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.
Mitochondria and reactive oxygen species (ROS) are important factors regulating the response of human body to environmental stress. We propose to develop chemical reagents and methods to find and track mitochondrial proteins that are directly modified by ROS in response to two environmental factors: silver nanoparticles and ionizing radiation. We will combine these new chemical tools with state-of-the-art technologies to gain a better overall understanding of processes integral to environmental-associated diseases such as lung injury.
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