Studies in Oxidative Damage-induced Signaling: A Focus Outside the Nucleus.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry & Biophysics, 2011.Oxidative stress, or increased reactive oxygen species (ROS), damages cellular macromolecules and activates signaling cascades that modulate cell fate. Oxidative damage and induction of these pathways are associated with numerous diseases. Therefore, pathways activated by oxidative stress and their mechanisms of initiation are active areas of research. Here, oxidative stress-induced activation of the unfolded protein response (UPR), which increases protein folding and degradation capacity, was investigated. Although the UPR was previously shown to be attenuated by antioxidants, evidence provided here demonstrates that hyperoxia, an inducer of chronic oxidative stress, was unable to activate the three ER stress initiating proteins IRE1, PERK, and ATF6. However, hyperoxia did sensitize cells to tunicamycin induced cell death. Collectively, these results suggest that chronic oxidative stress alone is insufficient to activate the UPR, but exacerbates ER stress-induced cell death. Next, we aimed to identify novel macromolecular damage species capable of initiating known stress response pathways. Nuclear DNA damage activates multiple signaling pathways. However, RNA and mitochondrial DNA (mtDNA) are more sensitive to oxidative damage than nuclear DNA. Therefore, we hypothesized that these damaged species activate stress response signaling. As an example, activation of p53 was investigated. P53 regulates many proteins that protect against the consequences of oxidative damage, including those regulating proliferation, cell death, and redox status. Our results demonstrate that transfection of exogenously oxidized RNA was insufficient to activate p53 in cells. Conversely, damage to mitochondrial DNA, introduced by the mitochondrial targeted restriction enzyme PstI, increased p53 dependent transcription and slowed proliferation. Additionally, p53 activation preceded detectable loss of mitochondrial function as indicated by no change in oxidative phosphorylation or superoxide production. Further, four days of PstI expression inhibited oxidative phosphorylation and this defect was exacerbated by knockdown of p53. These studies support the hypothesis that damage to readily oxidized macromolecules, specifically mtDNA, preemptively activates signaling to manage continual oxidative insult. A clear characterization of the signals activated by mtDNA damage could present avenues for therapeutic intervention in oxidative stress associated diseases