Protein sumoylation in synaptosomes, effect on glutamate release and presynaptic Ca2+ infux
SUMO-1 is a protein with similar structure to ubiquitin. SUMOylation modifi es proteins structure effecting their molecular mechanisms. SUMO-1 is present in neurons and in the synapse. Here we are investigating whether SUMO-1 is a presynaptic protein and whether SUMOylation modulates glutamate release. Ultrasynaptic separation approach was used to investigate the distribution of SUMO-1 in peri-, post- and presynaptic compartments in synaptosomes. Presynaptic protein SUMOylation is augmented by KCl or AMPA stimulus and is decreased by kainate. NMDA has no infl uence presynaptic protein SUMOylation level. Synaptosomes glutamate release was measured. Recombinant SUMO-1 and SENP-1, a SUMO-specifi c proteases which cleave SUMO from its substrates, where used with their mutated recombinant variant in synaptosomes as entrapped proteins. KCl stimulated glutamate release is increased when synaptosomes contain SENP-1 but is decreased when synaptosomes contain full-length SUMO-1. It was observed decrease in kainate evoked glutamate release from synaptosomes preloaded with SENP-1 but a marked increase in release from synaptosomes preloaded with SUMO-1. Intracellular Ca2+ concentration was also measured. Wild-type SENP-1 increased Ca2+ infl ux evoked by KCl or AMPA and decreased it following a kainate challenge. Conjugatable SUMO-1(GG), on the other hand, reduced Ca2+ infl ux evoked by KCl or AMPA and increased the levels of Ca2+ infl ux elicited by kainate.