Studies in Oxidative Damage-induced Signaling: A Focus Outside the Nucleus.
- Publication date
- Publisher
Abstract
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