiratory Syndrome Signal Pathway Signal Transduction Site Structure System Techniques Testing Ubiquitin Ubiquitination Viral Virus Virus Diseases Work cancer immunotherapy crosslink experimental study gain of function global health immune function influenzavirus innate immune pathways microbial multicatalytic endopeptidase complex mutant novel pandemic disease receptor receptor function structural biology synergism therapeutic target ubiquitin ligase ubiquitin-protein ligase

项目摘要

SUMMARY Effective immune defense against microbial infection depends upon efficient detection of pathogens by innate immune receptors. Among the proteins that ensure proper functioning of these immune receptors are ubiquitin (Ub) and E3 ligases that work through both proteasome-dependent and -independent mechanisms. In this grant, we explore the molecular mechanism of RIPLET, an E3 ligase that plays a proteasome-independent function in activating antiviral innate immune receptor, RIG-I. This grant builds upon our previous work on RIG- I and our recent findings on RIPLET. RIG-I is a conserved cytosolic innate immune receptor that recognizes RNAs from a broad range of viruses. RIG-I contains an N-terminal signaling domain (tandem CARD or 2CARD) and C-terminal RNA binding domain. Studies from our lab, and others, have identified at least three steps involved in the activation of RIG-I: (i) RNA binding, (ii) release of 2CARD auto-repression, and (iii) tetramerization of 2CARDs. The 2CARD tetramer then activates the downstream adaptor, MAVS, which in turn stimulates the antiviral signaling pathways. In particular, the third step of 2CARD tetramerization is stimulated by K63-linked polyubiquitin chains (K63-Ubn), which binds and stabilizes the 2CARD tetramer, as demonstrated by our crystal structures.! Despite the detailed understanding of the action of K63-Ubn on RIG-I, much remains debated about how and when K63-Ubn is placed on RIG-I. Accumulating evidence suggests that RIPLET, a poorly understood E3 ligase, plays an essential role in conjugating K63-Ubn required for 2CARD tetramerization. We found that RIPLET recognizes the RNA-binding domain of RIG-I, but only when it is pre-oligomerized on dsRNA in a filamentous form. We further revealed that RIPLET binds RIG-I filaments through two distinct binding modes: intra-filament binding and inter-filament bridging. The latter dominates for RIG-I filaments on longer dsRNAs, leading to RIG-I clustering and further amplification of RIG-I signaling in a dsRNA length-dependent manner. These findings showed the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination (Cadena et al, under revision, available in BioRxiv). These findings of ours now raise new and deeper questions about the RIG-I mechanism from the fresh perspective of RIPLET: precisely how RIG-I is ubiquitinated by RIPLET (Aim 1), how RIG-I is recognized by RIPLET (Aim 2), how the oligomeric state of RIG-I is altered by RIPLET (Aim 3), and whether RIPLET can be utilized to identify ligands for RIG-I (Aim 4). We here propose a combination of biochemistry, structural biology and cell biology to answer these questions, which we believe are the key to resolving the next layers of complexity in the RIG-I signaling pathway. The four aims will be pursued independently, but are highly synergistic. These four aims build upon our strong preliminary data, an established network of collaboration and biochemical and functional assays that our lab has developed over the last several years. !

概括针对微生物感染的有效免疫防御取决于先天对病原体的有效检测免疫受体。确保这些免疫受体正常发挥作用的蛋白质包括泛素 (Ub) 和 E3 连接酶通过蛋白酶体依赖性和独立机制发挥作用。在这个授予，我们探索了 RIPLET 的分子机制，RIPLET 是一种 E3 连接酶，发挥蛋白酶体独立作用激活抗病毒先天免疫受体 RIG-I 的功能。这笔赠款建立在我们之前在 RIG 上的工作基础上我和我们最近关于 RIPLET 的发现。 RIG-I 是一种保守的胞质先天免疫受体，可识别多种 RNA 病毒。 RIG-I 包含 N 端信号结构域（串联 CARD 或 2CARD）和 C 端 RNA 结合域。我们实验室和其他实验室的研究已经确定了激活过程中至少涉及三个步骤 RIG-I 的：(i) RNA 结合，(ii) 2CARD 自抑制的释放，以及 (iii) 2CARD 的四聚化。这然后 2CARD 四聚体激活下游接头 MAVS，进而刺激抗病毒信号传导途径。特别是，2CARD 四聚化的第三步是由 K63 连接的多聚泛素刺激的链 (K63-Ubn)，它结合并稳定 2CARD 四聚体，如我们的晶体结构所示。尽管对 K63-Ubn 对 RIG-I 的作用有了详细的了解，但关于如何当 K63-Ubn 放置在 RIG-I 上时。越来越多的证据表明 RIPLET，一个人们知之甚少的 E3 连接酶在 2CARD 四聚化所需的 K63-Ubn 缀合中起着重要作用。我们发现 RIPLET 可以识别 RIG-I 的 RNA 结合结构域，但前提是它在 dsRNA 上预寡聚化。丝状形式。我们进一步揭示 RIPLET 通过两种不同的结合模式结合 RIG-I 丝：丝内结合和丝间桥接。后者在较长 dsRNA 上的 RIG-I 丝中占主导地位，导致 RIG-I 聚类并以 dsRNA 长度依赖性方式进一步放大 RIG-I 信号传导。这些发现表明 E3 连接酶作为直接参与的共受体具有意想不到的作用受体寡聚化和配体区分（Cadena 等人，正在修订中，可在 BioRxiv 中找到）。我们的这些发现现在对 RIG-I 机制提出了新的、更深层次的问题。 RIPLET 的观点：RIG-I 到底是如何被 RIPLET 泛素化的（目标 1），RIG-I 是如何被 RIPLET 识别的 RIPLET（目标 2），RIPLET 如何改变 RIG-I 的寡聚状态（目标 3），以及 RIPLET 是否可以用于鉴定 RIG-I 的配体（目标 4）。我们在这里提出生物化学、结构生物学的结合和细胞生物学来回答这些问题，我们认为这是解决下一层问题的关键 RIG-I 信号通路的复杂性。这四个目标将独立追求，但高度相关协同作用。这四个目标建立在我们强大的初步数据和已建立的合作网络的基础上以及我们实验室在过去几年中开发的生化和功能测定。！