Genetic intervention and biochemical analysis of antioxidant enzyme function and their post-translational modifications in yeast

Antioxidant enzymes play a critical role in stress response and cell viability by controlling intracellular levels of reactive oxygen species (ROS). While at low concentrations they trigger stress response and extend chronological lifespan, high ROS levels are toxic since they damage DNA, lipids and proteins. Thus, cells must rapidly sense changes in ROS levels and synchronize the activities of antioxidant enzymes to prevent harmful oxidative modifications of cellular components. This thesis reports on how genetic manipulation of the mitochondrial antioxidant enzyme cytochrome c peroxidase (Ccp1) alters the synchronization of the activity of antioxidant enzymes during the chronological aging of yeast. Ccp1 is identified as a mitochondrial H2O2 sensor that prevents mitohormesis and lifespan extension by increasing catalase A (Cta1) activity and attenuating the H2O2-dependent stress response during aging. On the other hand, Ccp1 signaling protects cells against acute H2O2 stress by increasing peroxiredoxin and catalase activity. The latter activity was found to be a critical defense against exogenous H2O2, since blocking the induction of catalase activity by genetic intervention or low nutrient availability impairs adaptation on H2O2 challenge. The critical antioxidant enzyme copper-zinc superoxide dismutase (Sod1) from 7-day, stationary phase yeast cells was observed to be oxidized, which induced the formation of soluble high-molecular weight Sod1-containing aggregates. These species resemble the non-amyloid aggregates seen in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). Combined, the presented work provides molecular insights into how antioxidant defenses are synchronized and modified during acute stress and during aging. Such synchronization modulates ROS levels, stress response and longevity, which in yeast was found to correlate with oxidative damage to mitochondrial proteins.