Protein-S-glutathionylation in Caenorhabditis elegans - with focus on glutaredoxins and omega-class glutathione S-transferases
Animal Physiology and Biochemistry
Final Report Abstract
Our project aimed to identify target proteins of (de)glutathionylation under normal and oxidative stress conditions, focusing on dithiol glutaredoxins (GLRX) and the omegaclass glutathione S-transferase GSTO-1 from Caenorhabditis elegans. It was planned to verify S-glutathionylation and to analyze selected target proteins in vitro by pulldown assay and in vivo interaction studies. Although we could not complete all proposed tasks, we still made significant progress. Using GLRX-22 as a proof-of-principle, we optimized our co-immunoprecipitation/mass spectrometry workflow, resulting in a highly efficient and robust protocol that provides strong correlation of replicate experiments. The identification of potential target proteins revealed versatile functions of GLRX-22. Moving forward, our efforts will focus on validating target proteins of GLRX-22 and identifying target proteins of GLRX-21. Functional analyses of glrx-21 and glrx-22 deletion mutants and reporter strains showed that GLRX-21 and GLRX-22 are involved in the response to heat stress, with GLRX-22 also affecting the lifespan and reproduction of C. elegans. The two proteins appear to work in tandem, with GLRX-22 active under standard conditions and GLRX-21 activated under stress conditions. To gain an insight into the functions of the Omega GSTs in C. elegans, the GST-44 was studied in more detail. We found that the GST-44 has thioltransferase and peroxidase activity. gst-44 deletion mutants had a shortened lifespan and increased sensitivity toward arsenic stress and ER stress. In agreement with this, an increased expression of transcriptional reporter strains in response to these stressors was observed. Via RNAi assays, an activating impact of the transcription factors SKN-1 and PHA-4 on the expression of gst-44 could be shown.
