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

基本信息

批准号：
10570903
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
项目类别：
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/10570903
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 Communicable Diseases Complex Cryoelectron Microscopy Disease Electron Microscopy Elements Endotoxins Filament Future Goals Gram-Negative Bacteria Hospitalization 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 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 biophysical properties design drug development experience experimental study improved in vivo inflammatory marker insight microbial novel strategies novel therapeutic intervention oxidized phosphatidyl choline programs receptor reconstitution recruit sensor septic patients trial design

项目摘要

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.

项目概要/摘要 Caspase-4/-5/-11 非典型炎症小体已知在多种炎症中发挥关键作用和传染病，例如败血症。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，例如氧化磷脂（例如 OxPAPC），通过其 N 端半胱天冬酶分别是激活和招募结构域 (CARD) 和 C 端催化结构域。然而， 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结合配体的情况仍然未知。在这个提案中，我们的目标是阐明非规范的结构基础通过表征 caspase-4/-5/-11 和配体（LPS 和）之间的相互作用来研究炎症小体信号传导 OxPAPC，并确定 caspase/-4/-5/-11 与配体复合物的高分辨率结构。我们的结构发现将为脓毒症和其他非典型炎症小体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） caspase-4/-5/-11 和 LPS 之间的相互作用 - 包括基本结构元件的鉴定 LPS 分子中以及 caspase-4/-5/-11 CARD 上 caspase-4/-5/-11 所需的关键残基中激活; 2) 确定 caspase-4/-5/-11 CARD 的非活性形式和非活性形式的高分辨率结构与 LPS 复合； 3)表征caspase-4/-11和OxPAPC之间的相互作用，并确定与 OxPAPC 复合的 caspase-4/-11 催化结构域的高分辨率结构。我们将追求这些目标是使用尖端实验方法，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。拟议的研究将显着扩展了我们目前对非典型炎症小体信号传导机制的了解，并且为设计控制非激活的新策略提供理论基础和结构基础规范炎症小体，以更好地治疗相关疾病。