KINETIC CHARACTERIZATION OF A NON-CANONICAL THIOREDOXIN SPECIFIC FOR CYSTINE REDUCTION FROM ENTAMOEBA HISTOLYTICA

ARIAS, DIEGO G; BIROCCO, FRANCO; GONZALEZ, LN; VILLAR, S; GUERRERO, SA; IGLESIAS, AA; FERRER-SUETA, G

Congreso; Reunión Conjunta SAIB-SAMIGE 2020; 2020

Entamoeba histolytica, a unicellular parasite, usually lives and multiplies within the human gut, under reduced oxygen pressure. During tissue invasion, the parasite is exposed to increased amounts of highly toxic reactive oxygen species. E. histolytica lacks or has insignificant amounts of glutathione, with cysteine being the major intracellular thiol. Previously, we characterized in E. histolytica its functional thioredoxin system, composed of thioredoxins (TRXs) and thioredoxin reductase (TRXR). In this work, we present the kinetics characterization of non-canonical thioredoxin (EhTRX212) with high specificity for cystine reduction from E. histolytica. By steady-state kinetics assays, we observed that EhTRX212 was unable to accept reduction equivalents from EhTRXR directly and reduce protein disulfides. However, the EhTRX212 was able to catalyze the reduction of cystine, CySNO, and cysteine-derivate low molecular mass disulfides via EhTRX8 (a canonical TRX) and EhTRXR. Besides, EhTRX212 exhibited GSH-dependent cysteine reductase activity via a coupled enzymatic assay. Kinetic data indicated that regeneration of EhTRX212 to its reduced form is more efficient using reduced EhTRX8 than GSH as a reducing substrate. The obtained results are novelty because the EhTRX212 is one of the first cases where it is observed a specific and efficient reduction equivalent transfer from a canonical TRX to another TRX. In a complementary way, by pre-steady-state kinetics (using stopped-flow methodology), we observed that the cystine-reduction by EhTRX212 follows a biphasic temporal progression, consistent with a thiol-disulfide exchange mechanism. The first phase (the nucleophilic cysteine attack on cystine disulfide) followed second-order kinetics (k = 2.4 ·106 M-1s-1) and the second phase (the mixed disulfide resolution) followed first-order kinetics (k = 11 s-1). The chemical substitutions (for example, the N-acetylation) in cysteine moiety impair the reduction by EhTRX212 of derivative low molecular mass disulfides, indicating a substrate specificity by cysteine-moiety in disulfide substrates. Finally, EhTRX212 has been localized in the cytosol of trophozoites by confocal microscopy experiments. This work strongly supports the occurrence of a new class of TRX. Our results extend the knowledge regarding TRX function in E. histolytica and suggest that these proteins have critical roles in the redox metabolism of this pathogen parasite.Granted by ANPCyT (PICT2016-1778 and PICT2017-2268)