Redox control of cellular signalling

Abstract

Reactive oxygen species (ROS) are natural by-products of cellular energy production. Consequently, mammalian cells encounter them on a daily basis. Increased ROS levels are associated with the onset of cancer and accelerated ageing, and historically, ROS are therefore considered harmful molecules. However, recent research has indicated that low levels of ROS play important roles in the regulation of several cellular processes and are in fact required for cell viability. The goal of the research described in this thesis is to understand how ROS are able to control healthy cellular signalling. Redox signalling is the cells means to translate changes in the intracellular redox environment into a cellular response. Oxidative conditions (due to increased ROS concentrations) result in the reversible oxidation of specific cysteines, which can lead to the formation of covalent disulphide-bonds. This thesis gives insight into how disulphide-dependent protein-protein interactions control classical (i.e. kinase-mediated) signal transduction pathways. A major part of the research was dedicated to revealing novel redox signalling pathways converging on the Forkhead box O transcription factors FOXO3 and FOXO4. These tumour suppressive proteins are inhibited through the action of the insulin signalling pathway, and activated upon increased ROS levels. The molecular mechanism of the latter has remained unknown. We have used a combination of advanced quantitative proteomics, redox biochemistry and live cell imaging to elucidate novel ROS-dependent pathways involved in the regulation of FOXO proteins. To study the role of disulphide-dependent protein-protein interactions, we have developed a novel label-free mass-spectrometry-based assay, and used this to analyse the cysteine-dependent interactome of FOXO3 and FOXO4. In these studies, we show that ROS-induced activation of FOXO requires the disulphide-dependent interaction with a nuclear import protein. Interestingly, we find that two different nuclear import proteins are required for the ROS-induced nuclear import of FOXO paralogues FOXO3 and FOXO4. Phylogenetic analysis combined with biochemical analysis suggests that evolutionary acquisition of cysteines has contributed to functional divergence of the FOXO paralogues. Besides increasing the understanding of redox control of FOXO transcription factors, the results of this research can also be viewed of as a paradigm for how redox signalling affects signal transduction in general. The findings presented in this thesis suggest that redox signalling through the formation of mixed disulphide-dependent complexes is a general phenomenon that plays an important role in cellular signalling. Long-term disturbance of the cellular redox state has been observed in ageing cells, as well as in tumour cells. It will therefore be interesting to see if the regulation of the disulphide-dependent signalling pathways described in this thesis is disrupted in these cells and whether this contributes to cancer and ageing.