Among reactive oxygen species (ROS), HO alone acts as a signaling molecule that promotes diverse phenotypes depending on the intracellular concentration. Mitochondria have been suggested as both sources and sinks of cellular HO, and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically encoded HO generator, d-amino acid oxidase (DAAO), was targeted to the mitochondria of human cells, and its utility in investigating cellular response to a range of HO doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased HO flux due to the activity of DAAO. Cell death was observed in a concentration- and time-dependent manner, and protein oxidation shifted in localization as the dose increased. This work presents the first systematic study of HO-specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in HO in comparison with prior studies that targeted the cytosol.
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