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. 2015 May 29;348(6238):1027-30.
doi: 10.1126/science.aaa6986. Epub 2015 Apr 9.

Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound

Yusuke Sekine  1 , Alisa Zyryanova  2 , Ana Crespillo-Casado  2 , Peter M Fischer  3 , Heather P Harding  2 , David Ron  1
Affiliations

Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound

Yusuke Sekine et al. Science. .

Abstract

The integrated stress response (ISR) modulates messenger RNA translation to regulate the mammalian unfolded protein response (UPR), immunity, and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action, we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the amino-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wild-type cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity toward its substrate, the translation initiation factor eIF2, in vitro. Thus, ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR.

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Figures

Fig. 1
Fig. 1. ISRIB reverses attenuated translation and accelerates eIF2B GEF activity towards eIF2(αP) in vitro
(A) Immunoblot of newly-synthesized puromycinylated proteins in extracts of untreated CHO cells or cells exposed to the ISR-inducing agent thapsigargin (Tg 300 nM, 30 minutes) in the presence or absence of trans ISRIB (100 nM). Phosphorylated (P-eIF2α) and total eIF2α were detected in the immunoblots below. Quantified signal intensities are shown in Fig. S5. (B) Dose-response of ISRIB-stimulation of translation in reticulocyte lysate fitted to a non-linear trace. Shown are mean ± SEM (n = 3) and EC50 (for active trans-ISRIB). Note the inactivity of cis-ISRIB. (C) GEF activity as reflected in time dependent decrease in fluorescence of weakly and heavily phosphorylated eIF2 loaded with Bodipy-FL-GDP and incubated with unlabeled GDP in the presence or absence of cell lysate (μg). Shown is a mean of three independent measurements. (D) Relation between the initial velocities of the release of Bodipy-FL-GDP from heavily phosphorylated eIF2 and ISRIB concentration, fitted to a non-linear trace. Shown are mean ± SEM (n = 3) and EC50 for trans-ISRIB. (E) GEF activity reflected in the initial velocities of GDP release reactions with CHO cell lysate (samples 1-4), wildtype or mutant eIF2αS51A/S51A mouse embryonic fibroblast lysate (MEFs, samples 5-8) and Bodipy-FL-GDP loaded eIF2 of the indicated eIF2α genotype. Shown are mean ± SEM (n = 3 for samples 1-4 and n = 6 for samples 5-8). *P < 0.05, **P < 0.01 (Student’s t test). (F) As in “E”, but with purified eIF2B and Bodipy-FL-GDP loaded non-phosphorylated and phosphorylated eIF2. Shown are mean ± SEM (n = 8). *P = 0.012, **P = 0.0054 (Student’s t test). (G) Coomassie-stained SDS-PAGE of the purified eIF2B used in “F”. The five subunits of eIF2B and PRMT5 (*, a non-specific contaminant) are noted.
Fig. 2
Fig. 2. Selection of ISRIB resistant (ISRIBr) mutations
(A) Histograms of the distribution of GFP fluorescence arising from an ISR-inducible CHOP::GFP reporter gene in parental CHO-C30 cells and clones bearing the indicated mutations. The cells were left untreated or treated with histidinol (His; 0.5 mM), ISRIB (100 nM) or both. EMS1M-5 exemplifies a class of clones with a weak and EMS1H-4 a class with a strong ISRIBr phenotype. (B) Immunoblot of puromycinylated proteins in extracts of parental CHO-C30 cells or a representative strong ISRIBr clone (EMS1H-4) following exposure to thapsigargin (Tg) in the presence or absence of ISRIB (as in Fig. 1A). The images are representative of all three independent experiments that yielded similar results. Quantified signal intensities are shown in (Fig. S5). (C) Bar diagram, displaying the reversal of translation attenuation by ISRIB in “B” above: Reversal = [(PTg+ISRIB-PTg) ÷ (PUT-PTg)] × 100, (PTg+ISRIB, PTg and PUT are the puro signal from the sample treated with Tg and ISRIB (Lanes 3 or 6), Tg alone (Lanes 2 or 5) and the untreated sample (Lanes 1 or 3), respectively. Shown are mean ± SEM (n = 3). * P < 0.05 (Student’s t test). (D) Bar diagram of the GEF activity of lysates from parental and strong ISRIBr mutant cells with Bodipy-FL-GDP-loaded eIF2(αP) as a substrate in the absence or presence of ISRIB, as indicated. Shown are mean ± SEM of the initial velocity of the decline in Bodipy-FL-GDP fluorescence upon adding lysate, normalized to the rate in the untreated sample (n = 4).
Fig. 3
Fig. 3. Clustered mutations in Eif2b4 impart ISRIB resistance
(A) Schema of eIF2Bδ with the position of the mutations associated with an ISRIBr phenotype showing. These are clustered at the unique N-terminal region that is not conserved in the other regulatory subunits (α, β) of eIF2B. (B-E) Distribution of CHOP::GFP reporter gene activity in parental CHO-C30 cells and derivative sub-clones bearing the indicated mutations (induced by CRISPR-Cas9 targeted homologous recombination at the Eif2b4 locus). The cells were left untreated or treated for 24 hours with histidinol (His; 0.5 mM) alone or with ISRIB (100 nM). (F-G) Bar diagram of the GEF activity of lysates from parental or CRISPR-Cas9-induced ISRIBr mutant cells with Bodipy-FL-GDP-loaded eIF2 or eIF2(αP) as substrates in the absence or presence of ISRIB, as indicated. Shown are mean ± SEM (n = 6, for “F”; n=5 for “G”). * P < 0.05, n.s.; not significant (Student’s t test).
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