One-pot stereospecific detection of methionine sulfoxide reduction in native environments

BRUNO MANTA; NATALIA LABADIÉ; ANDRÉS BINOLFI; VERÓNICA LOMBARDO; FRANCO BIGLIONE; CAROLINA SÁNCHES-LOPÉZ

Berlin

Workshop; EMBO Workshop_In situ Methods in Cell Biology and Cellular Biophysics; 2018

Oxidation of methionine (Met) side chains, which has been traditionally perceived as damage derived from oxidative stress, is now emerging as a post-translational modification capable of regulating protein activity and cellular processes, such as the activation of Ca2+-calmodulin dependent protein kinase II or the remodeling of the actin cytoskeleton. Oxidation of Met leads to two diasteroisomers known as the R and S forms of methionine sulfoxide (MetOx). In vivo, MetOx is reduced back to Met by two families of stereospecific and conserved methionine sulfoxide reductases, MSRA and MSRB, which specifically target MetOx-S and -R isomers, respectively. In order to assess the role of MSRs in cellular function we ought to monitor their activities in vivo with high resolution. MetOx display different sets of NMR signals for its R and S isomers that are readily identified and we exploited this feature to design an NMR based assay to simultaneously delineate MSRA and MSRB reductase activity in vitro and in vivo. Using chemically modified isotopically enriched L-Met and the protein gamma-synuclein as MSR substrates we showed that bacteria have increased MSRB activity towards the free aminoacid while MSRA and MSRB activity on protein-bound MetOx are comparable. In higher organisms MSRA and MSRB activities are comparable for the different substrates. Our results suggest that there exist functional differences between prokaryotic and eukaryotic MSRs that have emerged during evolution to increase the signaling repertoire of higher organisms.