Abstract
Hydrogen peroxide (H₂O₂) is a ubiquitous oxidant produced in a regulated manner in mammalian cells. At low concentrations H₂O₂ can reversibly oxidise thiol groups of cysteine residues to mediate intracellular signalling.
Peroxiredoxin (Prdx) enzymes are the key reductants of H₂O₂ in cells. Prdx1 and Prdx2, the cytoplasmic isoforms of Prdx, react so rapidly with H₂O₂ that they are predicted to deplete ∼ 99 % of H₂O₂ in the cytoplasm. For this reason, they are being investigated as potential modulators of H₂O₂ signalling.
Several specific protein targets of reversible H₂O₂ oxidation have been described, however, the global response of cells to H₂O₂ treatment is not well understood. This Thesis aimed to investigate the role of H₂O₂ in cell signalling by addressing three broad aims. Firstly, by investigating the transcriptomic response of Jurkat cells to a physiologically relevant treatment with H₂O₂. Secondly, by investigating the role of Prdxs in H₂O₂-induced gene expression using Prdx1 knock-out (KO) and Prdx2 KO cell lines. Finally, by investigating the phosphorylation changes induced by treatment of Jurkat cells with H₂O₂.
H₂O₂ was found to predominantly induce, rather than repress, gene expression. Pathway analysis indicated that pathways associated with forkhead box O (FOXO) and neurotrophic tyrosine receptor kinase (NTRK) signalling were specifically activated by H₂O₂. The use of software to identify potential transcriptional regulators indicated that gene activation by H₂O₂ was mediated by binding to specific enhancer sequences.
Both the basal role of Prdxs and the role of Prdxs in H₂O₂-induced gene expression were explored using two Prdx1 KO and two Prdx2 KO cell lines. Analysis of the basal changes in gene expression indicated that Prdx1 is involved in glucose metabolism, whereas Prdx2 is involved in autophagy. Prdx1-dependent and Prdx2-dependent changes in H₂O₂-induced gene expression were identified, supporting current evidence that these proteins modulate intracellular H₂O₂ signalling.
A pilot phosphoproteomics study was completed to investigate changes in phosphorylation that are induced by H₂O₂ treatment in Jurkat cells. Fewer than expected phosphorylation events were detected indicating that the method requires further optimisation to establish biological insight. However, the preliminary results suggest that H₂O₂ is involved in phosphorylation of mitogen activated protein kinase (MAPK) and cell cycle proteins which supports published data.
Overall, this work advances our knowledge of physiologically relevant pathways that are activated by H₂O₂. The results strongly support the concept that H₂O₂ is a bona fide signalling molecule, activating pathways that ultimately lead to changes in gene expression. The snapshot of gene expression changes in Prdx1 KO and Prdx2 KO cells in response to H₂O₂ treatment will need to be combined with further studies to gain insight into the role of these enzymes in H₂O₂ signalling.