Glutathione Reductase and Glutamate Dehydrogenase of Plasmodium Falciparum , The Causative Agent of Tropical Malaria

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Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in an oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. Here, we dissected the in vivo roles of both redox pathways by gene targeting of the respective NADPHdependent disulfide reductases. We show that Plasmodium berghei glutathione reductase and thioredoxin reductase are dispensable for proliferation of the pathogenic blood stages. Intriguingly, glutathione reductase is vital for extracellular parasite development inside the insect vector, whereas thioredoxin reductase is dispensable during the entire parasite life cycle. Our findings suggest that glutathione reductase is the central player of the parasite redox network, whereas thioredoxin reductase fulfils a specialized and dispensable role for P. berghei. These results also indicate redundant roles of the Plasmodium redox pathways during the pathogenic blood phase and query their suitability as promising drug targets for antimalarial intervention strategies.

Section: Discussionmentioning

confidence: 99%

Molecular Genetics Evidence for the in Vivo Roles of the Two Major NADPH-dependent Disulfide Reductases in the Malaria Parasite

Buchholz

Putrianti

Rahlfs

et al. 2010

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“…vant for stages of P. falciparum, such as the merozoite stage, which contain glutathione but practically no glutathione reductase (41). When comparing the Trx system with glutathione reductase, less GR than TrxR was required for a given turnover at high enzyme concentrations.…”

Section: Resultsmentioning

confidence: 99%

The Thioredoxin System of the Malaria Parasite Plasmodium falciparum

Kanzok¹,

Schirmer²,

Türbachova³

et al. 2000

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234

193

In most living cells, redox homeostasis is based both on the glutathione and the thioredoxin system. In the malaria parasite Plasmodium falciparum antioxidative proteins represent promising targets for the development of antiparasitic drugs. We cloned and expressed a thioredoxin of P. falciparum (pftrx), and we improved the stable expression of the thioredoxin reductase (PfTrxR) of the parasite by multiple silent mutagenesis. Both proteins were biochemically characterized and compared with the human host thioredoxin system. Intriguingly, the 13-kDa protein PfTrx is a better substrate for human TrxR (K m ‫؍‬ 2 M, k cat ‫؍‬ 3300 min ؊1 ) than for P. falciparum TrxR (K m ‫؍‬ 10.4 M, k cat ‫؍‬ 3100 min ؊1 ). Possessing a midpoint potential of ؊270 mV, PfTrx was found to reduce the disease-related metabolites S-nitrosoglutathione and GSSG. The rate constant k 2 for the reaction between reduced P. falciparum thioredoxin and GSSG was determined to be 0.039 M ؊1 min ؊1 at 25°C and pH 7.4. The k 2 for thioredoxins from man, Drosophila melanogaster, and Escherichia coli was ϳ5 times lower. Our data suggest that GSSG reduction can be supported at a high rate by the TrxR/Trx system in glutathione reductase-deficient cells; this may be relevant for certain stages of the malarial parasite but also for cells containing high [GSSG] of other organisms like dormant forms of Neurospora, glutathione reductasedeficient yeast mutants, or CD4 ؉ lymphocytes of AIDS patients.

“…In parallel, a functional glutathione system comprising NADPH, an FAD-dependent homodimeric glutathione reductase (PfGR), and glutathione exists in P. falciparum. PfGR has been studied in detail (27)(28)(29). Here we describe for the first time the presence of a functional glutaredoxin in P. falciparum, and demonstrate the presence of a second, glutaredoxin-like protein, which shares similarities with PICOT-HD-containing proteins.…”

mentioning

confidence: 88%

Plasmodium falciparum Possesses a Classical Glutaredoxin and a Second, Glutaredoxin-like Protein with a PICOT Homology Domain

Rahlfs

Fischer

Becker

2001

101

The genes coding for two different proteins with homologies to glutaredoxins have been identified in the genome of the malarial parasite Plasmodium falciparum. Both genes were amplified from a gametocytic cDNA and overexpressed in Escherichia coli. The smaller protein (named PfGrx-1) with 12.4 kDa in size exhibits the typical glutaredoxin active site motif "CPYC," shows glutathione-dependent glutaredoxin activity in the ␤-hydroxyethyl disulfide (HEDS) assay, and reduces Trypanosoma brucei ribonucleotide reductase. Glutathione:HEDS transhydrogenase activity (approximately 60 milliunits/mg of protein) was clearly detectable in trophozoite extracts from eight different P. falciparum strains and did not differ between chloroquine-resistant and -sensitive parasites. Five different antimalarial drugs at 100 M did not significantly influence isolated PfGrx-1 activity. In contrast, the second protein (deduced mass 19.9 kDa) with homology to glutaredoxins (31% identity to Schizosaccharomyces pombe in a 140-amino acid overlap) was not active in the HEDS assay; however, its general dithiol reducing activity was demonstrated in the insulin assay in the presence of dithiothreitol. Interestingly, the sequence contains a PICOT (for protein kinase C-interacting cousin of thioredoxin) homology domain, which might suggest regulatory functions of the protein. We named this protein PfGLP-1, for P. falciparum 1-Cys-glutaredoxin-like protein-1. In contrast to glutaredoxins, PfGLP-1 could not be reduced by glutathione. This is the first report on glutaredoxin-like proteins in the family of Plasmodia.