Malaria, one of the most severe infectious diseases, is caused by five pathogenic Plasmodium species: P. malariae, P. vivax, P. knowlesi, P. berghei and P. falciparum. The latter, causing severe anaemia and cerebral malaria, accounts for the majority of malaria infections in Sub-Saharan Africa. The malarial parasite possesses two redox systems: the thioredoxin system and the glutathione system, both comprising a cascade of redox-active proteins. As P. falciparum lacks catalase and glutathione peroxidase, the parasite is in need of maintaining the intracellular redox balance which is provided by enzymes such as peroxiredoxins. Peroxiredoxins (Prxs) are thiol peroxidases involved in fundamental processes of life by providing antioxidant defense and by contributing to cellular redox regulation. Prxs are tightly regulated, occur in different oligomerization states, and reduce hydrogen peroxide, organic hydroperoxides, and peroxynitrite by thiol-based enzyme-substitution mechanisms. The unicellular eukaryotic malaria parasite Plasmodium falciparum possesses five Prxs including cytosolic Prx1a and Prx6, mitochondrial Prx1m, apicoplast Prx5, and nuclear PrxQ. Furthermore, the parasite imports human erythrocyte Prx2 into its cytosol to enhance its antioxidant capacity.In this thesis, the contributions of conserved amino acids located closely to the active site, at the dimer/dimer interface, or at the monomer/monomer interface to the catalytic cycle of Plasmodium peroxiredoxins were assessed. In-depth site directed mutagenesis studies on PfPrx1a indicated that the substitution of the conserved residue Tyr42 results in a complete loss of peroxidase activity, presumably because the mutated enzyme cannot form the catalytic essential higher oligomers anymore. Loss of Arg125 also led to a drastic decrease in activity, although the dynamic shift from decamer to dimer was intact. P. falciparum possesses a large repertoire of small redox-active proteins which have a thioredoxin sequence similarity and belong to the group of the thioredoxin superfamily. Plasmoredoxin (Plrx), has been identified by the Becker group and on the basis of databank information, Plrx seems to be unique for Plasmodium species. To continue, the accessibility of individual cysteine residues of PfPrxs and PfPlrx to S-glutathionylation and S-nitrosation, post-translational modification involved in regulation of protein structure and function, was studied. PfPrx1a, PfPrx1m, PfPrx5, PfPrxQ, and PfPlrx are identified as targets for post-translational modifications. For further in-depth analysis of the susceptible cysteine residues, cysteine mutants of PfPrx1a (C50S/C74A, C74A/C170S, C50S/C170S, C50S/C74A/C170S), PfPrx5 (C117S, C143S, C117S/C143S), and PfPrxQ (C56S, C103S, C56S/C103S) were glutathionylated. For PfPrx1a, all cysteines were found to be glutathionylated with particular susceptibility of Cys74 and Cys50. Both cysteines of PfPrx5 were glutathionylated, with a slight preference for Cys117 (CP of Prx5) over Cys143. For PfPrxQ, solely the peroxidatic cysteine Cys56 was found to bind glutathione. All wild type Prxs were analyzed for their glutathionylation sites via mass spectrometry. This method confirmed glutathionylation of all three cysteines in PfPrx1a, and both cysteines in PfPrx5. Although Western Blot analysis had indicated only Cys56 of PfPrxQ as target of glutathionylation, mass spectrometry (MS) showed that also the resolving cysteine Cys103 might be accessible. For PfPrx1m, MS indicated the peroxidatic (C152) and the resolving (C187) cysteine as glutathionylation sites.PfPlrx wild type enzyme and cysteine mutants PfPlrxC3S, PfPlrxC63S, and PfPlrxC115S were analyzed for their glutathionylation sites via mass spectrometry and the S-glutathionylation of the two active site cysteines C60 and C63 could be shown. Furthermore, PfPrxs and PfPlrx are accessible for S-nitrosation.This data provides further insight into the complex catalysis of these redox-active enzymes and points to potential novel target sites for specific enzyme inhibitors.
Verknüpfung zu Publikationen oder weiteren Datensätzen
