Monitoring Methionine Sulfoxide Reductase Activity in Native Environments by NMR

BINOLFI, A.; LABADIE, N.; LOMBARDO, V.; LOMBARDO, V.; MANTA, B.; MANTA, B.; BINOLFI, A.; LABADIE, N.

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Oxidation of methionine side chains, which has been traditionally perceived as damage derived from oxidative stress, is now emerging as a post-translational modifcation capable of regulating protein activity and cellular processes such as the activation of Ca2+/Calmodulin dependent kinase II or remodeling of the acting cytoskeleton. Methionine oxidation leads to two diastereomers known as the R and S forms of methionine sulfoxide (MetOx). In vivo, MetOx is reduced back to methionine by two families of stereospecifc and conserved methionine sulfoxide reductases, MSRA and MSRB, which specifcally target protein-bound MetOx-R and -S isomers, respectively. Methionine oxidation is amongst the most diffcult-to-study post-translational protein modifcation in vivo because sulfoxides are not easily detected with chemical probes or antibodies and because they spontaneously form during sample preparation for mass spectrometry. By contrast, MetOx display unique NMR signals, including different sets of resonances for its R and S isomers that are readily identifed in the NMR spectra. In order to assess the role of MSRs in cellular function we ought to monitor their activities in vivo with high resolution. In this work we designed an NMR based assay to simultaneously delineate MSRA and MSRB reductase activity in vitro and in cell extracts and validated its use for further in vivo studies using In-cell NMR methodologies. Our results showed that MSRA is moreactive than MSRB in E. coli cells and those bacteria have free MetOx reduction activity, in line with previous reports. We expect that our results will open new research paths to expand the repertoire of MetOx-regulated signaling pathways and to further understand the role of MSRs in physiology and pathology.