Oxidative Cysteine Modification by Thiol Isomerases in Sickle Cell Disease

项目摘要/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 尽管目前的治疗方法包括使用羟基尿素来增加胎儿血红蛋白和 以P-选择素为细胞黏附靶点的Crizanlizumab。SCD中的氧化应激增加了血管病变的风险 血管闭塞和目前的抗氧化治疗，包括L-谷氨酰胺，在减少这些事件方面显示出有效性。 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 镰状细胞病需要建立在对氧化还原机制的更好理解的基础上。硫醇异构酶 属于一类氧化还原酶，由血小板和内皮细胞分泌，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中的血栓性炎症对氧化还原环境很敏感，可以通过类黄酮类药物进行靶向治疗。 就是异槲皮素。我们的初步数据表明，异槲皮素可降低SCD小鼠的细胞间黏附 PDI可能成为血管闭塞的潜在靶点。然而，PDI促进血管紧张素转换酶的机制。 闭塞情况尚不清楚。这一提议将检验硫醇异构酶促进氧化还原敏感性这一中心假设。 镰状细胞病中半胱氨酸电子传递事件引起的血管闭塞。我们将评估氧化还原压力- 应用细胞和化学生物学方法在SCD的三个综合目标中介导血管闭塞 这种疾病的小鼠模型。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。这一目标将决定 还原或氧化PDI通过催化电子促进SCD中血小板和中性粒细胞的激活 从他们已知的氧化还原目标撤出。在目标2中，我们将把我们的研究转移到评估 用活体显微镜观察硫醇异构酶催化的电子撤回机制 SCD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除对体内白细胞-细胞黏附事件的影响。最后，目标3将利用 标记特定半胱氨酸硫氧化物的碳亲核探针探测电子的整体功能 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点 以确定血管是否需要半胱氨酸二硫化物断裂或形成的特征集。 遮挡。这些探针还将从机械上识别半胱氨酸电子转移事件在 血红蛋白对红细胞的镰刀作用和白细胞介导的细胞-细胞黏附治疗血管闭塞 事件。K99阶段将侧重于目标1和2，而R00阶段将侧重于目标3。 这个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 独特的定位让我建立一个独立的研究计划，专注于氧化半胱氨酸修饰在 氧化还原调节的SCD血管闭塞事件。

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.