Stress-Induced Protein S-Glutathionylation and S-Trypanothionylation in African Trypanosomes-A Quantitative Redox Proteome and Thiol Analysis.
OBJECTIVE
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, H2O2or 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. H2O2causes 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)2and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification.


METHODS
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.


CONCLUSIONS
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.
1 Biochemie-Zentrum der Universität Heidelberg (BZH) , Heidelberg, Germany .
Kathrin
K
Ulrich
1 Biochemie-Zentrum der Universität Heidelberg (BZH) , Heidelberg, Germany .
Caroline
C
Finkenzeller
2 Zentrum für Molekularbiologie der Universität Heidelberg (ZMBH) , Heidelberg, Germany .
Sabine
S
Merker
3 Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh , Edinburgh, United Kingdom .
Federico
F
Rojas
3 Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh , Edinburgh, United Kingdom .
Keith
K
Matthews
2 Zentrum für Molekularbiologie der Universität Heidelberg (ZMBH) , Heidelberg, Germany .
Thomas
T
Ruppert
1 Biochemie-Zentrum der Universität Heidelberg (BZH) , Heidelberg, Germany .
R Luise
RL
Krauth-Siegel
D003958:Diamide / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology; D018384:Oxidative Stress; D017293:Protein S / Q000378:metabolism*; D020543:Proteome / Q000032:analysis; D015800:Protozoan Proteins / Q000032:analysis; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D017293:Protein S; D020543:Proteome; D015800:Protozoan Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D006997:Hypochlorous Acid; C044809:trypanothione; D006861:Hydrogen Peroxide; D005978:Glutathione; D013095:Spermidine
Trypanosoma; oxidative stress; protein S-glutathionylation; proteome; trypanothione