Differential Association of 4E-BP2-Interacting Proteins Is Related to Selective Delayed Neuronal Death after Ischemia

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.

Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

“…It is established that the availability of eIF4E depending on the eIF4E/eIF4G association contributes to the progress of translation during cellular stress response. Thus, while the specific inhibition of eIF4E by eIF4E-binding proteins (4E-BPs) is a well-known control mechanism of translation inhibition, including cerebral ischemia [11,26,28,[39][40][41], the knowledge of eIF4G regulation is incomplete.…”

Stroke Proteomics: From Discovery to Diagnostic and Therapeutic Applications

“…84 To reveal other potentially important proteins in this regulation, 4E-BP2–interacting proteins were identified by proteomics in the ischemia-resistant cortex and vulnerable hippocampus in sham and animals that underwent bilateral common carotid artery occlusion. 85 Interestingly, the 4E-BP2 interaction profile in the ischemia-resistant cortex compared with the susceptible hippocampus was already vastly different at baseline, which could explain differing vulnerabilities of these areas. Of note, HSC70, DRP2, α-enolase, UCH-L1, adenosine kinase-1, GAPDH, and phosphoglycerate kinase-1, maintained their region-specific interaction with 4E-BP2 after ischemia.…”

“…Of note, HSC70, DRP2, α-enolase, UCH-L1, adenosine kinase-1, GAPDH, and phosphoglycerate kinase-1, maintained their region-specific interaction with 4E-BP2 after ischemia. 85 Although proteomic association studies led to the discovery of many potential new regulators of PKC, PPP1, and 4E-BP2, follow-up studies on the functional implications of modified interactions in the context of ischemia and preconditioning are lacking.…”

“…Of note, HSC70, DRP2, α-enolase, UCH-L1, adenosine kinase-1, GAPDH, and phosphoglycerate kinase-1, maintained their region-specific interaction with 4E-BP2 after ischemia. 85…”

Dihydropyrimidinase-Related Protein 2 Is a New Partner in the Binding between 4E-BP2 and eIF4E Related to Neuronal Death after Cerebral Ischemia

“…In previous studies by our group, we demonstrated that 4E-BP2 phosphorylation-or other post-translational modifications-is not the mechanism that operates on 4E-BP2 regulation under ischemiareperfusion stress, though it is the changes in the 4E-BP2-eIF4E association that have consequences on protein-synthesis recovery or inhibition in the resistant and vulnerable brain regions, respectively [20,21]. In our goal to determine whether the protein synthesis recovery in resistant areas could be dependent on specific modulators involved in the association/dissociation of 4E-BP2-eIF4E, using a proteomic approach we recently reported new 4E-BP2-interacting proteins, which has been the first described 4E-BP2 interactome in the cerebral cortex to date, and we compared it with that of the vulnerable CA1 region [22]. The study found seven 4E-BP2-interacting proteins with changes in their association between the resistant and vulnerable brain regions after IR (Supplementary Figure S1).…”