Role of FIP200 in RIG-I-mediated innate immunity

项目摘要 视黄酸诱导基因I（RIG-I）是识别病毒双链RNA的细胞内传感器 （dsRNA）和5 '三磷酸RNA。在RNA识别后，RIG-I诱导I型抗体的稳健产生， 干扰素（IFN）。为了防止IFN的异常表达，RIG-I的激活受到几个步骤的调控，包括 RIG-I的CARD结构域的去磷酸化和寡聚化。CARD结构域的磷酸化 RIG-I在静息细胞中保持不活性。在RNA配体接合后，RIG-I被RNA配体去磷酸化。 蛋白磷酸酶1（PP 1），其允许随后的寡聚化用于活化。然而，PP 1 细胞中的许多底物以及决定PP 1对RIG-I的特异性的因素尚不清楚。 此外，去磷酸化如何促进RIG-I寡聚化尚不清楚。因此， 确定RIG-I去磷酸化和寡聚化的调节机制。阐明 这些机制不仅揭示了一种新的宿主防御调控机制， 用于开发新的治疗策略以防止异常IFN活化。 我们实验室的长期目标是了解核酸诱导的先天性 免疫力本提案的总体目标是研究RIG-I介导的调节机制 先天免疫我们的初步蛋白质组学研究发现，RIG-I与FIP 200（FAK-family interacting 200 kDa的蛋白），一种众所周知的自噬蛋白。自噬与先天免疫有交叉作用， 一些自噬基因抑制RIG-I信号通路。然而，令人惊讶的是，FIP 200的不足之处在于， 消除5 ′-三磷酸RNA和dsRNA诱导的I型IFN表达并促进RNA病毒复制， 如水泡性口炎病毒（VSV）。有趣的是，FIP 200也被称为PP 1调节亚基， 131（PPP 1 R131），但其在PP 1去磷酸化中的作用尚不清楚。根据现有的文献和我们的 根据初步数据，我们提出了中心假设，即FIP 200对dsRNA介导的先天性 通过调节RIG-I去磷酸化和寡聚化来增强免疫力。我们将研究以下目标： 目的1：确定FIP 200在RIG-I信号通路中作用的分子基础。 目的2：确定FIP 200促进RIG-I激活的机制。 目的3：使用小鼠模型确定FIP 200和RIG-I之间的体内相互作用。 干扰素应答的失调可导致对病毒感染的抵抗力下降或由于干扰素应答的失调而产生有害影响。 过度活跃的免疫系统该提案研究了FIP 200的分子机制， 控制对病毒感染的先天免疫反应。从这一建议中得出的机械概念将 不仅填补了RIG-I调控的知识空白，而且为抗病毒治疗提供了新的见解。

Project summary Retinoic acid-inducible gene I (RIG-I) is an intracellular sensor for recognition of viral double-stranded RNA (dsRNA) and 5` triphosphate RNA. Following RNA recognition, RIG-I induces a robust production of type I interferons (IFN). To prevent aberrant IFN expression, RIG-I activation is regulated by several steps, including dephosphorylation and oligomerization of the CARD domain of RIG-I. Phosphorylation of the CARD domain retains RIG-I inactive in the resting cells. Upon RNA ligand engagement, RIG-I is dephosphorylated by the protein phosphatase 1 (PP1), which permits subsequent oligomerization for activation. However, PP1 has numerous substrates in the cell and what determines the specificity of PP1 towards RIG-I is unknown. Furthermore, how dephosphorylation facilitates RIG-I oligomerization is not clear. Thus, there is a critical need to define the regulatory mechanisms for the dephosphorylation and oligomerization of RIG-I. Elucidation of these mechanisms will not only reveal a novel regulatory mechanism of host defense, but also provide insights for developing novel therapeutic strategies to prevent aberrant IFN activation. The long-term goal of our lab is to understand the regulatory mechanisms of nucleic acid-elicited innate immunity. The overall objective of this proposal is to investigate the regulatory mechanisms for RIG-I-mediated innate immunity. Our pilot proteomics study found that RIG-I interacted with FIP200 (FAK-family interacting protein of 200 kDa), a well-known autophagy protein. Autophagy has a crosstalk with innate immunity and several autophagy genes suppress RIG-I signaling pathway. However, to our surprise, deficiency of FIP200 abolishes 5` triphosphate RNA and dsRNA-induced type I IFN expression and promotes RNA virus replication, such as the vesicular stomatitis virus (VSV). Interestingly, FIP200 is also known as the PP1 regulatory subunit 131 (PPP1R131), but its role in PP1 dephosphorylation is unknown. Based on the existing literature and our preliminary data, we propose the central hypothesis that FIP200 is essential for dsRNA-mediated innate immunity by regulating RIG-I dephosphorylation and oligomerization. We will investigate the following aims: Aim 1: Determine the molecular basis for the role of FIP200 in RIG-I signaling pathway. Aim 2: Determine the mechanisms by which FIP200 promotes RIG-I activation. Aim 3: Determine the in vivo interaction between FIP200 and RIG-I using a mouse model. Dysregulation of IFN responses can result in decreased resistance to viral infection or detrimental effects due to an overactive immune system. This proposal investigates the molecular mechanisms by which FIP200 controls innate immune responses to viral infection. The mechanistic concepts derived from this proposal will not only fill the knowledge gap on RIG-I regulation, but also provide insights for antiviral therapeutics.