title: Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example.
abstract: Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.
authors: Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria.
authors: Sarah
authors: S
authors: Schlosser
authors: David
authors: D
authors: Leitsch
authors: Michael
authors: M
authors: Duchêne
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology; D015153:Blotting, Western; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004748:Entamoeba histolytica / Q000187:drug effects / Q000201:enzymology*; D013058:Mass Spectrometry; D008795:Metronidazole / Q000494:pharmacology; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D054464:Peroxiredoxins / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D051050:Serine O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000981:Antiprotozoal Agents; D004220:Disulfides; D015800:Protozoan Proteins; D008795:Metronidazole; D013879:Thioredoxins; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase; D051050:Serine O-Acetyltransferase