Investigating the role of peroxiredoxin in hydrogen peroxide signalling
Carrad, Rose Jane
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Carrad, R. J. (2017). Investigating the role of peroxiredoxin in hydrogen peroxide signalling (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/7717
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Abstract:
Hydrogen peroxide (H2O2) was viewed as an unwanted by-product of aerobic respiration for many years but it is now well-established as being involved in many different signalling pathways. H2O2 is reduced to water by peroxiredoxins (Prdx), which make up a family of antioxidant enzymes. Due to high rate constants and the abundancy of Prdx in cells, H2O2 is quickly reduced to water. Therefore, signalling proteins have to compete with Prdx to be oxidised by H2O2.
This has resulted in two conflicting theories for how Prdx are involved in H2O2 signalling. The first is the floodgate model, suggesting that Prdx are inactivated by high levels of H2O2, allowing for direct H2O2 oxidation of signalling proteins. The second model, signal peroxidase model, suggests that Prdx are more directly involved in the reversible oxidative activation of specific non-peroxidase thiol-containing proteins. Prdx1, a ubiquitously expressed cytoplasmic enzyme, is one of six isoforms of the Prdx family. Prdx1 has been characterised as acting in a redox relay mechanism in the H2O2 activation of the apoptosis signal-regulating kinase 1 (ASK1) apoptotic pathway. ASK1 activates apoptosis via the phosphorylation and activation of p38. This set of experiments aims to characterise the role of Prdx1 in H2O2 signal transduction, looking specifically at H2O2 induced apoptosis via the phosphorylation and activation of p38.
The model systems used for this set of experiments were Hap1 wild type and Prdx1 knockout cells. Experiments carried out to measure the effect that Prdx1 knockout has on cell growth were done through the observation of Prdx1 knockout and WT growth rates. There was no observed difference in growth rates providing evidence that Prdx1 knockout does not affect cell growth. Secondly the effect that Prdx1 has on H2O2 induced phosphorylation and activation of p38 was determined through the comparison of p-p38 in WT and Prdx1 knockout cell lines that had been treated with H2O2. Due to nonspecific binding of the anti-phoshpo-p38 antibody these experiments remain inconclusive. Therefore, H2O2 induced activation of apoptosis was measured in Prdx1 knockout and WT cell lines by caspase-3 activity. Again, there was no observed difference of H2O2 induced apoptosis between the Prdx1 knockout and WT cell lines. This result gives evidence that Prdx1 does not have a critical role in the H2O2 signalling cascade that activates apoptosis. Further work to supplement these experiments is to observe H2O2 induced phosphorylation of a broad range of human kinases between Prdx1 knockout and WT cell lines. There were various kinases that were seen to be differentially phosphorylated in Prdx1 knockout cells. C-Jun, Hck, HSP60, PYK2, STAT2, STAT5a and STAT5a/b were seen to be differentially phosphorylated in Prdx1 knockout cells when treated with H2O2.
In conclusion, this set of experiments gives evidence that Prdx1 does not have a crucial role in cell growth or H2O2 induced apoptosis in Hap1 cells. Therefore, these results do not support either theory for how Prdx are involved in H2O2 signalling in the cell. Preliminary phospho-kinase array results show a possible role of Prdx1 in signal transduction as per the signal peroxidase model.
Date:
2017
Advisor:
Ledgerwood, Elizabeth Clare
Degree Name:
Bachelor of Biomedical Sciences with Honours
Degree Discipline:
Biochemistry
Publisher:
University of Otago
Keywords:
Peroxiredoxin; hydrogen peroxide; Prdx1
Research Type:
Thesis
Languages:
English
Collections
- Biochemistry collection [227]
- Thesis - Honours [339]