An NMR-active chemical probe to measure stereospecific methionine sulfoxide reductase activity in vitro and in vivo

FRANCO A. BIGLIONE; VERÓNICA A. LOMBARDO; ANDRES BINOLFI; CAROLINA SÁNCHES-LOPÉZ; BRUNO MANTA PORTEIRO

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica - SAB 2019; 2019

Sociedad Argentina de Biofísica

Oxidation of methionines is a hallmark of oxidative stress but it is now emerging as a post-translational modification capable of regulating protein activity and cellular processes. The oxidation of methionine side chains is a stereoselective oxidation of sulphur giving rise to two diastereomers designated S- and R-methionine sulfoxide (MetOx). This covalent modification can be reversed by the stereospecific enzymes MSRA and MSRB that specifically reduce MetOx S- and R-diastereomers, respectively. The stereospecific activity of these enzymes has been widely studied in vitro, nevertheless the analytical tools available to study reductases activities in native environments are scarce. The few available are limited to analyze just the activity of a single reductase and they do not allow discriminating between MSRA and MSRB in the same reaction. In this work, we developed a MetOx-derived small molecule called CarMetOx, as an NMR-active chemical probe to monitor stereospecific MSRs activity in native environments. We exploited the idea that sulfoxides display unique NMR signals that include different sets of resonances for their S- and R-diastereomers that are readily identified in the NMR spectra. Therefore, the progressive disappearance of cross-peaks corresponding to each diastereomer of 15N-labelled CarMetOx can be analyzed by NMR to describe MSRA and MSRB enzymatic activity in vitro and in vivo. Reduction kinetics of an in vitro mixture of S- and R-diastereomers of CarMetOx spiked with pure MSRA or MSRB, showed that our chemical probe behaves like an oxidized protein substrate and allowed us to unambiguously assign each diastereoisomer. Studies in cell extracts demonstrated that CarMetOx is a good biosensor to monitor the activity of endogenous bacterial and mammalian MSRA and MSRB. Lastly, we tested CarmetOx in developing zebrafish embryos as a proof-of-principle for its in vivo use. Real-time NMR measurements of CarMetOx microinjected in the entire organism showed endogenous enzymatic MSRs activity and the analysis of each diastereomer demonstrated that zebrafish MSRAs enzymes are more efficient than MSRBs. Overall, we introduce CarMetOx, a novel NMR-based biosensor to study MSRs activities in native environments and at the same time introduces an analytical tool to explore the structural roles and functions of biomolecules in multicellular organisms by NMR.