UCSF

The mammalian integrated stress response (ISR) is a major translational control point activated in response to diverse cellular stresses, such as unfolded proteins in the ER, viral infection, heme-deficiency and amino-acid starvation. The ISR is mediated by four kinases that activate upon stress and phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). This phosphorylation event renders eIF2 an inhibitor of its dedicated guanine exchange factor, eIF2B, leading to the attenuation of global protein synthesis and selective translation of uORF-containing mRNAs, e.g. the stress-responsive transcription factor ATF4. Using a cell-based screen, our lab discovered the small molecule ISRIB (integrated stress response inhibitor) which reverses the effects of eIF2α-phosphorylation, restores protein synthesis and remarkably enhances cognition in both wild-type mice and corrects cognitive deficits after brain injury. In this work a reporter-based shRNA screen identified eIF2B as a potential molecular target of ISRIB. We found that ISRIB stabilizes an eIF2B heterodecamer and enhances the GEF activity of eIF2B independent of eIF2 phosphorylation. We also designed new analogs of ISRIB that improve its EC50 to 600pM in cell culture. Furthermore, to gain mechanistic and structural insight into ISRIB's activity we established a robust recombinant system for eIF2B and solved a 2.8Å cryo-EM structure of ISRIB-bound human eIF2B. The structure revealed ISRIB's binding site at the symmetric core of the eIF2B heterodecamer. Structural and biochemical analyses further revealed that the fully active eIF2B heterodecamer depends on assembly of two identical tetrameric subcomplexes, and that ISRIB promotes this step by bridging the symmetry interface. Thus, regulation of eIF2B assembly appears as a new translational control point central to the integrated stress response.