Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins thioredoxin (Trx) and thioredoxin reductase (TrxR). 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 thioredoxin-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. Based on the disulphide/dithiol-exchange mechanism we constructed an active site mutant of Trx, capable of binding interacting proteins as a stable, mixed disulphide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, serine acetyltransferase-1 (EhSat1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far Western analysis. Trx was able to restore 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-treate...
Source: BJ Energy - Category: Biochemistry Authors: Tags: BJ Energy Source Type: research