The dynamic function of the cytosolic redox regulatory network and the sensory role of the type II peroxiredoxins B/C/D.

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The presence of a thiol redox-regulatory network is a common feature of all plasmatic cell compartments including the cytosol. It co-controls many processes such as metabolism and signaling. In plants, the redox network is best understood for chloroplasts. However, its specific features and functions in the cytosol remain mostly to be explored, in particular with respect to the role of type II peroxiredoxins (PRXIIB/C/D) as redox sensors in comparison to glutathione-peroxidase-like proteins (GPXL). The proposed work on A. thaliana will address the competition for electrons, specificity of interaction, and kinetics of redox transmission. The propagation of an oxidative stimulus will be monitored in a novel reconstituted redox system consisting of various network components. Using high resolution quantitative mass spectrometry (MS)-based proteomics, this setup will distinguish thiols, disulphides between peptides, and S-glutathionylation and dissect the roles of each type of redox element in the system. The function of the three PRXIIB/C/D isoforms will be explored in knock out lines using CRISPR/Cas and compared with mutants lacking GPXL2/8, e.g., after light shifts or RBOH activation. The interactome of PRXIIB will be determined in vitro using affinity chromatography-based pull-down of interactors followed by MS identification and in vivo using Turbo-ID-PRXII constructs. The significance of the network will be further scrutinized in two case studies namely the redox regulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) and vacuolar H+-ATPase. Overall, the project is planned to test the concept of “redoxosomes” as regulatory units. Advanced quantitative understanding of the cytosolic redox subnetworks will be attempted by mathematical modelling and dynamic simulation in order to deliver novel insight into the kinetic coupling between the network components.