title: Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced by Ischemic Preconditioning.
abstract: OBJECTIVE
  The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation.
  
  
  METHODS
  Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay.
  
  
  RESULTS
  We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning.
  
  
  CONCLUSIONS
  Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Ornella
authors.initials: O
authors.lastname: Cuomo
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pignataro
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Rossana
authors.initials: R
authors.lastname: Sirabella
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Pasquale
authors.initials: P
authors.lastname: Molinaro
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Serenella
authors.initials: S
authors.lastname: Anzilotti
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Antonella
authors.initials: A
authors.lastname: Scorziello
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Maria Josè
authors.initials: MJ
authors.lastname: Sisalli
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.).
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Di Renzo
authors.affiliation: From the Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatological Sciences, School of Medicine, Federico II University of Naples, Naples, Italy (O.C., G.P., P.M., A.S., M.J.S., G.D.R., L.A.); and SDN IRCCS, Naples, Italy (R.S., S.A.). lannunzi@unina.it.
authors.forename: Lucio
authors.initials: L
authors.lastname: Annunziato
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism* / Q000473:pathology; D019194:Ischemic Preconditioning; D008297:Male; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D000066829:Neuroprotection / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D025842:SUMO-1 Protein / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D058207:Sumoylation
chemical_list: D025842:SUMO-1 Protein; C491102:Slc8a3 protein, rat; D019831:Sodium-Calcium Exchanger
keywords: hibernation; ischemia; neuroprotection; sumoylation; ubiquitin