Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

Thiol redox proteomics identifies differential targets of cytosolic and mitochondrial glutaredoxin-2 isoforms in Saccharomyces cerevisiae. Reversible S-glutathionylation of DHBP synthase (RIB3).

Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms.