Peroxiredoxin 6 (PRDX6) has been associated with tumor progression and cancer metastasis. Its acting on phospholipid hydroperoxides and its phospholipase-A2 activity are unique among the peroxiredoxin family and add complexity to its action mechanisms. As a first step towards the study of PRDX6 involvement in cancer, we have constructed a human hepatocarcinoma HepG2PRDX6-/- cell line using the CRISPR/Cas9 technique and have characterized the cellular response to lack of PRDX6.

Annotation of protein-coding genes is a key step in sequencing projects. Protein functions are mainly assigned on the basis of the amino acid sequence alone by searching of homologous proteins. However, fully automated annotation processes often lead to wrong prediction of protein functions, and therefore time-intensive manual curation is often essential. Here we describe a fast and reliable way to correct function annotation in sequencing projects, focusing on surface proteomes. We use a proteomics approach, previously proven to be very powerful for identifying new vaccine candidates against Gram-positive pathogens. It consists of shaving the surface of intact cells with two proteases, the specific cleavage-site trypsin and the unspecific proteinase K, followed by LC/MS/MS analysis of the resulting peptides. The identified proteins are contrasted by computational analysis and their sequences are inspected to correct possible errors in function prediction.

Aims: A three-step catalytic cycle is common to all peroxiredoxins (Prxs), despite structural and kinetic differences. The second step in 1-Cys type Prxs is a matter of debate since they lack an additional cysteine to play the resolving role, as happens with the 2-Cys Prxs. The aim of this study was to elucidate the role of glutathione (GSH) in the thioredoxin-dependent peroxidase activity of Saccharomyces cerevisiae mitochondrial Prx1p, a 1-Cys type Prx. Results: The peroxidatic Cys91 residue of two Prx1p peptides can be linked by a disulfide, which can be reduced by thioredoxin and by GSH (Km=6.1 μ M ). GSH forms a mixed disulfide with the peroxidatic cysteine spontaneously in vitro and in vivo . Mitochondrial Trx3p deglutathionylates Prx1p without formation of GSSG so that GSH is not consumed in the process. The structural unit of native Prx1p is a dimer whose subunits are not covalently linked, but a hexameric assembly of three disulfide-bound dimers can also be formed. Innovation: GSH is presented as a protective cofactor of Prx1p, which is not consumed during the peroxidase reaction, but provides a robust mechanism as the resolving cysteine and efficiently prevents Prx1p overoxidation. GSH exerts these roles at concentrations well below those commonly considered necessary for its antioxidant and redox buffering functions. Conclusion: A 1-Cys peroxide scavenging mechanism operates in yeast mitochondria involving an autonomous glutathione molecule and the thioredoxin system, which could have universal validity. Prx1p is fairly well protected from overoxidation, questioning its role in a floodgate mechanism for H ₂ O ₂ signaling. Antioxid. Redox Signal. 24, 115–128.

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.