Role of TBK1 polyubiquitination in innate antiviral immunity

描述（由申请人提供）：先天免疫是所有类型的细胞用来抵御入侵微生物的第一道防线。防御微生物攻击对整个身体至关重要，并涉及广泛的医学领域。适当调节抗菌反应可控制感染并限制损伤和刺激的影响。当这些过程出错时——因为反应太弱、太强或错位——结果可能是免疫缺陷、自身免疫或癌症。先天抗病毒免疫应答的研究受到ikk相关激酶TBK1的发现的高度影响。TBK1的底物包括转录因子干扰素调节因子(IRF)-3，它控制1型干扰素（IFN）的转录。本研究旨在探讨IFN和其他抗微生物细胞因子tbk1依赖性转录的分子机制。我们的初步数据确定了两个E3泛素连接酶，它们靶向TBK1进行K63-linked多泛素化（pUb），控制TBK1激酶活性，调节IRF3信号传导，并保护病毒感染。Specific Aim 1分析TBK1 pUb，包括E3连接酶的表征和TBK1 Ub受体位点的鉴定。作为背景，我们提供的证据表明TBK1在翻译后被k63连接的pUb链以配体依赖的方式修饰。我们还提供了数据，表明心理炸弹（MIB）蛋白在调节TBK1 pUb和IFN产生中的作用。这是E3连接酶MIB1和MIB2参与IRF依赖性反应的第一个证据。特异性目的2研究MIB1和/或MIB2缺乏对IFN产生和对病毒复制的保护的影响。最初的实验令人放心地支持了mib基因在预防水疱性口炎病毒（VSV）中的作用。特异性目标3将检查在成纤维细胞、巨噬细胞和树突状细胞中，mb介导的TBK1 pUb在响应RLR和TLR配体或响应dsDNA时的需求。最后的特异性目标将确定TBK1相关分子，这些分子调节RLR、TLR3、TLR4或dsDNA信号通路。控制TBK1信号体组装的分子机制将被确定。我们最近的数据提出了一个新的假设，说明mavs依赖性TBK1激活；拟议的研究将测试和扩展这一模式。详细描述调节TBK1活性的分子相互作用是很重要的，因为这种激酶的失调与病毒感染和其他疾病的易感性有关。拟议研究的见解可能最终有助于设计可能减少慢性炎症疾病中ikk相关途径或在需要增强免疫力时增强TBK1活性的疗法。

DESCRIPTION (provided by applicant): Innate immunity is the first-line of defense used by all types of cells to protect against invading microbes. Defense against microbial attack is critically important for the entire body and touches far-ranging medical domains. Properly regulated antimicrobial responses control infection and limit the effects of injury and irritation. When these processes go awry - because the response is too weak, too strong or misplaced - the result may be immunodeficiency, autoimmunity, or cancer. Studies of innate antiviral immune responses have been highly influenced by the discovery of the IKK-related kinase, TBK1. Substrates for TBK1 include the transcription factor interferon regulatory factor (IRF)-3, which controls transcription of type 1 interferons (IFN). This proposal investigates the molecular mechanisms underlying TBK1-dependent transcription of IFN and other antimicrobial cytokines. Our preliminary data identify two E3 ubiquitin ligases which target TBK1 for K63-linked polyubiquitination (pUb), control TBK1 kinase activity, regulate IRF3 signaling, and protect against viral infection. Specific Aim 1 analyzes TBK1 pUb including characterization of the E3 ligases and identification of TBK1 Ub acceptor sites. As background we provide evidence that TBK1 is posttranslationally modified by K63-linked pUb chains in a ligand dependent fashion. We also supply data indicating a role for mind bomb (MIB) proteins in regulating TBK1 pUb and IFN production. This is the first evidence that the E3 ligases MIB1 and MIB2 participate in IRF dependent responses. Specific Aim 2 investigates the consequences of MIB1 and/or MIB2 deficiency on IFN production and protection against viral replication. Initial experiments have reassuringly supported a role for mib genes in protection against vesicular stomatitis virus (VSV). Specific Aim 3 will examine the requirements for MIB-mediated TBK1 pUb in response to RLR and TLR ligands or in response to dsDNA in fibroblasts, macrophages, and dendritic cells. The final Specific Aim will identify TBK1 associated molecules which regulate the RLR, TLR3, TLR4, or dsDNA signaling pathways. The molecular mechanisms controlling the assembly of TBK1 signalosomes will be determined. Our recent data suggest a novel hypothesis accounting for MAVS-dependent TBK1 activation; the proposed studies will test and expand upon this model. Detailed descriptions of the molecular interactions that regulate TBK1 activity are important since dysregulation of this kinase is associated with susceptibility to viral infection and other diseases. Insights from the proposed studies may ultimately facilitate design of therapies that may curtail IKK-related pathways in chronic inflammatory diseases or enhance TBK1 activity when needed to boost immunity.