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

线性泛素组装体对 NLRP1 炎症小体的负调控

• 批准号: 8202704
• 负责人: MARY RODGERS
• 金额: $ 4.84万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8202704
• 关键词: Address; Alleles; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biological; Caspase-1; Cell Culture Techniques; Cells; Complex; Crohn' s disease; Cytoplasm; Data; Disease; Disease susceptibility; Etiology; Family; Family member; Goals; Gout; Immune; Immune System Diseases; Immune response; Immunologic Deficiency Syndromes; In Vitro; Infection Control; Inflammation; Inflammatory; Inflammatory Response; Insulin-Dependent Diabetes Mellitus; Invaded; Knowledge; Ligand Binding; Ligands; Lupus; Maps; Measures; Mediating; Molecular; Mus; Mutation; Protein Family; Proteins; Receptor Activation; Regulation; Research; Role; Signal Transduction; Testing; Therapeutic; Therapeutic Intervention; Tissues; Ubiquitin; Ubiquitination; Vitiligo; Work; base; cytokine; design; human disease; improved; in vivo; innovation; insight; member; mutant; novel; novel therapeutics; overexpression; pathogen; prevent; procaspase-1; receptor; research study; response; ubiquitin-protein ligase; weapons

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. PUBLIC HEALTH RELEVANCE: Inflammation is an essential component of the immune response against invading pathogens that must be tightly regulated to prevent inappropriate responses, which have been implicated in many autoinflammatory immune diseases. It is critical to fully understand natural negative regulation of inflammation to treat these diseases and my research will directly test whether the linear ubiquitin assembly complex (LUBAC), is a novel negative regulator of inflammation. By studying the mechanism and biological relevance of this potential negative regulator of inflammation in vitro and in vivo, we will improve our understanding of several autoinflammatory diseases and provide a basis for designing new therapeutic strategies.

描述（由申请人提供）：病原体识别受体（PRR）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，PRR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的NLR（Nod样受体）和RLR（RIG-I样受体）家族的许多成员与自身炎症性疾病有关，因此检查这些蛋白质的负调节因子以了解逆转PRR激活的现有机制将尤为重要。在我们最近的工作中已经鉴定了PRR的RLR家族的主要负调节因子，证明线性泛素化组装复合物（LUBAC）是RIG-I的关键负调节因子（Inn KS等人，Molecular Cell，In Press）。我们已经表明，HOIL-1 L亚基LUBAC通过靶向RIG-I的正调节因子，通过线性泛素化降解和直接结合RIG-I来阻断RIG-I的活化。特别是，我的初步数据表明，HOIL-1 L还结合NLRP 1（NACHT LRR和PYD结构域蛋白1），NLR家族PRR的成员，其在细胞质中结合配体后寡聚化形成“炎性小体”复合物，以激活促炎性半胱天冬酶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的主调节因子;然而，与NLRP 1相互作用的生物学后果尚不清楚。为了解决这一知识缺口，我的目标是测试的假设，LUBAC负调控NLRP 1介导的炎症通过线性泛素化降解NLRP 1，这将在三个特定的目标进行测试。在第一个目标中，我将确定NLRP 1是否是LUBAC泛素化的底物。在第二个目标中，我将确定HOIL-1 L/NLRP 1相互作用是否通过HOIL-1 L过表达、突变和敲低负调控细胞培养中NLRP 1-炎性体诱导的信号传导。在第三个目的中，将在HOIL-1 L-/-小鼠中检查NLR家族成员的表达和NLRP 1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强烈相关的NLRP 1 L115 H等位基因的HOIL-1 L结合，以确定NLRP 1/HOIL-1 L相互作用对人类疾病分子基础的意义。总的来说，所有三个目标的实验将检查HOIL-1 L和NLRP 1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中的差距，并为NLRP 1介导的疾病的潜在分子机制提供新的见解。由于NLRP 1是PRRs的NLR家族中的20多种蛋白质之一，这些蛋白质与广泛的自身炎症性疾病有关，因此我的工作可能会更广泛地解决其他NLR的调节问题，并为设计操纵NLR-炎性体激活以治疗自身炎症性疾病的治疗方法提供信息。 公共卫生关系：炎症是针对入侵病原体的免疫应答的重要组成部分，必须严格调节以防止不适当的应答，这与许多自身炎性免疫疾病有关。充分理解炎症的自然负调节对治疗这些疾病至关重要，我的研究将直接测试线性泛素组装复合物（LUBAC）是否是一种新型的炎症负调节因子。通过在体外和体内研究这种潜在的炎症负调节因子的机制和生物学相关性，我们将提高对几种自身炎症性疾病的理解，并为设计新的治疗策略提供基础。