Characterization of an atypical thioredoxin from Entamoeba histolytica with specificity for cystine reduction

GUERRERO SA; BIROCCO F; ARIAS DG; IGLESIAS AA

Salta

Congreso; SAIB LV Reunión Anual; 2019

SAIB

Entamoebahistolytica, a unicellular parasite,usually lives and multiplies within the human gut, under reduced oxygenpressure. During tissue invasion, it is exposed to increased amounts ofreactive oxygen species, which are highly toxic for the parasite. The metabolicpathways used by this organism to cope with such environmental changes andredox homeostasis are a matter of our work. Recently, we characterized in E.histolytica its functional thioredoxin system, composed by thioredoxins(TRXs) and thioredoxin reductase (TRXR). In this work, we present thefunctional characterization of an atypical thioredoxin (EhTRX212) withspecificity for cystine reduction from E. histolytica. By in vitroassays we observed that EhTRX212 was unable to accept reductionequivalents from EhTRXR directly. However, the protein was able to catalysethe reduction of cystine, CySNO and cysteine-derivate low molecular massdisulfides via EhTRXR in presence of EhTRX8 (a canonical TRX). Interestingly,chemical substitutions (for example, N-acetylation) in cysteine moiety preventthe reduction by EhTRX212 of derivative low molecular mass disulfides,indicating substrate specificity by cysteine-moiety in disulfide substrates. Inaddition, the protein catalysed GSH-dependant cystine reduction, similar to classicglutaredoxin activity. In line with the above, EhTRX212 was able tocoordinate iron-sulfur cluster (ISC) by an in vitro reconstitutionassay. By gel filtration chromatography and UV-Vis spectroscopy experimentswere detected EhTRX212-ISC complexes. Complementarily, by biotin-switch technique, we evaluated thecapacity of EhTRX212 to reduce S-cysteinylated proteins from E. histolytica cells. Finally, weperformed confocal microscopy experiments and EhTRX212 has been immunolocalized in cytosol of trophozoites. This work strongly supportsthe occurrence in E. histolytica of a new TRX, which were not previouslydescribed in the parasite. Our results extend the knowledge regarding to EhTRXfunction and suggest that these proteins have important functions in redox metabolismof this pathogen parasite.Granted by ANPCyT (PICT2016-1778 and PICT2017-2268).