Structural and functional analyses of the RIG-I filament in innate immunity

描述(由申请人提供)：配体诱导的受体寡聚是免疫系统中普遍存在的信号激活机制。了解这些受体如何在配体识别过程中寡聚并激活下游信号通路是了解它们的功能的基础，也是这些受体治疗应用的先决条件。在这项建议中，我们描述了我们的策略，以研究保守的天然免疫受体维甲酸诱导基因-1(RIG-I)的寡聚机制、寡聚体结构和信号激活机制。RIG-I及其类似物MDA5代表一个主要的受体家族，识别广泛细胞类型细胞质中的病毒RNA。RIG-I和MDA5都含有一个串联的caspase激活募集结构域(2CARD)，用于信号激活，以及一个解旋酶结构域和一个C-末端结构域，用于RNA结合和依赖于RNA的ATP水解。2RIG-I和MDA5的CARD通过促进其单体到细丝的转变来激活其下游的接头分子MAV。MAVS丝反过来招募更多的下游信号分子来激活干扰素？/？信号通路。我们先前已经证明MDA5沿着dsRNA的长度协同形成一条细丝，它的形成对于与dsRNA的高亲和力相互作用、2CARD的寡聚以及dsRNA长度依赖的信号活性调节是重要的。与MDA5不同，RIG-I的寡聚一直不清楚，一直被认为严格需要一个辅助因子，K63连接的多泛素链。最近，我们发现RIG-I在ATP水解过程中组装成一个细丝，并且在没有多泛素链的情况下，该细丝可以直接激活MAV，这为RIG-I识别RNA和激活信号提供了一种新的机制。我们在这里建议确定RIG-I组装成细丝(目标1)并刺激独立于或与多泛素链一起形成MAV细丝的精确机制(目标2)，以及RIG-I细丝如何在病毒感染的背景下形成和发挥作用(目标3)。这一建议建立在我们之前对MDA5细丝的研究基础上(Wu等人，Cell，2013；Peisley等人，PNAS，2012&PNAS，2011；Rice等人，NAT。遗传学，在印刷中)，我们对RIG-I细丝的发现(Peisley等人，Mol.细胞，2013)，一个最新的RIG-I 2CARD晶体结构 与K63-UBN结合的四聚体(Peisley等人，自然，Epub)，最后是MAVS细丝的原子结构以及RIG-I 2CARD：MAVS卡片复合体(手稿正在准备中)。我们相信，拟议的研究将提供RIG-I功能的全面图景，并帮助我们剖析RIG-I和MDA5在病毒RNA检测和信号激活机制方面的共同点和差异。此外，对RIG-I细丝的详细机制了解可能为在抗病毒和抗癌治疗中调节RIG-I的活性的新治疗策略提供潜在的见解。

DESCRIPTION (provided by applicant): Ligand-induced receptor oligomerization is a ubiquitous mechanism for signal activation in the immune system. Understanding how these receptors oligomerize during ligand recognition and activate downstream signaling pathways is fundamental to understanding their functions and is pre-requisite to therapeutic application of these receptors. In this proposal, we describe our strategies to investigate the oligomerization mechanism, oligomer architecture and signal activation mechanism of a conserved innate immune receptor, retinoic acid inducible gene-1 (RIG-I). RIG-I and its paralog, MDA5, represent a major receptor family that recognizes viral RNAs in the cytoplasm of a broad range of cell types. RIG-I and MDA5 both contain a tandem caspase activation recruitment domain (2CARD) for signal activation and a helicase domain and a C-terminal domain for RNA binding and RNA- dependent ATP hydrolysis. 2CARDs of RIG-I and MDA5 activate their downstream adaptor molecule, MAVS, by promoting its monomer-to-filament transition. MAVS filaments, in turn, recruit further downstream signaling molecules to activate the IFN?/? signaling pathways. We have previously shown that MDA5 cooperatively forms a filament along the length of dsRNA, and that its formation is important for high affinity interaction with dsRNA, oligomerization of 2CARD and dsRNA length dependent regulation of signaling activity. Unlike MDA5, oligomerization of RIG-I has been unclear, and has been thought to strictly require a co-factor, K63-linked polyubiquitin chains. Recently, we found that RIG-I assembles into a filament during ATP hydrolysis, and that the filament can directly activate MAVS in the absence of polyubiquitin chains, suggesting a novel mechanism for RNA recognition and signal activation by RIG-I. We here propose to determine the precise mechanisms by which RIG-I assembles into a filament (Aim 1) and stimulates MAVS filament formation independent of or together with polyubiquitin chains (Aim 2), and how RIG-I filaments form and function in the context of viral infection (Aim 3). This proposal builds upon our previous research on the MDA5 filament (Wu et al, Cell, 2013; Peisley et al, PNAS, 2012 & PNAS, 2011; Rice et al, Nat. Genetics, In press), our discovery of the RIG-I filament (Peisley et al, Mol. Cell, 2013), a very recent crystal structure of RIG-I 2CARD tetramer bound by K63-Ubn (Peisley et al, Nature, Epub) and finally the atomic structures of the MAVS filament as well as the RIG-I 2CARD:MAVS CARD complex (manuscript in preparation). We believe that the proposed research will provide a comprehensive picture of functions of RIG-I and help us dissect commonalities and divergences between RIG-I and MDA5 in viral RNA detection and signal activation mechanisms. Furthermore, detailed mechanistic understanding of the RIG-I filament could potentially offer insights into novel therapeutic strategies to modulat the activity of RIG-I in antiviral and anticancer therapies.