Abstract
The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites.
Highlights
Dithiol Trx and Grx belong to the thioredoxin superfamily whose members characteristically share the ‘thioredoxin-fold’ consisting of a central four-stranded b-sheet surrounded by ahelices [15], and an active site with two conserved cysteine residues that specify the biological activity of the protein
Protection from oxidative stress and efficient redox regulation are essential for malarial parasites which have to grow and multiply rapidly in various environments
As shown by glucose-6 phosphate dehydrogenase deficiency, a genetic variation protecting from malaria, the parasite– host cell unit is very susceptible to disturbances in redox equilibrium
Summary
Dithiol Trx and Grx belong to the thioredoxin superfamily whose members characteristically share the ‘thioredoxin-fold’ consisting of a central four-stranded b-sheet surrounded by ahelices [15], and an active site with two conserved cysteine residues that specify the biological activity of the protein. Besides Trx with the classical active site sequence CGPC and Grx possessing a CPYC-motif, tryparedoxin, protein disulfide isomerase, glutathione peroxidase, glutathione S-transferase, and DsbA (a disulfide bond forming protein of bacteria) belong to the thioredoxin superfamily [15,16,17]. The gene encoding Plrx was found to be non-essential for Plasmodium berghei and Plrx knock out parasites did not reveal a significant phenotype throughout the complete life-cycle [19]
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