Protein-S-glutathionylation in Caenorhabditis elegans - with focus on glutaredoxins and omega-class glutathione S-transferases

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Protein S-glutathionylation, the reversible post-translational formation of a mixed-disulfide bridge between glutathione and an accessible free thiol on a protein, has emerged as a crucial mechanism involved in the regulation of kinase signaling pathways, glycolysis and energy metabolism, antioxidant enzymes, calcium homeostasis, protein folding, ubiquitin-proteasome and apoptosis. It is therefore not surprising that S-glutathionylation of specific proteins is discussed in conjunction with cancer, cardiovascular and lung diseases, diabetes and neurodegerative diseases. Glutaredoxins (GRXs) and possibly the omega-class glutathione S-transferases (GSTOs) play a major role in these (de)glutathionylation processes. The proposal is part of our comprehensive investigation of glutathione-dependent enzymes with the long-term goal of contributing to the understanding of their role in (i) redox homeostasis, (ii) sensing and signaling of environmental stress and (iii) adaptation to environmental changes. C. elegans is an excellent model system to study the physical interaction, localization and native expression dynamics of redox proteins and antioxidant detoxification systems that are unique to multicellularity in general and animals in particular. We plan to identify target proteins of S-glutathionylation under normal and oxidative stress conditions. Here we will focus on identifying, verifying and analyzing the specific targets of three cytosolic dithiol GRXs and the GSTO-1. A novel 'mutant protein trapping strategy' will be used to identify target proteins. This in vitro method is based on the reduction of glutathione-mixed disulfides by CXXS-mutated GRX or GSTOs, their labeling followed by enrichment and proteomic identification. We have upgraded and fine-tuned the conventional method by (i) using the recombinant C. elegans CXXS-mutated GRXs and GSTOs in the deglutathionylation step, thereby selectively trapping their specific target proteins, (ii) using worm mutants where the corresponding endogenous GRX is knocked out. This will greatly enhance the interaction between the exogenous GRX and target proteins. In our in vivo approach we want to directly identify the targets of our redox enzymes by overexpressing them in their physiological location in the available grx-mutant worms. This enables the in situ trapping of the target proteins. Following verification of S-glutathionylation of selected target proteins, in vitro pull-down assays will be performed to validate target interactions. Finally, a novel GFP-recruitment assay will be used in C. elegans that enables the in vivo analysis of selected GRX (GSTO) - target protein interactions. Functional consequences of these interactions will be analysed and we expect that the identification of novel targets of S-glutathionylation will lead to an advancement in the knowledge of this fundamental cellular process and perhaps builds the basis for new therapeutic strategies.