Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B

AbstractThe Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the β and δ regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congeners in vivo and ISRIB-binding in vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

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The β/Gcd7 Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2B), a Guanine Nucleotide Exchange Factor, Is Crucial for Binding eIF2 In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.00265-10 ◽

2010 ◽

Vol 30 (21) ◽

pp. 5218-5233 ◽

Cited By ~ 23

Author(s):

Kamal Dev ◽

Hongfang Qiu ◽

Jinsheng Dong ◽

Fan Zhang ◽

Dominik Barthlme ◽

...

Keyword(s):

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Nucleotide Exchange ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Eukaryotic Translation Initiation

ABSTRACT Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(αP)]. While eIF2B contains five subunits, the ε/Gcd6 subunit is sufficient for GEF activity in vitro. The δ/Gcd2 and β/Gcd7 subunits function with α/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(αP)-GDP, but the essential functions of δ/Gcd2 and β/Gcd7 are not well understood. We show that the depletion of wild-type β/Gcd7, three lethal β/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in β/Gcd7 and eIF2α all impair eIF2 binding to eIF2B without reducing ε/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, β/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2α (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, β/Gcd7 can overcome the toxicity of eIF2(αP) and rescue native eIF2B function when overexpressed with δ/Gcd2 or γ/Gcd1. In aggregate, these findings provide compelling evidence that β/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (αP).

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Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.6876 ◽

1997 ◽

Vol 17 (12) ◽

pp. 6876-6886 ◽

Cited By ~ 52

Author(s):

S Z Tarun ◽

A B Sachs

Keyword(s):

Translation Initiation ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation ◽

Stimulation Of

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

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Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

Molecular and Cellular Biology ◽

10.1128/mcb.02254-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2384-2397 ◽

Cited By ~ 40

Author(s):

Jeanne M. Fringer ◽

Michael G. Acker ◽

Christie A. Fekete ◽

Jon R. Lorsch ◽

Thomas E. Dever

Keyword(s):

Translation Initiation ◽

Ribosomal Subunit ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation ◽

Cell Extracts ◽

Eukaryotic Translation Initiation ◽

Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

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Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4618 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4618-4631 ◽

Cited By ~ 31

Author(s):

J L Bushman ◽

M Foiani ◽

A M Cigan ◽

C J Paddon ◽

A G Hinnebusch

Keyword(s):

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Eukaryotic Translation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Initiation Factor 2 ◽

Eukaryotic Translation Initiation

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

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eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

10.1101/2020.12.28.424567 ◽

2020 ◽

Author(s):

Michael Schoof ◽

Morgane Boone ◽

Lan Wang ◽

Rosalie Lawrence ◽

Adam Frost ◽

...

Keyword(s):

Stress Response ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Α Subunit ◽

Rocking Motion

AbstractThe 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 two-fold 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 an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

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eIF2B conformation and assembly state regulates the integrated stress response

eLife ◽

10.7554/elife.65703 ◽

2021 ◽

Vol 10 ◽

Author(s):

Michael Schoof ◽

Morgane Boone ◽

Lan Wang ◽

Rosalie Lawrence ◽

Adam Frost ◽

...

Keyword(s):

Stress Response ◽

Binding Sites ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Α Subunit ◽

Rocking Motion

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 two-fold 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-EM 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.

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Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4618-4631.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4618-4631

Author(s):

J L Bushman ◽

M Foiani ◽

A M Cigan ◽

C J Paddon ◽

A G Hinnebusch

Keyword(s):

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Eukaryotic Translation ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Initiation Factor 2 ◽

Eukaryotic Translation Initiation

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

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Cytoplasmic eIF6 promotes OSCC malignant behavior through AKT pathway

Cell Communication and Signaling ◽

10.1186/s12964-021-00800-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Zechen Zhao ◽

Weiming Chu ◽

Yang Zheng ◽

Chao Wang ◽

Yuemei Yang ◽

...

Keyword(s):

Western Blot ◽

Direct Interaction ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Migration And Invasion ◽

Eukaryotic Translation ◽

Mesenchymal Transformation

Abstract Background Eukaryotic translation initiation factor 6 (eIF6), also known as integrin β4 binding protein, is involved in ribosome formation and mRNA translation, acting as an anti-association factor. It is also essential for the growth and reproduction of cells, including tumor cells. Yet, its role in oral squamous cell carcinoma (OSCC) remains unclear. Methods The expression characteristics of eIF6 in 233 samples were comprehensively analyzed by immunohistochemical staining (IHC). Effects of eIF6 over-expression and knockdown on cell proliferation, migration and invasion were determined by CCK-8, wound healing and Transwell assays. Western blot, immunofluorescence (IF) and co-immunoprecipitation (co-IP) were performed for mechanical verification. Results We found that cytoplasmic eIF6 was abnormally highly expressed in OSCC tissues, and its expression was associated with tumor size and the clinical grade. Amplification of eIF6 promoted the growth, migration and invasion capabilities of OSCC cell lines in vitro and tumor growth in vivo. Through Western blot analysis, we further discovered that eIF6 significantly promotes epithelial-mesenchymal transformation (EMT) in OSCC cells, while depletion of eIF6 can reverse this process. Mechanistically, eIF6 promoted tumor progression by activating the AKT signaling pathway. By performing co-immunoprecipitation, we discovered a direct interaction between endogenous eIF6 and AKT protein in the cytoplasm. Conclusion These results demonstrated that eIF6 could be a new therapeutic target in OSCC, thus providing a new basis for the prognosis of OSCC patients in the future.

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Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

Science ◽

10.1126/science.aaq0939 ◽

2018 ◽

Vol 359 (6383) ◽

pp. eaaq0939 ◽

Cited By ~ 63

Author(s):

Jordan C. Tsai ◽

Lakshmi E. Miller-Vedam ◽

Aditya A. Anand ◽

Priyadarshini Jaishankar ◽

Henry C. Nguyen ◽

...

Keyword(s):

Competitive Inhibitor ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Cryo Electron Microscopy ◽

Nucleotide Exchange Factor ◽

Initiation Factor Eif2 ◽

Resolution Structure

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo–electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

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