Oxidative Cysteine Modification by Thiol Isomerases in Sickle Cell Disease

点击翻译按钮获取中文摘要

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.