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Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.

Eukaryotic initiation factor 2Be (eIF2Be) plays a critical role in the initiation of mRNA translation and its expression and guanine nucleotide exchange activity are major determinants of the rate of protein synthesis. In this work we provide evidence that the catalytic epsilon subunit of eIF2B is subject to ubiquitination and proteasome-mediated degradation. Lysates of C2C12 myoblasts treated with proteasome inhibitor were subjected to sequential immunoprecipitations for eIF2Be followed by ubiquitin. Tandem mass spectrometry (LC–MS/MS) analysis of immunoprecipitated proteins resulted in the identification of five peptides containing ubiquitin (diglycine) modifications on eIF2Be. The specific lysine residues containing the ubiquitin modifications were localized as Lys-56, Lys-98, Lys-136, Lys-212 and Lys-500 (corresponding to the rat protein sequence). In addition three novel phosphorylation sites were identified including Ser-22, Ser-125, and Thr-317. Moreover, peptides corresponding to the amino acid sequence of the E3 ligase NEDD4 were also detected in the LC–MS/MS analysis, and an interaction between endogenous eIF2Be with NEDD4 was confirmed by co-immunoprecipitation.

Peptide sequence data were obtained from rabbit protein synthesis initiation factor subunit eIF2B gamma. Searching the database of expressed sequence tags (dbEST) revealed nucleotide sequences potentially encoding human eIF2B gamma that contained peptides corresponding to those from the rabbit subunit. PCR primers were derived from these sequences and used to generate a probe. This was used to screen a rat skeletal muscle cDNA library, and a clone encoding rat eIF2B gamma was isolated. This cDNA gave a product in coupled transcription/translation that co-migrated with the gamma-subunit of purified eIF2B under SDS/PAGE. The sequence of this rat eIF2B gamma cDNA is reported. The protein sequence shows homology with that of yeast eIF2B gamma (the GCD1 gene product). We have also identified an open reading frame from the Caenorhabditis elegans genome project that probably encodes the gamma-subunit of C. elegans eIF2B. All these sequences show similarity to nucleotidyl- and acyltransferases, as previously reported for GCD1 [Koonin (1995) Protein Sci. 4, 1608-1617], and contain conserved motifs potentially involved in nucleotide binding. They also contain "I-patch' motifs: isoleucine-rich hexamer repeats that have been associated with the binding of acyl groups in bacterial acyltransferases. The roles of these motifs are discussed in relation to the known properties of eIF2B.

Vanishing white matter is a leukoencephalopathy that usually affects young children. Five genes were found recently for this disease, allowing a DNA-based diagnosis. The authors describe six patients homozygous for the Arg113His mutation in eIF2Bε. Only one had a childhood onset; four had a later onset and a protracted disease course; one adult still has no symptoms. Our data suggest that the Arg113His mutation is particularly mild and should be considered in the differential diagnosis of adult diffuse leukoencephalopathies, independent of whether there are associated clinical signs, an episodic course, or MRI shows white matter rarefaction/cystic degeneration.

See corresponding article on page 286. More than 40 y ago, Morgan et al (1, 2) reported that in the isolated perfused rat heart, elevating amino acid concentrations in the perfusate to 5 times the concentrations observed in the plasma of a fasted animal stimulated protein synthesis rates by enhancing the initiation phase of mRNA translation. Since that time, a plethora of studies have focused on delineating the mechanism or mechanisms through which amino acids act to upregulate translation initiation. The results of those studies have identified 2 steps in initiation as targets of amino acid signaling: the binding of initiator methionyl-tRNA (met-tRNAi)4 to the 40S ribosomal subunit to form the 43S preinitiation complex and the binding of mRNA to the 43S preinitiation complex. The binding of met-tRNAi to the 40S ribosomal subunit is mediated by eukaryotic initiation factor (eIF) 2 and regulated by the guanine nucleotide exchange factor (GEF) eIF2B. Both proteins are phosphorylated in cells in culture on specific subunits (eIF2 on its α-subunit and eIF2B on its ϵ-subunit) in response to changes in amino acid concentrations. Specifically, the deprivation of essential amino acids leads to activation of the protein kinase general control non-derepressing 2, which phosphorylates eIF2α, converting it from a substrate into a competitive inhibitor of eIF2B (3). Amino acid deprivation also leads to increased phosphorylation of eIF2B on its catalytic ϵ-subunit, resulting in inhibition of its GEF activity toward eIF2 (4). Together, phosphorylation of eIF2α and eIF2Bϵ leads to repressed rates of translation initiation by perturbing the assembly of the eIF2·GTP·met-tRNAi complex that mediates transfer of met-tRNAi to the 40S ribosomal subunit. The deprivation of essential amino acids also results in development of an impairment in the binding of mRNA to the 43S preinitiation complex, an event mediated by the eIF4F complex composed of eIF4A, eIF4E, and eIF4G (5). For most mRNAs, binding to the 43S preinitiation complex occurs through the interaction of eIF4E with the m7GTP cap at the 5′-end of the message and the subsequent binding of eIF4G to eIF3 that is part of the 43S preinitiation complex. The deprivation of essential amino acids leads to the disassembly of the eIF4F complex by promoting the interaction of eIF4E with eIF4E binding proteins (eg, 4E-BP1) and the interaction of eIF4A with programmed cell death 4 (PDCD4), thereby preventing their interaction with eIF4G. The association of eIF4E with 4E-BP1 and eIF4A with PDCD4 is regulated by phosphorylation of 4E-BP1 and PDCD4 in response to the activation of a protein kinase complex known as mechanistic target of rapamycin complex 1 (mTORC1). Amino acids, and in particular the branched-chain amino acid leucine, activate mTORC1, which leads to phosphorylation of 4E-BP1 and PDCD4, releasing their companion proteins and allowing them to bind to eIF4G to form the active eIF4F complex. Thus, in response to amino acid deprivation, mTORC1 is inhibited and eIF4E and eIF4A are sequestered away from eIF4G, resulting in impaired binding of mRNA to the 43S preinitiation complex. Studies that investigated the effects of amino acids on protein synthesis in vivo have focused primarily on skeletal muscle. In skeletal muscle, oral administration of either leucine alone or a protein-containing meal leads to a stimulation of the rate of protein synthesis concomitant with activation of mTORC1 and increased assembly of the eIF4F complex (eg, references 6 and 7). Treatment with the mTORC1 inhibitor rapamycin blocks both the amino acid–induced stimulation of protein synthesis and activation of mTORC1 signaling in animals and humans (6–8). Notably, neither phosphorylation of eIF2α nor changes in eIF2B GEF activity are typically observed in skeletal muscle in response to feeding or oral administration of leucine (9, 10), suggesting that activation of mTORC1 is the primary target responsible for the stimulation of protein synthesis by amino acids in vivo. In a study reported in this issue of the Journal, Coeffier et al (11) present data showing that enteral delivery of a mixture of protein and maltodextrin stimulates protein synthesis in the duodenal mucosa compared with provision of maltodextrin alone. Surprisingly, they do not observe an elevation in phosphorylation of 2 direct targets of mTORC1, 4E-BP1 and p70S6K1, suggesting that, in contrast to skeletal muscle, mTORC1 was not activated by the protein mixture even though elevated plasma concentrations of the branched-chain amino acids persisted throughout the 5-h infusion period. The authors conclude that enteral delivery of the protein mixture stimulated duodenal mucosa protein synthesis in humans through an mTORC1-independent pathway. If this conclusion is correct, it would represent an important new mechanism in cell biology and one that would stimulate elucidation of potentially novel means by which amino acids modulate protein synthesis. It is therefore imperative that future studies not only confirm the results of the present one but also test possible alternative pathways that might be involved in the effect. For example, in the Coeffier et al study (11), measurements were taken at a single time point, 5 h after the start of enteral delivery of the protein mixture and intravenous infusion of [13C]leucine and [2H]phenylalanine. To exclude the possibility that the protein-induced increase in incorporation of [13C]leucine and [2H]phenylalanine into protein may have occurred transiently at a time when mTORC1 signaling was activated, and that both may have returned to basal values by the time biopsies were taken, a time course analysis would be necessary. Moreover, a study that uses rapamycin to inhibit mTOR signaling could be performed, as has been used by others to assess the role of mTORC1 in the amino acid– and exercise-induced stimulation of protein synthesis in humans (8, 12). It is important that Western blot analysis, rather than dot-blot analysis, be used to assess potential changes in p70S6K1 and mTOR phosphorylation, and changes in protein phosphorylation should be expressed relative to the respective target protein, rather than GAPDH, to account for potential changes in expression. Finally, possible mTORC1-independent pathways, such as those involving eIF2 and eIF2B, which were not assessed in the Coeffier et al study (11), should be evaluated in future studies. It is possible that such measurements were performed and not reported in the present study or are planned for a future report. If not, future studies should address such possibilities to increase confidence that an mTORC1-independent pathway is indeed involved in the observed changes in duodenal mucosa protein synthesis. Overall, the data reported by Coeffier et al (11) are interpreted to support a model in which amino acids act to stimulate protein synthesis in the duodenal mucosa through a mechanism distinct from the established one involving activation of mTORC1. Future studies should aim to confirm the initial conclusion and to identify the mTORC1-independent pathway involved in regulating protein synthesis in the duodenal mucosa.

Background A prerequisite of hypertrophic response of the myocardium is an increase in protein synthesis. A central regulator of translation initiation is Eukaryotic initiation factor 2B (eIF2B). Here we assessed the hypothesis that regulation of protein synthesis via eIF2Bε is essential to cardiac hypertrophic response in vivo. Methods Two transgenic mouse lines were generated with cardiac restricted overexpression of eIF2Bε or its mutant eIF2Bε-eIFS535A, which cannot be inactivated by phosphorylation through GSK-3β. Results (1) Under baseline conditions eIF2Bε transgenic mice showed no difference in cardiac phenotype compared to wild type, whereas in the mutant eIF2Bε-S535A an increase in LV/tibia length (7.5±0.4 mg/mm vs. 6.2±0.2 mg/mm, p

Background Mutations in the bone morphogenetic protein type II receptor (BMPR-II) underlie the majority (>80%) of familial and up to 25% of ‘sporadic’ cases of idiopathic pulmonary arterial hypertension (PAH). Recently, homozygous recessive mutations in GCN2 (also known as EIF2AK4) were identified as causative in a rare cause of PAH, pulmonary veno-occlusive disease (PVOD) and pulmonary capillary haemangiomatosis (PCH). The mechanisms by which GCN2 deficiency contributes to the development of PAH remain unknown. GCN2 is a serine/threonine protein kinase, one of a family of 4 kinases that phosphorylate the α-subunit of the translation initiation factor eIF2α. Phosphorylation of eIF2α at serine 51 results in an inhibition of eIF2B (the eIF2 guanine nucleotide exchange factor), which ultimately leads to loss of eIF2α function. This results in inhibition of protein synthesis while enhancing the translation of a small number of mRNAs encoding proteins involved in the response to cellular stress. Methods We characterised the pulmonary vascular phenotype of the homozygous null Gcn2 ( Gcn2−/− ) and wild-type controls at 6 months of age. This mouse was chosen because it mimics the homozygous loss-of-function genotype of patients with familial PVOD/PCH. In addition, we investigated the phenotype of Gcn2−/− mice crossed with bmpr2 -deficient mice. In complementary in vitro studies, pulmonary artery smooth muscle cells (PASMCs) were extracted from patients with PVOD and idiopathic/heritable PAH and studied with wild-type controls. Findings The Gcn2−/− mouse displayed mild pulmonary hypertension. Mean right ventricular systolic pressure (RVSP) was 28.3 ± 1.2 mmHg in Gcn2−/− mice compared with 24.7 ± 1.1 mmHg in wild-type mice. GCN2−/− mice crossed with bmpr2 -deficient mice demonstrated a further increase in RVSP (32.8 ± 4.1 mmHg). Exposure of human PASMCs derived from a PVOD patient, and patients with mutations in BMPR2, to L-histidinol, which increases the phosphorylation of eIF2α, resulted in proliferation of these cells but had no effect on control PASMCs. Conclusions GCN2 deficiency promotes the development of pulmonary hypertension in mice, an effect that is exaggerated by BMPR2 deficiency. Increased phosphorylation of eIF2α resulted in PASMC proliferation. Further elucidation of these mechanisms may reveal new treatments for this devastating disease.

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm.

BACKGROUND: Mutations in any of the five subunits of eukaryotic translation initiation factor 2B (eIF2B) can lead to an inherited chronic-progressive fatal brain disease of unknown aetiology termed leucoencephalopathy with vanishing white matter (VWM). VWM is one of the most prevalent childhood white matter disorders, which markedly deteriorates after inflammation or exposure to other stressors. eIF2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. A previous study demonstrated that Eif2b5(R132H/R132H) mice suffer delayed white matter development and fail to recover from cuprizone-induced demyelination, although eIF2B enzymatic activity in the mutant brain is reduced by merely 20%. PRINCIPAL FINDINGS: Poor astrogliosis was observed in Eif2b5(R132H/R132H) mice brain in response to systemic stress induced by peripheral injections of lipopolysaccharide (LPS). Even with normal rates of protein synthesis under normal conditions, primary astrocytes and microglia isolated from mutant brains fail to adequately synthesise and secrete cytokines in response to LPS treatment despite proper induction of cytokine mRNAs. CONCLUSIONS: The mild reduction in eIF2B activity prevents the appropriate increase in translation rates upon exposure to the inflammatory stressor LPS. The data underscore the importance of fully-functional translation machinery for efficient cerebral inflammatory response upon insults. It highlights the magnitude of proficient translation rates in restoration of brain homeostasis via microglia-astrocyte crosstalk. This study is the first to suggest the involvement of microglia in the pathology of VWM disease. Importantly, it rationalises the deterioration of clinical symptoms upon exposure of VWM patients to physiological stressors and provides possible explanation for their high phenotypic variability.

Eukaryotic initiation factor 2B (eIF2B) plays a key role in protein synthesis and in its control. It comprises five different subunits, α-ϵ, of which eIF2Bϵ contains the catalytic domain. Formation of the complete complex is crucial for full activity and proper control of eIF2B. Mutations in the genes for eIF2B cause an often severe neurological disorder, “vanishing white matter.” eIF2Bγ and eIF2Bϵ contain homologous and conserved domains with sequence similarity to nucleotidyl transferases (NTs) and acyl transferases and can form a binary complex. The latter contain a hexad repeat that mainly comprises isoleucyl residues (hence termed the “I-patch” region). These data reveal that certain residues in the NT domains of eIF2Bγ/ϵ, which are highly conserved throughout eukaryotes, play key roles in the interactions between subunits in the eIF2B complex. Our data show that the I-patch regions are important in the interactions between the catalytic eIF2Bγϵ complex and the other subunits. We also studied the functional effects of vanishing white matter mutations in the NT and I-patch domains. Lastly, our data show that eIF2Bγ promotes the expression of eIF2Bϵ, providing a mechanism for achieving correct stoichiometry of these eIF2B subunits in the cell.

Activation of Toll-like receptors (TLRs) induces the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. However, despite increased levels of phosphorylated eIF2α (p-eIF2α), a TLR-TRIF-dependent pathway assures that the cells avoid CHOP induction, apoptosis and translational suppression of critical proteins. As p-eIF2α decreases the functional interaction of eIF2 with eIF2B, a guanine nucleotide exchange factor (GEF), we explored the hypothesis that TLR-TRIF signalling activates eIF2B GEF activity to counteract the effects of p-eIF2α. We now show that TLR-TRIF signalling activates eIF2B GEF through PP2A-mediated serine dephosphorylation of the eIF2B ε-subunit. PP2A itself is activated by decreased Src-family-kinase-induced tyrosine phosphorylation of its catalytic subunit. Each of these processes is required for TLR-TRIF-mediated CHOP suppression in ER-stressed cells in vitro and in vivo. Thus, in the setting of prolonged, physiologic ER stress, a unique TLR-TRIF-dependent translational control pathway enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis while still benefiting from the protective arms of the UPR. © 2012 Macmillan Publishers Limited. All rights reserved., link_to_subscribed_fulltext

Background Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker. Methodology/Principal Findings We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders. Conclusion Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.

CACH, also termed vanishing white matter (VWM) leukoencephalopathy, is caused by mutations in the eukaryotic translation factor. There are five different subunits in this factor (which play an important role in cellular protein synthesis). Here, ⇓Fogli et al. describe an acute, fatal infantile form and report the molecular defect in the e subunit. Activation of eIF2 by eIF2B. The translation initiation factor eIF2 is activated by exchange of GDP for GTP by the activated factor eIF2B-GTP (composed of five different subunits). The e-subunit of eIF2B permits the GDP/GTP exchange (GEF activity: Guanine Exchange Factor); the β-subunit binds the GTP. see page 1966 Commentary by Michael J. Noetzel, MD Clinicians have approached neurologic disorders using the conceptual framework of lumping and splitting for decades. In the current age of molecular genetics, these old constructs have new meaning. Nowhere is this better illustrated than in childhood leukodystrophies. Despite the advances of biochemistry, neuroradiology, and histopathology, a large number of these leukodystrophies remain “undiagnosed.” Relying on the concept of splitting, clinicians and researchers defined relatively homogeneous groups of these undiagnosed leukodystrophy patients. As a result, new …

Leukodystrophy with vanishing white matter (VWM) is a neurodegenerative disorder with autosomal recessive traits that is caused by alteration of the eukaryotic translation initiation factor-2B (EIF2B). An 11-month-old patient with distinctive features began to exhibit progressive developmental deterioration associated with intractable epilepsy, which was triggered by recurrent acute infectious diseases. Brain magnetic resonance imaging (MRI) revealed abnormal white matter intensity. Chromosomal microarray testing identified a submicroscopic deletion at 14q24.3 that included EIF2B2, the gene encoding one of the subunits of EIF2B. Because the patient's clinical findings were distinctive for VWM, compound heterozygous mutations of EIF2B2 were suspected, and subsequent sequencing analysis of the remaining allele unmasked the existence of a novel missense mutation of EIF2B2 (V85W). Some distinctive features including small palpebral fissures, bushy eyebrows, ear abnormalities, small upturned nose, downturned corners of the mouth, and micrognathia may be the common features of the patients with 14q24.3 deletions. © 2012 Wiley Periodicals, Inc.

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.

The maintenance of muscle mass is critical for health and issues associated with the quality of life. Over the last decade, extensive progress has been made with regards to our understanding of the molecules that regulate skeletal muscle mass. Not surprisingly, many of these molecules are intimately involved in the regulation of protein synthesis and protein degradation [e.g. the mammalian target of rapamycin (mTOR), eukaryotic initiation factor 2B (eIF2B), eukaryotic initiation factor 3f (eIF3f) and the forkhead box O (FoxO) transcription factors]. It is also becoming apparent that molecules which sense, or control, the energetic status of the cell play a key role in the regulation of muscle mass [e.g. AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator-1 α (PGC1α)]. In this review we will attempt to summarize the current knowledge of how these molecules regulate skeletal muscle mass.

Translation initiation factor 2 (IF2) is one of key components of the translation initiation system in living cells. In bacteria IF2 is a multidomain monomeric protein, while in eukaryotic and archaean cells e/aIF2 is heterotrimer (αβγ). Data, including our own, on eukaryotic type translation initiation factor 2 (e/aIF2) structure and functioning are presented. There are also new data on initiation factors eIF5 and eIF2B that directly interact with eIF2 and control its participation in nucleotide exchange.

Vanishing white matter disease (VWM) is the first human hereditary disease known to be caused by defects in initiation of protein synthesis. Gene defects in each of the five subunits of eukaryotic translation initiation factor 2B (eIF2B α-ɛ) are responsible for the disease, although the mechanism of the pathogenesis is not well understood. In our previous study, four novel eIF2Bɛ mutations were found in Chinese patients: p.Asp62Val, p.Cys335Ser, p.Asn376Asp and p.Ser610-Asp613del. Functional analysis was performed on these mutations and the recently reported p.Arg269X. Our data showed that all resulted in a decrease in the guanine nucleotide exchange (GEF) activity of the eIF2B complex. p.Arg269X and p.Ser610-Asp613del mutants displayed the lowest activity, followed by p.Cys335Ser, p.Asn376Asp and p.Asp62Val. p.Arg269X and p.Ser610-Asp613del could not produce stable eIF2Bɛ, leading to almost complete loss-of-function. No evidence was obtained for the three missense mutations in changes in eIF2Bɛ protein level or eIF2BɛSer(540) phosphorylation, and disruption of holocomplex assembly, or binding to eIF2. All patients in our study had the classical phenotype. p.Asp62Val and p.Asn376Asp mutations caused only mildly decreased GEF activity, were probably responsible for relatively mild phenotype in cases of classical VWM.

Autosomal recessive mutations in eukaryotic initiation factor 2B (eIF2B) cause leukoencephalopathy vanishing white matter with a wide clinical spectrum. eIF2B comprises five subunits (α-ε; genes EIF2B1, 2, 3, 4 and 5) and is the guanine nucleotide-exchange factor (GEF) for eIF2. It plays a key role in protein synthesis. Here, we have studied the functional effects of selected VWM mutations in EIF2B2-5 by coexpressing mutated and wild-type subunits in human cells. The observed functional effects are very diverse, including defects in eIF2B complex integrity; binding to the regulatory α-subunit; substrate binding; and GEF activity. Activity data for recombinant eIF2B complexes agree closely with those for patient-derived cells with the same mutations. Some mutations do not affect these parameters even though they cause severe disease. These findings are important for three reasons; they demonstrate that measuring eIF2B activity in patients' cells has limited value as a diagnostic test; they imply that severe disease can result from alterations in eIF2B function other than defects in complex integrity, substrate binding or GEF activity, and last, the diversity of functional effects of VWM mutations implies that seeking agents to manage or treat VWM should focus on downstream effectors of eIF2B, not restoring eIF2B activity. © 2011 Wiley-Liss, Inc.

Background Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM), which is associated with a clinical pathology of brain myelin loss upon physiological stress. eIF2B is the guanine nucleotide exchange factor (GEF) of eIF2, which delivers the initiator tRNAMet to the ribosome. We recently reported that a R132H mutation in the catalytic subunit of this GEF, causing a 20% reduction in its activity, leads under normal conditions to delayed brain development in a mouse model for CACH/VWM. To further explore the effect of the mutation on global gene expression in the brain, we conducted a wide-scale transcriptome analysis of the first three critical postnatal weeks. Methodology/Principal Findings Genome-wide mRNA expression of wild-type and mutant mice was profiled at postnatal (P) days 1, 18 and 21 to reflect the early proliferative stage prior to white matter establishment (P1) and the peak of oligodendrocye differentiation and myelin synthesis (P18 and P21). At each developmental stage, between 441 and 818 genes were differentially expressed in the mutant brain with minimal overlap, generating unique time point-specific gene expression signatures. Conclusions The current study demonstrates that a point mutation in eIF2B, a key translation initiation factor, has a massive effect on global gene expression in the brain. The overall changes in expression patterns reflect multiple layers of indirect effects that accumulate as the brain develops and matures. The differentially expressed genes seem to reflect delayed waves of gene expression as well as an adaptation process to cope with hypersensitivity to cellular stress.

Background Eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response. Methods We used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of ATF4, and transcriptional induction of stress-specific mRNAs (ATF4, CHOP, ASNS, GRP78) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL). Results Despite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls. Conclusions Taken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.

Glycogen synthase kinase 3β (GSK3β) activity is regulated by phosphorylation processes and regulates in turn through phosphorylation several proteins, including eukaryotic initiation factor 2B (eIF2B). Serine 9 phosphorylation of GSK3β (pGSK3βSer9), usually promoted by activation of the PI3K/Akt survival pathway, triggers GSK3β inhibition. By contrast, tyrosine 216 phosphorylation of GSK3β (pGSK3βTyr216) increases under apoptotic conditions, leading to GSK3β activation. Lithium chloride (LiCl) is usually described to increase pGSK3βSer9 through the PI3K/Akt pathway, resulting in GSK3β inhibition. The purpose of this study is to demonstrate that in some cases LiCl is also able to increase pGSK3βTyr216, resulting in GSK3β activation. For this, we used SH-SY5Y cells and primary neuronal cultures and investigated the effects of LiCl on the two phosphorylated forms of GSK3β under staurosporine (STS)-intoxicated conditions. The ratios between the phosphorylated and total forms of GSK3β and eIF2B were determined by Western blotting. Our results revealed that, besides its ability to increase pGSK3βSer9, LiCl is also able to increase pGSK3βTyr216 greatly in STS-intoxicated SH-SY5Y cells but not in STS-intoxicated primary neuronal cultures. This accumulation of both Ser9 and Tyr216 phosphorylation results in GSK3β activation in STS-intoxicated SH-SY5Y cells in spite of the presence of LiCl. These findings indicate that LiCl treatment is not necessarily correlated with GSK3β inhibition even though it generates Ser9 phosphorylation. Consequently, the ratio pGSK3βSer9/pGSK3βTyr216, which takes into account the balance between the two inactive (Ser9) and active (Tyr216) forms of GSK3β, could be more useful for predicting GSK3β inhibition.

Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2·GDP to eIF2·GTP. The eIF2B is composed of five subunits, α, β, γ, δ and ɛ, within which the ɛ subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2Bɛ (eIF2Bɛ-CTD) at 2.0-A resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2Bɛ-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2Bɛ-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif.

Interaction between the 40S ribosomal subunit and the IRES of hepatitis C virus (HCV) is thought to be independent of initiation proteins, while joining of the 60S ribosomal subunit, and initiation of translation is dependent upon components of the translation machinery. An established in vivo functional assay for internal initiation mediated by the HCV IRES was used to identify proteins needed for IRES dependent translation in Saccharomyces cerevisiae strains possessing alterations of the translation machinery. Internal initiation dependent upon the HCV IRES was abrogated in strains lacking eIF5B, and reduced in strains with altered eIF3, either lacking the Hcr1p subunit, a component of eIF3 not previously known to interact with HCV RNA, or possessing an amino acid change in the Rpg1p subunit. The HCV RNA-induced conformational change in the 40S subunit might affect positioning of eIF3 and lead to different interactions between the ribosome, eIF3, and the multifactor complex. HCV IRES dependent initiation was unaffected in strains in which the concentration of the initiating tRNA was reduced. Alteration of the δ subunit of eIF2B, which leads to inefficient recycling, or substitution of aspartic acid for serine 51 of eIF2α had no effect on internal initiation. Production of human Pkr inhibited HCV IRES dependent initiation in yeast. The synthesis of Pkr in yeast is known to result in high levels of eIF2α phosphorylation, increased Gcn4p synthesis, and reduced ribosomal protein production. These alterations may explain the effect of Pkr synthesis on HCV IRES dependent initiation in yeast.

A key challenge in clinics is the identification of sensitive and specific biomarkers for early detection, prognostic evaluation, and surveillance of disease. A biomarker is defined as a biological substance that can be used to specifically detect a disease, measure its progression, or the effect of a treatment. A biomarker should be easily accessible, and ideally sensitivity and specificity must be sufficient to distinguish between false positives, false negatives, and true positives. To be useful for routine clinical evaluation, a biomarker should be detectable in body fluids (e.g., plasma, serum, urine). A biomarker can be a metabolite, a specific post-translational modification, a lipid, a phospholipid, or a protein. Due to technical advances in the analysis of biomolecules by mass spectrometry (MS), investigations of peptide biomarkers have increased. In contrast to genome, the peptidome is dynamic and constantly changing. Elucidating how the peptides complement changes in a cell type in diseases is crucial to understand how these processes occur at a molecular level. Lymphoblastoid cell lines, derived from blood lymphocytes, represent suitable models for biochemical investigations and biomedical applications because of their stability, the ease of amplification, and long-term preservation. Technological improvements of MS and liquid chromatography (LC) during the last 10 years resulted in the development of highly sensitive approaches for proteomic and peptidomic analyses. Here we provide guidelines for the preparation of the lymphoblastoid cell lines, the extraction of the peptides and their purification. We describe a number of technologies which we developed for the peptidomic profiling of lymphoblastoid cell extracts from patients with leukodystrophies, linked to mutations in the genes encoding the eukaryotic initiation factor 2B (eIF2B; eIF2B-related disorders).

Protein synthesis (also termed mRNA translation) is a key step in the expression of a cell's genetic information, in which the information contained within the coding region of the mRNA is used to direct the synthesis of the new protein, a process that is catalysed by the ribosome. Protein synthesis must be tightly controlled, to ensure the right proteins are made in the right amounts at the right time, and must be accurate, to avoid errors that could lead to the production of defective and potentially damaging proteins. In addition to the ribosome, protein synthesis also requires proteins termed translation factors, which mediate specific steps of the process. The first major stage of mRNA translation is termed ‘initiation’ and involves the recruitment of the ribosome to the mRNA and the identification of the correct start codon to commence translation. In eukaryotic cells, this process requires a set of eIFs (eukaryotic initiation factors). During the second main stage of translation, ‘elongation’, the ribosome traverses the coding region of the mRNA, assembling the new polypeptide: this process requires eEFs (eukaryotic elongation factors). Control of eEF2 is important in certain neurological processes. It is now clear that defects in eIFs or in their control can give rise to a number of diseases. This paper provides an overview of translation initiation and its control mechanisms, particularly those examined in neuronal cells. A major focus concerns an inherited neurological condition termed VHM (vanishing white matter) or CACH (childhood ataxia with central nervous system hypomyelination). VWM/CACH is caused by mutations in the translation initiation factor, eIF2B, a component of the basal translational machinery in all cells.

BACKGROUND:In recent years, the phenotypes of leukodystrophies linked to mutations in the eukaryotic initiation factor 2B genes have been extended, classically called CACH/VWM (Childhood ataxia with cntral hypomyélination/vanishing white matter disorder). The large clinical spectrum observed from the more severe antenatal forms responsible for fetal death to milder adult forms with an onset after 16 years old and restricted to slow cognitive impairment have lead to the concept of eIF2B-related disorders. The typical MRI pattern with a diffuse CSF-like aspect of the cerebral white matter can lack particularly in the adult forms whereas an increasing number of patients with clinical and MRI criteria for CACH/VWM disease but without eIF2B mutations are found. Then we propose the use of biochemical markers to help in this difficult diagnosis. The biochemical diagnosis of eIF2B-related disorder is difficult as no marker, except the recently described asialotransferrin/transferrin ratio measured in cerebrospinal fluid, has been proposed and validated until now. Decreased eIF2B GEF activity has been previously reported in lymphoblastoid cell lines from 30 eIF2B-mutated patients. Our objective was to evaluate further the utility of this marker and to validate eIF2B GEF activity in a larger cohort as a specific diagnostic test for eIF2B-related disorders. METHODOLOGY/PRINCIPAL FINDINGS:We performed eIF2B GEF activity assays in cells from 63 patients presenting with different clinical forms and eIF2B mutations in comparison to controls but also to patients with defined leukodystrophies or CACH/VWM-like diseases without eIF2B mutations. We found a significant decrease of GEF activity in cells from eIF2B-mutated patients with 100% specificity and 89% sensitivity when the activity threshold was set at < or =77.5%. CONCLUSION:These results validate the measurement of eIF2B GEF activity in patients' transformed-lymphocytes as an important tool for the diagnosis of eIF2B-related disorders.

Eukaryotic initiation factor 2 (eIF2) B is a guanine nucleotide exchange factor that plays a central role in translation initiation and its control, especially in response to diverse cellular stresses. In addition, inherited mutations in human eIF2B subunits cause a fatal brain disorder commonly called childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter. In yeast, inhibiting activity of eIF2B up-regulates expression of the transcriptional activator general control nondepressible (GCN) 4. We report here evaluation of high-throughput screening (HTS) using a yeast-based reporter gene assay, in which strains containing either wild-type or a mutant eIF2B were screened in parallel to identify compounds modifying eIF2B-dependent responses. The goals of the HTS were twofold: first, to discover compounds that restore normal function to mutant eIF2B, which may have therapeutic utility for the fatal human disease; and second, to identify compounds that activate a GCN4 response, which might be useful experimental tools. The HTS assay measured cell growth by absorbance, and activation of gene expression via a beta-galactosidase reporter gene fusion. Because mutant eIF2B activates GCN4 in the absence of stress inducers, the mutant strain was screened for reduction in GCN4 activation. HTS revealed apparent mutant-selective inhibitors but did not reliably predict selectivity as these hits affected both wild-type and mutant strains equally on dose-response confirmation. Wild-type strain results from the HTS identified two GCN4 response activators, both of which were confirmed to be wild-type selective in dose-response testing, suggesting that these compounds may activate GCN4 by a mechanism that down-regulates eIF2B activity.

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the alpha subunit of human eIF2B at 2.65 A resolution. The eIF2Balpha structure consists of the N-terminal alpha-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 A in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 A in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Balpha. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Balpha. Interestingly, the yeast eIF2Balpha missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Balpha missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The beta and delta subunits of eIF2B are homologous to eIF2Balpha and may have tertiary structures similar to the present eIF2Balpha structure. The sulfate-ion-binding residues of eIF2Balpha are well conserved in eIF2Bbeta/delta. The abovementioned yeast and human missense mutations of eIF2Bbeta/delta were also mapped on the eIF2Balpha structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.

Vanishing white matter (VWM) disease, inherited in an autosomal recessive manner, is one of the most prevalent inherited leukoencephalopathies in childhood. It is a hereditary human disease resulting from the direct defects during protein synthesis, with the gene defects in EIF2B1-5 (identified in 2001-2002) encoding the five subunits of eukaryotic translation initiation factor (eIF2B alpha, beta, gamma, delta and epsilon), respectively. Most of the published studies were carried out in the white population. The analysis of clinical features and EIF2B mutation screening were performed in 11 Chinese patients for the first time. Mutations were identified exclusively in EIF2B5 and EIF2B3 in these patients, with six novel mutations, including five missense mutations (EIF2B5: c.185A>T, p.D62V; c.1004G>C, p.C335S; c.1126A>G, p.N376D; EIF2B3: c.140G>A, p.G47E; c.1037T>C, p.I346T) and one deletion leading to amino-acid deletion (EIF2B5: c.1827-1838del, p.S610-D613del). EIF2B3 mutation, accounting for 20% of the total number of mutations found in this study, is more prevalent than expected according to an earlier report (7%). A hot spot mutation in EIF2B3 was identified in this study. A unique EIF2B mutation spectrum in Chinese VWM patients was shown. A systemic study to assess mutation spectrum in different populations needs to be carried out.

Eukaryotic initiation factor 2B (eIF2B), a five-subunit guanine nucleotide exchange factor, plays a key role in the regulation of mRNA translation. Expression of its ε-subunit is specifically up-regulated in certain conditions associated with increased cell growth. Therefore, the purpose of the present study was to examine the effect of repressing eIF2Bε expression on growth rate, protein synthesis, and other characteristics of two tumorigenic cell lines that display up-regulated expression of the ε-subunit. Experiments were designed to compare spontaneously transformed fibroblasts to transformed mouse embryonic fibroblasts infected with a lentivirus containing a short hairpin RNA directed against eIF2Bε. Cells expressing the short hairpin RNA displayed a reduction in eIF2Bε abundance to 30% of the value observed in uninfected transformed mouse embryonic fibroblasts, with no change in the expression of any of the other four subunits. The repression of eIF2Bε expression was accompanied by reductions in guanine nucleotide exchange factor activity and global rates of protein synthesis. Moreover, repressed eIF2Bε expression led to marked reductions in cell growth rate in culture, colony formation in soft agar, and tumor progression in nude mice. Similar results were obtained in MCF-7 human breast cancer cells in which eIF2Bε expression was repressed through transient transfection with a small interfering RNA directed against the ε-subunit. Overall, the results support a role for eIF2Bε in the regulation of cell growth and suggest that it might represent a therapeutic target for the treatment of human cancer. [Cancer Res 2008;68(21):8752–60]

Background Mutations in eukaryotic translation initiation factor 2B (eIF2B) cause Childhood Ataxia with CNS Hypomyelination (CACH), also known as Vanishing White Matter disease (VWM). The disease is manifested by loss of brain myelin upon physiological stress. In a previous study, we showed that fibroblasts isolated from CACH/VWM patients are hypersensitive to pharmacologically-induced endoplasmic reticulum (ER) stress. Since brain cells from affected individuals are not available for research, we wished to assess the effect of eIF2B mutation on oligodendroglial-derived cells. Methodology/Principal Findings A rat oligodendroglial-derived cell line was used for a stable knock-down of eIF2B5 followed by stable expression of mutated eIF2B5(R195H) cDNA. In response to a pharmacological ER-stress agent, eIF2B5(R195H) expressing cells exhibited heightened ER-stress response demonstrated by hyper induction of ATF4, GADD34, Bip, PDIA1, PDIA3, PDIA4 and PDIA6 proteins. Moreover, even in the absence of a pharmacological stress agent, eIF2B5(R195H)-expressing cells exhibited high basal levels of ATF4, GADD34 and ER-associated Bip, PDIA1 and PDIA3. Significance The data provide evidence that oligodendroglial-derived cells expressing a mutated eIF2B constantly use their stress response mechanism as an adaptation mean in order to survive. The current study is the first to demonstrate the effects of eIF2B5 mutation on ER homeostasis in oligodendroglial-derived cells.

Major improvements in the yield of recombinant proteins produced by animal cell cultures are likely to result from an extension of productive cell lifetime. Genetic engineering of cell cycle and death mechanisms and /or improved fed-batch processes have been used to enhance cell productivity. In contrast, by direct genetic manipulation of the rate of protein synthesis in animal cells we intend to augment both cell specific production, and productive cell lifetime in batch culture. This approach also provides a means to increase the transient expression of recombinant proteins by ‘transient’ host cell engineering. mRNA translation initiation factors control the overall rate of protein synthesis. The phosphorylation state of a subset of these factors; 4E-BP1, eIF2B, eIF4G, eIF2 and eIF4E are important in the regulation of protein synthesis (Figure 1.). An increase in the phosphorylation state of the a subunit of eIF2 is associated with an inhibition of protein synthesis in response to stimuli such as heat shock (Duncan & Hershey, 1987), amino acid, glucose, or serum deprivation (Scorsone et al., 1987). The impairment of translation seen in response to heat shock is partially overcome by expression of a non-phosphorylatable mutant of eIF2α, Ser5lAla (Murtha-Riel et al., 1993). Thus, reduced rates of protein synthesis seen when cells encounter other such conditions are also expected to be attenuated by expression of this mutant, which is otherwise functional in translation.

Purified recombinant human subunits of eukaryotic initiation factor 2 (eIF2) expressed in bacteria are found to interact with each other to form alphabeta, alphagamma, and betagamma complexes in a pull-down experiment. Recombinant phosphorylated human eIF2alpha that cannot interact with purified eIF2B, the GDP/GTP exchange factor of eIF2, however interacts efficiently with eIF2B along with the beta-subunit of eIF2 of the rabbit reticulocyte lysates and also with the purified recombinant beta-subunit. These findings therefore suggest that the beta-subunit of eIF2 mediates the productive and non-productive interactions between eIF2 and 2B. Recombinant alpha and beta-subunits serve as substrates for not only kinases but also for caspase 3 and interestingly phosphorylated subunits resist caspase action. Phosphorylation also modifies the beta-subunit's interaction with Nck1, a cofactor of eIF2alpha phosphatase, but not with eIF5, the GTPase activating protein. These findings suggest that subunits of mammalian eIF2 interact with each other and the beta-subunit plays a critical role both in the regulation and function of eIF2.

The CACH/VWM syndrome is an autosomal recessive leukodystrophy characterized by a broad spectrum of clinical presentations and by diffuse cavitary degeneration of the white matter on MRI. Mutations responsible for this disorder are missense or frameshift mutations occurring in the five genes (EIF2B1-5) that encode the translation eukaryotic initiation factor eIF2B. We found that a patient with infantile CACH/VWM carries a mutation in the acceptor splice site of EIF2B5 exon 6. In lymphoblastoid cells of the patient, we detected an abnormal EIF2B5 transcript in which exon 6 was absent, however, the predicted protein product lacking part of the non-catalytic domain encoded by exon 6 was not detected. The eIF2B GEF activity was severely decreased. These data support the importance of the non-catalytic domain of the eIF2Bepsilon subunit in the eIF2B complex formation and activity.

Eukaryotic initiation factor 2B (eIF2B)-related disorders are heritable white matter disorders with a variable clinical phenotype (including vanishing white matter disease and ovarioleukodystrophy) and an equally heterogeneous genotype. We report 9 novel mutations in the EIF2B genes in our subject population, increasing the number of known mutations to more than 120. Using homology modeling, we have analyzed the impact of novel mutations on the 5 subunits of the eIF2B protein. Although recurrent mutations have been found at CpG dinucleotides in the EIF2B genes, the high incidence of private or low frequency mutations increases the challenge of providing rapid genetic confirmation of this disorder, and limits the application of EIF2B screening in cases of undiagnosed leukodystrophy.

In a previous study we demonstrated a requirement for activation of mTORC1 in the stimulation of eIF2Bepsilon mRNA translation in skeletal muscle in response to resistance exercise. Although that study established the necessity of mTORC1 activation, the experimental model used did not lend itself readily to address the question of whether or not mTORC1 activation was sufficient to produce the response. Therefore, the present study was designed to address the sufficiency of mTORC1 activation, using cultures of Rat2 fibroblasts in which mTORC1 signaling was repressed by serum/leucine-depletion and stimulated by repletion of leucine and/or IGF-1. Repletion with leucine and IGF-1 caused a shift of eIF2Bepsilon mRNA into actively translating polysomes and a stimulation of new eIF2Bepsilon protein synthesis, but had no effect on mRNAs encoding the other four eIF2B subunits. Stimulation of eIF2Bepsilon translation was reversed by pre-treatment with the mTORC1 inhibitor rapamycin. Exogenous overexpression of FLAG-Rheb, a proximal activator of mTORC1, also caused a re-distribution of eIF2Bepsilon mRNA into polysomes and a stimulation of eIF2Bepsilon protein synthesis. The stimulation of eIF2Bepsilon mRNA translation occurred in the absence of any effect on eIF2Bepsilon mRNA abundance. RNAi-mediated knockdown of eIF2Bepsilon resulted in reduced cellular proliferation, a result that phenocopied the known cytostatic effect of mTORC1 repression. Overall the results demonstrate that activation of mTORC1 is both necessary and sufficient to stimulate eIF2Bepsilon mRNA translation and that this response may represent a novel mechanism through which mTORC1 can affect mRNA translation initiation, rates of protein synthesis, and cellular growth/proliferation.

Eukaryotic initiation factor 2B (eIF2B) plays a key role in controlling the initiation of mRNA translation. eIF2B is heteropentamer whose catalytic (epsilon) subunit promotes GDP/GTP exchange on eIF2. We show here that depriving human cells of amino acids rapidly results in the inhibition of eIF2B, independently of changes in eIF2 phosphorylation. Although amino acid deprivation also inhibits signaling through the mammalian target of rapamycin complex 1 (mTORC1), the inhibition of eIF2B activity by amino acid starvation is independent of mTORC1. Instead, amino acids repress the phosphorylation of a novel site in eIF2Bepsilon. We identify this site as Ser525, located adjacent to the known phosphoregulatory region in eIF2Bepsilon. Mutation of Ser525 to Ala abolishes the regulation of eIF2B and protein synthesis by amino acids. This indicates that phosphorylation of this site is crucial for the control of eIF2B and protein synthesis by amino acids. These findings identify a new way in which amino acids regulate a key step in translation initiation and indicate that this involves a novel amino acid-sensitive signaling mechanism.

Mutations in the eukaryotic translation initiation factor 2B (eIF2B) represent a heterogenous group of autosomal recessive leucodystrophy characterized by a diffuse CSF-like aspect of the white matter at MRI designed as vanishing white matter (VWM) and episodes of acute deterioration after stresses. The mild juvenile and adult forms are often associated with primary ovarian failure, a syndrome referred to as ovarioleukodystrophy (OLD). We reported case of a woman with OLD who successfully underwent in vitro fertilization with donated oocytes and embryo transfer. Pregnancy was complicated by a non-convulsive epileptic status leading to the identification of compound heterozygous EIF2B5 mutation (p.Arg113His and p.Arg299His). The patient gave birth to a healthy child by Caesarean section. In conclusion, we report for the first time that in vitro fertilization and embryo transfer can lead to a successful procreation in patients with OLD related to EIF2B mutations. However this procedure must be considered with cautiousness, because of its potential neurological risks.

A new association of Vanishing White Matter (VWM) and premature ovarian failure (POF) was recently described as a sole entity called ovarioleukodystrophy. Seven out of eight patients reported by Fogli et al. had translation initiation factor (eIF2B) mutations, specific to the VWM. The only patient without mutations had a distinctive neurological presentation that included cognitive deterioration without motor signs and white matter abnormalities restricted to the frontal lobe. We describe here a case suggestive of ovarioleukodistrophy carrying no eIF2B mutations.

Vanishing white matter (VWM) is a childhood leukoencephalopathy with central hypomyelination, white matter rarefaction, and cystic degeneration. Adult onset, variable phenotype, and high frequency characterize Arg113His mutation caused by G338A polymorphism associated with VWM. A patient with trauma-associated onset, and clinical features compatible with multiple sclerosis (MS), was homozygous for G338A mutation of eukaryotic translation initiation factor (eIF2B5). The authors checked a cohort of 101 MS patients, including 19 with head/neck trauma-associated onset, and failed to find the mutation, described above, in MS chromosomes. Our report does not exclude the presence in MS chromosomes of other mutations in the eIF2B gene family. Multiple Sclerosis 2007; 13: 424-427. http://msj.sagepub.com

A decline in relative levels and phosphorylation of many of the eukaryotic initiation factors (eIFs) including S6, the 40S ribosomal subunit protein in many of the rat tissues during chronological aging is accompanied by elevated levels of eIF2alpha kinases, such as PKR and PERK, but not their activity. Concomitant with increased eIF2alpha phosphorylation, young tissues displayed a higher level of eIF2B to tolerate the toxic effect of eIF2alpha phosphorylation on translation, ATF4, a b-zip transcriptional factor that is produced as part of the gene expression programme in response to eIF2alpha phosphorylation, and BiP, an endoplasmic reticulum (ER) molecular chaperone and regulator of ER stress sensors. Decline in eIF2alpha phosphorylation in aged tissues is associated with a higher level of GADD34, a subunit of eIF2alpha phosphatase, and proapoptotic proteins like CHOP/GADD153 and phospho JNK, suggesting that young tissues possess an efficient ER stress adaptive mechanism that declines with aging.

The eukaryotic initiation factor 2B (eIF2B) is a five-subunit guanine nucleotide exchange factor, that functions during translation initiation to catalyze the otherwise slow exchange of GDP for GTP on its substrate eIF2. Assays to measure substrate interaction and guanine nucleotide release ability of eIF2B require the complex to be purified free of interacting proteins. We have also found that a subcomplex of two subunits, gamma and epsilon or the largest one, epsilon alone, promotes this activity. Within eIF2Bepsilon, the catalytic center requires the C-terminal 200 residues only. Here, we describe our protocols for purifying the Saccharomyces cerevisiae eIF2B complexes and the catalytic subunit using FLAG-tagged proteins overexpressed in yeast cells. Using commercially available FLAG-affinity resin and high salt buffer, we are able to purify active eIF2B virtually free of contaminants.

The discovery of Green Fluorescent Protein (GFP) and the development of technology that allows specific proteins to be tagged with GFP has fundamentally altered the types of question that can be asked using cell biological methods. It is now possible not only to study where a protein is within a cell, but also feasible to study the precise dynamics of protein movement within living cells. We have exploited these technical developments and applied them to the study of translation initiation factors in yeast, focusing particularly on the key regulated guanine nucleotide exchange step involving eIF2B and eIF2. This chapter summarizes current methodologies for the tagging and visualization of GFP-tagged proteins involved in translation initiation in live yeast cells.

Famine and viral infection, as well as interferon therapy have been reported to increase the risk of developing bipolar disorder. In addition, almost 100 polymorphic genes have been associated with this disease. Several form most of the components of a phosphatidyl-inositol signalling/AKT1 survival pathway (PIK3C3, PIP5K2A, PLCG1, SYNJ1, IMPA2, AKT1, GSK3B, TCF4) which is activated by growth factors (BDNF, NRG1) and also by NMDA receptors (GRIN1, GRIN2A, GRIN2B). Various other protein products of genes associated with bipolar disorder either bind to or are affected by phosphatidyl-inositol phosphate products of this pathway (ADBRK2, HIP1R, KCNQ2, RGS4, WFS1), are associated with its constituent elements (BCR, DUSP6, FAT, GNAZ) or are downstream targets of this signalling cascade (DPYSL2, DRD3, GAD1, G6PD, GCH1, KCNQ2, NOS3, SLC6A3, SLC6A4, SST, TH, TIMELESS). A further pathway relates to endoplasmic reticulum-stress (HSPA5, XBP1), caused by problems in protein glycosylation (ALG9), growth factor receptor sorting (PIK3C3, HIP1R, SYBL1), or aberrant calcium homoeostasis (WFS1). Key processes relating to these pathways appear to be under circadian control (ARNTL, CLOCK, PER3, TIMELESS). DISC1 can also be linked to many of these pathways. The growth factor pathway promotes protein synthesis, while the endoplasmic reticulum stress pathway, and other stress pathways activated by viruses and cytokines (IL1B, TNF, Interferons), oxidative stress or starvation, all factors associated with bipolar disorder risk, shuts down protein synthesis via control of the EIF2 alpha and beta translation initiation complex. For unknown reasons, oligodendrocytes apper to be particularly prone to defects in the translation initiation complex (EIF2B) and the convergence of these environmental and genomic signalling pathways on this area might well explain their vulnerability in bipolar disorder.