Biochemical and structural analysis of the NLRP3 inflammasome

NLRP3炎症小体的生化和结构分析

• 批准号: 504829198
• 负责人: Professor Dr. Matthias Geyer
• 金额: --
• 依托单位: Institut für Strukturbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/504829198?language=en
• 关键词: Biochemical; structural; analysis; NLRP3; inflammasome

Mammals possess a very effective and complex immune system to counteract pathogens threatening their physical health. Inflammasomes are cytosolic multi-protein complexes that form in response to a wide range of pathogens, tissue damage and other harmful stimuli in order to trigger innate immune responses. Members of the family of NOD-like receptors (NLRs) sense these pathogen and danger associated molecular patterns, which leads to their activation and oligomerization. NLRP3 is possibly the most prominent inflammasomal receptor protein as it senses a broad variety of stimuli and is involved in multiple autoinflammatory diseases. Yet, the structural basis of inflammasome function and the molecular transition from an autoinhibited state into a fully activated state is only poorly understood. In preliminary work we have purified human full length, wild type NLRP3 protein from baculo virus infected insect cells to homogeneity. Unexpectedly, the protein elutes at a size of 1.15 MDa indicating a large oligomeric assembly. Using single particle cryo-EM, we determined the structure of NLRP3 in the inactive state bound to the cytokine release inhibitory drug CRID3 at 3.9 Ang resolution. ADP-bound NLRP3 is a decamer composed of homodimers of intertwined LRR domains that assemble back-to-back as pentamers. The NACHT domain is located at the apical axis of this spherical structure. We now want to address two major aims: (1) The cryo-EM structure determination of the oligomeric NLRP3 complex in an active state, and (2) the functional characterization of the different conformational states of NLRP3 by biochemical means. Besides state of the art cryo-EM imaging and structure reconstruction methods, the research aims shall be achieved in eight work packages, which includes conversion of the NLRP3 decamer into an active state, implications of the kinase NEK7 on the conformational states of NLRP3, the set-up of an NLRP3-stimulated ASC filament seeding assay, the structural and functional characterization of a membrane associated NLRP3 species, analysis of ATP hydrolysis, characterization of disease mutations and autoinhibition, the interaction with model membranes, and the monitoring of conformational changes upon in vitro phosphorylation of NLRP3. These analyses will help elucidating the structure–function relationship in NLRP3 and provide insights into the conformational states of this medicinally important inflammasome.

哺乳动物有潜在的一种非常有效且复杂的免疫系统来抵消威胁其身体健康的病原体。炎症是胞质多蛋白质复合物，响应广泛的病原体，组织损伤和其他有害刺激，以触发先天的免疫反应。 NOD样受体（NLR）家族的成员感知了这些病原体和舞蹈家相关的分子模式，从而导致它们的激活和寡聚化。 NLRP3可能是最突出的炎性症受体蛋白，因为它感觉到了各种刺激，并且参与了多种自发疾病。然而，炎症功能的结构基础和从自身抑制状态到完全激活状态的分子过渡仅被鲜为人知。在初步工作中，我们已经纯化了人类全长，野生型NLRP3蛋白，从baculo病毒感染的昆虫细胞到同质性。出乎意料的是，蛋白质洗脱量为1.15 mDa，表明较大的寡聚组件。使用单个粒子冷冻EM，我们确定了与细胞因子释放抑制性药物CRID3在3.9 ANG分辨率下结合的NLRP3的结构。 ADP结合的nlrp3是一个decamer，由互联的LRR域的同型二聚体组成，它们是五角星的背对背组装。 NACHT结构域位于该球形结构的顶端轴上。现在，我们想解决两个主要目的：（1）活性状态下低聚物NLRP3复合物的冷冻EM结构测定，以及（2）通过生化手段对NLRP3的不同浓度态的功能表征。除了艺术的冷冻成像和结构重建方法外，该研究的目的还应在八个工作包中实现，其中包括将NLRP3 Decamer转换为活跃状态，激酶NEK7在NLRP3会议上的含义，在NLRP3会议上，NLRP3刺激的Ascrament Asseating Assay和功能分析的结构和功能的分析，是A的结构和功能的分析。水解，疾病突变和自身抑制作用的表征，与模型膜的相互作用以及NLRP3体外磷酸化后构象变化的监测。这些分析将有助于阐明NLRP3中的结构 - 功能关系，并提供对这种具有药物重要炎症体的构象状态的见解。