Structural and mechanistic elucidation of non-canonical inflammasome signaling

非典型炎症小体信号传导的结构和机制阐明

• 批准号: 10179803
• 负责人: Jianbin Ruan
• 金额: $ 41万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-17 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10179803
• 关键词: Adult; Bacterial Infections; Binding; Biochemical; Biological Process; C-terminal; CASP1 gene; Caspase; Catalytic Domain; Cause of Death; Cell Death; Cell membrane; Child; Cleaved cell; Communicable Diseases; Complex; Cryoelectron Microscopy; Disease; Electron Microscopy; Elements; Endotoxins; Filament; Future; Goals; Gram-Negative Bacteria; Human; Immunologic Surveillance; In Vitro; Infection; Inflammasome; Inflammatory; Intervention; Knowledge; Ligand Binding; Ligands; Lipids; Lipopolysaccharides; Mass Spectrum Analysis; Membrane; Modeling; Molecular; Multiprotein Complexes; Mus; Mutagenesis; N-terminal; Newborn Infant; Oxides; Patients; Phase; Phospholipids; Play; Process; Research; Resolution; Role; Sepsis; Sequence Alignment; Signal Pathway; Signal Transduction; Site; Structure; Testing; Therapeutic; VDAC1 gene; Variant; X-Ray Crystallography; base; biophysical properties; design; drug development; experience; experimental study; improved; in vivo; inflammatory marker; insight; microbial; novel strategies; novel therapeutic intervention; programs; receptor; reconstitution; recruit; sensor; septic patients; trial design

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.

项目摘要/摘要 已知caspase-4/-5/-11非典型性炎症体在各种炎性反应中起关键作用。 以及感染性疾病，如败血症。目前的研究表明，caspase-4/-5/-11是由 直接感知细胞内微生物感染，如脂多糖(LPS，也称为内毒素)，或 内源性产物，如氧化磷脂(如OxPAPC)，通过其N-末端半胱氨酸氨基转移酶 激活和募集结构域(CARD)和C末端催化结构域。然而， Caspase-4/-5/-11识别配体和caspase-4/-5/-11被激活的分子机制 在结合时，配体仍然未知。在这项建议中，我们旨在阐明非正则的结构基础 通过表征caspase-4/-5/-11与配体-内毒素和 OxPAPC，并测定了caspase/-4/-5/-11与配体形成的络合物的高分辨结构。我们的 结构发现将为脓毒症和其他非典型炎症性疾病提供新的治疗策略。 相关疾病。我们提出了三个具体目标来实现我们的目标：1)生物化学表征 Caspase-4/-5/-11与内毒素的相互作用--包括基本结构元件的鉴定 在caspase-4/-5/-11所需的内毒素分子和caspase-4/-5/-11卡上的关键残基中 激活；2)确定caspase-4/-5/-11卡的非活性形式和非活性形式的高分辨率结构 3)研究caspase-4/-11与OxPAPC的相互作用，并确定 Caspase-4/-11催化结构域在OxPAPC络合物中的高分辨结构我们将致力于实现这些目标 旨在使用尖端实验方法，包括生化和生物物理表征、X射线 结晶学、质谱学、电子显微镜和细胞实验。拟议的研究将 显著扩展我们目前关于非典型炎症体信号机制的知识，以及 为设计新的策略来控制非脑组织的激活提供理论基础和结构基础 用于更好地治疗相关疾病的典型炎症性小刀。