Oxidative Cysteine Modification by Thiol Isomerases in Sickle Cell Disease

镰状细胞病中硫醇异构酶的氧化半胱氨酸修饰

• 批准号: 10505477
• 负责人: Moua Yang
• 金额: $ 16.45万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-30 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10505477
• 关键词: Adhesions; Antioxidants; Biological Assay; Biology; Blood Platelets; Carbon; Cell Adhesion; Cell-Cell Adhesion; Cells; Characteristics; Chemicals; Clinical; Clinical Research; Complement; Cysteine; Data; Disease; Disease model; Disulfides; Electron Transport; Electrons; Endothelial Cells; Environment; Event; Fetal Hemoglobin; Flavonoids; Glutamine; Goals; Hemoglobin; Hemoglobin concentration result; Hemorrhage; Hemostatic function; Injury; Isomerase; K-Series Research Career Programs; Lasers; Leukocytes; Mediating; Methods; Modeling; Modification; Monitor; Mus; Neutrophil Activation; Organ failure; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductase; P-Selectin; Patients; Phase; Platelet Activation; Positioning Attribute; Protein Disulfide Isomerase; Research; Risk; Role; Sickle Cell; Sickle Cell Anemia; Stress; Sulfhydryl Compounds; Sulfur; Techniques; Testing; Thrombosis; Thrombus; Training; Withdrawal; base; extracellular; hydroxyurea; imaging system; improved; in vivo; intravital microscopy; mouse model; neutrophil; oxidant stress; prevent; programs; thromboinflammation; treatment strategy; vaso-occlusive crisis

Project Summary/Abstract Vaso-occlusive events represent a major clinical burden in sickle cell disease (SCD). Vaso-occlusive events recur in patients despite current treatments, including the use of hydroxyurea to increase fetal hemoglobin and crizanlizumab that targets P-selectin for cellular adhesion. Oxidative stress in SCD increases the risk for vaso- occlusion and current anti-oxidative treatments, including L-glutamine, show efficacy in decreasing these events. However, antioxidants do not ameliorate vaso-occlusive crises. New treatment strategies for vaso-occlusion in sickle cell disease based on an improved understanding of the redox mechanisms are required. Thiol isomerases belong to a class of oxidoreductases that are secreted from platelets and endothelial cells and are required for thrombus formation. The archetypal thiol isomerase, protein disulfide isomerase (PDI), promotes thromboinflammation in SCD, is sensitive to the redox environment, and can be targeted with flavonoids such as isoquercetin. Our preliminary data that isoquercetin decreases cell-cell adhesion in SCD mice suggests that PDI could be a potential target for vaso-occlusion. However, the mechanism by which PDI promotes vaso- occlusion is unclear. This proposal will test the central hypothesis that thiol isomerases promote redox-sensitive vaso-occlusion through cysteine electron transferring events in sickle cell disease. We will evaluate redox stress- mediated vaso-occlusion in SCD in three integrated aims using cell and chemical biology approaches with murine models of the disease. In Aim 1, we will mechanistically examine the capacity of PDI to sense the redox environment in SCD to promote electron transfers in the form of cysteine disulfides. This aim will determine whether reduced or oxidized PDI promotes platelet and neutrophil activation in SCD by catalyzing electron withdrawal from their known redox targets. In Aim 2, we will transition our studies to evaluate the function of electron withdrawal mechanisms catalyze by thiol isomerases using intravital microscopy to observe thrombosis, hemostasis, and vaso-occlusion in SCD. We will also complement the studies by observing the function of thiol isomerase-mediated electron withdrawal on leukocyte cell-cell adhesion events in vivo. Lastly, Aim 3 will utilize carbon nucleophilic probes that tag specific cysteine sulfur oxoforms to probe the global function of electron transferring events in SCD. The probes will identify new targets of thiol isomerases in an unbiased manner in order to determine whether a characteristic set of cysteine disulfide scission or formation is required for vaso - occlusion. The probes will also identify mechanistically the role of cysteine electron transferring events on hemoglobin function for red blood cell sickling and leukocyte-mediated cell-cell adhesion for vaso-occlusive events. The K99 phase will focus on Aims 1 and 2 whereas the R00 phase will focus on Aim 3. The additional training afforded by this career development award will not only enable me to expand my skillsets, but will also uniquely position me to build an independent research program focused on oxidative cysteine modification in the redox-regulated vaso-occlusive events of SCD.

项目概要/摘要 血管闭塞事件是镰状细胞病（SCD）的主要临床负担。血管闭塞事件 尽管目前的治疗包括使用羟基脲来增加胎儿血红蛋白和 crizanlizumab 以 P-选择素为靶点进行细胞粘附。 SCD 中的氧化应激会增加血管疾病的风险 闭塞和当前的抗氧化治疗（包括 L-谷氨酰胺）在减少这些事件方面显示出功效。 然而，抗氧化剂并不能改善血管闭塞危机。血管闭塞的新治疗策略 镰状细胞病需要基于对氧化还原机制的更好理解。硫醇异构酶 属于一类由血小板和内皮细胞分泌的氧化还原酶，是 血栓形成。原型硫醇异构酶，蛋白质二硫键异构酶（PDI），促进 SCD 中的血栓炎症，对氧化还原环境敏感，可以用黄酮类化合物作为靶标，例如 如异槲皮素。我们的初步数据表明，异槲皮素可降低 SCD 小鼠的细胞间粘附力，表明 PDI 可能是血管闭塞的潜在目标。然而，PDI 促进血管生成的机制 遮挡不清楚。该提案将检验硫醇异构酶促进氧化还原敏感性的中心假设 镰状细胞病中半胱氨酸电子转移事件引起的血管闭塞。我们将评估氧化还原压力- 使用细胞和化学生物学方法以三个综合目标介导 SCD 中的血管闭塞 该疾病的小鼠模型。在目标 1 中，我们将机械地检查 PDI 感知氧化还原的能力 SCD 中的环境促进半胱氨酸二硫化物形式的电子转移。这个目标将决定 还原或氧化的PDI是否通过催化电子促进SCD中血小板和中性粒细胞的活化 从已知的氧化还原目标中撤出。在目标 2 中，我们将把我们的研究转向评估 电子撤回机制由硫醇异构酶催化，使用活体显微镜观察血栓形成， SCD 中的止血和血管闭塞。我们还将通过观察硫醇的功能来补充研究 异构酶介导的电子撤回对体内白细胞细胞间粘附事件的影响。最后，目标 3 将利用 标记特定半胱氨酸硫氧代物以探测电子整体功能的碳亲核探针 在 SCD 中传输事件。这些探针将以公正的方式识别硫醇异构酶的新靶标 为了确定血管是否需要半胱氨酸二硫化物裂解或形成的特征集 - 闭塞。这些探针还将从机械上识别半胱氨酸电子转移事件对 血红蛋白对红细胞镰状化的功能和白细胞介导的血管闭塞的细胞间粘附的功能 事件。 K99 阶段将重点关注目标 1 和 2，而 R00 阶段将重点关注目标 3。 该职业发展奖提供的培训不仅使我能够扩展我的技能，而且还将 使我能够建立一个独立的研究项目，重点关注氧化半胱氨酸修饰 SCD 的氧化还原调节血管闭塞事件。