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.