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化是免疫系统中普遍存在的信号激活机制。了解这些受体在配体识别过程中如何寡聚并激活下游信号通路是理解其功能的基础，也是这些受体治疗应用的先决条件。在这篇文章中，我们描述了我们的策略来研究一个保守的先天免疫受体，维甲酸诱导基因-1 （RIG-I）的寡聚化机制、寡聚物结构和信号激活机制。rig - 1及其类似物MDA5代表了一个主要的受体家族，可以识别多种细胞类型细胞质中的病毒rna。rig - 1和MDA5都含有一个用于信号激活的串联半胱天冬酶激活招募结构域（2CARD），以及一个用于RNA结合和RNA依赖性ATP水解的解旋酶结构域和c端结构域。rig - 1和MDA5的2CARDs通过促进其单体到细丝的转变来激活其下游适配分子MAVS。MAVS细丝反过来招募下游的信号分子来激活IFN?/？信号通路。我们之前已经证明MDA5沿着dsRNA的长度协同形成细丝，并且它的形成对于与dsRNA的高亲和力相互作用，2CARD的寡聚化以及dsRNA长度依赖性的信号活性调节非常重要。与MDA5不同，rig - 1的寡聚化一直不清楚，并且被认为严格需要一个辅助因子，即k63连接的多泛素链。最近，我们发现RIG-I在ATP水解过程中组装成细丝，并且该细丝可以在缺乏多泛素链的情况下直接激活MAVS，这提示了RIG-I识别RNA和信号激活的新机制。我们在此提出确定RIG-I组装成细丝（Aim 1）和刺激MAVS细丝独立或与多泛素链一起形成的精确机制（Aim 2），以及RIG-I细丝在病毒感染背景下的形成和功能（Aim 3）。该建议建立在我们之前对MDA5丝的研究（Wu等人，Cell, 2013； Peisley等人，PNAS， 2012和PNAS, 2011； Rice等人，Nat Genetics, In press），我们发现rig - 1丝（Peisley等人，Mol. Cell, 2013）， rig - 1 2CARD的最新晶体结构的基础上

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.