Functional Characterization of Suppressor of IKKepsilon

描述(申请人提供)：病原体挑战引起先天免疫系统的即时反应。多种天然免疫信号通路汇聚在一起，激活了一种普遍表达的蛋白水解酶结合蛋白1(Tak1)。事实证明，TBK1的底物在协调宿主防御方面起着关键作用，决定着下游先天免疫反应的形状。我们定义了一种新的Tbk1底物--IKKϵ抑制因子(SIKE)。SIKE首先被鉴定为一种TBK1抑制剂。在确定SIKE介导的TBK1抑制作用的研究中，我们发现SIKE被TBK1磷酸化，并且TK1：SIKE相互作用受SIKE磷酸化的调节。对于其他的TBK1底物，SIKE作为TBK1的纳米分子混合型抑制剂。虽然SIKE作为一种高亲和力底物影响了TBK1的活性，但SIKE的主要功能仍不清楚。为了解决这一知识差距，我们研究了SIKE的本地化模式和互动网络。大体定位研究表明，GFP-SIKE是胞质的，在细胞骨架结构中蓄积，胞质颗粒致密。免疫沉淀的SIKE复合体的串联MS/MS数据表明，SIKE与核糖核蛋白复合体(RNPs)相关的细胞骨架、翻译机制和mRNA转运蛋白相互作用。RNP是在病毒感染后形成的，以减少病毒复制的翻译机制的可用性。这种关系表明，RNPs形成了宿主抗病毒防御的一部分，但天然免疫信号和这些RNA介导的防御之间的联系尚不明确。根据我们的初步数据，我们假设SIKE在细胞质RNPs中作为先天免疫反应和转录后mRNA池之间的桥梁，在翻译水平上介导宿主的快速反应。在目标1中，我们将在上皮细胞中建立sike相互作用，形成对入侵病原体的防御屏障，并在 RNP的形成可能是一种关键的防御机制，吞噬免疫细胞负责隔离/清除病原体，其中快速的细胞骨架重排和细胞迁移是必不可少的功能。我们将使用dsRNA以多肌苷-多胞酸的形式评估病原体特异性的SIKE相互作用，dsRNA模拟病毒感染的关键方面，RNPs的诱导者，以及鼠伤寒沙门氏菌，它被吞噬细胞摄取并激活TBK1依赖的通路。在目标2中，我们将研究Sike的磷酸化状态对其在细胞类型和病原体刺激下定位的影响。我们将通过细胞迁移、吞噬、含有空泡的病原体的形成以及RNP的形成和分解等功能分析来研究SIKE在细胞外围与细胞骨架相关的功能与其在胞浆颗粒中的作用。这些研究旨在确定SIKE在连接TBK1介导的天然免疫反应和RNA颗粒方面的功能，将为理解TBK1如何塑造我们的天然免疫防御系统提供关键的新见解，并为建立关于TBK1介导的天然免疫反应的功能、调节和病毒颠覆的长期研究奠定基础。

DESCRIPTION (provided by applicant): Pathogen challenge elicits an immediate response by the innate immune system. Multiple innate immune signaling pathways converge to activate TANK binding kinase 1 (TBK1), a ubiquitously expressed kinase. Substrates of TBK1 have proven to be critical players in coordinating host defenses, dictating the shape of the downstream innate immune response. We have defined a new TBK1 substrate, Suppressor of IKKϵ (SIKE). SIKE was first identified as a TBK1 inhibitor. In our studies to define SIKE-mediated TBK1 inhibition, we discovered that SIKE was phosphorylated by TBK1 and the TBK1:SIKE interaction was modulated by SIKE phosphorylation. With respect to other TBK1 substrates, SIKE acted as a nanomolar, mixed-type inhibitor of TBK1. Although SIKE influenced TBK1 activity as a high affinity substrate, SIKE's primary function remains unknown. To address this gap in knowledge, we examined SIKE's localization pattern and interaction network. Gross localization studies indicated that GFP-SIKE was cytosolic, accumulated in cytoskeletal structures, and dense cytosolic granules. Tandem MS/MS data from immunoprecipitated SIKE complexes revealed that SIKE interacted with the cytoskeleton, translation machinery, and mRNA transport proteins associated with ribonucleoprotein complexes (RNPs). RNPs are formed following viral infection to reduce the availability of translation machinery for viral replication. This relationship suggests that RNPs form part of the host's antiviral defenses, yet the linkage between innate immune signaling and these RNA-mediated defenses is poorly defined. Based upon our preliminary data, we hypothesize that SIKE functions as a bridge between the innate immune response and post-transcriptional mRNA pools in cytosolic RNPs to mediate a rapid host response at the level of translation. In Aim 1, we will establish SIKE interactions in epithelial cells that form a defensive barrier to invading pathogen and where rapid RNP formation may represent a key defense mechanism, and phagocytic immune cells responsible for sequestering/removing pathogen where rapid cytoskeletal rearrangement and cell migration are essential functions. We will assess pathogen-specific SIKE interactions using dsRNA in the form of polyinosinic-polycytidylic acid, which mimics key aspects of viral infection, an inducer of RNPs, and S. typhimurium, which is taken up by phagocytic cells and activates TBK1-dependent pathways. In Aim 2, we will examine the impact of SIKE's phosphorylation state on its localization with respect to cell-type and pathogen stimuli. We will investigate SIKE' function at the cell periphery in association with the cytoskeleton versus its role in cytosolic granules through functional assays including cell migration, phagocytosis, formation of pathogen containing vacuoles, and RNP formation and resolution. These studies to determine SIKE's function in bridging TBK1-mediated innate immune responses and RNA granules will provide critical new insight into how TBK1 shapes our innate immune defenses and establish a foundation for building long-term studies on the function, regulation and viral subversion of TBK1-mediated innate immune responses.