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EIF2B conformation and assembly state regulate the integrated stress response.

ABSTRACT: The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a twofold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-electron microscopy structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

SUBMITTER: Schoof M

PROVIDER: S-EPMC7990499 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Purpose: The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We performed RNA-seq analysis of cerebellum collected from mice harboring a homozygous point mutation in eIF2B5 (HO R191H), a mutation that causes a severe form of VWM in humans and compared gene expression to wildtype age-matched littermates to identify transcriptomic differences at early, mid and late time points during disease progression. We also performed RNA-seq on cerebellum collected from animals treated with the eIF2B activator 2BAct for 1 month to determine to what extent small molecule treatment could normalize the VWM transcriptome. Methods: Samples were collected at 2, 5, and 7 months of age. In addition, HO R191H and WT mice treated with and without with the eIF2B activator 2BAct for 1 month were profiled. RNA-seq libraries were prepared using purified RNA isolated from frozen tissue using the RNeasy Mini kit. RNA quality and concentration were assayed using a Fragment Analyzer instrument. RNA-seq libraries were prepared using the TruSeq Stranded Total RNA kit paired with the Ribo-Zero rRNA removal kit. Libraries were sequenced on an Illumina HiSeq 4000 instrument. For the experiment comparing different ages, N = 3 males/genotype/time point. For the 4-week 2BAct treatment experiment, N = 3 females/condition. Conclusions: HO R191H mutant brains show an elevated gene expression signature of the Integrated Stress Reponse in mice as early as 2 months, preceding the appearance of overt pathology in these animals. The expression of the ISR-related genes does not significantly increases at 5 months and 7 months of age. One month of treatment with 2BAct completely prevented the expression of ISR-related in genes in 2 month old animals.

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EIF2B conformation and assembly state regulate the integrated stress response.

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