Rip Proteins in Innate Immune Signaling

描述（由申请人提供）：细胞因子TNF、病原体识别toll样受体（TLRs）和核苷酸结合寡聚结构域（NOD）蛋白部分通过激活转录因子NF-kB介导宿主对感染的防御。我们发表的研究表明，Rip1是TNF-和TLR3/4、trif依赖的NF-kB通路的关键介质，Rip1的泛素修饰对于TNF-诱导的NF-kB激活和细胞因子产生至关重要。我们发现Rip1缺陷细胞对病毒感染的I型干扰素反应受损，表明Rip1有助于rig - 1 /Mda5抗病毒信号传导。我们最近的研究揭示了Rip1在干扰素调控因子7 （IRF-7）的泛素化和激活中的新调控作用，IRF-7是一种对I型干扰素产生至关重要的转录因子。因此，我们假设Rip1和潜在的多泛素化Rip1通过调节NF-kB和IRF-7活性介导抗病毒先天免疫反应。为了支持这一假设，我们将在体内测试Rip1缺陷是否会损害先天抗病毒反应，并将用多种病毒感染Rip1缺陷的巨噬细胞和树突状细胞，以确定Rip1如何调节IRF-7的转录活性（Aim 1）。TNF、Trif或rig - 1 /Mda5通路的激活刺激Rip1多泛素化，因此我们将测试病毒感染细胞中NF-kB和/或IRF-7的激活是否需要Rip1多泛素化，并将确定不能泛素修饰Rip1是否会导致先天抗病毒反应受损（目的2）。同样，我们对相关Rip1蛋白Rip2的研究发现，内源性Rip2在mdp刺激的巨噬细胞中多泛素化，提示Nod2通路受泛素调控。我们假设多泛素化的Rip2是nod2介导的NF-kB激活和先天抗菌免疫反应所必需的，并预测泛素失调有助于nod2相关的人类炎症性疾病。为了验证这一假设，我们将确定Rip2上的关键泛素位点，并将测试nod介导的NF-kB激活是否需要多泛素化Rip2，并将确定与人类炎症疾病相关的NOD2等位基因的表达如何影响Rip2的募集和多泛素化（Aim3）。总的来说，我们的研究表明，先天免疫反应是由泛素调节的，这提高了负责Rip蛋白泛素修饰的酶可能被靶向治疗感染性疾病或慢性炎症性疾病的可能性。公共卫生相关性：细菌和病毒被设计用来抵抗感染的宿主受体识别。这些受体通过产生具有抗菌和抗病毒活性的可溶性因子作出反应。我们的研究重点是Rip蛋白如何促进宿主对感染的反应，目标是在人类疾病中，Rip蛋白的活性可以根据需要被药物刺激或减弱。

DESCRIPTION (provided by applicant): The cytokine TNF, the pathogen recognition Toll-like receptors (TLRs) and the nucleotide binding oligomerization domain (NOD) proteins mediate host defense against infection in part by activating the transcription factor NF-kB. Our published work reveals Rip1 as a critical mediator of the TNF- and TLR3/4, Trif-dependent NF-kB pathways and the ubiquitin modification of Rip1 is essential for TNF- induced NF-kB activation and cytokine production. We find Rip1-deficient cells impaired in their type I interferon response to viral infection, revealing that Rip1 contributes to Rig-I/Mda5 anti-viral signaling. Our recent studies reveal a novel regulatory role for Rip1 in the ubiquitination and activation of the interferon regulatory factor 7 (IRF-7), a transcription factor critical for type I interferon production. Therefore, we hypothesize that Rip1 and potentially polyubiquitinated Rip1 mediate anti-viral innate immune responses by regulating NF-kB and IRF-7 activity. To support this hypothesis, we will test whether a Rip1-deficiency impairs innate anti-viral responses in vivo and will infect Rip1-deficient macrophages and dendritic cells with multiple classes of viruses to determine how Rip1 regulates the transcriptional activity of IRF-7 (Aim 1). Activation of the TNF, Trif or Rig-I/Mda5 pathways stimulates Rip1 polyubiquitination, hence we will test whether polyubiquitinated Rip1 is required for the activation of NF-kB and/or IRF-7 in virally infected cells and will determine whether an inability to ubiquitin modify Rip1 results in impaired innate anti-viral responses (Aim 2). Similarly, our studies on the related Rip1 protein Rip2 find endogenous Rip2 polyubiquitinated in MDP-stimulated macrophages, suggesting that the Nod2 pathway is ubiquitin-regulated. We hypothesize that polyubiquitinated Rip2 is required for Nod2-mediated NF-kB activation and for innate anti-bacterial immune responses and predict that ubiquitin deregulation contributes to NOD2-associated human inflammatory diseases. To test this hypothesis, we will identify the critical ubiquitin site on Rip2 and will test whether polyubiquitinated Rip2 is required for Nod-mediated NF-kB activation and will determine how expression of NOD2 alleles associated with human inflammatory diseases affect the recruitment and polyubiquitination of Rip2 (Aim3). Collectively, our studies suggest that innate immune responses are ubiquitin regulated, raising the possibility that the enzymes responsible for the ubiquitin modification of Rip proteins may be targeted therapeutically to treat infectious disease or chronic inflammatory disease. PUBLIC HEALTH RELEVANCE: Bacteria and viruses are recognized by host receptors designed to fight infection. These receptors respond by producing soluble factors that have anti-bacterial and anti-viral activity. Our research is focused on how Rip proteins contribute to host responses against infection with the goal that the activity of Rip proteins can be stimulated or attenuated by drugs as needed, in human disease.