linoleic-acid-hydroperoxide and trypanothione

Hydroperoxide detoxification in African trypanosomes is achieved by 2-Cys-peroxiredoxin (TXNPx)- and non-selenium glutathione peroxidase (Px)-type enzymes which both obtain their reducing equivalents from the unique trypanothione/tryparedoxin system. Previous RNA interference approaches revealed that the cytosolic TXNPx and the Px-type enzymes are essential for Trypanosoma brucei. Because of partially overlapping in vitro substrate specificities and subcellular localisation the physiological function of the individual enzymes was not yet clear. As shown here, TXNPx and Px are expressed at comparable levels and in their active reduced state. Px-overexpressing parasites were less sensitive toward linoleic acid hydroperoxide but not hydrogen peroxide. Kinetic studies confirmed that Px-but not TXNPx-reduces lipophilic hydroperoxides including phospholipids with high efficiency. Most interestingly, the severe proliferation defect of Px-depleted bloodstream cells could be rescued by Trolox, but not by hydrophilic antioxidants, in the medium. This allowed us to knock-out the three Px genes individually and thus to distinguish their in vivo role. Deletion of the cytosolic Px I and II resulted in extremely fast membrane peroxidation followed by cell lysis. Cells lacking specifically the mitochondrial Px III showed a transient growth retardation and cardiolipin peroxidation but adapted within 24h to normal proliferation.

Topics: Antioxidants; Chromans; Gene Knockdown Techniques; Glutathione; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Oxidation-Reduction; Protozoan Proteins; Spermidine; Thioredoxins; Transgenes; Trypanosoma brucei brucei; Trypanosomiasis, African

In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18 kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide>cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.

Topics: Amino Acid Sequence; Animals; Glutathione; Glutathione Peroxidase; Hydrogen Peroxide; Linoleic Acids; Lipid Peroxides; Molecular Sequence Data; Recombinant Fusion Proteins; Sequence Alignment; Spermidine; Substrate Specificity; tert-Butylhydroperoxide; Trypanosoma cruzi