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Background Vanishing white matter (VWM) is a leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes (EIF2B1–EIF2B5); 80% are missense mutations. Clinical severity is highly variable, with a strong, unexplained genotype–phenotype correlation. Materials and Methods With information from a recent natural history study, we severity‐graded 97 missense mutations. Using in silico modeling, we created a new human eIF2B model structure, onto which we mapped the missense mutations. Mutated residues were assessed for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity. Results Over 50% of mutations have (ultra‐)severe phenotypic effects. About 60% affect the ε‐subunit, containing the catalytic domain, mostly with (ultra‐)severe effects. About 55% affect subunit cores, with variable clinical severity. About 36% affect subunit interfaces, mostly with severe effects. Very few mutations occur on the external eIf2B surface, perhaps because they have minor functional effects and are tolerated. One external surface mutation affects eIF2B‐substrate interaction and is associated with ultra‐severe phenotype. Conclusion Mutations that lead to (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and function of eIF2B. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and in the future for individualized therapy decisions., Vanishing white matter (VWM) is a clinically highly variable leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)‐subunit genes, mostly missense mutations. To gain insight into the strong genotype‐phenotype correlation, we severity‐graded 97 missense mutations using information from a clinical natural history study, created a new human eIF2B model structure by in silico modeling, and assessed mutated residues for location in subunits, eIF2B complex, and functional domains, and for information on biochemical activity. We demonstrated that mutations associated with (ultra‐)severe disease mostly affect amino acids with pivotal roles in complex formation and eIF2B function, while mutations on the external eIf2B surface are mostly associated with mild phenotypes, probably because of minor functional effects. Therapies for VWM are emerging and reliable mutation‐based phenotype prediction is required for propensity score matching for trials and individualized therapy decisions.

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 poorly understood. Using a genomic mutagenesis screen for longevity in Caenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinase kin-35 is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging., Aging is associated with declining protein homeostasis. Here, using a chemical mutagenesis screen for lifespan extension in C. elegans, the authors report that inhibition of the integrated stress response enhances longevity and protein homeostasis in a manner dependent on kin-35, without reducing protein synthesis.

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.

Summary eIF2B is the guanine nucleotide exchange factor (GEF) required for cytoplasmic protein synthesis initiation in eukaryotes and its regulation within the integrated stress response (ISR). It activates its partner factor eIF2, thereby promoting translation initiation. Here we provide evidence through biochemical and genetic approaches that eIF2B can bind directly to GTP and this can enhance its rate of GEF activity toward eIF2–GDP in vitro. GTP binds to a subcomplex of the eIF2Bγ and ε subunits. The eIF2Bγ amino-terminal domain shares structural homology with hexose sugar phosphate pyrophosphorylase enzymes that bind specific nucleotides. A K66R mutation in eIF2Bγ is especially sensitive to guanine or GTP in a range of functional assays. Taken together, our data suggest eIF2Bγ may act as a sensor of purine nucleotide availability and thus modulate eIF2B activity and protein synthesis in response to fluctuations in cellular nucleotide levels., Graphical abstract, Highlights • eIF2B, the GDP exchange factor for eIF2 in translation and its control, binds GTP • GTP binding enhances the rate of eIF2B GEF activity toward eIF2–GDP in vitro • A K66R mutation in yeast eIF2Bγ is sensitive to guanine in vivo or GTP in vitro • eIF2B may act as a sensor of purine nucleotide availability, Biological sciences; Molecular biology; Cell biology; Biomechanics

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.

We report two unrelated patients with infantile onset leukoencephalopathy with vanishing white matter (VWM) and hyperinsulinaemic hypoglycaemia. To our knowledge, this association has not been described previously. Both patients had compound heterozygous pathogenic variants in EIF2B4 detected on exome sequencing and absence of other variants which might explain the hyperinsulinism. Hypoglycaemia became apparent at 6 and 8 months, respectively, although in one patient, transient neonatal hypoglycaemia was also documented. One patient responded to diazoxide and the other was managed with continuous nasogastric feeding. We hypothesise that the pathophysiology of hyperinsulinism in VWM may involve dysregulation of transcription of genes related to insulin secretion.

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.

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex., During the integrated stress response, translation is modulated through the phosphorylation of translation initiation factor eIF2 and the formation of a complex with eIF2B. Here the authors present structures of the eIF2:eIF2B complex with and without phosphorylation, shedding light on how eIF2 phosphorylation regulates translation.

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.

Translation initiation factor 2B (eIF2B) is a master regulator of global protein synthesis in all cell types. The mild genetic Eif2b5(R132H) mutation causes a slight reduction in eIF2B enzymatic activity which leads to abnormal composition of mitochondrial electron transfer chain complexes and impaired oxidative phosphorylation. Previous work using primary fibroblasts isolated from Eif2b5(R132H/R132H) mice revealed that owing to increased mitochondrial biogenesis they exhibit normal cellular ATP level. In contrast to fibroblasts, here we show that primary astrocytes isolated from Eif2b5(R132H/R132H) mice are unable to compensate for their metabolic impairment and exhibit chronic state of low ATP level regardless of extensive adaptation efforts. Mutant astrocytes are hypersensitive to oxidative stress and to further energy stress. Moreover, they show migration deficit upon exposure to glucose starvation. The mutation in Eif2b5 prompts reactive oxygen species (ROS)-mediated inferior ability to stimulate the AMP-activated protein kinase (AMPK) axis, due to a requirement to increase the mammalian target of rapamycin complex-1 (mTORC1) signalling in order to enable oxidative glycolysis and generation of specific subclass of ROS-regulating proteins, similar to cancer cells. The data disclose the robust impact of eIF2B on metabolic and redox homeostasis programs in astrocytes and point at their hyper-sensitivity to mutated eIF2B. Thereby, it illuminates the central involvement of astrocytes in Vanishing White Matter Disease (VWMD), a genetic neurodegenerative leukodystrophy caused by homozygous hypomorphic mutations in genes encoding any of the 5 subunits of eIF2B.

11 Pág. Centro de Biotecnología y Genómica de Plantas, Protein synthesis is a fundamental process for life and, as such, plays a crucial role in the adaptation to energy, developmentaland environmental conditions. For these reasons, and despite the general conservation of the eukaryotic translational machinery, it is not surprising that organisms with different lifestyles have evolved distinct mechanisms of regulation to adapt translation initiation to their intrinsic growth and development. Plants have clear peculiarities compared with other eukaryotes that have also extended to translation control. This review describes the plant-specific mechanisms for regulation of translation initiation, with a focus on those that modulate the eIF4F complexes, central translational regulatory hubs in all eukaryotes, and highlights the latest discoveries on the signaling pathways that regulate their constituents and activity., Our research related to translation is supported by Grants S2013-ABI2734 (CAM, Spain) and RTI2018-095946-Our research related to translation is supported by Grants S2013-ABI2734 (CAM, Spain) and RTI2018-095946-B100 (MICIU, Spain) to M.M.C. and Grants BIO2017-82720-P and RYC2017-22323 (MINECO, Spain) and P18-RT-1218 (Junta de Andalucía, Spain) to C.M.

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.

Vanishing white matter (VWM) disease is a genetic leukodystrophy leading to severe neurological disease and early death. VWM is caused by bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (EIF2B). Previous studies have attempted to investigate the molecular mechanism of VWN by constructing models for each subunit of EIF2B that causes VWM disease. The underlying molecular mechanisms of the way in which mutations in EIF2B3 result in VWM are largely unknown. Based on our recent results, we generated an eif2b3 knockout (eif2b3−/−) zebrafish model and performed quantitative proteomic analysis between the wild-type (WT) and eif2b3−/− zebrafish, and identified 25 differentially expressed proteins. Four proteins were significantly upregulated, and 21 proteins were significantly downregulated in eif2b3−/− zebrafish compared to WT. Lon protease and the neutral amino acid transporter SLC1A4 were significantly increased in eif2b3−/− zebrafish, and crystallin proteins were significantly decreased. The differential expression of proteins was confirmed by the evaluation of mRNA levels in eif2b3−/− zebrafish, using whole-mount in situ hybridization analysis. This study identified proteins which candidates as key regulators of the progression of VWN disease, using quantitative proteomic analysis in the first EIF2B3 animal model of VWN disease.

Neuronal protein synthesis is essential for long-term memory consolidation, and its dysregulation is implicated in various neurodegenerative disorders, including Alzheimer's disease (AD). Cellular stress triggers the activation of protein kinases that converge on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which attenuates mRNA translation. This translational inhibition is one aspect of the integrated stress response (ISR). We found that postmortem brain tissue from AD patients showed increased phosphorylation of eIF2α and reduced abundance of eIF2B, another key component of the translation initiation complex. Systemic administration of the small-molecule compound ISRIB (which blocks the ISR downstream of phosphorylated eIF2α) rescued protein synthesis in the hippocampus, measures of synaptic plasticity, and performance on memory-associated behavior tests in wild-type mice cotreated with salubrinal (which inhibits translation by inducing eIF2α phosphorylation) and in both β-amyloid-treated and transgenic AD model mice. Thus, attenuating the ISR downstream of phosphorylated eIF2α may restore hippocampal protein synthesis and delay cognitive decline in AD patients.

Eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within protein synthesis and regulates translation initiation in response to cellular stress. Mutations within eIF2B result in the fatal disease, leukoencephalopathy with vanishing white matter (VWM). Previous biochemical studies on VWM mutations have illustrated that changes in the activity of eIF2B poorly correlate with disease severity. This suggests that there may be additional characteristics of eIF2B contributing to VWM pathogenesis. Here, we investigated whether the localization of eIF2B to eIF2B bodies was integral for function and whether this localization could provide insight into the pathogenesis of VWM. We demonstrate that the regulatory subunit, eIF2Bα, is required for the assembly of eIF2B bodies in yeast and that loss of eIF2B bodies correlates with an inability of cells to regulate eIF2B activity. Mutational analysis of eIF2Bα showed that missense mutations that disrupt the regulation of eIF2B similarly disrupt the assembly of eIF2B bodies. In contrast, when eIF2Bα mutations that impact the catalytic activity of eIF2B were analyzed, eIF2B bodies were absent and instead eIF2B localized to small foci, termed microfoci. Fluorescence recovery after photobleaching analysis highlighted that within these microfoci, eIF2 shuttles more slowly indicating that formation of eIF2B bodies correlates with full eIF2B activity. When eIF2Bα VWM mutations were analyzed, a diverse impact on localization was observed, which did not seem to correlate with eIF2B activity. These findings provide key insights into how the eIF2B body assembles and suggest that the body is a fundamental part of the translational regulation via eIF2α phosphorylation.

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.

Objective To examine the effect of pSer9-GSK-3β on breast cancer and to determine whether the underlying metabolic and immunological mechanism is associated with ROS/eIF2B and natural killer (NK) cells. Methods We employed TWS119 to inactivate GSK-3β by phosphorylating Ser9 and explored its effect on breast cancer and NK cells. The expression of GSK-3β, natural killer group 2 member D (NKG2D) ligands, eIF2B was quantified by PCR and Western blot. We measured intracellular reactive oxygen species (ROS) and mitochondrial ROS using DCFH-DA and MitoSOXTM probe, respectively, and conducted quantitative analysis of cellular respiration on 4T1 cells with mitochondrial respiratory chain complex I/III kits. Results Our investigation revealed that TWS119 downregulated NKG2D ligands (H60a and Rae1), suppressed the cytotoxicity of NK cells, and promoted the migration of 4T1 murine breast cancer cells. Nevertheless, LY290042, which attenuates p-GSK-3β formation by inhibiting the PI3K/Akt pathway, reversed these effects. We also found that higher expression of pSer9-GSK-3β induced higher levels of ROS, and observed that abnormality of mitochondrial respiratory chain complex I/III function induced the dysfunction of GSK-3β-induced electron transport chain, naturally disturbing the ROS level. In addition, the expression of NOX3 and NOX4 was significantly up-regulated, which affected the generation of ROS and associated with the metastasis of breast cancer. Furthermore, we found that the expression of pSer535-eIF2B promoted the expression of NKG2D ligands (Mult-1 and Rae1) following by expression of pSer9-GSK-3β and generation of ROS. Conclusions The PI3K/Akt/GSK-3β/ROS/eIF2B pathway could regulate NK cell activity and sensitivity of tumor cells to NK cells, which resulted in breast cancer growth and lung metastasis. Thus, GSK-3β is a promising target of anti-tumor therapy.

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.

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.

Vanishing White Matter disease (VWM) is a severe leukodystrophy of the central nervous system caused by mutations in subunits of the eukaryotic initiation factor 2B complex (eIF2B). Current models only partially recapitulate key disease features, and pathophysiology is poorly understood. Through development and validation of zebrafish (Danio rerio) models of VWM, we demonstrate that zebrafisheif2bmutants phenocopy VWM, including impaired somatic growth, early lethality, impaired myelination, loss of oligodendrocyte precursor cells, increased apoptosis in the CNS, and impaired motor swimming behavior. Expression of humanEIF2B2in the zebrafisheif2b2mutant rescues lethality and CNS apoptosis, demonstrating conservation of function between zebrafish and human. In the mutants, intron 12 retention leads to expression of a truncatedeif2b5transcript. Expression of the truncatedeif2b5in wild-type larva impairs motor behavior and activates the ISR, suggesting that a feed-forward mechanism in VWM is a significant component of disease pathophysiology.

Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.

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.

Aim: Vanishing White Matter (VWM) is a devastating leucoencephalopathy without effective treatment options. Patients have mutations in the EIF2B1-5 genes, encoding the five subunits of eIF2B, a guanine exchange factor that is an important regulator of protein translation. We recently developed mouse models for VWM that replicate the human disease. To study disease improvement after treatment in these mice, it is essential to have sensitive biomarkers related to disease stage. The Bergmann glia of the cerebellum, an astrocytic subpopulation, translocate into the molecular layer in symptomatic VWM mice and patients. This study looked at the prospects of using Bergmann glia pathology as an objective disease marker for VWM. Methods: We defined a new quantitative measurement of Bergmann glia pathology in the cerebellum of VWM mice and patients. To test the sensitivity of this new marker for improvement, VWM mutant mice received long-term treatment with Guanabenz, an FDA-approved anti-hypertensive agent affecting eIF2B activity. Results: Bergmann glia translocation was significantly higher in symptomatic VWM mice and VWM patients than in controls and worsened over the disease course. Both Bergmann glia pathology and cerebellar myelin pathology improved with Guanabenz treatment in mice, showing that Bergmann glia translocation is a sensitive measurement for improvement. Conclusions: Bergmann glia translocation can be used to objectively assess effects of treatment in VWM mice. Future treatment strategies involving compounds regulating eIF2 phosphorylation might benefit VWM patients.