Development of an in vitro system to study heme-induced caspase activation

开发用于研究血红素诱导的半胱天冬酶激活的体外系统

• 批准号: 10723823
• 负责人: Lisa Bouchier-Hayes
• 金额: $ 8.03万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723823
• 关键词: Algorithms; Amino Acids; Apical; Bacteria; Binding; Biological Assay; Biophysics; CASP1 gene; CASP5 gene; Caspase; Catalytic Domain; Cells; Clinical; Communicable Diseases; Complex; Data; Development; Dimerization; Disease; Drug Design; Drug Screening; Erythrocytes; Functional disorder; Future; Genetic Transcription; Goals; Heme; Hemolysis; Hemorrhage; Immune System Diseases; In Vitro; Incubated; Individual; Inflammasome; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interleukin-1 beta; Length; Measures; Mediating; Mission; Molecular; Multiprotein Complexes; Mutate; National Institute of Allergy and Infectious Disease; Organ; Outcome; Pain; Pathway interactions; Pattern; Pharmaceutical Preparations; Plant Extracts; Proteins; Public Health; Radiolabeled; Recombinants; Regulation; Reporting; Research; Sepharose; Sepsis; Sickle Cell Anemia; Site-Directed Mutagenesis; Source; Structure; System; Testing; Translating; Work; biophysical properties; candidate identification; cell type; cofactor; cytokine; experimental study; extracellular; improved; in vitro Assay; insight; monomer; prevent; reconstitution; response; scale up

PROJECT SUMMARY Excessive intravascular hemolysis of red blood cells (RBCs) leads to numerous complications associated with uncontrolled inflammation including pain, vaso-occlusion, and organ damage that underlie the pathophysiology of several disease states including sepsis, intravascular hemorrhage, and sickle cell disease (SCD). The primary mediator of this inflammation is heme released from damaged RBCs. The applicant’s goup recently reported that heme activates the inflammatory caspases, caspase-1, caspase-4, and caspase-5 to coordinate the cleavage and release of the apical pro-inflammatory cytokine IL-1β. Importantly, unlike caspase-1, caspase-4 and caspase-5 are activated independently of inflammasome assembly. Thus, heme is one of the first examples of a DAMP (damage associated molecular pattern) that activates the non-canonical inflammasome pathway. The applicant has shown that heme binds caspase-4 and caspase-5 and induces oligomerization of these caspases. The central hypothesis is that heme directly binds to caspase-4 and caspase-5 to induce oligomerization and activation. The hypothesis will be tested by the following Aims: 1) to determine the biophysical composition of heme-induced inflammatory caspase complexes and 2) to identify the protein components required for heme-induced inflammatory caspase activation. Under the first Aim full length recombinant caspase proteins will be produced and the ability of heme to bind caspases and to induce caspase oligomerization and cleavage will be assessed. Under the second aim the amino acid residues required for heme binding, oligomerization and cleavage will be identified as well as the type of cytosolic factors, if any, contribute heme-induced caspase activation. This work will provide an in vitro assay that will be the basis for future rational drug design approaches and a source of purified caspases that can be used for future studies to determine the structures of the heme-induced caspase complexes. Understanding the mechanisms of inflammatory caspase activation with provide justification for targeting these proteins to improve clinical outcomes in hemolytic conditions.

项目摘要 红细胞（RBC）过度血管内溶血导致许多并发症 不受控制的注射包括疼痛，血管咬合和器官损伤，这些损害是病理生理学的基础 在几种疾病状态中，包括败血症，血管内出血和镰状细胞病（SCD）。主要 这种炎症的介体从损坏的RBC中释放出血红素。申请人的GOUP最近报道了 血红素激活炎症性caspase，caspase-1，caspase-4和caspase-5，以协调 清洁和顶端促炎细胞因子IL-1β的释放。重要的是，与caspase-1，caspase-4不同 和caspase-5独立于炎性体组装而激活。那是血红素是第一个例子之一 激活非典型炎性体途径的潮湿（损伤相关的分子模式） 申请人表明，血红素结合caspase-4和caspase-5并诱导这些寡聚化 caspase。中心假设是血红素直接与caspase-4和caspase-5结合以诱导 寡聚和激活。该假设将通过以下目的检验：1）确定 血红素诱导的炎症性胱天蛋白酶复合物的生物物理组成和2）鉴定蛋白质 血红素诱导的炎症胱天蛋白酶激活所需的成分。在第一个目标下全长 将产生重组胱天冬酶蛋白，以及血红素结合胱天蛋白酶并诱导caspase的能力 将评估寡聚化和裂解。在第二个目标下，氨基酸保留了血红素所需的 将确定结合，寡聚和裂解以及胞质因子的类型（如果有的话） 血红素诱导的胱天蛋白酶激活。这项工作将提供体外评估，这将成为未来理性的基础 药物设计方法和纯化的胱天蛋白酶的来源，可用于以后的研究来确定 血红素诱导的caspase复合物的结构。了解炎症性胱天蛋白酶的机制 激活具有靶向这些蛋白质以改善溶血疗法的临床结局的合理性 状况。