Student-Faculty Collaborative Research Grant Report
Analytical Chemistry | Biochemical Phenomena, Metabolism, and Nutrition | Biochemistry | Biology | Systems Biology
Mitochondria are essential organelles in most eukaryotic cells because of their role in metabolism and the production of ATP by the oxidative phosphorylation (OXPHOS) pathway, as well as other key cellular processes. Metal cofactors, such as copper and iron, are incorporated into the OXPHOS protein complexes of yeast, and misincorporation or modulation of these available metals in mitochondria leads to the production of reactive oxygen species (ROS). ROS are reactive molecules containing oxygen such as peroxides, superoxide, and hydroxyl radicals. Yeast are a good model for studying aging and the effect of ROS on lifespan because they are easy to grow, and many of the genes and proteins involved in determining yeast lifespan are conserved in humans and other mammals. Specifically, chronological lifespan assays in yeast rely on promoting a quiescent stationary phase by calorie restriction, a phase characterized by curtailed overall metabolic rate and a shift from fermentation to mitochondrial respiration. This shift in glucose utilization can be accomplished by reduced TOR (Target of Rapamycin) pathway signaling, a complex regulator of cell growth and cell cycle. Many downstream effects of TOR signaling can be silenced by cell treatment with Rapamycin, a drug that binds to Tor1p and inhibits kinase domain function, drastically increasing a cell culture’s chronological lifespan.
Mitochondrial OXPHOS complexes, whose activity and expression are modulated by TOR signaling, is the primary site of superoxide formation. A primary defense of free radical damage is the neutralization of superoxide by the enzyme superoxide dismutase 1 (Sod1), which requires copper and zinc as cofactors. Copper is additionally utilized in the OXPHOS complex cytochrome c oxidase as an electron transferring group. Our first aim was to investigate the role of copper in the production of mitochondrial reactive oxygen species (ROS) as a part of normal aerobic respiration utilizing a yeast model. Specifically, we worked to quantify the effect of exogenous copper treatment on the relative protein expression of copper-dependent cytochrome c oxidase (COX) subunits and overall COX complex assembly. Our previous work has indicated the protective behavior of copper treatment on yeast lifespan, and we propose this is due to copper inducing more robust electron movement through more functional electron transport chain (ETC) complexes. Improved efficacy of the ETC is thought to minimize premature electron leakage to oxygen and lessen mitochondrial ROS levels. This work hopes to contribute to our understanding of the copper-utilizing components of this mitochondrial pathway and this metal’s impact on local ROS production. Our second aim was to determine how copper levels, ROS levels, and enzyme activity are related during yeast chronological aging. We are able to use biochemical staining assays utilizing fluorescent molecules to quantify ROS levels and track live cells. Specifically, we can track extracellular hydrogen peroxide via Amplex Red, and superoxide generation in the mitochondria via dihydroethidium (DHE). Our initial results indicate that we are able to track superoxide production using DHE in wild type cells and sod1Δ yeast strains spectroscopically. Ultimately, we will use both spectroscopy and live cell imaging via microscopy to assess superoxide levels in multiple yeast strains as well as in the presence and absence of copper. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan.
Bestwick, Megan, "Student-Faculty Collaborative Research Grant Report" (2018). Post-Grant Reports. Report. Submission 137.