The interactome of 2-Cys peroxiredoxins in Plasmodium falciparum.

Peroxiredoxins (Prxs) are crucially involved in maintaining intracellular H2O2 homeostasis via their peroxidase activity. However, more recently, this class of proteins was found to also transmit oxidizing equivalents to selected downstream proteins, which suggests an important function of Prxs in the regulation of cellular protein redox relays. Using a pull-down assay based on mixed disulfide fishing, we characterized the thiol-dependent interactome of cytosolic Prx1a and mitochondrial Prx1m from the apicomplexan malaria parasite Plasmodium falciparum (Pf). Here, 127 cytosolic and 20 mitochondrial proteins that are components of essential cellular processes were found to interact with PfPrx1a and PfPrx1m, respectively. Notably, our data obtained with active-site mutants suggests that reducing equivalents might also be transferred from Prxs to target proteins. Initial functional analyses indicated that the interaction with Prx can strongly impact the activity of target proteins. The results provide initial insights into the interactome of Prxs at the level of a eukaryotic whole cell proteome. Furthermore, they contribute to our understanding of redox regulatory principles and thiol-dependent redox relays of Prxs in subcellular compartments.

Kinetic characterization of wild-type and mutant human thioredoxin glutathione reductase defines its reaction and regulatory mechanisms.

In most cells, the thioredoxin (Trx) and glutathione systems are essential in maintaining redox homeostasis. The selenoprotein thioredoxin glutathione reductase (TGR) is a hybrid enzyme in which a glutaredoxin (Grx) domain is linked to a thioredoxin reductase (TrxR). Notably, the protein is also capable of reducing glutathione disulfide (GSSG), thus representing an important link between the two redox systems. In this study, we recombinantly produced human TGR (hTGR wild-type) by fusing its open reading frame with a bacterial selenocysteine insertion sequence element and co-expressing the construct in Escherichia coli together with the selA, selB, and selC genes. Additionally, the Sec→Cys mutant (hTGRU642C ) of the full-length protein, the isolated TrxR domain (hTGR151-643 ) and the Grx domain containing a monothiol active site (hTGR1-150 ) were produced and purified. All four proteins were kinetically characterized in direct comparison using Trx, DTNB, HED, or GSSG as the oxidizing substrate. Interestingly, the HED reduction activity was Sec independent and comparable in the full-length protein and the isolated Grx domain, whereas the TrxR and glutathione reductase reactions were clearly selenocysteine dependent, with the GR reaction requiring the Grx domain. Site-directed mutagenesis studies revealed novel insights into the mechanism of GSSG reduction. Furthermore, we identified several glutathionylation sites in hTGR, including Cys93, Cys133, and Cys619, and an inhibitory effect of these modifications on enzyme activity. In contrast to other TGRs, for example, from platyhelminth parasites, hTGR did not exhibit hysteretic behavior. These findings provide new insights into the reaction mechanism and regulation of monothiol Grx-containing TGRs. DATABASE: EC numbers: 1.8.1.9; 1.8.1.B1.