Molecular characterization and interactome analysis of Trypanosoma cruzi Tryparedoxin 1

Abstract

Trypanosoma cruzi tryparedoxin 1 (TcTXN1) is an oxidoreductase belonging to the thioredoxin superfamily, which mediates electron transfer between trypanothione and peroxiredoxins. In trypanosomes TXNs, and not thioredoxins, constitute the oxido-reductases of peroxiredoxins. Since, to date, there is no information concerning TcTXN1 substrates in T. cruzi, the aim of this work was to characterize TcTXN1 in two aspects: expression throughout T. cruzi life cycle and subcellular localization; and the study of TcTXN1 interacting-proteins. We demonstrate that TcTXN1 is a cytosolic and constitutively expressed protein in T. cruzi. In order to start to unravel the redox interactome of T. cruzi we designed an active site mutant protein lacking the resolving cysteine, and validated the complex formation in vitro between the mutated TcTXN1 and a known partner, the cytosolic peroxiredoxin. Through the expression of this mutant protein in parasites with an additional 6xHis-tag, heterodisulfide complexes were isolated by affinity chromatography and identified by 2-DE/MS. This allowed us to identify fifteen TcTXN1 proteins which are involved in two main processes: oxidative metabolism and protein synthesis and degradation. Our approach led us to the discovery of several putatively TcTXN1-interacting proteins thereby contributing to our understanding of the redox interactome of T. cruzi.

TcI Isolates of Trypanosoma cruzi Exploit the Antioxidant Network for Enhanced Intracellular Survival in Macrophages and Virulence in Mice

“…The increase in tryparedoxin perdoxidases occurred in infective trypomastigote and replicative amastigote stages irrespective of the cell types (e.g., muscle cells in this study, and LLC-MK2 and macrophages in other studies [34,50]) used for culturing the parasites. Regeneration of the reduced TXNPx depends on cytosolic (TXN I)-and membrane (TXN II)-localized thioredoxin substrates (31,51). Consistent with the increased abundance of TXNPx, the TXN I level was also increased in COL and SYL trypomastigotes (Fig.…”

“…Parasite lysates were probed by using antibodies against cytosolic (cTXNPx) and mitochondrial (mTXNPx) isoforms of tryparedoxin peroxidase, tryparedoxin isoforms TXN I and TXN II, and iron superoxide dismutase isoforms FeSODa and FeSODb (31,32). Due to the finding of a differential abundance of ␣-tubulin and ␤-actin in T. cruzi isolates (Table 1 and Table 2), we could not use these proteins for normalizing the antioxidant protein content.…”

“…Polyclonal antibodies against T. cruzi cytosolic tryparedoxin peroxidase (cTXNPx) and mitochondrial tryparedoxin peroxidase (mTXNPx) were obtained as described previously (31,32). Polyclonal antibodies against the iron superoxide dismutase (FeSOD) isoforms, FeSODa and FeSODb, and the tryparedoxin isoforms, TXN I and TXN II, were produced as described previously (31,32).…”

Expresión diferencial entre estadios de Trypanosoma cruzi I en el aislamiento de un paciente con cardiomiopatía chagásica crónica de zona endémica de Santander, Colombia

“…Dada su relevancia en los mecanismos de evasión de la respuesta inmunitaria, estas enzimas se han considerado factores de virulencia importantes y están siendo objeto de estudio para utilizarlas en el diseño racional de fármacos (21). En un estudio de análisis de expresión diferencial entre parásitos obtenidos a partir de un caso agudo comparados con los de uno crónico, obtenidos en la zona endémica de Santander en Colombia, se encontró sobreexpresión de la peroxidasa triparredoxina, la cual podría estar asociada con el desarrollo de la forma aguda de la enfermedad (22).…”

Biochemistry and Physiology of Reactive Oxygen Species in Euglena

“…4.4.3 for T(SH) 2 and TRYR). Previous studies have demonstrated that TXNs function as electron donors for the Prxs required for ROS metabolism, DNA and protein synthesis through protein-protein interactions, thus making them essential for parasite survival (Piñeyro et al 2011;Flohé 2012). Four TXN homologous genes have been found in Euglena, with three isoforms being predicted to be localized in the cytosol and the other in mitochondria (Table 4.1, Fig.…”