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 (Cu) and iron (Fe), are incorporated into OXPHOS protein complexes of yeast located within the inner membrane of the mitochondria. Misincorporation or modulation of these available metals in mitochondrial enzymes 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.
Mitochondrial OXPHOS protein complexes are 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 (Zn) 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 a protective behavior of copper treatment on yeast lifespan, and we propose this is due to copper inducing more robust electron movement through a 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. During this granting period we showed that addition of 0.25 mM copper to yeast media increases lifespan of wild type (WT) and lys7∆ cells, but the addition of 0.25 mM copper to the media only affects the growth of sod1∆ yeast cells in the short term. As expected, the higher concentration of 2.0 mM copper addition to media was too high and became toxic, killing most of the yeast cells. We began performing protein analysis of mitochondrial proteins as shown in figure 1 (see Comments).
Future directions are to increase the yeast lifespan experimental length and to determine effects of copper on protein expression (i.e., improve the western blot loading), protein activity, and at the level of transcription level for respiratory proteins. 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 sod1∆ 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. We are currently working on utilizing the fluorescent dye MitoSox (Molecular Probes) to specifically identify and quantify superoxide species generated within the mitochondria. 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" (2019). Post-Grant Reports. Report. Submission 167.