New Oxidation-Sensing Probes to Evaluate Mitochondrial Dysfunction in Lung Injury

 描述（由申请人提供）：环境相关疾病中的线粒体功能障碍和活性氧（ROS）蓄积已得到充分证实。然而，我们只有一个有限的欣赏这些过程中的细胞表型变化的分子机制，强调环境压力的发病机制。在NIH IMAT计划的支持下，维克森林的这个研究小组率先开发了高度特异性的化学探针，可以检测和鉴定氧化蛋白（ROS的分子靶点）。虽然这些探针已成功地用于鉴定在许多疾病条件下（例如，癌症、衰老、炎症），但它们尚未靶向细胞内的特定细胞器或应用于研究细胞对环境应激源的反应。目前的提案描述了实现这些重要任务的新策略，首先关注（R21阶段）蛋白质氧化的靶向化学探针的开发和验证，然后关注（R33阶段）这些氧化传感探针和分析方法的应用，以研究电离辐射（IR）和银纳米颗粒（AgNP）诱导的肺损伤机制。新的探针将能够选择性标记线粒体中的亲电和亲核蛋白亚磺酸（-SOH）。将采用将靶向DNA和氧化传感探针与针对感兴趣蛋白质的抗体组合的联合收割机的新成像方法来可视化选择性蛋白质-SOH原位修饰和氧化蛋白质在细胞内的移动（例如，线粒体和细胞核之间）。然后将机械地使用探针来研究线粒体功能障碍和环境肺损伤之间的关系。新的计算方法（COSMro）将被用来推断特定途径的依赖性上调或下调，然后将使用细胞和肺损伤动物模型的研究进行验证。这些研究将在幼龄和老龄动物中进行，使用单一和组合环境应激源，尽可能模拟人类群体的环境暴露。成功完成这个项目将产生很大的影响，使线粒体和氧化还原控制的细胞内过程参与生物反应，在我们的日常生活中遇到的环境压力。

 DESCRIPTION (provided by applicant): Mitochondrial dysfunction and accumulation of reactive oxygen species (ROS) in environmentally associated diseases is well established. Yet we have only a limited appreciation for the molecular mechanisms linking these processes to changes in cellular phenotype underlining the pathogenesis of environmental stressors. With previous support from the NIH IMAT program, this research team at Wake Forest has pioneered the development of highly specific chemical probes, which enable detection and identification of oxidized proteins (molecular targets of ROS). While these probes have been used successfully to identify global targets of oxidation within cellular proteins under numerous disease conditions (e.g., cancer, aging, inflammation), they have not yet been targeted to specific organelles within the cells or applied to study cellular response to environmental stressors. The current proposal describes new strategies to achieve these important tasks by focusing first (R21 phase) on the development and validation of mitochondria-targeted chemical probes for protein oxidation and then (R33 phase) on the application of these oxidation-sensing probes and methods of analysis to investigate mechanisms of lung Injury induced by ionizing radiation(IR) and silver nanoparticles (AgNP). The new probes will enable selective labeling of electrophilic and nucleophilic protein sulfenic acids (-SOH) in mitochondria. New imaging methods that combine the mitochondria-targeted and oxidation-sensing probes with an antibody against the protein of interest will be employed to visualize selective protein -SOH modification in situ and movement of the oxidized protein within the cell (e.g., between mitochondria and nucleus). The probes will then be employed mechanistically to investigate the relationship between mitochondrial dysfunction and environmental lung injury. New computational methods (COSMro) will be employed to infer mitochondria-dependent up or downregulation of specific pathways, which will then be validated using studies in cells and animal models of lung injury. These studies will be performed in young and old animals using single and combined environmental stressors to mimic to the extent possible the environmental exposure in a human population. Successful completion of this project will have high impact, enabling a much deeper understanding of mitochondria- and redox-controlled intracellular processes involved in the biological response to environmental stressors encountered in our daily lives.