Abstract
Cerebral ischemia induces an inhibition of protein synthesis and causes cell death and neuronal deficits. These deleterious effects do not occur in resilient areas of the brain, where protein synthesis is restored. In cellular stress conditions, as brain ischemia, translational repressors named eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) specifically bind to eIF4E and are critical in the translational control. We previously described that 4E-BP2 protein, highly expressed in brain, can be a molecular target for the control of cell death or survival in the reperfusion after ischemia in an animal model of transient cerebral ischemia. Since these previous studies showed that phosphorylation would not be the regulation that controls the binding of 4E-BP2 to eIF4E under ischemic stress, we decided to investigate the differential detection of 4E-BP2-interacting proteins in two brain regions with different vulnerability to ischemia-reperfusion (IR) in this animal model, to discover new potential 4E-BP2 modulators and biomarkers of cerebral ischemia. For this purpose, 4E-BP2 immunoprecipitates from the resistant cortical region and the vulnerable hippocampal cornu ammonis 1 (CA1) region were analyzed by two-dimensional (2-D) fluorescence difference in gel electrophoresis (DIGE), and after a biological variation analysis, 4E-BP2-interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Interestingly, among the 4E-BP2-interacting proteins identified, heat shock 70 kDa protein-8 (HSC70), dihydropyrimidinase-related protein-2 (DRP2), enolase-1, ubiquitin carboxyl-terminal hydrolase isozyme-L1 (UCHL1), adenylate kinase isoenzyme-1 (ADK1), nucleoside diphosphate kinase-A (NDKA), and Rho GDP-dissociation inhibitor-1 (Rho-GDI), were of notable interest, showing significant differences in their association with 4E-BP2 between resistant and vulnerable regions to ischemic stress. Our data contributes to the first characterization of the 4E-BP2 interactome, increasing the knowledge in the molecular basis of the protection and vulnerability of the ischemic regions and opens the way to detect new biomarkers and therapeutic targets for diagnosis and treatment of cerebral ischemia.
Highlights
Eukaryotic initiation factor 4E-binding protein 2 (4E-BP2) belongs to a group of 4E-binding translational repressor proteins (4E-BP1, 4E-BP2, and 4E-BP3), which binds to eIF4E and inhibit its function
The results showed that 4E-BP1 was not detected in the immunoprecipitates, but it remained mainly in the non-precipitated fraction, along with almost undetectable amounts of 4E-BP2 (Figure S1)
In this study we identified the 4E-BP2 interactome in a transient global cerebral ischemia model
Summary
Eukaryotic initiation factor (eIF) 4E-binding protein 2 (4E-BP2) belongs to a group of 4E-binding translational repressor proteins (4E-BP1, 4E-BP2, and 4E-BP3), which binds to eIF4E and inhibit its function. Active (hypo- or de-phosphorylated) forms of 4E-BPs translational repressors compete with eIF4G factor in their union with eIF4E, avoiding eIF4F complex formation and inhibiting cap-dependent translation [2,3]. It is well-known that 4E-BP1 is a substrate of mammalian target of rapamycin (mTOR), a serine/threonine-protein kinase in the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or Akt)/mTOR signaling pathway, which is the main catalytic component of mTOR complex 1 (mTORC1) [4,5]. While translation regulation by 4E-BP1 phosphorylation has been deeply characterized [7,8,10,11], 4E-BP2 regulation mechanism is still unclear, and other mechanisms different to phosphorylation are involved, despite 4E-BP2 shares with 4E-BP1 the phosphorylation regulation sites, [13,14]
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