CONICET | Buscador de Institutos y Recursos Humanos

Calcium/calmodulin-dependent protein kinase 2 delta isoform (CaMKII) is a highly abundant myocardial enzyme that plays a central role in cellular signaling.1 CaMKII transmits Ca2+ signals to downstream substrates through the activation of its serine/threonine kinase activity in a calcium-bound calmodulin (CaM) dependent manner. Under physiological conditions, CaMKII activity level is finely tuned by the complex interplay between a series of ligand binding, structural and post-translational modification (PTM) events acting on its regulatory domain.2 In this sense, regulation imbalances leading to CaMKIICa2+-independent hyperactivation have been related to several cardiac pathologies.1,3 Although some of the control mechanisms such as Ca2+/CaM concentrations, CaMKII autophosphorylation and autoinhibitory structural arrangements have been extensively characterized2, oxidative regulation by methionine sulfoxide (MetOx) formation has been recently identified as a PTM capable of modulating the switch-like properties of this kinase.4 In this work, we introduce a new NMR-based approach to delineate CaMKII regulatory phenomena in vitro and in vivo with atomic resolution. We have designed an isotopically enriched peptide probe (rCaMKII consisting of the regulatory domain of CaMKII in its reduced or methione-oxidized forms and studied it using high-resolution multidimensional NMR. Our studies suggest that rCaMKII can be used to thoroughly characterize the oxidative regulation in an integrative and sitespecific manner. Furthermore, we tested the use of rCaMKII as an in vivo NMR reporter in living zebrafish (Danio rerio) embryos. We monitored the endogenous reduction of multiple peptide MetOx sites in the native cellular context of a multicellular organism. Altogether, we have developed a novel strategy that will allow us to delineate the regulatory pathways controlling CaMKII activity and their cross-talk under physiological and pathological conditions.