title: Validation of Trypanosoma brucei trypanothione synthetase as drug target.
abstract: In trypanosomes, the parasite-specific thiol trypanothione [T(SH)2] fulfills various functions, the best established being detoxification of H2O2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH)2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH)2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH)2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H2O2 and tert.-butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-conjugated thiols (Gsp and T(SH)2) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites.
authors.affiliation: Department of Biochemistry, Technical University of Braunschweig, Braunschweig, Germany.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Comini
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Guerrero
authors.forename: Simon
authors.initials: S
authors.lastname: Haile
authors.forename: Ulrich
authors.initials: U
authors.lastname: Menge
authors.forename: Heinrich
authors.initials: H
authors.lastname: Lünsdorf
authors.forename: Leopold
authors.initials: L
authors.lastname: Flohé
mesh_terms: D019732:Amide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002470:Cell Survival; D015536:Down-Regulation; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D034622:RNA Interference; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism; D013489:Suppression, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D000964:Antimetabolites, Antineoplastic; D016877:Oxidants; C012333:glutathionylspermidine; D019328:Buthionine Sulfoximine; C044809:trypanothione; D006861:Hydrogen Peroxide; D019732:Amide Synthases; C064407:trypanothione synthetase; D005978:Glutathione; D013095:Spermidine