CHARACTERIZATION OF AN ATYPICAL THIOREDOXIN FROM ENTAMOEBA HISTOLYTICA WITH SPECIFICITY FOR CYSTINE REDUCTION

BIROCCO, FRANCO; GUERRERO, SERGIO A.; IGLESIAS, ALBERTO A.; ARIAS, DIEGO G.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular y Pan-American Association for Biochemistry and Molecular Biology

Entamoeba histolytica, a unicellular parasite, usually lives and multiplies within the human gut, under reduced oxygen pressure. During tissueinvasion, it is exposed to increased amounts of reactive oxygen species, which are highly toxic for the parasite. The metabolic pathways used bythis organism to cope with such environmental changes and redox homeostasis are a matter of our work. Recently, we characterized in E. histolyticaits functional thioredoxin system, composed by thioredoxins (TRXs) and thioredoxin reductase (TRXR). In this work, we present the functionalcharacterization of atypical thioredoxin (EhTRX212) with specificity for cystine reduction from E. histolytica. By in vitro assays, we observedthat EhTRX212 was unable to accept reduction equivalents from EhTRXR directly. However, the protein was able to catalyze the reduction ofcystine, CySNO and cysteine-derivate low molecular mass disulfides via EhTRXR in presence of EhTRX8 (a canonical TRX). Interestingly,chemical substitutions (for example, N-acetylation) in cysteine moiety prevent the reduction by EhTRX212 of derivative low molecular massdisulfides, indicating substrate specificity by cysteine-moiety in disulfide substrates. In addition, the protein catalyzed GSH-dependant cystinereduction, similar to classic glutaredoxin activity. In line with the above, EhTRX212 was able to coordinate the iron-sulfur cluster (ISC) by an invitro reconstitution assay. By gel filtration chromatography and UV-Vis spectroscopy experiments, we detected EhTRX212-ISC complexes.Complementarily, by the biotin-switch technique, we evaluated the capacity of EhTRX212 to reduce S-cysteinylated proteins from E. histolyticacells. Finally, we performed confocal microscopy experiments and EhTRX212 has been immunolocalized in the cytosol of trophozoites. Thiswork strongly supports the occurrence in E. histolytica of a new TRX, which was not previously described in the parasite. Our results extend theknowledge regarding EhTRX function and suggest that these proteins have important functions in the redox metabolism of this pathogen parasite.Granted by ANPCyT (PICT2016-1778 and PICT2017-2268)