eIF2B-capturing viral protein NSs suppresses the integrated stress response

Kashiwagi, Kazuhiro; Shichino, Yuichi; Osaki, Tatsuya; Sakamoto, Ayako; Nishimoto, Madoka; Takahashi, Mari; Mito, Mari; Weber, Friedemann; Ikeuchi, Yoshiho; Iwasaki, Shigeo; Ito, Takuhiro

doi:10.1101/2021.06.07.447466

Cited by 3 publications

(4 citation statements)

References 44 publications

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“…Deep sequencing data processing was performed as described previously 134,135 with modifications. For paired-end sequencing, fastp (version 0.21.0) 136 was used for base correction and read 1 was processed for downstream analysis.…”

Section: Discussionmentioning

confidence: 99%

The complexity and dynamics ofin organellotranslation assessed by high-resolution mitochondrial ribosome profiling

Wakigawa,

Mito,

Yamashiro

et al. 2023

Preprint

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Eukaryotes house an additional protein synthesis system within the mitochondria. Given that mitochondrial translation dictates OXPHOS complex abundance and ATP levels in cells, exhaustive, quantitative, and high-resolution delineation of mitoribosome traversal is needed. Here, we developed a technique for high-resolution mitochondrial ribosome profiling and unveiled the tight regulation of mammalian in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the absolute numbers of mitoribosomes on a transcript and the elongation rate, initiation rate, and lifetime rounds of translation of individual transcripts. We also surveyed the impacts of modifications at anticodon stem loop in mt-tRNAs, including all possible decorations at 34th position, by deleting the corresponding enzymes and harnessing patient-derived mtDNA A3243G cells. Moreover, retapamulin-assisted profiling and disome profiling unveiled cryptic translation initiation sites at subcognate codons and programmed mitoribosome collision sites across the mitochondrial transcriptome, respectively. Our work provides a useful resource for delineating protein synthesis within this indispensable organelle.

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Section: Discussionmentioning

confidence: 99%

The complexity and dynamics ofin organellotranslation assessed by high-resolution mitochondrial ribosome profiling

Wakigawa,

Mito,

Yamashiro

et al. 2023

Preprint

Self Cite

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“…Figs. 1–2 ) 13,18,21,22,26,27 . We observed broad agreement between regions of HDX-MS protection and experimental secondary structure assignment based on the cryo-EM structure of apo-eIF2B ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…All experiments performed prior to March 1, 2022 were performed using human eIF2 purified as previously described 27 . Some experiments were performed using purified eIF2 that was a generous gift of Calico Life Sciences LLC.…”

Section: Purification Of Heterotrimeric Human Eif2mentioning

confidence: 99%

“…In the absence of binding partners, cryo-EM data suggests that eIF2B primarily samples the A-state 14 . The eIF2B A-State can be further stabilized by binding to eIF2 substrate, ISRIB and its analogs, or the viral ISR-inhibiting protein NSs 26,27 . The two states are negatively coupled: the A-State and I-State have alternatively accessible binding sites, such that the A-State has a properly formed eIF2-binding pocket, and the I-State has a properly formed eIF2-P-binding pocket 13,14 .…”

Section: Introductionmentioning

confidence: 99%

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A central helical fulcrum in eIF2B coordinates allosteric regulation of Integrated Stress Response signaling

Lawrence

Shoemaker

Deal

et al. 2022

Preprint

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The Integrated Stress Response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling down-regulates translation and activates expression of stress-responsive factors that promote return to homeostasis, and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions are unknown. Using hydrogen deuterium exchange-mass spectrometry and cryo-EM, we identified a single alpha-helix whose orientation allosterically controls eIF2B conformation and assembly. Biochemical and signaling assays show that this 'Switch-Helix' controls eIF2B activity and signaling in cells. In sum, the Switch-Helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a novel allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.

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A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

Wang

Boone

Lawrence

et al. 2021

Preprint

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In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.

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eIF2B-capturing viral protein NSs suppresses the integrated stress response

Cited by 3 publications

References 44 publications

The complexity and dynamics ofin organellotranslation assessed by high-resolution mitochondrial ribosome profiling

The complexity and dynamics ofin organellotranslation assessed by high-resolution mitochondrial ribosome profiling

A central helical fulcrum in eIF2B coordinates allosteric regulation of Integrated Stress Response signaling

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

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