NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

Eukaryotic translation initiation factor 4E family member 2 (eIF4E2) has recently been identified as an interacting protein of GSK3β and regulates its proline-directed kinase activity. eIF4E2 undergoes ISGylation at K134 and K222, a conserved posttranslational modification mediated by interferon-stimulated gene 15. In this study, we engineered a novel Nanobody-based ISGylation Targeting Chimera (NITAC) tool to specifically activate eIF4E2 ISGylation and investigate its role in the eIF4E2-GSK3β signaling pathway. By integrating eIF4E2-specific nanobodies Nb.30C7 with the catalytic E3 ligase domain HECT from HERC5, we constructed the NITAC (Nb.30C7-HECT). This NITAC tool mediates site-specific ISGylation of eIF4E2, enhancing the eIF4E2-GSK3β interaction and unexpectedly suppressing proline-directed serine/threonine phosphorylation across multiple crucial targets within the eIF4E2-GSK3β pathway. Importantly, NITAC treatment exerted cytoprotection against oxygen-glucose deprivation/reoxygenation stress, a commonly used in vitro model to simulate ischemic conditions in cell cultures. Furthermore, NITAC treatment reduced reactive oxygen species in neurons and microglia and promoted an anti-inflammatory phenotype in microglia by suppressing proline-directed serine/threonine phosphorylation. In summary, we created a novel NITAC to specifically activate eIF4E2 ISGylation, which showed cytoprotective effects under oxygen-glucose deprivation/reoxygenation stress by inhibiting GSK3β proline-directed kinase activity.