Investigating the Role of Peroxiredoxin 1 in Hydrogen Peroxide Signalling
|dc.identifier.citation||Powell, M. (2017). Investigating the Role of Peroxiredoxin 1 in Hydrogen Peroxide Signalling (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/7218||en|
|dc.description.abstract||Cells have elaborate responses to cues provided to them. Hydrogen peroxide (H2O2) is often produced as a second messenger in response to various growth factors binding to their receptors. Cells contain a range of peroxidases to rapidly react with H2O2 to prevent H2O2 induced damage to the cell. Peroxiredoxins are highly abundant and catalytically efficient thiol peroxidases that will react with the majority of H2O2 produced during a signalling event. Despite the effectiveness of the cellular peroxidases, some non-peroxidases are susceptible to cysteine oxidation during growth factor or H2O2 treatment. There are two broad models that attempt to describe how non-peroxidase proteins become oxidised in the presence of efficient and abundant peroxidases. The direct oxidation model proposes that a high concentration of H2O2 inactivates peroxiredoxins and thus overcomes the barrier of oxidising non-peroxidase proteins. The indirect oxidation model proposes that it is the peroxiredoxins that react with the low concentrations of H2O2 produced during signalling events and transfer the peroxide signal via disulfide exchange reactions onto the target proteins. In both models thioredoxin is suspected to reduce the oxidised target proteins to turn off the peroxide signal. The overall goal of this project was to provide insight into the role of Prx1 in the indirect oxidation model of H2O2 signalling. HeLa cells with inducible Prx1 knockdown were used to investigate the role of Prx1 in H2O2 signalling. The first aim of the project was to investigate the effect of Prx1 knockdown on H2O2 metabolism. By monitoring the oxidation of a redox sensitive fluorescent protein, HyPer, I was able to assess the change in H2O2 metabolism when Prx1 was knocked down in HeLa cells. I demonstrated that there is no change in H2O2 metabolism when Prx1 is knocked down in HeLa cells. This suggests that the primary role of Prx1 is not as a general H2O2 metaboliser, and supports a more specific role of Prx1 in signalling. The second aim of the project was to investigate the effect of Prx1 knockdown on Trx1 oxidation. I hypothesised that we would see a different pattern of Trx1 oxidation when Prx1 was knocked down depending on whether H2O2 was signalling via the direct or indirect model. To enable monitoring of Trx1 oxidation, I needed to label oxidised cysteine residues to allow separation of oxidised and reduced Trx1 by SDS-PAGE. I was able to establish labelling of recombinant Trx1 but due to methodological challenges we were unable to investigate Trx1 oxidation when Prx1 was knocked down in HeLa cells.|
|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||Investigating the Role of Peroxiredoxin 1 in Hydrogen Peroxide Signalling|
|thesis.degree.name||Master of Science|
|thesis.degree.grantor||University of Otago|
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