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.

项目摘要/摘要 血管熟悉的事件代表了镰状细胞病（SCD）的主要临床负担。血管熟悉的事件 尽管患者当前治疗，包括使用羟基脲来增加胎儿血红蛋白和 靶向P-选择素的Crizanlizumab用于细胞粘附。 SCD中的氧化应激增加了血管的风险 包括L-谷氨酰胺在内的闭塞和当前的抗氧化处理在减少这些事件方面有效。 但是，抗氧化剂不会改善血管熟悉的危机。在 基于对氧化还原机制的了解，镰状细胞疾病是基于对氧化还原机制的提高的。硫醇异构酶 属于一类从血小板和内皮细胞分泌的氧化还原酶，需要 血栓形成。原型硫醇异构酶，蛋白二硫异构酶（PDI）促进 SCD中的血栓炎对氧化还原环境敏感，可以用类黄酮靶向这样 作为isoquercetin。我们的初步数据降低了SCD小鼠的细胞细胞粘附，这表明 PDI可能是血管封闭的潜在目标。但是，PDI促进血管的机制 闭塞尚不清楚。该建议将检验硫醇异构酶促进氧化还原敏感的中心假设 通过半胱氨酸电子转移事件在镰状细胞疾病中的气管批准。我们将评估氧化还原应力 - 使用细胞和化学生物学方法的三个综合目的的SCD中介导的血管牙合 该疾病的鼠模型。在AIM 1中，我们将机械学检查PDI感知氧化还原的能力 SCD中的环境以半胱氨酸二硫化物的形式促进电子传输。这个目标将决定 降低或氧化的PDI是否通过催化电子促进SCD中的血小板和中性粒细胞激活 退出已知的氧化还原靶标。在AIM 2中，我们将过渡我们的研究以评估 电子戒断机制使用浸润显微镜催化硫醇异构酶来观察血栓形成， SCD中的止血和血管封闭。我们还将通过观察硫醇的功能来补充研究 异构酶介导的电子在体内的白细胞细胞 - 细胞粘附事件上提取电子。最后，AIM 3将使用 碳亲核探针标记特异性半胱氨酸硫氧型以探测电子的全球功能 在SCD中转移事件。探针将以公正的方式识别硫醇异构酶的新靶 为了确定是否需要一组特征性的半胱氨酸二硫化物分裂或形成。 阻塞。探针还将从机械上识别半胱氨酸电子转移事件在 红细胞病态的血红蛋白功能和白细胞介导的细胞 - 细胞粘附量 事件。 K99阶段将重点放在目标1和2上，而R00阶段将专注于目标3。 这个职业发展奖提供的培训不仅可以使我扩大自己的技能，而且还将 独特地将我定位为建立一个独立的研究计划，以氧化性半胱氨酸修饰 SCD的氧化还原调节的血管熟悉事件。