Regeneration of cytosolic thiol peroxidases

Abstract

Three soluble type two peroxiredoxins (PRXIIB, C, D) and two glutathione peroxidase‐like enzymes (GPXL2, 8) reside in the cytosol of Arabidopsis thaliana cells and function both as thiol‐dependent antioxidants and redox sensors. Their primary substrate is H2O2, but they also accept other peroxides with a distinct preference between PRXII and GPXL. Less known is their regeneration specificity in the light of the large set of thiol reductases, namely eight annotated thioredoxin h isoforms (TRXh1‐5, 7–9), a few TRX‐like proteins, including CxxS1 (formerly TRXh6) and several glutaredoxins (GRX) associated with the cytosol. This study addressed this open question by in vitro enzyme tests using recombinant protein. GPXL2 and 8 exclusively accepted electrons from the TRX system, namely TRXh1‐5 and TDX, while PRXIIB/C/D were efficiently regenerated with GRXC1 and C2 but not the TRX‐like protein Picot1. They showed significant but low activity (<3% of GRXC2) with TRXh1‐5 and TDX. A similar reduction efficiency with TRX was seen in the insulin assay, only TDX was less active. Finally, the reduction of oxidized cytosolic malate dehydrogenase 1, as measured by regained activity, showed an extremely broad ability to accept electrons from different TRXs and GRXs. The results demonstrate redundancy and specificity in the redox regulatory network of the cytosol.

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution

“…Glutathione oxidation was increased and reached a higher maximal oxidation level if GPXL2/8 were absent. This result was fully in line with the reported coupling of PRXII to the GRX/GSH-system and of GPXL to the TRX system [ 11 ]. The H 2 O 2 -induced oxidation of the roGFP2-Orp1-sensor increased in speed and maximum value in the absence of GPXL2/8 ( Fig.…”

“…1 Choice of proteins for the reconstituted network. Thioredoxins such as TRXh7-9, which were inactive as electron donors for cytosolic thiol peroxidases [ 11 ], were excluded from the network. If tryptic digestion of protein isoforms resulted in identical peptides, the less active or less abundant isoform was omitted from the network, such as PRXIIC or TRXh4.…”

“…Table S2 ) and cloned into pET28a. The expression vectors for PRXIID (AT1G60740), GPXL2 (AT2G31570), TRXh1 (AT3G51030), TRXh2 (AT5G39950), TDX (AT3G17880) and GRXC1 (AT5G63030) were cloned into pET28a as described in Vogelsang & Dietz [ 11 ]. PRXIIB (AT1G65980), GPXL8 (AT1G63460), TRXh3 (AT5G42980), TRXh5 (AT1G45145), GRXC2 (AT5G40370), NTRA (AT2G17420, without the sequence coding for the transit peptide), GR (AT3G24170) and PGK3 (AT1G79550) were cloned into pET28a as described in Knieper et al [ 24 ].…”

“…Fundamental questions concern the role of and the coupling between the thiol peroxidases peroxiredoxin (PRX) and glutathione peroxidase-like (GPXL), the thioredoxin (TRX) and glutaredoxin (GRX) isoforms and the redox-regulated protein targets. In the cytosol, the type II PRXIIB/C/D are most efficiently coupled to GRX, GSH, glutathione reductase (GR) and NADPH, while GPXL2/8 retrieve electrons from the TRX and NADPH-dependent TRX reductase A/B (NTRA/B) and NADPH [ 11 ].…”

The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux

“…Cellular GSSG accumulation is limited by the fact that cells employ multiple redundant reductive pathways to reduce cytosolic GSSG or to actively export GSSG to the extracellular environment, for example by MRP1 in mammalian cells or to alternative subcellular compartments, for example by Ycf1 in yeast [21,59]. Moreover, GSH has been shown to be able to act as a source of reductive power for typical 2-Cys peroxiredoxins, including human PRDX2 [60] and Arabidopsis PRXIIB, C and D [61]. The relative importance of the glutathione system compared to the more important thioredoxin system may be further amplified under acute oxidative challenge as thioredoxins or thioredoxin reductases may readily become limiting and/or oxidised and thus inactive [62], which would strongly limit the capacity of the thioredoxin pathway to act as a conduit of anti-oxidative electron flux.…”