Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced By Ischemic Preconditioning

Ornella Cuomo, Giuseppe Pignataro, Rossana Sirabella, Pasquale Molinaro, Serenella Anzilotti, Antonella Scorziello, Maria Josè Sisalli, Gianfranco Di Renzo, Lucio Annunziato

Research output: Contribution to journalArticle

Abstract

BACKGROUND AND PURPOSE—: 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.

Original languageEnglish
JournalStroke
DOIs
Publication statusAccepted/In press - Mar 15 2016

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Sumoylation
Ischemic Preconditioning
Brain
Middle Cerebral Artery Infarction
Post Translational Protein Processing
Brain Ischemia
Sodium-Calcium Exchanger
Ubiquitins
Cell Membrane
Hibernation
Neuroprotection
Ubiquitin
Site-Directed Mutagenesis
Immunoprecipitation
Lysine
Blood Proteins
Membrane Proteins
Homeostasis
Hot Temperature
Western Blotting

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Clinical Neurology
  • Advanced and Specialised Nursing

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Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced By Ischemic Preconditioning. / Cuomo, Ornella; Pignataro, Giuseppe; Sirabella, Rossana; Molinaro, Pasquale; Anzilotti, Serenella; Scorziello, Antonella; Sisalli, Maria Josè; Di Renzo, Gianfranco; Annunziato, Lucio.

In: Stroke, 15.03.2016.

Research output: Contribution to journalArticle

Cuomo, Ornella ; Pignataro, Giuseppe ; Sirabella, Rossana ; Molinaro, Pasquale ; Anzilotti, Serenella ; Scorziello, Antonella ; Sisalli, Maria Josè ; Di Renzo, Gianfranco ; Annunziato, Lucio. / Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced By Ischemic Preconditioning. In: Stroke. 2016.
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AU - Cuomo, Ornella

AU - Pignataro, Giuseppe

AU - Sirabella, Rossana

AU - Molinaro, Pasquale

AU - Anzilotti, Serenella

AU - Scorziello, Antonella

AU - Sisalli, Maria Josè

AU - Di Renzo, Gianfranco

AU - Annunziato, Lucio

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AB - BACKGROUND AND PURPOSE—: 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.

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