Biochemical and structural analysis of the NLRP3 inflammasome

哺乳动物拥有一个非常有效和复杂的免疫系统来对抗威胁它们身体健康的病原体。炎性小体是一种胞质多蛋白复合物，在对多种病原体、组织损伤和其他有害刺激的反应中形成，以触发先天免疫反应。nod样受体（NLRs）家族的成员感知这些病原体和危险相关的分子模式，从而导致它们的激活和寡聚化。NLRP3可能是最重要的炎症体受体蛋白，因为它能感知多种刺激，并参与多种自身炎症性疾病。然而，炎性小体功能的结构基础和分子从自抑制状态到完全激活状态的转变尚不清楚。在前期工作中，我们从杆状病毒感染的昆虫细胞中纯化了人全长野生型NLRP3蛋白，使其具有均匀性。出乎意料的是，该蛋白以1.15 MDa的大小被洗脱，这表明一个大的低聚物组装。我们利用单颗粒冷冻电镜（cryo-EM），以3.9 Ang的分辨率测定了NLRP3与细胞因子释放抑制药物CRID3结合时失活状态下的结构。adp结合的NLRP3是由交织在一起的LRR结构域的同型二聚体组成的十聚体，它们作为五聚体背对背组装。NACHT结构域位于这个球形结构的顶轴。我们现在想解决两个主要目标：(1)低聚NLRP3复合物在活性状态下的低温电镜结构测定，以及(2)通过生化手段对NLRP3不同构象状态的功能表征。除了最先进的冷冻电镜成像和结构重建方法，研究目标将在八个工作包中实现，包括将NLRP3十聚体转化为活性状态，激酶NEK7对NLRP3构象状态的影响，建立NLRP3刺激的ASC灯丝种子试验，膜相关NLRP3物种的结构和功能表征，ATP水解分析，以及NLRP3的结构和功能。表征疾病突变和自身抑制，与模型膜的相互作用，以及监测NLRP3体外磷酸化后的构象变化。这些分析将有助于阐明NLRP3的结构-功能关系，并为这种具有重要医学意义的炎性体的构象状态提供见解。

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.