Gene Knockdown of γ-Glutamylcysteine Synthetase by RNAi in the Parasitic Protozoa Trypanosoma brucei Demonstrates That It Is an Essential Enzyme

摘要: The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in biosynthesis GSH. To evaluate importance thiol metabolism parasite, RNAi methods were used knock down gene expression gamma-GCS procyclic T. cells. Induction with tetracycline led cell death within 4-6 days post-induction. Cell was preceded by depletion protein RNA loss pools T(SH)2. addition (80 microM) cultures rescued phenotype restored intracellular wild-type levels. Treatment cells buthionine sulfoximine (BSO), an enzyme-activated inhibitor gamma-GCS, also resulted death. However, toxicity not reversed GSH, suggesting that BSO has more than one target. depletes thiols similar extent as RNAi; however, did restore These data suggest acts inhibit transport or its peptide metabolites into cell. ability both synthesis likely makes it effective cytotoxic agent single mode action. Finally potential for T(SH)2 biosynthetic enzymes be regulated response reduced levels studied. ornithine decarboxylase S-adenosylmethionine decarboxylase, two essential spermidine biosynthesis, remained constant induced lines.