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于抵御入侵微生物的第一道防线。防御微生物的攻击对整个身体至关重要，涉及广泛的医学领域。适当调节的抗菌反应可以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、太强或放错了位置--结果可能是免疫缺陷、自身免疫或癌症。先天性抗病毒免疫应答的研究受到IKK相关激酶TBK 1的发现的高度影响。TBK 1的底物包括转录因子干扰素调节因子（IRF）-3，其控制1型干扰素（IFN）的转录。本研究旨在探讨TBK 1依赖性IFN和其他抗微生物细胞因子转录的分子机制。我们的初步数据确定了两种E3泛素连接酶，它们靶向TBK 1进行K63连接的多聚泛素化（PUB），控制TBK 1激酶活性，调节IRF 3信号传导，并保护免受病毒感染。Specific Aim 1分析TBK 1 pUb，包括E3连接酶的表征和TBK 1 Ub受体位点的鉴定。作为背景，我们提供的证据表明，TBK 1是后修饰K63连接的pUb链在配体依赖性的方式。我们还提供了数据，表明头脑炸弹（MIB）蛋白在调节TBK 1 pUb和IFN生产的作用。这是E3连接酶MIB 1和MIB 2参与IRF依赖性反应的第一个证据。具体目标2研究MIB 1和/或MIB 2缺陷对IFN产生和针对病毒复制的保护的后果。最初的实验已经令人放心地支持了mib基因在预防水泡性口炎病毒（VSV）中的作用。具体目标3将检查成纤维细胞、巨噬细胞和树突状细胞中MIB介导的TBK 1 pUb对RLR和TLR配体的反应或对dsDNA的反应的需求。最终的特异性目的将鉴定调节RLR、TLR 3、TLR 4或dsDNA信号通路的TBK 1相关分子。将确定控制TBK 1信号体组装的分子机制。我们最近的数据表明，一种新的假设占MAVS依赖TBK 1激活;拟议的研究将测试和扩展这个模型。对调节TBK 1活性的分子相互作用的详细描述是重要的，因为这种激酶的失调与对病毒感染和其他疾病的易感性有关。从拟议的研究中获得的见解可能最终有助于设计治疗方法，这些治疗方法可能会在慢性炎症性疾病中减少IKK相关通路，或在需要增强免疫力时增强TBK 1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.