FUNCTIONAL CHARACTERIZATION OF A HYBRID FMSR PROTEIN FROM TRYPANOSOMA CRUZI DM28C

GONZALEZ, NADIA L.; BIROCCO, FRANCO; SASONI, NATALIA; 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

Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzedby methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems hasbeen mainly focused on the involvement of trypanothione, a specific redox component in these organisms. Trypanosoma cruzi, the etiologicalagent of Chagas disease, is auxotroph by methionine. Through an analysis of its genome project, we identified a coding sequence of free methioninesulfoxide reductase (fMSR), a protein only present in unicellular organisms. In T. cruzi, this protein is constituted by a GAF-like domain fused tothe TIP41 domain (homologous to yeast TIP41 protein involved in the TOR pathway negative regulation). The encoding sequence for the GAFdomain was expressed in Escherichia coli, and the corresponding recombinant protein was purified and functionally characterized. Therecombinant protein exhibited MSR activity with L-Met(R)SO and T. cruzi tryparedoxins and thioredoxin as the reducing substrates. Our resultssupported that this enzyme has non-saturation ping-pong kinetics. In addition, based on the fact that the GAF domain is present in proteins capableto bind nucleotides, we evaluated the effect of AMP, ADP or ATP on its MSR activity. We observed an inhibitory effect at low substrateconcentration (in the presence of MgCl2), mainly by ATP. On the other hand, we performed a yeast complementation assay using a Δtip41 mutant.The results showed that TcfMSR (both isolated TIP41 domain as the full protein) could compensate the sensitive to rapamycin phenotype. Thisresult indicates that the TcfMSR is active acting in the yeast TOR pathway. These results suggest that the TcfMSR protein is a possible linkbetween the redox metabolism and the TOR pathway in Trypanosoma cruzi. Granted by ANPCyT (PICT2016-1778 and PICT2017-2268)