An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes

Dissecting the catalytic mechanism of Trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modelling.

Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form trypanothione. Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing the entire kinetic profile. Conclusion: Trypanothione synthetase is affected by substrate and product inhibition. Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this parasite-specific enzyme. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Unsaturated mannich bases active against multidrug-resistant <i>Trypanosoma brucei brucei</i> strains

Unsaturated Mannich bases with potent antitrypanosomal action against multidrug-resistant strains of T. brucei brucei were identified. Their observed activities correlated well with their high Michael acceptor properties but not with their affinities to the P2 purine transporter. A series of unsaturated Mannich bases possessing two electrophilic sites was recently identified as irreversible inhibitors of trypanothione reductase from Trypanosoma cruzi. New derivatives were synthesized by modifying the substitution pattern on the aromatic ring and by incorporating the melamine motif of melarsoprol. Their affinity to P2 transporter and their trypanocidal properties have been studied using three strains expressing various purine transporters. While the melamine derivatives showed some affinity to the P2 transporter, unsaturated Mannich bases without the melamine motif showed excellent potencies against pentamidine-resistant strains of T. brucei brucei suggesting alternative drug uptake routes. The Michael acceptor properties of the three most active compounds towards glutathione correlated with the observed trypanocidal activities

Molecular characterization of glutathione reductase cDNAs from pea (Pisum sativum L.).

A cDNA for pea glutathione reductase has been cloned and sequenced. The derived amino acid sequence of 562 residues shows a high degree of homology to the previously published GR sequences from human erythrocytes and from two prokaryotes: Escherichia coli and Pseudomonas aeruginosa. The pea enzyme differs from other GRs in having an M-terminal leader sequence of about 60–70 residues which may be a chloroplast transit peptide and a 20 amino acid C-terminal extension of unknown function