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

项目摘要 红细胞（RBC）的过度血管内溶血导致与以下相关的许多并发症： 不受控制的炎症，包括疼痛、血管闭塞和病理生理学基础的器官损伤 包括败血症、血管内出血和镰状细胞病（SCD）在内的几种疾病状态。主 这种炎症的介质是从受损的红细胞释放的血红素。申请人的小组最近报告说， 血红素激活炎性半胱天冬酶，半胱天冬酶-1，半胱天冬酶-4和半胱天冬酶-5， 顶端促炎细胞因子IL-1β的裂解和释放。重要的是，与caspase-1不同，caspase-4 和胱天蛋白酶-5的活化独立于炎性小体组装。因此，血红素是最早的例子之一， DAMP（损伤相关分子模式）激活非经典炎症体途径。 申请人已经表明，血红素结合半胱天冬酶-4和半胱天冬酶-5，并诱导这些半胱天冬酶的寡聚化。 半胱天冬酶。中心假设是血红素直接与caspase-4和caspase-5结合， 寡聚化和活化。假设将通过以下目的进行检验：1）确定 血红素诱导的炎性半胱天冬酶复合物的生物物理组成和2）鉴定蛋白质 血红素诱导的炎症性半胱天冬酶激活所需的组分。在第一个目标下全长 将产生重组半胱天冬酶蛋白，并且血红素结合半胱天冬酶和诱导半胱天冬酶的能力降低。 将评估寡聚化和裂解。在第二个目标下，血红素所需的氨基酸残基 结合、寡聚化和裂解以及胞质因子的类型（如果有的话）将被鉴定， 血红素诱导的半胱天冬酶激活。这项工作将提供一个体外测定，这将是未来合理的基础。 药物设计方法和纯化的半胱天冬酶的来源，可用于未来的研究，以确定 血红素诱导的caspase复合物的结构。了解炎症性caspase的机制 激活为靶向这些蛋白质以改善溶血性疾病的临床结局提供了依据。 条件

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.