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

描述（由申请人提供）：病原体识别受体（PRRs）的基本任务是启动针对入侵病原体的即时炎症反应，以快速控制感染；然而，不当或长时间的PRRs激活会导致免疫缺陷、自身免疫和严重的组织损伤。由于PRRs的NLR （nod样受体）和RLR （rig - i样受体）家族的许多成员都与自身炎症性疾病有关，因此研究这些蛋白的负调节因子以了解逆转PRR激活的现有机制将变得尤为重要。我们最近的工作已经确定了PRRs RLR家族的一个主要负调控因子，表明线性泛素化组装复合体（LUBAC）是rig - 1的一个关键负调控因子（Inn KS等，Molecular Cell, in Press）。我们已经证明，LUBAC的HOIL-1L亚基通过靶向rig -1的正调节因子，通过线性泛素化和直接结合rig -1来阻止rig -1的激活。特别是，我的初步数据表明，HOIL-1L还结合NLRP1 (NACHT LRR和PYD结构域蛋白1)，NLRP1是PRRs NLR家族的成员，它在细胞质中结合配体后寡聚形成“炎性体”复合物，激活促炎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的主要调节剂；然而，与NLRP1相互作用的生物学后果尚不清楚。为了解决这一知识空白，我的目标是验证LUBAC通过线性泛素化靶向NLRP1降解来负调控NLRP1介导的炎症的假设，这将在三个具体目标中进行测试。在第一个目标中，我将确定NLRP1是否是LUBAC泛素化的底物。在第二个目标中，我将通过HOIL-1L过表达、突变和敲低来确定HOIL-1L/NLRP1相互作用是否负调控NLRP1炎性小体诱导的细胞培养信号。在第三个目标中，我们将在HOIL-1L-/-小鼠中检测NLR家族成员的表达和nlrp1介导的炎症，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等位基因与HOIL-1L结合的情况，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，以解决我们对炎症负调控的理解空白，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PRRs NLR家族中20多种蛋白中的一种，这些蛋白与广泛的自身炎症性疾病有关，因此我的工作可能更广泛地解决其他NLR的调控问题，并为设计操纵NLR炎性小体激活来治疗自身炎症性疾病提供信息。

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.