Stress-Induced ProteinS-Glutathionylation andS-Trypanothionylation in African Trypanosomes—A Quantitative Redox Proteome and Thiol Analysis

Abstract

Aims: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation.Results: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification.Innovation: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides.Conclusion: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517–533.

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites

“…The reverse reaction, namely the reduction of mixed disulfides of GSH with proteins, is the primary function of glutaredoxins and Grx1 is not the exception (28). In the infective form of T. brucei, Grx1 accounts for about 40 -50% of the deglutathionylase activity measured in cell extracts (27), which was then confirmed by the slow reversion of protein S-thiolation in Grx1-KO parasites exposed to diamide (67). The current evidence shows that Grx1 does not function as a major protein oxidoreductase in the protection against oxidative stress (66, 67) but as key redox regulator of the activity of yet unknown partner(s) involved in parasite thermotolerance (27).…”

“…A recent study shows that Grx1 does not contribute to the overall protein S-thiolation of T. brucei (i.e. the level of protein-bound GSH was almost identical in WT and Grx1-deficient cells (67)). This is likely explained by the fact that Grx1 is rapidly oxidized to its disulfide by GSSG and that the glutathionylated intermediate could only be detected at low concentrations of GSSG.…”

A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes

“…The level of free thiols, free thiols and disulfides and protein-bound thiols was determined as described previously [64] . Shortly, about 1 × 10 8 BS T. brucei were grown at 37 °C or 39 °C, washed with PBS and an aliquot was removed for counting.…”

“…Despite the absence of a classical glutathione system, trypanosomatids contain appreciable concentrations of free GSH as well as a repertoire of distinct Grxs [12] , [33] . Recently we showed that as response to exogenous and endogenous oxidative stresses, the mammalian bloodstream (BS) form of T. brucei can undergo protein S-glutathionylation and S-trypanothionylation [64] .…”

“…Both T. brucei Grxs and T. cruzi Grx catalyze the reduction of the mixed disulfide between GSH and either 2-mercaptoethanol or cysteine residues of various model proteins, a reaction that is not taken over, at least to a physiological competent degree, by Tpx [9] , [43] , [46] . Indeed, the cytosolic Grx1 has been shown to contribute to about 50% of the deglutathionylation capacity of infective T. brucei [49] and facilitates the reversion of stress-induced protein S-thiolation [64] . Nonetheless, at variance with Tpx [13] , parasites lacking Grx1 are fully proliferative in vitro and in vivo , and even display a marked thermoresistance when grown at 39 °C [49] .…”