Peroxiredoxin 3 and Oxidative Stress in Mitochondria
|dc.identifier.citation||Poynton, R. (2014). Peroxiredoxin 3 and Oxidative Stress in Mitochondria (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/5101||en|
|dc.description.abstract||Peroxiredoxin 3 (Prx 3) is a major antioxidant protein located in the mitochondrial matrix. Prx 3 decomposes hydroperoxides to water, but intriguingly, it is also sensitive to oxidative inactivation by H2O2. In the catalytic cycle the sulfenylated peroxidatic cysteine condenses with a resolving cysteine on an opposing subunit to form a disulfide bond. This requires localised unfolding at the C-terminus of the protein, allowing time for H2O2 to oxidise the sulfenic acid to a sulfinic acid (hyperoxidation). Unfolding occurs more slowly in eukaryote Prxs due to a C-terminal extension, which renders them more susceptible to hyperoxidation than prokaryote Prxs. The functional consequences of Prx hyperoxidation are unclear. Previous studies using semi-quantitative western blotting have indicated that Prx 3 is less susceptible to hyperoxidation than the cytosolic family member Prx 2. The methods used to measure hyperoxidation of Prxs in biological systems were investigated and it became evident that they may not be suitable for monitoring subtle changes to hyperoxidation of individual Prxs. A new liquid chromatography/mass spectrometry (LC/MS) method was used to quantify Prx redox states. Wild type recombinant human Prx 2 and 3 were treated with H2O2, and dimerization and hyperoxidation was quantified by LC/MS. Kinetic modelling of catalase competition assays were used to estimate the rate constants of the disulfide bond formation and hyperoxidation reactions. The reactivity of the Prx 2 and 3 sulfenic acids with H2O2 were determined to be identical (12,000 M-1 s-1). However, the rate of dimerization and thus unfolding at the C-terminus was 10 times faster for Prx 3 (20 s-1 versus 2 s-1), thereby explaining its reduced sensitivity to hyperoxidation. C-terminus features of eukaryotic Prxs are hypothesised to influence the sensitivity to hyperoxidation. Four mutations at the C-terminus Prx 3 were introduced to match the amino acids found in Prx 2 (N232G, T234K, D236G and P238D), and vice versa. The introduction of these C-terminal mutations decreased the susceptibility of Prx 2 to hyperoxidation, while mutations at the C-terminus of Prx 3 increased the sensitivity of Prx 3 to hyperoxidation in vitro. The C-terminal mutations altered the sensitivity to hyperoxidation by changing the rate of disulfide bond formation. The Prx 3 C-terminal mutant was transiently expressed in HeLa cells and was more susceptible to hyperoxidation in comparison to the transiently expressed wild type Prx 3. The Prx 3 proteins were tagged at the N-terminus to distinguish them from the endogenously expressed Prx 3. Surprisingly, N-terminal tags themselves appeared to increase the susceptibility of the Prx 3 to hyperoxidation. N-terminus modifications may therefore regulate of hyperoxidation of Prx 3 in cells. We are now in a position where the biological consequences associated with the expression of a Prx 3 with increased susceptibility to hyperoxidation can be assessed. Overall, this thesis has increased our understanding of the Prx 3 hyperoxidation reaction and the features of the proteins which influence its susceptibility to hyperoxidation.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Peroxiredoxin 3 and Oxidative Stress in Mitochondria|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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