Negative Regulation of NLRP1-inflammasomes by the linear ubiquitin assembly compl

描述(申请人提供)：病原体识别受体(PRRs)的基本任务是启动对入侵病原体的即时炎症反应，以快速控制感染；然而，PRRs的不适当或长期激活会导致免疫缺陷、自身免疫和严重的组织损伤。由于PRR家族中的许多成员NLR(NOD样受体)和RLR(RIG-I样受体)与自身炎症性疾病有关，因此研究这些蛋白的负调控因子以了解现有的逆转PRR激活的机制将尤为重要。在我们最近的工作中，我们已经确定了RLR家族PRRs的一个主要负调控因子，证明线性泛素化组装复合体(LUBAC)是RIG-I的关键负调节因子(Inn KS等人，分子细胞，在出版社)。我们已经证明，LUBAC的HOIL-1L亚基通过靶向RIG-I的正调控因子线性泛素化降解和直接与RIG-I结合来阻止RIG-I的激活。特别是，我的初步数据表明，HOIL-1L还与NLRP1(Nacht LRR和PYD结构域包含蛋白1)结合，NLRP1是NLR家族PRRs的成员，它在细胞质中的配体结合时进行寡聚，形成炎症体复合体，激活促炎症caspase 1和细胞因子IL-1b。LUBAC与PRRs的RLR和NLR家族成员之间的相互作用表明，LUBAC是PRRs的主要调节者；然而，与NLRP1相互作用的生物学后果尚不清楚。为了解决这一知识差距，我的目标是测试LUBAC通过线性泛素化靶向NLRP1降解来负向调节NLRP1介导的炎症的假设，这将在三个特定的目标中进行测试。在第一个目标中，我将确定NLRP1是否为LUBAC泛素化的底物。在第二个目的中，我将确定HOIL-1L/NLRP1相互作用是否通过HOIL-1L的过度表达、突变和敲除来负向调节NLRP1-炎症体诱导的细胞培养信号。第三个目的是在HOIL-1L-/-小鼠体内检测NLR家族成员的表达和NLRP1介导的炎症反应，以确定LUBAC在体内的调节作用。此外，将评估与白癜风、I型糖尿病和Addison自身免疫性疾病易感性密切相关的NLRP1 L115H等位基因的HOIL-1L结合，以确定NLRP1/HOIL-1L相互作用对人类疾病分子基础的意义。总的来说，这三个目标的实验将检查HOIL-1L和NLRP1之间相互作用的意义和后果，以解决我们对炎症的负面调控的理解差距，并为NLRP1介导的疾病的潜在分子机制提供新的见解。由于NLRP1是NLR家族中的20多个蛋白质之一，参与了广泛的自体炎症疾病，我的工作可能更广泛地解决其他NLR的调节，并为操纵NLR-炎症体激活来治疗自体炎症疾病的疗法的设计提供信息。

DESCRIPTION (provided by applicant): The fundamental task of pathogen recognition receptors (PRRs) is to initiate an immediate inflammatory response against invading pathogens to quickly control infection; however, inappropriate or prolonged activation of PRRs leads to immunodeficiency, autoimmunity, and severe tissue damage. Since many members of the NLR (Nod-like receptor) and RLR (RIG-I-like receptor) families of PRRs have been implicated in autoinflammatory diseases, it will be especially important to examine the negative regulators of these proteins to understand the existing mechanisms for reversing PRR activation. A primary negative regulator of the RLR family of PRRs has been identified in our recent work demonstrating that the linear ubiquitination assembly complex (LUBAC) is a critical negative regulator of RIG-I (Inn KS, et al, Molecular Cell, In Press). We have shown that the HOIL-1L subunit of LUBAC blocks RIG-I activation by targeting a positive regulator of RIG-I for degradation by linear ubiquitination and by directly binding to RIG-I. Particularly, my preliminary data demonstrates that HOIL-1L also binds NLRP1 (NACHT LRR and PYD domain-containing protein 1), a member of the NLR family of PRRs, which oligomerizes upon ligand binding in the cytoplasm to form 'inflammasome' complexes to activate proinflammatory caspase 1 and cytokine IL- 1b. The interaction between LUBAC and members of both the RLR and NLR families of PRRs suggests that LUBAC is a master regulator of PRRs; however, the biological consequences of the interaction with NLRP1 are unknown. To address this gap in knowledge, I aim to test the hypothesis that LUBAC negatively regulates NLRP1- mediated inflammation by targeting NLRP1 for degradation via linear ubiquitination, which will be tested in three specific aims. In the first aim, I will determine whether NLRP1 is a substrate for ubiquitination by LUBAC. In the second aim, I will establish whether the HOIL-1L/NLRP1 interaction negatively regulates NLRP1-inflammasome induced signaling in cell culture by HOIL-1L overexpression, mutation, and knockdown. In the third aim, the expression of NLR family members and NLRP1-mediated inflammation will be examined in the HOIL-1L-/- mouse to determine the regulatory role of LUBAC in vivo. Additionally, the NLRP1 L115H allele strongly associated with vitiligo, type I diabetes, and Addison's autoimmune disease susceptibility will be assessed for HOIL-1L binding to determine significance of the NLRP1/HOIL-1L interaction for the molecular basis of human diseases. Collectively, the experiments in all three aims will examine the significance and consequences of the interaction between HOIL-1L and NLRP1 to address the gap in our understanding of the negative regulation of inflammation and provide new insight for the underlying molecular mechanisms of NLRP1-mediated diseases. Since NLRP1 is one of over 20 proteins in the NLR family of PRRs, which have been implicated in a wide range of autoinflammatory diseases, my work may more broadly address the regulation of other NLRs and inform the design of therapeutics that manipulate NLR-inflammasome activation to treat autoinflammatory disease.