Role of TBK1 polyubiquitination in innate antiviral immunity

描述(申请人提供)：先天免疫是所有类型的细胞用来防御入侵微生物的第一道防线。对微生物攻击的防御对整个身体至关重要，涉及广泛的医学领域。适当调节的抗微生物反应可控制感染，并限制伤害和刺激的影响。当这些过程出错时--因为反应太弱、太强或放错了地方--结果可能是免疫缺陷、自身免疫或癌症。对先天抗病毒免疫反应的研究受到了IKK相关激酶TBK1的发现的极大影响。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(干扰素)的转录。这项建议研究了干扰素和其他抗微生物细胞因子依赖于TBK1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，它们针对K63连锁的多泛素化(PUB)的TBK1，控制TBK1的活性，调节IRF3信号，并保护病毒感染。具体目的1分析TBK1pub，包括E3连接酶的特性和TBK1Ub受体位点的鉴定。作为背景，我们提供的证据表明，TBK1是由K63连接的Pub链以配体依赖的方式翻译后修饰的。我们还提供了数据，表明心灵炸弹(MIB)蛋白在调节TBK1 pub和干扰素的产生中发挥了作用。这是E3连接酶MIB1和MIB2参与IRF依赖反应的第一个证据。具体目的2研究MIB1和/或MIB2缺乏对干扰素的产生和对病毒复制的保护作用。初步实验已令人放心地支持MIB基因在预防水疱性口炎病毒(VSV)中的作用。具体目标3将研究MIB介导的TBK1 pub响应RLR和TLR配体或响应成纤维细胞、巨噬细胞和树突状细胞中dsDNA的需求。最终的特定目标是识别调节RLR、TLR3、TLR4或dsDNA信号通路的TBK1相关分子。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种新的假设，解释了MAVS依赖的TBK1激活；拟议的研究将在此模型的基础上进行测试和扩展。对调节TBK1活性的分子相互作用的详细描述是重要的，因为该激酶的失调与病毒感染和其他疾病的易感性有关。来自拟议研究的见解可能最终有助于设计治疗方案，这些疗法可能会减少慢性炎症性疾病中与IKK相关的通路，或者在需要提高免疫力时增强TBK1的活性。

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.