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Vanishing white matter disease (VWMD) due to eIF2B mutations is a common leukodystrophy characterised by childhood onset, autosomal recessive inheritance, and progressive clinical course with episodic worsening. There are no reports of genetically confirmed adult patients from India. We describe the phenotype of two adults with genetically confirmed VWMD and typical radiological findings. Both had spastic ataxia and cognitive and behavioural disturbances. Other neurological features included myoclonic jerks and parkinsonism. At the last follow-up (duration: 2–9 years), one patient was wheelchair-bound. VWMD is rare in adults but should be suspected based on radiological findings and confirmed by eIF2B mutation.

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

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.

Eukaryotic translation initiates upon recruitment of the EIF2-GTP·Met-tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, β, γ subunits) is a GTPase. The GDP to GTP exchange within EIF2 is facilitated by the guanine nucleotide exchange factor EIF2B (α-e subunits). During stress-induced conditions, phosphorylation of the α-subunit of EIF2 turns EIF2 into an inhibitor of EIF2B. In turn, inhibition of EIF2B decreases TC formation and triggers the internal stress response (ISR), which determines the cell fate. Deregulated ISR has been linked to neurodegenerative disorders and cancer, positioning EIF2B as a promising therapeutic target. Hence, a better understanding of the mechanisms/factors that regulate EIF2B activity is required. Here, combining transcript and protein level analyses, we describe an intronically polyadenylated (IPA) transcript of EIF2B's γ-subunit. We show that the IPA mRNA isoform is translated into a C-terminus truncated protein. Using structural modeling, we predict that the truncated EIF2Bγ protein has unfavorable interactions with EIF2γ, leading to a potential decrease in the stability of the nonproductive EIF2:EIF2B complex. While we discovered and confirmed the IPA mRNA isoform in breast cancer cells, the expression of this isoform is not cancer-specific and is widely present in normal tissues. Overall, our data show that a truncated EIF2Bγ protein co-exists with the canonical protein and is an additional player to regulate the equilibrium between productive and nonproductive states of the EIF2:EIF2B complex. These results may have implications in stress-induced translation control in normal and disease states. Our combinatorial approach demonstrates the need to study noncanonical mRNA and protein isoforms to understand protein interactions and intricate molecular mechanisms.

The eukaryotic translation initiation factor eIF2 is a GTPase, which brings the initiator Met-tRNAi to the ribosome as the eIF2-GTP·Met-tRNAi ternary complex (TC). TC regeneration is catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2 phosphorylation by several stress-induced kinases converts it into a competitive inhibitor of eIF2B. Inhibition of eIF2B activity lowers cellular TC concentrations, which in turn triggers the integrated stress response (ISR). Depending on its degree of activation and duration, the ISR protects the cell from the stress or can itself induce apoptosis. ISR dysregulation is a causative factor in the pathology of multiple neurodegenerative disorders, while ISR inhibitors are neuroprotective. The realization that eIF2B is a promising therapeutic target has triggered significant interest in its structure and its mechanisms of action and regulation. Recently, four groups published the cryo-electron microscopy structures of eIF2B with its substrate eIF2 and/or its inhibitor, phosphorylated eIF2 [eIF2(α-P)]. While all three structures of the nonproductive eIF2B·eIF2(α-P) complex are similar to each other, there is a sharp disagreement between the published structures of the productive eIF2B·eIF2 complex. One group reports a structure similar to that of the nonproductive complex, whereas two others observe a vastly different eIF2B·eIF2 complex. Here, we discuss the recent reports on the structure, function, and regulation of eIF2B; the preclinical data on the use of ISR inhibitors for the treatment of neurodegenerative disorders; and how the new structural and biochemical information can inform and influence the use of eIF2B as a therapeutic target.

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1-month-old Eif2b5I98M brain. Histological analyses indicated up-regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751.

Objective: Vanishing white matter (VWM) is a fatal, stress-sensitive leukodystrophy that mainly affects children and is currently without treatment. VWM is caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B) that is crucial for initiation of mRNA translation and its regulation during the integrated stress response (ISR). Mutations reduce eIF2B activity. VWM pathomechanisms remain unclear. In contrast with the housekeeping function of eIF2B, astrocytes are selectively affected in VWM. One study objective was to test our hypothesis that in the brain translation of specific mRNAs is altered by eIF2B mutations, impacting primarily astrocytes. The second objective was to investigate whether modulation of eIF2B activity could ameliorate this altered translation and improve the disease. Methods: Mice with biallelic missense mutations in eIF2B that recapitulate human VWM were used to screen for mRNAs with altered translation in brain using polysomal profiling. Findings were verified in brain tissue from VWM patients using qPCR and immunohistochemistry. The compound ISRIB (for “ISR inhibitor”) was administered to VWM mice to increase eIF2B activity. Its effect on translation, neuropathology, and clinical signs was assessed. Results: In brains of VWM compared to wild-type mice we observed the most prominent changes in translation concerning ISR mRNAs; their expression levels correlated with disease severity. We substantiated these findings in VWM patients’ brains. ISRIB normalized expression of mRNA markers, ameliorated brain white matter pathology and improved motor skills in VWM mice. Interpretation: The present findings show that ISR deregulation is central in VWM pathomechanisms and a viable target for therapy.

Cells respond to viral infections through sensors that detect non-self-molecules, and through effectors, which can have direct antiviral activities or adapt cell physiology to limit viral infection and propagation. Eukaryotic translation initiation factor 2 alpha kinase 2, better known as PKR, acts as both a sensor and an effector in the response to viral infections. After sensing double-stranded RNA molecules in infected cells, PKR self-activates and majorly exerts its antiviral function by blocking the translation machinery and inducing apoptosis. The antiviral potency of PKR is emphasized by the number of strategies developed by viruses to antagonize the PKR pathway. In this review, we present an update on the diversity of such strategies, which range from preventing double-stranded RNA recognition upstream from PKR activation, to activating eIF2B downstream from PKR targets.

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor., Here the authors show that a viral protein interferes with the binding of phosphorylated eIF2 to eIF2B, thereby suppressing the host integrated stress response (ISR). This suppression of the ISR abrogates translational changes of the host and ameliorates neurite degradation under stress.

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.

Significance The integrated stress response (ISR) protects cells from a variety of harmful stressors by temporarily halting protein synthesis. However, chronic ISR activation has pathological consequences and is linked to several neurological disorders. Pharmacological inhibition of chronic ISR activity emerges as a powerful strategy to treat ISR-mediated neurodegeneration but is typically linked to adverse effects due to the ISR’s importance for normal cellular function. Paradoxically, the small-molecule ISR inhibitor ISRIB has promising therapeutic potential in vivo without overt side effects. We demonstrate here that ISRIB inhibits low-level ISR activity, but does not affect strong ISR signaling. We thereby provide a plausible mechanism of how ISRIB counteracts toxic chronic ISR activity, without disturbing the cytoprotective effects of a strong acute ISR., Activation of the integrated stress response (ISR) by a variety of stresses triggers phosphorylation of the α-subunit of translation initiation factor eIF2. P-eIF2α inhibits eIF2B, the guanine nucleotide exchange factor that recycles inactive eIF2•GDP to active eIF2•GTP. eIF2 phosphorylation thereby represses translation. Persistent activation of the ISR has been linked to the development of several neurological disorders, and modulation of the ISR promises new therapeutic strategies. Recently, a small-molecule ISR inhibitor (ISRIB) was identified that rescues translation in the presence of P-eIF2α by facilitating the assembly of more active eIF2B. ISRIB enhances cognitive memory processes and has therapeutic effects in brain-injured mice without displaying overt side effects. While using ISRIB to investigate the ISR in picornavirus-infected cells, we observed that ISRIB rescued translation early in infection when P-eIF2α levels were low, but not late in infection when P-eIF2α levels were high. By treating cells with varying concentrations of poly(I:C) or arsenite to induce the ISR, we provide additional proof that ISRIB is unable to inhibit the ISR when intracellular P-eIF2α concentrations exceed a critical threshold level. Together, our data demonstrate that the effects of pharmacological activation of eIF2B are tuned by P-eIF2α concentration. Thus, ISRIB can mitigate undesirable outcomes of low-level ISR activation that may manifest neurological disease but leaves the cytoprotective effects of acute ISR activation intact. The insensitivity of cells to ISRIB during acute ISR may explain why ISRIB does not cause overt toxic side effects in vivo.

Neuronal protein synthesis is essential for long-term memory consolidation. Conversely, dysregulation of protein synthesis has been implicated in a number of neurodegenerative disorders, including Alzheimer’s disease (AD). Several types of cellular stress trigger the activation of protein kinases that converge on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α-P). This leads to attenuation of cap-dependent mRNA translation, a component of the integrated stress response (ISR). We show that AD brains exhibit increased eIF2α-P and reduced eIF2B, key components of the eIF2 translation initiation complex. We further demonstrate that attenuating the ISR with the small molecule compound ISRIB (ISR Inhibitor) rescues hippocampal protein synthesis and corrects impaired synaptic plasticity and memory in mouse models of AD. Our findings suggest that attenuating eIF2α-P-mediated translational inhibition may comprise an effective approach to alleviate cognitive decline in AD.

Summary The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the β-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.

Protein 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.

Vanishing white matter disease (VWMD) is a rare leukodystrophy involving loss of function mutations of the guanine exchange factor eIF2B and typically presenting with juvenile onset. We aimed to identify repurposable FDA approved drugs in anin vitrodrug screen using patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived astrocytes. Dysregulated GADD34 and CHOP were identified in patient fibroblasts and iPSC-derived astrocytes under proteasomal stress conditions. A drug screen from a 2400 FDA approved drug library withEIF2B5disease patient fibroblasts identified 113 anti-inflammatory drugs as a major class of hits with cytoprotective effects. A panel of potential candidate drugs including berberine, deflazacort, ursodiol, zileuton, guanabenz and Anavex 2-73, and preclinical ISRIB, increased cell survival of MG132-stressedEIF2B2andEIF2B5disease VWMD astrocytes, and were further investigated for their effect on the integrated stress response and mitochondrial stress. ISRIB but not other drugs significantly affected eIF2α phosphorylation and GADD34 expression. Ursodiol demonstrated capacity to reduce complex I subunit upregulation, ameliorate oxidative stress, loss of mitochondrial membrane potential and upregulation of eIF2B subunits in VWMD astrocytes, highlighting its potential as a cytoprotective compound for VWMD.

Background Eukaryotic initiation factor 2B (eIF2B) initiates and regulates translation initiation in eukaryotes. eIF2B gene mutations cause leukoencephalopathy called vanishing white matter disease (VWM) in humans and slow growth (Slg−) and general control derepression (Gcd−) phenotypes in Saccharomyces cerevisiae. Results To suppress eIF2B mutations, S. cerevisiae genomic DNA library was constructed in high-copy vector (YEp24) and transformed into eIF2B mutant S. cerevisiae strains. The library was screened for wild-type genes rescuing S. cerevisiae (Slg−) and (Gcd−) phenotypes. A genomic clone, Suppressor-I (Sup-I), rescued S. cerevisiae Slg− and Gcd− phenotypes (gcd7-201 gcn2∆). The YEp24/Sup-I construct contained truncated TAN1, full length EMC4, full length YGL230C, and truncated SAP4 genes. Full length EMC4 (chaperone protein) gene was sub-cloned into pEG (KG) yeast expression vector and overexpressed in gcd7-201 gcn2∆ strain which suppressed the Slg− and Gcd− phenotype. A GST-Emc4 fusion protein of 47 kDa was detected by western blotting using α-GST antibodies. Suppression was specific to gcd7-201 gcn2∆ mutation in eIF2Bβ and Gcd1-502 gcn2∆ in eIF2Bγ subunit. Emc4p overexpression also protected the wild type and mutant (gcd7-201 gcn2∆, GCD7 gcn2∆, and GCD7 GCN2∆) strains from H2O2, ethanol, and caffeine stress. Conclusions Our results suggest that Emc4p is involved in eIF2B-mediated translational regulation under stress and could provide an amenable tool to understand the eIF2B-mediated defects.

Summary Aims Vanishing white matter disease (VWM) is an inherited leukoencephalopathy in children attributed to mutations in EIF2B1–5, encoding five subunits of eukaryotic translation initiation factor 2B (eIF2B). Although the defects are in the housekeeping genes, glial cells are selectively involved in VWM. Several studies have suggested that astrocytes are central in the pathogenesis of VWM. However, the exact pathomechanism remains unknown, and no model for VWM induced pluripotent stem cells (iPSCs) has been established. Methods Fibroblasts from two VWM children were reprogrammed into iPSCs by using a virus‐free nonintegrating episomal vector system. Control and VWM iPSCs were sequentially differentiated into neural stem cells (NSCs) and then into neural cells, including neurons, oligodendrocytes (OLs), and astrocytes. Results Vanishing white matter disease iPSC‐derived NSCs can normally differentiate into neurons, oligodendrocytes precursor cells (OPCs), and oligodendrocytes in vitro. By contrast, VWM astrocytes were dysmorphic and characterized by shorter processes. Moreover, δ‐GFAP and αB‐Crystalline were significantly increased in addition to increased early and total apoptosis. Conclusion The results provided further evidence supporting the central role of astrocytic dysfunction. The establishment of VWM‐specific iPSC models provides a platform for exploring the pathogenesis of VWM and future drug screening.

Yan Jiao,1 Zhuo Fu,2 Yanqing Li,3 Lingyu Meng,1 Yahui Liu1 1Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, People’s Republic of China; 2Department of Hand and Foot Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, People’s Republic of China; 3Department of Pathophysiology, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130021, People’s Republic of China Purpose: Liver cancer is a high mortality disease with no curable treatments. Posttranscriptional modifications play essential roles in the occurrence and the progression of liver cancer. EIF2B5 is a subunit of EIF2B that regulates the initiation and the rate of translation and participates in several diseases including tumors. This study aims to elucidate the prognostic significance of EIF2B5 in liver cancer. Materials and methods: We used The Cancer Genome Atlas database to analyze the expression of EIF2B5 in liver cancer. Then we used chi-squared and Fisher exact tests to test the correlation between clinical characteristics and EIF2B5 expression. Finally, we assessed the role of EIF2B5 in prognosis by Kaplan–Meier curves and Cox analysis. Gene set enrichment analysis was performed by using The Cancer Genome Atlas data set. Results: The results showed that EIF2B5 was upregulated in liver cancer, and the expressionwas related to histologic grade, clinical stage, and vital status. Moreover, Kaplan–Meier curves and Cox analysis implicated that highly expressed EIF2B5 correlated with poor prognosis, and EIF2B5 was an independent risk factor for liver cancer. Gene set enrichment analysis showed that ATR and BRCA pathway, cell cycle pathway, DNA repair, myc signaling pathway, and E2F targets are differentially enriched in EIF2B5 high-expression phenotype. Conclusion: Our results suggest that EIF2B5 participated in cancer progression and could become a biomarker for the prognosis of patients with liver cancer. Keywords: liver cancer, EIF2B5, prognosis, diagnosis

Hearing loss is a significant public health concern, affecting over 250 million people worldwide. Both genetic and environmental etiologies are linked to hearing loss, but in many cases the underlying cellular pathophysiology is not well understood, highlighting the importance of further discovery. We found that inactivation of the gene Tmtc4 (transmembrane and tetratricopeptide repeat 4), which was broadly expressed in the mouse cochlea, caused acquired hearing loss in mice. Our data showed Tmtc4 enriched in the endoplasmic reticulum, and that it functioned by regulating Ca2+ dynamics and the unfolded protein response (UPR). Given this genetic linkage of the UPR to hearing loss, we demonstrated a direct link between the more common noise-induced hearing loss (NIHL) and the UPR. These experiments suggested a novel approach to treatment. We demonstrated that the small-molecule UPR and stress response modulator ISRIB (integrated stress response inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout of Tmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing loss disorders.

A key site of translation control is the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which reduces the rate of GDP to GTP exchange by eIF2B, leading to altered translation. The extent of eIF2α phosphorylation within neurons can alter synaptic plasticity. Phosphorylation of eIF2α is triggered by four stress-responsive kinases, and as such eIF2α is often phosphorylated during neurological perturbations or disease. Moreover, in some cases decreasing eIF2α phosphorylation mitigates neurodegeneration, suggesting that this could be a therapeutic target. Mutations in the γ subunit of eIF2, the guanine exchange factor eIF2B, an eIF2α phosphatase, or in two eIF2α kinases can cause disease in humans, demonstrating the importance of proper regulation of eIF2α phosphorylation for health.

VWM is one of the most prevalent leukodystrophies with unique clinical, pathological and molecular features. It mostly affects children, but may develop at all ages, from birth to senescence. It is dominated by cerebellar ataxia and susceptible to stresses that act as factors provoking disease onset or episodes of rapid neurological deterioration possibly leading to death. VWM is caused by mutations in any of the genes encoding the five subunits of the eukaryotic translation initiation factor 2B (eIF2B). Although eIF2B is ubiquitously expressed, VWM primarily manifests as a leukodystrophy with increasing white matter rarefaction and cystic degeneration, meager astrogliosis with no glial scarring and dysmorphic immature astrocytes and increased numbers of oligodendrocyte progenitor cells that are restrained from maturing into myelin-forming cells. Recent findings point to a central role for astrocytes in driving the brain pathology, with secondary effects on both oligodendroglia and axons. In this, VWM belongs to the growing group of astrocytopathies, in which loss of essential astrocytic functions and gain of detrimental functions drive degeneration of the white matter. Additional disease mechanisms include activation of the unfolded protein response with constitutive predisposition to cellular stress, failure of astrocyte-microglia crosstalk and possibly secondary effects on the oxidative phosphorylation. VWM involves a translation initiation factor. The group of leukodystrophies due to defects in mRNA translation is also growing, suggesting that this may be a common disease mechanism. The combination of all these features makes VWM an intriguing natural model to understand the biology and pathology of the white matter.

Leukoencephalopathy with vanishing white matter (LEVWM) is one of the most common hereditary leukoencephalopathies with characteristic MRI picture of diffuse white matter lesions with cystic degeneration. The disease is associated with EIF2B1-5 genes, encoding five subunits of translation initiation factor EIF2B. There are infantile, Childhood (the most frequent one), and adult-onset forms. Adult-onset LEVWM accounts for 15–20% of all cases and is characterized by significant clinical variability. In addition to neurological and cognitive disorders, this disease is characterized by ovarian failure. The review presents the clinical and molecular genetic aspects of adult-onset LEVWM.

We characterized the transcriptional response accompanying maintenance of long-term sensitization (LTS) memory in the pleural ganglia of Aplysia californica using microarray (N = 8) and qPCR (N = 11 additional samples). We found that 24 h after memory induction there is strong regulation of 1198 transcripts (748 up and 450 down) in a pattern that is almost completely distinct from what is observed during memory encoding (1 h after training). There is widespread up-regulation of transcripts related to all levels of protein production, from transcription (e.g., subunits of transcription initiation factors) to translation (e.g., subunits of eIF1, eIF2, eIF3, eIF4, eIF5, and eIF2B) to activation of components of the unfolded protein response (e.g., CREB3/Luman, BiP, AATF). In addition, there are widespread changes in transcripts related to cytoskeleton function, synaptic targeting, synaptic function, neurotransmitter regulation, and neuronal signaling. Many of the transcripts identified have previously been linked to memory and plasticity (e.g., Egr, menin, TOB1, IGF2 mRNA binding protein 1/ZBP-1), though the majority are novel and/or uncharacterized. Interestingly, there is regulation that could contribute to metaplasticity potentially opposing or even eroding LTS memory (down-regulation of adenylate cyclase and a putative serotonin receptor, up-regulation of FMRFa and a FMRFa receptor). This study reveals that maintenance of a “simple” nonassociative memory is accompanied by an astonishingly complex transcriptional response.

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.

Cell-nonautonomous effects of signaling in the nervous system of animals can influence diverse aspects of organismal physiology. We previously showed that phosphorylation of Ser49 of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) in two chemosensory neurons by PEK-1/PERK promotes entry of Caenorhabditis elegans into dauer diapause. Here, we identified and characterized the molecular determinants that confer sensitivity to effects of neuronal eIF2α phosphorylation on development and physiology of C. elegans. Isolation and characterization of mutations in eif-2Ba encoding the α-subunit of eIF2B support a conserved role, previously established by studies in yeast, for eIF2Bα in providing a binding site for phosphorylated eIF2α to inhibit the exchange factor eIF2B catalytic activity that is required for translation initiation. We also identified a mutation in eif-2c, encoding the γ-subunit of eIF2, which confers insensitivity to the effects of phosphorylated eIF2α while also altering the requirement for eIF2Bγ. In addition, we show that constitutive expression of eIF2α carrying a phosphomimetic S49D mutation in the ASI pair of sensory neurons confers dramatic effects on growth, metabolism, and reproduction in adult transgenic animals, phenocopying systemic responses to starvation. Furthermore, we show that constitutive expression of eIF2α carrying a phosphomimetic S49D mutation in the ASI neurons enhances dauer entry through bypassing the requirement for nutritionally deficient conditions. Our data suggest that the state of eIF2α phosphorylation in the ASI sensory neuron pair may modulate internal nutrient sensing and signaling pathways, with corresponding organismal effects on development and metabolism.

Eukaryotic translation initiation factor 2B (eIF2B) is a master regulator of protein synthesis under normal and stress conditions. Mutations in any of the five genes encoding its subunits lead to vanishing white matter (VWM) disease, a recessive genetic deadly illness caused by progressive loss of white matter in the brain. In this study we used fibroblasts, which are not involved in the disease, to demonstrate the involvement of eIF2B in mitochondrial function and abundance. Mass spectrometry of total proteome of mouse embryonic fibroblasts (MEFs) isolated from Eif2b5R132H/R132H mice revealed unbalanced stoichiometry of proteins involved in oxidative phosphorylation and of mitochondrial translation machinery components, among others. Mutant MEFs exhibit 55% decrease in oxygen consumption rate per mtDNA content and 47% increase in mitochondrial abundance (p 3-fold lower ATP-linked respiration per cell despite a 2-fold increase in mtDNA content (p

Background & Aims Chronic amino acid (AA) deficiency, as in kwashiorkor, reduces the size of the pancreas through an effect on mammalian target of rapamycin complex 1 (mTORC1). Because of the physiological importance of AAs and their role as a substrate, a stimulant of mTORC1, and protein synthesis, we studied the effect of acute protein and AA deficiency on the response to feeding. Methods ICR/CD-1 mice were fasted overnight and refed for 2 hours with 4 different isocaloric diets: control (20% Prot); Protein-free (0% Prot); control (AA-based diet), and a leucine-free (No Leu). Protein synthesis, polysomal profiling, and the activation of several protein translation factors were analyzed in pancreas samples. Results All diets stimulated the Protein Kinase-B (Akt)/mTORC1 pathway, increasing the phosphorylation of the kinase Akt, the ribosomal protein S6 (S6) and the formation of the eukaryotic initiation factor 4F (eIF4F) complex. Total protein synthesis and polysome formation were inhibited in the 0% Prot and No Leu groups to a similar extent, compared with the 20% Prot group. The 0% Prot diet partially reduced the Akt/mTORC1 pathway and the activity of the guanine nucleotide exchange factor eIF2B, without affecting eIF2α phosphorylation. The No Leu diet increased the phosphorylation of eIF2α and general control nonderepressible 2, and also inhibited eIF2B activity, without affecting mTORC1. Essential and nonessential AA levels in plasma and pancreas indicated a complex regulation of their cellular transport mechanisms and their specific effect on the synthesis of digestive enzymes. Conclusions These studies show that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition., Graphical abstract

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament assembly is an efficient mechanism that allows yeast cells to adapt to fluctuating environments., Summary: Filament formation by eIF2B is an adaptive mechanism that allows for efficient shutdown of translation in starved yeast cells.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression and play roles in a wide range of physiological processes, including ontogenesis. Herein, we discovered a novel microRNA, novel miR-26, which inhibits translation of the phosphofructokinase (PFK) gene by targeting the 3’ untranslated region (UTR) of pfk directly, thereby inhibiting the molting and body length growth of the freshwater shrimp (Neocaridina heteropoda). Lowering expression of the PFK gene by RNA interference (RNAi) led to a longer ecdysis cycle and smaller individuals. This phenotype was mirrored in shrimps injected with novel miR-26 agomirs, but the opposite phenotype occurred in shrimps injected with novel miR-26 antagomirs (i.e., the ecdysis cycle was shortened and body length was increased). After injection of 20-hydroxyecdysone (ecdysone 20E), expression of the novel miR-26 was decreased, while expression of the PFK gene was up-regulated, and the fructose-1,6-diphosphate metabolite of PFK accumulated correspondingly. Furthermore, expression of eIF2 (eukaryotic initiation factor 2) increased under stimulation of fructose-1,6-diphosphate, suggesting that protein synthesis was stimulated during this period. Taken together, our results suggest that the novel miR-26 regulates expression of the PFK gene and thereby mediates the molting and growth of N. heteropoda.

A mechanism based on reversible phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) has been confirmed as an important regulatory pathway for the inhibition of protein synthesis in mammalian and yeast cells, while plants constitute the significant exception. We studied the induction of TaeIF2α phosphorylation in germinated wheat (Triticum aestivum) embryos subjected to different adverse conditions. Data confirmed that formation of TaeIF2(αP) was not a general response, as no phosphorylation was observed under salt, oxidative, or heat stress. Nevertheless, treatment by salicylic acid, UV-light, cold shock and histidinol did induce phosphorylation of TaeIF2α of wheat as has been established previously for AteIF2α in Arabidopsis (Arabidopsis thaliana). The influence of TaeIF2α phosphorylation on translation of reporter mRNA with different 5′-untranslated regions (5′UTRs) was studied in wheat germ cell-free system (WG-CFS), in which TaeIF2α was first phosphorylated either by heterologous recombinant human protein kinase, HsPKR (activated by double-stranded (ds)RNA), or by endogenous protein kinase TaGCN2 (activated by histidinol). Pretreatment of WG-CFS with HsPKR in the presence of dsRNA or with histidinol resulted in intense phosphorylation of TaeIF2α; however, the translation levels of all tested mRNAs decreased by only 10–15% and remained relatively high. In addition, factor OceIF2 from rabbit (Oryctolagus cuniculus) bound GDP much more strongly than the homologous factor TaeIF2 from wheat germ. Furthermore, factor OceIF2B was able to stimulate guanine nucleotide exchange (GDP→GTP) on OceIF2 but had no effect on a similar exchange on TaeIF2. These results suggest that the mechanism of stress response via eIF2α phosphorylation is not identical in all eukaryotes, and further research is required to find and study in detail new plant-specific mechanisms that may inhibit overall protein synthesis in plants under stress.

© 2019 Regulator of G protein signaling 2 (RGS2) is upregulated by multiple forms of stress and can augment translational attenuation associated with the phosphorylation of the initiation factor eIF2, a hallmark of several stress-induced coping mechanisms. Under stress-induced translational inhibition, key factors, such as ATF4, are selectively expressed via alternative translation mechanisms. These factors are known to regulate molecular switches that control cell fate by regulating pro-survival and pro-apoptotic signals. The molecular mechanisms that balance these opposing responses to stresses are unclear. The present results suggest that RGS2 may be an important regulatory component in the cellular stress response through its translational control abilities. Previously, we have shown that RGS2 can interact with the translation initiation factor, eIF2B, and inhibit de novo protein synthesis. Here, we demonstrate that the expression of either full length RGS2 or its eIF2B-interacting domain (RGS2 eb ) significantly increases levels of ATF4 and CHOP, both of which are linked to stress-induced apoptosis. Furthermore, we show that these effects are translationally regulated and independent of eIF2 phosphorylation. The present results thus point to a novel function of RGS2 in the stress response directly related to its ability to reduce global protein synthesis.

Vanishing white matter (VWM) disease (OMIM#306896) is an autosomal recessive neurodegenerative leukodystrophy caused by hypomorphic mutations in any of the five genes encoding the subunits of eukaryotic translation initiation factor 2B (eIF2B). The disease is manifested by loss of cerebral white matter and progressive deterioration upon exposure to environmental and physiological stressors. "Foamy" oligodendrocytes (OLG), increased numbers of oligodendrocytes precursor cells (OPC), and immature defective astrocytes are major neuropathological denominators. Our recent work using Eif2b5R132H/R132H mice uncovered a fundamental link between eIF2B and mitochondrial function. A decrease in oxidative phosphorylation capacity was observed in mutant astrocytes and fibroblasts. While an adaptive increase in mitochondria abundance corrects the phenotype of mutant fibroblasts, it is not sufficient to compensate for the high-energy demand of astrocytes, explaining their involvement in the disease. To date, astrocytes are marked as central for the disease while eIF2B-mutant OLG are currently assumed to lack a cellular phenotype on their own. Here we show a reduced capacity of eIF2B-mutant OPC isolated from Eif2b5R132H/R132H mice to conduct oxidative respiration despite the adaptive increase in their mitochondrial abundance. We also show their impaired ability to efficiently complete critical differentiation steps towards mature OLG. The concept that defective differentiation of eIF2B-mutant OPC could be a consequence of mitochondrial malfunction is in agreement with numerous studies indicating high dependency of differentiating OLG on accurate mitochondrial performance and ATP availability.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Objective Anti-nuclear autoantibodies are known to occur in 85-99% of Systemic Sclerosis (SSc) patients, with each SSc autoantibody correlating with a distinct clinical subset of patients. The objective of this study was to investigate novel SSc autoantibodies in the remaining autoantibody negative patients and establish clinical associations. Methods Serum samples and clinical data were collected from 548 SSc patients. Sera were tested for known SSc autoantibodies by routine serological techniques, with negative samples being further investigated by radiolabelled protein immunoprecipitation (IPP). Sera that immunoprecipitated a novel 30 kDa band were analysed by indirect immunofluorescence and IPP using depleted cell extracts to establish a common reactivity. Mass spectrometry (MS) was used to identify the novel autoantigen and findings were confirmed using commercial antibodies. Sera from 426 patients with other forms of connective tissue disease, 103 patients with rheumatoid arthritis, 114 patients with idiopathic ILD and 150 healthy controls were serotyped as controls. Results A novel autoantigen with a molecular weight of ∼30 kDa was recognised by seven sera with SSc, six of whom had interstitial lung disease (ILD) and by no controls. Six of the patients had diffuse cutaneous involvement and four had overlap features with other autoimmune diseases. Immunodepletion experiments indicated that all samples targeted the same autoantigen and MS identified the novel autoantigen as eIF2B (Eukaryotic Initiation Factor 2B). Conclusion We report a novel autoantibody (anti-eIF2B) in a small number of patients with SSc (approximately 1%) that is closely associated with diffuse cutaneous manifestations and the presence of ILD. This article is protected by copyright. All rights reserved.

Eukaryotic translation initiation factor 2B (eIF2B), a heterodecameric complex of two sets of the α, β, γ, δ, and e subunits, is the guanine nucleotide exchange factor (GEF) specific for eIF2, a heterotrimeric G protein consisting of the α, β, and γ subunits. The eIF2 protein binds GTP on the γ subunits and delivers an initiator methionyl-tRNA (Met-tRNAiMet ) to the ribosome. The GEF activity of eIF2B is inhibited by stress-induced phosphorylation of Ser51 in the α subunit of eIF2, which leads to lower amounts of active eIF2 and a limited quantity of Met-tRNAiMet for the ribosome, resulting in global repression of translation. However, the structural mechanism of the GEF activity inhibition remained enigmatic, and therefore the three-dimensional structure of the entire eIF2B molecule had been awaited. Recently, we determined the crystal structure of Schizosaccharomyces pombe eIF2B. In this Structural Snapshot, we present the structural features of eIF2B and the mechanism underlying the GEF activity inhibition by the phosphorylation of eIF2α, elucidated from structure-based in vitro analyses.

Ovarioleukodystrophy-the co-occurrence of leukodystrophy and premature ovarian failure-is a rare presentation now recognised to be part of the clinical spectrum of vanishing white matter disease. We describe a woman with epilepsy and neuroimaging changes consistent with leukoencephalopathy who presented with non-convulsive status epilepticus after starting hormone replacement therapy in the context of premature ovarian failure. Genetic testing confirmed her to be a compound heterozygote for EIF2B5 mutations; the gene encodes a subunit of eukaryotic translation initiation factor 2B. Mutations in EIF2B1-5 result in vanishing white matter disease. We highlight the importance of ovarian failure as a diagnostic pointer to eukaryotic translation initiation factor 2B (eIF2B)-related ovarioleukodystrophy and present a brief literature review of ovarioleukodystrophy.

Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved ability to replicate in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles., Author summary Rhesus macaque CMV (RhCMV) is an important model for human CMV (HCMV) pathogenesis and vaccine development. Therefore, it is important to understand the similarities and differences in infectivity and interaction of these viruses with their host species. In contrast to the strict species-specificity of HCMV, RhCMV is able to cross species barriers to replicate in human cells. We know from past work that a component of this broader host range is RhCMV’s ability to counteract both the rhesus and human versions of a key antiviral factor. Here we delve further into the mechanisms by which RhCMV can adapt to counteract human cellular defenses. We find that RhCMV appears to be poised to undergo a specific genomic rearrangement that facilitates increased replication efficiency in human cells. Besides providing insights into CMV species-specificity and host barriers to cross-species transmission, this work also provides more generalized clues about viral adaptative mechanisms.

Summary The small molecule ISRIB antagonizes 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 for eIF2. We have found that ISRIB-mediated acceleration of eIF2B’s nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (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., Graphical Abstract, Highlights • Mutually antagonistic binding of phosphorylated eIF2 and ISRIB to eIF2B • Opposing structural rearrangement of eIF2B by binding ISRIB or phosphorylated eIF2 • Phosphorylated eIF2 exposes ISRIB’s ability to enhance eIF2B activity in vitro • Weak ISR-inhibitory activity of ISRIB in cells lacking phosphorylated eIF2, ISRIB is a drug-like compound that restores memory and cognitive function in mouse models of certain human diseases. Its beneficial properties derive from interference with a stress-response pathway that hinges on regulating rates of protein synthesis. Here, Zyryanova, Kashiwagi et al. dissect the molecular basis of ISRIB action in cells.

The eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within translation initiation. eIF2B initiates translation by exchanging GDP for GTP on eIF2. However, this mechanism may be attenuated in response to cellular stress by the phosphorylation of eIF2, which paradoxically enhances the translation of stress-specific mRNA to instigate a stress response. Autosomal recessive mutations within eIF2B result in the disease leukoencephalopathy with vanishing white matter (VWM). Current models of VWM disease suggest that abnormal eIF2B results in the cell being in a constant state of stress. However, previous biochemical studies have illustrated that VWM mutations not only decrease the catalytic activity of eIF2B, but can increase activity or not affect it at all. Furthermore, changes in the activity eIF2B do not correlate with disease severity. These observations suggest that there may be additional characteristics of eIF2B contributing to VWM pathogenesis which remain to be elucidated.\ud In this study, we investigated whether the localisation of eIF2B to eIF2B bodies was integral for function. Using the yeast S. cerevisiae, we show that the regulatory subunit eIF2Bα is required for localisation. Interestingly, catalytic mutations in eIF2Bα localised eIF2B to several smaller foci, termed microbodies. In vivo FRAP analysis highlighted that eIF2 shuttles through microbodies slower than eIF2B bodies, indicating that localisation to eIF2B bodies may be required for full eIF2B activity. Surprisingly, phenotypes from eIF2Bα VWM missense mutations were highly variable in localisation, possibly recapitulating variable phenotypes observed in humans.\ud The putative membrane proteins Mst27p, Erp4p and Sac1p have been shown to associate with eIF2B. We therefore investigated whether they were required for the formation of eIF2B bodies. Deletion of membrane proteins did not significantly alter eIF2B localisation. Surprisingly however, deletion of the PI(4)P Sac1p reduced rates of translation initiation independently of eIF2B, highlighting a link between membrane integrity and translational control.

Eukaryotic initiation factor 2 (eIF2) is a G protein critical for translation. It is tightly regulated in the integrated stress response (ISR) via phosphorylation of eIF2α and the subsequent control of eukaryotic initiation factor 2B (eIF2B), a multisubunit guanine nucleotide exchange factor. Through studying the localization of eIF2B subunits, we identified cytoplasmic eIF2B bodies in mammalian cells. We highlight a relationship between body size and the eIF2B subunits localizing to them; larger bodies contain all subunits and smaller bodies contain predominantly catalytic subunits. eIF2 localizes to eIF2B bodies and shuttles within these bodies in a manner that correlates with eIF2B activity. On stress, eIF2α-P localizes predominately to larger bodies and results in a decreased shuttling of eIF2. Interestingly, drugs that inhibit the ISR can rescue eIF2 shuttling in a manner correlating to levels of eIF2α-P. In contrast, smaller bodies show increased eIF2 shuttling in response to stress, which is accompanied by the localization of eIF2Bδ to these bodies, suggesting the formation of a novel trimeric complex of eIF2B. This response is mimicked by ISR-inhibiting drugs, providing insight into their potential mechanism of action. This study provides evidence that the composition and function of mammalian eIF2B bodies are regulated by the ISR and the drugs that control it.

The integrated stress response (ISR) regulates protein synthesis under conditions of stress. Phosphorylation of translation initiation factor eIF2 by stress-sensing kinases converts eIF2 from substrate to competitive inhibitor of its dedicated nucleotide exchange factor, eIF2B, arresting translation. A drug-like molecule called integrated stress response inhibitor (ISRIB) reverses the effects of eIF2 phosphorylation and restores translation by targeting eIF2B. When administered to mice, ISRIB enhances cognition and limits cognitive decline due to brain injury. To determine ISRIB's mechanism of action, we solved an atomic structure of ISRIB bound to the human eIF2B decamer. We found that ISRIB acts as a molecular staple, pinning together tetrameric subcomplexes of eIF2B along the assembly path to a fully active, decameric enzyme. In this Structural Snapshot, we discuss ISRIB's mechanism, its ability to rescue disease mutations in eIF2B and conservation of the enzyme and ISRIB-binding pocket.

Activation of the integrated stress response (ISR) by a variety of stresses triggers phosphorylation of the α-subunit of translation initiation factor eIF2. P-eIF2α inhibits eIF2B, the guanine nucleotide exchange factor that recycles inactive eIF2•GDP to active eIF2•GTP. eIF2 phosphorylation thereby represses translation. Persistent activation of the ISR has been linked to the development of several neurological disorders, and modulation of the ISR promises new therapeutic strategies. Recently, a small-molecule ISR inhibitor (ISRIB) was identified that rescues translation in the presence of P-eIF2α by facilitating the assembly of more active eIF2B. ISRIB enhances cognitive memory processes and has therapeutic effects in brain-injured mice without displaying overt side effects. While using ISRIB to investigate the ISR in picornavirus-infected cells, we observed that ISRIB rescued translation early in infection when P-eIF2α levels were low, but not late in infection when P-eIF2α levels were high. By treating cells with varying concentrations of poly(I:C) or arsenite to induce the ISR, we provide additional proof that ISRIB is unable to inhibit the ISR when intracellular P-eIF2α concentrations exceed a critical threshold level. Together, our data demonstrate that the effects of pharmacological activation of eIF2B are tuned by P-eIF2α concentration. Thus, ISRIB can mitigate undesirable outcomes of low-level ISR activation that may manifest neurological disease but leaves the cytoprotective effects of acute ISR activation intact. The insensitivity of cells to ISRIB during acute ISR may explain why ISRIB does not cause overt toxic side effects in vivo.

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 show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts., eLife digest Cells must be able to respond to their changing environment in order to survive. When cells encounter particularly unfavorable conditions, they often react by activating a so-called ‘stress’ response. A group of proteins collectively known as eIF2B helps to regulate this response. In a severe neurological condition called Vanishing White Matter (VWM), the genes that produce the eIF2B proteins contain mutations that make eIF2B less active. As a result, certain cells in people with VWM are always stressed. Six years ago, researchers discovered a molecule that boosts the activity of eIF2B. In 2018, they found that it also works on various mutant forms of eIF2B found in VWM. The molecule had so far only been tested in biochemical laboratory experiments. Now, Wong et al. – including some of the researchers involved in the 2018 study – have tested whether an improved version of the molecule treats VWM in mice. The trial treatment successfully halted all signs of the disease in the mice. The molecule blunted the persistent stress response of the cells in the brain and spinal cord, primarily in a cell type that is severely affected by the human form of VWM. Cells in other parts of the body were spared. Overall, the results of the experiments suggest that an eIF2B activator may prove to be an effective treatment for VWM in humans. It could similarly be used to treat other conditions that activate this abnormal cell stress response. The molecule Wong et al. used is not suitable for use in humans, so work is continuing to find a suitable variant.

11 páginas, 5 figuras, 1 tabla., One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi., This study was supported by the MRC-LMB EM Facility. This work was supported by grants from the Medical Research Council (MC_U105184332) and the Wellcome Trust (WT096570) to V.R. and by a grant BFU2017-85814-P from the Spanish government to J.L.L.

Integrated stress response on the brain During translation, regulation of protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is a common consequence of diverse stress stimuli, which leads to reprogramming of gene expression. This process, known as the integrated stress response, is one of the most fundamental mechanisms of translational control conserved throughout eukaryotes. It is also a promising therapeutic target in neurodegenerative diseases and traumatic brain injury. Kashiwagi et al. report the cryo–electron microscopy and crystal structures and Kenner et al. report the cryo–electron microscopy structure of the guanine nucleotide exchange factor eIF2B in complex with eIF2 or phosphorylated eIF2. The structures of the eIF2•eIF2B complex reveal that the single phosphorylation modification on eIF2 changes how eIF2 binds to eIF2B and locks this enzyme into an inhibited complex. Science , this issue p. 495 , p. 491

Vanishing white matter (VWM) is a fatal, stress-sensitive leukodystrophy that mainly affects children and is currently without treatment. VWM is caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B) that is crucial for initiation of mRNA translation and its regulation under stress conditions. Mice with bi-allelic missense mutations in eIF2B recapitulate human VWM. VWM pathomechanisms are unclear. Using polysomal profiling to screen for mRNAs with altered translation we observed most prominent changes in expression of integrated stress response (ISR) mRNAs in brains of mutant compared to wild-type mice; expression levels correlated with disease severity. We substantiated these findings in VWM patients’ brains. ISRIB, an ISR inhibitor, normalized expression of mRNA markers, ameliorated white matter pathology and improved motor skills in VWM mice, thus showing that the ISR is central in VWM pathomechanisms and a viable target for therapy.One Sentence SummaryISRIB ameliorates ISR deregulation and clinical signs in VWM mice

Eukaryotic initiation factor 2 (eIF2) is a central controller of the eukaryotic translational machinery. To sustain the on-going translation activity, eIF2 cycles between its GTP and GDP bound states. However, in response to cellular stresses, the phosphorylation of eIF2 takes place, which acts as an inhibitor of the guanine nucleotide exchange factor eIF2B and switches the translation activity on physiological timescales. The main objective of this paper is to investigate the stability of the regulatory system under nominal conditions, parametric fluctuations, and structural damages. In this paper, a mathematical model of eIF2-dependent regulatory system is used to identify the stability-conferring features within the system with the help of direct and indirect methods of Lyapunov stability theory. To investigate the impact of intrinsic fluctuations and structural damages on the stability of regulatory system, the mathematical model has been linearized around feasible equilibrium point and the variation of system poles has been observed. The investigations have revealed that the regulatory model is stable and able to tolerate the intrinsic stressors but becomes unstable when particular complex is targeted to override the undesirable interaction. Our analyses indicate that, the stability is a collective property and damage in the structure of the system changes the stability of the system.

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.