Abstract
Peroxiredoxin 3 (Prx 3) is an abundant mitochondrial protein that is very effective at scavenging hydroperoxides. The overexpression of Prx 3 has been shown to protect cells from oxidative stress and apoptosis, and knockout studies show an increase in intracellular ROS levels and oxidative stress. However, the knockout phenotype is mild and there has been no in-depth characterization of the effects of Prx 3 knockdown on mitochondrial function in response to oxidative stress. In this study, Prx 3 expression was knocked down to approximately 10-20% in a HeLa cell line, over a 5-6 day period. An inducible small hairpin RNA (shRNA) system was used to achieve this and no significant compensatory increase by other members of the Prx family was observed. These cells showed no difference in cell viability in response to exogenous H2O2, or during increased mitochondrial H2O2 production by the respiratory chain inhibitors; rotenone and antimycin A. Mitochondrial respiration was measured in intact cells with a Seahorse Extracellular Flux Analyzer and while the Prx 3 knockdown cells appeared to have normal rates of O2 consumption under basal conditions, mitochondrial stress was more detrimental in these cells. Overall, Prx 3 knockdown had a relatively limited phenotype, suggesting that low levels of Prx 3 are sufficient to protect cells from oxidative stress, or that Prx 3 may have a more important role as a sensor and signalling molecule than as a direct antioxidant protein.