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

2021 ◽  
Author(s):  
Lan Wang ◽  
Morgane Boone ◽  
Rosalie E Lawrence ◽  
Adam Frost ◽  
Peter Walter ◽  
...  
Keyword(s):  
Stress Response ◽  
Point Mutation ◽  
Translational Control ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor

AbstractIn 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.

scholarly journals Binding of the integrated stress response inhibitor, ISRIB, reveals a regulatory site in the nucleotide exchange factor, elF2B

2017 ◽  
Author(s):  
Alisa F. Zyryanova ◽  
Félix Weis ◽  
Alexandre Faille ◽  
Akeel Abo Alard ◽  
Ana Crespillo-Casado ◽  
...  
Keyword(s):  
Stress Response ◽  
Cellular Response ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Allosteric Site ◽  
Exchange Factor ◽  
Interaction Sites ◽  
Nucleotide Exchange Factor

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.

scholarly journals Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J. Zachery Cogan ◽  
Rosalie E. Lawrence ◽  
Morgane Boone ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Double Stranded Rna ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2

AbstractViral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.

scholarly journals Viral Evasion of the Integrated Stress Response Through Antagonistic eIF2-P Mimicry

2021 ◽  
Author(s):  
Michael Schoof ◽  
Lan Wang ◽  
J Zachery Cogan ◽  
Rosalie Lawrence ◽  
Morgane Boone ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Double Stranded Rna ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 43 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα; mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.;

scholarly journals Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Carmela Sidrauski ◽  
Jordan C Tsai ◽  
Martin Kampmann ◽  
Brian R Hearn ◽  
Punitha Vedantham ◽  
...  
Keyword(s):  
Stress Response ◽  
Translational Control ◽  
Control Point ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Eif2α Phosphorylation ◽  
Symmetric Molecule ◽  
Invaluable Tool

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.

scholarly journals Structural basis of eIF2B-catalyzed GDP exchange and phosphoregulation by the integrated stress response

2018 ◽  
Author(s):  
Aditya A Anand ◽  
Lillian R Kenner ◽  
Henry C Nguyen ◽  
Alexander G Myasnikov ◽  
Carolin J Klose ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Structural Basis ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2 ◽  
General Translation

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a GTPase, that becomes activated by eIF2B, a large two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from substrate into an inhibitor of eIF2B. We report cryoEM structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's catalytic centers to catalyze guanine nucleotide exchange. Phosphorylation refolds eIF2, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequesters it into a non-productive complex.

scholarly journals Structural Basis for eIF2B Inhibition in Integrated Stress Response

2018 ◽  
Author(s):  
Kazuhiro Kashiwagi ◽  
Takeshi Yokoyama ◽  
Madoka Nishimoto ◽  
Mari Takahashi ◽  
Ayako Sakamoto ◽  
...  
Keyword(s):  
Stress Response ◽  
Translational Control ◽  
Binding Mode ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Structural Basis ◽  
Dependent Manner ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Electron Microscopic

AbstractA core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers methionylated initiator tRNA to the ribosome in a GTP-dependent manner. In contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor eIF2B, which leads to a deficiency of active eIF2 and resultant global translation repression. To unveil the mechanism by which the eIF2 phosphorylation status regulates the eIF2B nucleotide exchange activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. Intriguingly, the unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active “productive” and nucleotide exchange-inactive “nonproductive” modes, respectively. The nonproductive-mode phosphorylated eIF2, extending from one of the two eIF2B “central cavities”, not only prevents nucleotide exchange on itself, but also sterically prevents unphosphorylated eIF2 from productively binding on the other central cavity of eIF2B, which explains how phosphorylated eIF2 inhibits eIF2B.One Sentence SummaryA drastic change in the binding mode of eIF2 to eIF2B induces translational control in stress.

scholarly journals eIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response

Science ◽  
2019 ◽  
Vol 364 (6439) ◽  
pp. 491-495 ◽  
Author(s):  
Lillian R. Kenner ◽  
Aditya A. Anand ◽  
Henry C. Nguyen ◽  
Alexander G. Myasnikov ◽  
Carolin J. Klose ◽  
...  
Keyword(s):  
Stress Response ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Cryo Electron Microscopy ◽  
Nucleotide Exchange Factor ◽  
Guanosine Triphosphatase ◽  
Initiation Factor Eif2 ◽  
General Translation

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2’s dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo–electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B’s bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2α, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex.

scholarly journals eIF2B extends lifespan through inhibition of the integrated stress response

2020 ◽  
Author(s):  
Maxime Derisbourg ◽  
Laura Wester ◽  
Ruth Baddi ◽  
Martin S. Denzel
Keyword(s):  
Stress Response ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Protein Homeostasis ◽  
Integrated Stress Response ◽  
Nucleotide Exchange

AbstractProtein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains unexplored. Using a genomic screen in Caenorhabditis elegans, we discovered a role of eIF2B and the eIF2 kinases in longevity. By limiting the ISR, these mutations enhanced protein homeostasis and increased lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolonged lifespan. Lifespan extension through ISR inhibition occurred without changes in overall protein synthesis, and depended on enhanced translational efficiency of the kinase KIN-35. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.

scholarly journals ISRIB blunts the integrated stress response by allosterically antagonising the inhibitory effect of phosphorylated eIF2 on eIF2B

2020 ◽  
Author(s):  
Alisa F. Zyryanova ◽  
Kazuhiro Kashiwagi ◽  
Claudia Rato ◽  
Heather P. Harding ◽  
Ana Crespillo-Casado ◽  
...  
Keyword(s):  
Stress Response ◽  
Binding Pocket ◽  
Inhibitory Effect ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor 2

AbstractThe small molecule ISRIB antagonises 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 (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). 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.

scholarly journals Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1920-1932 ◽  
Author(s):  
J L Bushman ◽  
A I Asuru ◽  
R L Matts ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Open Reading Frames ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Molecular Weight Complex ◽  
Exchange Factor

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.

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