Structural and mechanistic elucidation of non-canonical inflammasome signaling

项目总结/摘要 Caspase-4/-5/-11非典型炎性小体在多种炎症反应中起关键作用， 和感染性疾病，如败血症。目前的研究表明，caspase-4/-5/-11被激活， 直接感测细胞内微生物感染，例如脂多糖（LPS，也称为内毒素），或 内源性产物，如氧化磷脂（例如，OxPAPC），通过它们的N-末端半胱天冬酶 激活和募集结构域（CARD）和C-末端催化结构域。但 caspase-4/-5/-11如何识别其配体以及caspase-4/-5/-11如何被激活的分子机制 在结合配体时仍然未知。在这个建议中，我们的目标是阐明非典型的结构基础， 通过表征胱天蛋白酶-4/-5/-11和配体- LPS之间的相互作用来研究炎性体信号传导， OxPAPC，并确定与配体复合的caspase/-4/-5/-11的高分辨率结构。我们 结构发现将为脓毒症和其他非典型炎性小体提供新的治疗策略， 相关疾病。我们提出了三个具体的目标，以实现我们的目标：1）生化表征的 caspase-4/-5/-11和LPS之间的相互作用-包括基本结构元件的鉴定 在LPS分子和caspase-4/-5/-11 CARD上的caspase-4/-5/-11所需的关键残基中 2）确定半胱天冬酶-4/-5/-11 CARD在其失活形式和活化形式下的高分辨率结构。 3）研究caspase-4/-11与OxPAPC的相互作用，并测定其与LPS的相互作用。 与OxPAPC复合的胱天蛋白酶-4/-11催化结构域的高分辨率结构。我们将继续努力， 目的是使用尖端的实验方法，包括生物化学和生物物理表征，X射线， 晶体学、质谱、电子显微镜和细胞实验。拟议的研究将 显著扩展了我们目前对非经典炎性体信号传导机制的认识， 为设计新的策略来控制非- 典型的炎性小体，更好地治疗相关疾病。

Project Summary/Abstract Caspase-4/-5/-11 non-canonical inflammasomes have been known to play pivotal roles in various inflammatory and infectious diseases, such as sepsis. Current studies demonstrate that caspase-4/-5/-11 are activated by directly sensing intracellular microbial infections, such as lipopolysaccharide (LPS, also known as endotoxin), or endogenous products, such as oxidized phospholipids (e.g., OxPAPC), through their N-terminal caspase activation and recruitment domains (CARDs) and C-terminal catalytic domains, respectively. However, the molecular mechanisms of how caspase-4/-5/-11 recognize their ligands and how caspase-4/-5/-11 are activated upon binding ligands remain unknown. In this proposal, we aim to elucidate the structural basis of non-canonical inflammasome signaling by characterizing the interactions between caspase-4/-5/-11 and ligands - LPS and OxPAPC, and determining the high-resolution structures of caspase/-4/-5/-11 in complex with ligands. Our structural findings will provide new therapeutic strategies for sepsis and other non-canonical inflammasome- associated diseases. We propose three specific aims to achieve our goal: 1) Biochemical characterization of the interactions between caspase-4/-5/-11 and LPS-including the identification of the essential structural elements in the LPS molecule and key residues on caspase-4/-5/-11 CARDs that are required for caspase-4/-5/-11 activation; 2) Determine high-resolution structures of caspase-4/-5/-11 CARDs both in their inactive form and in complex with LPS; 3) Characterize the interactions between caspase-4/-11 and OxPAPC, and determine the high resolution structures of caspase-4/-11 catalytic domains in complex with OxPAPC. We will pursue these aims using cutting-edge experimental approaches including biochemical and biophysical characterization, X-ray crystallography, mass spectrometry, electron microscopy, and cellular experiments. The proposed studies will significantly expand our current knowledge on the mechanisms of non-canonical inflammasome signaling, and provide rationale and a structural basis for designing novel strategies to control the activation of the non- canonical inflammasome for better treatment of related diseases.