Abstract
SummaryThe small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B’s nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.
Highlights
Under diverse stressful conditions, the a-subunit of eukaryotic translation initiation factor 2 is phosphorylated on serine 51 in its N-terminal domain (NTD)
ISRIB Accelerates eIF2B Guanine Nucleotide Exchange Activity in Presence of eukaryotic translation initiation factor 2 (eIF2)(aP) To assess the effects of ISRIB on eIF2B guanine nucleotide exchange activity in isolation of phosphorylated eIF2, we loaded BODIPY-GDP onto eIF2(aS51A) isolated from 293-F cells expressing eIF2 with a non-phosphorylatable S51A mutation in the a-subunit
As reported previously (Tsai et al, 2018), ISRIB only minimally accelerated the exchange of nucleotide mediated by eIF2B in an assay devoid of eIF2(aP) (Figure 1A)
Summary
The a-subunit of eukaryotic translation initiation factor 2 (eIF2) is phosphorylated on serine 51 in its N-terminal domain (NTD). By depleting ternary complexes of eIF2, GTP, and Met-tRNAi in the cell, eIF2a phosphorylation attenuates the translation of most mRNAs, with important effects on protein synthesis. Translation of few mRNAs is increased in an eIF2 phosphorylation-dependent manner As the latter encode potent transcription factors, the production of phosphorylated eIF2 [eIF2(aP)] is coupled with a conserved gene expression program referred to as the integrated stress response (ISR) (Harding et al, 2003). The effect of the mutations on translational control in response to stress was assessed by measuring the incorporation of puromycin into newly synthesized proteins by immunoblotting lysates of untreated and thapsigargin (Sigma, T9033) (200 nM, 45’)-treated cells that had been exposed to 10 mg/mL puromycin (Sigma, P8833) 10 minutes before lysis.
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