Quantification of time-dependent redox signalling in the Tpx1/Pap1 pathway in Schizosaccharomyces pombe.

Abstract

Reactive oxygen species (ROS) can damage cellular components leading to cell death, but paradoxically, ROS also play essential roles in metabolism and signalling in eukaryotic cells. Dysregulation of this balance is associated with a range of host diseases and cells have consequently evolved sophisticated signalling networks to sense, detoxify and adapt to changes in ROS levels. Hydrogen peroxide, for example, is reduced by thiol-peroxidases which in turn, can trigger the oxidation of thiol-dependent redox transcription factors. However, the relationship between hydrogen peroxide stimuli and the level of redox transcription factor activation has largely been described in qualitative terms. Because quantitative measures of the redox signal have been lacking, we tested whether three signalling parameters viz. the signalling time, duration and amplitude could be used to quantify the hydrogen peroxide-dependent redox signal in the Tpx1/Pap1 pathway in Schizosaccharomyces pombe. We found significant differences in the signalling time and duration, but not signal amplitude as hydrogen peroxide concentrations were increased from 100 to 1000 μM in our assays. By way of comparison, we also found that the general oxidant, tert-butyl hydroperoxide at 200 μM, decreased signal time and duration in the Pap1 pathway when compared to an equivalent hydrogen peroxide concentration. This method was also used to compare the hydrogen peroxide signalling by OxyR in Escherichia coli and Yap1 in Saccharomyces cerevisiae showing that these measures could be used to characterize and compare redox signalling from different oxidants and in different species. Thus, quantification of time-dependent redox signalling revealed new insights into hydrogen peroxide signalling that could not be readily obtained by qualitative methods and, these measures are expected to facilitate a better understanding of the role of redox signalling in health and disease.