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: 10689329
• 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/10689329
• 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; Oxidoreductase; P-Selectin; Patients; Phase; Platelet Activation; Positioning Attribute; Protein Disulfide Isomerase; Recurrence; Research; Risk; Role; Sickle Cell; Sickle Cell Anemia; Stress; Sulfhydryl Compounds; Sulfur; Techniques; Testing; Thrombosis; Thrombus; Training; Withdrawal; extracellular; hydroxyurea; imaging system; improved; in vivo; intravital microscopy; isoquercetin; mouse model; neutrophil; novel therapeutic intervention; oxidant stress; prevent; programs; skills; thromboinflammation; thrombotic; 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.

项目总结/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，包括使用羟基脲增加胎儿血红蛋白， crizanlizumab靶向P-选择素用于细胞粘附。SCD中的氧化应激增加了血管性心脏病的风险。 闭塞和目前的抗氧化治疗，包括L-谷氨酰胺，显示出减少这些事件的功效。 然而，抗氧化剂不能改善血管闭塞危象。血管闭塞的新治疗策略 需要基于对氧化还原机制的改进理解的镰状细胞病。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 血栓形成原型巯基异构酶，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，并且可以用黄酮类化合物如 异槲皮素。我们的初步数据表明，异槲皮素降低SCD小鼠的细胞-细胞粘附， PDI可能是血管闭塞的潜在靶点。然而，PDI促进血管内皮细胞增殖的机制尚不清楚。 闭塞不清楚。这项建议将测试中心假设，硫醇异构酶促进氧化还原敏感 镰状细胞病中通过半胱氨酸电子转移事件的血管闭塞。我们会评估氧化还原压力- 使用细胞和化学生物学方法， 疾病的小鼠模型。在目标1中，我们将机械地检查PDI感测氧化还原的能力。 在SCD中的环境，以促进电子转移的形式半胱氨酸二硫化物。这一目标将决定 无论是还原型还是氧化型PDI都通过催化电子传递促进SCD中的血小板和中性粒细胞活化， 从它们已知的氧化还原目标中退出。在目标2中，我们将把我们的研究转移到评估 通过巯基异构酶催化的电子撤回机制使用活体显微镜观察血栓形成， SCD中的止血和血管阻塞。我们还将通过观察巯基的功能来补充研究 异构酶介导的电子撤回对体内白细胞-细胞粘附事件的影响。最后，目标3将利用 碳亲核探针，其标记特异性半胱氨酸硫氧代形式以探测电子的整体功能 在SCD中传输事件。这些探针将以无偏的方式鉴定巯基异构酶的新靶点， 为了确定半胱氨酸二硫键断裂或形成的特征性集合是否是血管内皮生长因子受体所必需的， 闭塞该探针还将从机制上鉴定半胱氨酸电子转移事件在细胞内的作用。 血红蛋白对红细胞镰状化作用和白细胞介导的细胞-细胞粘附对血管闭塞的作用 事件K99阶段将侧重于目标1和2，而R 00阶段将侧重于目标3。附加 这个职业发展奖所提供的培训不仅使我能够扩展我的技能， 独特的位置我建立一个独立的研究计划，重点是氧化半胱氨酸修饰在 SCD的氧化还原调节血管闭塞事件。

项目成果

Plant flavonoid inhibition of SARS-CoV-2 main protease and viral replication.

• DOI: 10.1016/j.isci.2023.107602
• 发表时间: 2023-09-15
• 期刊: ISCIENCE
• 影响因子: 5.8
• 作者: Lin, Lin;Chen, Da-Yuan;Scartelli, Christina;Xie, Huanzhang;Merrill-Skoloff, Glenn;Yang, Moua;Sun, Lijun;Saeed, Mohsan;Flaumenhaft, Robert
• 通讯作者: Flaumenhaft, Robert

Protocol to identify flavonoid antagonists of the SARS-CoV-2 main protease.

鉴定 SARS-CoV-2 主要蛋白酶的类黄酮拮抗剂的方案。

• DOI: 10.1016/j.xpro.2024.102990
• 发表时间: 2024
• 期刊: STAR protocols
• 作者: Yang,Moua;Lin,Lin;Flaumenhaft,Robert
• 通讯作者: Flaumenhaft,Robert