Protein post-translational modifications in the cell's redox and energy states

UnrestrictedThe increased generation of reactive oxygen and nitrogen species and glutathione depletion creates profound changes in the redox status of the cell and has been implicated in the progression of neurodegenerative diseases such as Parkinson's disease. Redox regulation has become exceedingly important in understanding the cellular adaptation to oxidative stress. However, it remains unclear how changes in the cellular and mitochondrial redox environment affect redox sensitive protein function during exposure to NO and dopamine and the consequences of such changes with respect to mitochondrial function and redox signaling (achieved through post-translational modifications).; The work in this dissertation demonstrates that glutathionylation of mitochondrial proteins occurs during oxidative stress. This modification functions as a store of GSH and is released in response to changes in mitochondrial redox and energy status. Protein sulfhydryl recovery (de-glutathionylation) of mitochondrial proteins in energized mitochondria increased the GSH buffering capacity of mitochondria against physiological and non-physiological concentrations of hydrogen peroxide (H2O2), thus preventing drastic changes in mitochondrial redox status. This is exemplified using a Parkinson's disease model with NO and dopamine as modifiers of the redox environment. The reaction between NO and dopamine oxidation produces ONOO-, a powerful oxidant that leads to a 72% inhibition of mitochondrial respiration in isolated mitochondria and 71% inhibition in PC12 cells.; Additionally, we identified aconitase and ATP synthase as targets of protein post-translational modifications. Nitration of aconitase by ONOO- resulted in a dose-dependent decrease in aconitase function. In the presence of citrate, a substrate for aconitase, a 66-fold higher concentration of ONOO- was required for half-maximal inhibition. Glutathionylation of aconitase and ATP synthase also resulted in an inhibition of enzyme function. De-glutathionylation of ATP synthase resulted in a 221% change in activity. It is well established that damage to proteins under conditions of oxidative and nitrosative stress is highly specific, and plays an important role in many diseases. Characterization of post-translational modifications of mitochondrial proteins provides a mechanism for .NO and dopamine elicited damage to mitochondrial function leading to apoptosis of dopaminergic neurons in Parkinson's disease