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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.

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

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

Summary The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B’s nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR., Graphical Abstract, Highlights • Mutually antagonistic binding of phosphorylated eIF2 and ISRIB to eIF2B • Opposing structural rearrangement of eIF2B by binding ISRIB or phosphorylated eIF2 • Phosphorylated eIF2 exposes ISRIB’s ability to enhance eIF2B activity in vitro • Weak ISR-inhibitory activity of ISRIB in cells lacking phosphorylated eIF2, ISRIB is a drug-like compound that restores memory and cognitive function in mouse models of certain human diseases. Its beneficial properties derive from interference with a stress-response pathway that hinges on regulating rates of protein synthesis. Here, Zyryanova, Kashiwagi et al. dissect the molecular basis of ISRIB action in cells.

The overall process of protein biosynthesis across all domains of life is similar; however, detailed insights reveal a range of differences in the proteins involved. For decades, the process of protein translation in archaea has been considered to be closer to eukaryotes than to bacteria. In archaea, however, several homologues of eukaryotic proteins involved in translation initiation have not yet been identified; one of them being the initiation factor eIF2B consisting of five subunits (α, β, γ, δ and ε). Three open reading frames (PH0440, PH0702 and PH0208) in Pyrococcus horikoshii have been proposed to encode for the α-, β- and δ-subunits of aIF2B, respectively. The crystal structure of PH0440 shows similarity toward the α-subunit of eIF2B. However, the capability of PH0702 and PH0208 to function as the β- and δ-subunits of eIF2B, respectively, remains uncertain. In this study, we have taken up the task of annotating PH0702 and PH0208 using bioinformatics methods. The phylogenetic analysis of protein sequences belonging to IF2B-like family along with PH0702 and PH0208 revealed that PH0702 belonged to methylthioribose-1-phosphate isomerase (MTNA) group of proteins, whereas, PH0208 was found to be clustered in the group of ribose-1,5-bisphosphate isomerase (R15PI) proteins. A careful analysis of protein sequences and structures available for eIF2B, MTNA and R15PI confirms that PH0702 and PH0208 contain residues essential for the enzymatic activity of MTNA and R15PI, respectively. Additionally, the protein PH0208 comprises of the residues required for the dimer formation which is essential for the biological activity of R15PI. This prompted us to examine all eIF2B-like proteins from archaea and to annotate their function. The results reveal that majority of these proteins are homologues of the α-subunit of eIF2B, even though they lack the residues essential for their functional activity. A better understanding of the mechanism of GTP exchange during translation initiation in archaea is henceforth required.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the nervous system with a profound genetic element. It is already known that alterations in Eukaryotic Translation Initiation Factor 2B (EIF2B) gene encoding the five subunits of eIF2B complex cause Vanishing White Matter (VWM) disease of the brain and emerging evidences have advocated certain resemblances between MS and VWM in terms of clinical and epidemiological characteristics, thus validating the association study between EIF2B and MS. Moreover, a recent study has implicated EIF2B5 Ile587Val (rs843358) polymorphism as a susceptibility factor for MS. In order to investigate the association of EIF2B5 Ile587Val polymorphism with MS susceptibility in Kashmir region in India, we screened EIF2B5 Exon 13 in 30 MS patients and 65 controls (a total of 95 participants). During the present course of study, we could not find statistically significant difference in the frequency of Ile587Val between MS patients and controls, thus indicating that such alteration does not appear to influence MS development in Kashmiri population. Our results provide evidence against a major role for Ile587Val polymorphism in MS susceptibility.

An endoparasitoid wasp, Cotesia plutellae, possesses its specific symbiotic virus called C. plutellae bracovirus (CpBV) and parasitizes young larvae of Plutella xylostella. CpBV encodes CpBV15α, which was previously shown to interfere with host protein translation. In vivo transient expression of CpBV15α induced a significant decrease in a storage protein level without its transcriptional level change. In vitro translation assay using rabbit reticulocyte lysate showed that CpBV15α suppressed translation efficiency of mRNAs extracted from fat body of P. xylostella. Transient expression of CpBV15α in nonparasitized P. xylostella suppressed humoral immunity and development to pupal and adult stages. Immunoprecipitation (IP) of CpBV15α co-precipitated eIF2 and eIF2B (a guanine nucleotide exchange factor of eIF2) in parasitized P. xylostella. Additionally, IP of eIF2 co-precipitated CpBV15α as well as eIF2B and eIF5 in parasitized larvae. IP with eIF5 antibody showed that relative amount of eIF2 bound to eIF5 was much decreased in parasitized larvae, while significant amount of eIF2 was bound to CpBV15α. These results suggest that CpBV15α inhibits some host mRNA translation by sequestering eIF2.

Vanishing white matter disease (VWM) was described by Van der Knaap in 1996. This association with premature ovarian failure is known as ovarioleukodystrophy. This is a rare entity caused by a mutation in one of the subunits of eukaryotic initiation factor 2B (EIF2B). The onset in adulthood or late in adolescence is very infrequent. A 41-years-old woman and her 37-years-old sister developed epilepsy in association with premature ovarian failure at the age of 13 and 18 respectively. The oldest-one started 17 years later progressive subcortical cognitive decline with predominant behavioural disorders and a progressive spastic paraparesis in association with symmetric cystic changes in the with matter of both hemispheres. In both patients we found the c.1117C>T (p.Arg373Cys) mutation in homozygosis in the EIF2B4 gen.

The eukaryotic translation initiation factor eIF2 delivers Met-tRNA i Met to the ribosomal small subunit in GTP-bound form associated with eIF1, eIF1A, eIF3 and eIF5, and dissociates together with eIF5 as eIF5-eIF2-GDP complex from the ribosomal small subunit after formation of start codon–anticodon base pairing between Met-tRNA i Met and mRNA. The inactive form eIF2-GDP is then exchanged for the active form eIF2-GTP by eIF2B for further initiation cycle. Previous studies showed that the C-terminal domains of eIF5 (eIF5-CTD) and eIF2Be (eIF2Be-CTD) have a common eIF2β-binding site for interacting with an N-terminal region of eIF2β (eIF2β-NTD). Here we have reconstructed the complexes of (eIF5-CTD)-(eIF2β-NTD) and (eIF2Be-CTD)-(eIF2β-NTD) i n vitro , and investigated binding mechanism by circular dichroism spectroscopy and small angle X-ray scattering in solution. The results showed the conformation of eIF2β-NTD was changed when bound to partner proteins, whereas the structures of eIF5-CTD and eIF2Be-CTD were similar in both isolated and complex states. We propose that eIF2β-NTD works as an intrinsically disordered domain which is disorder in the isolated state, but folds into a definite structure when bound to its partner proteins. Such flexibility of eIF2β-NTD is expected to be responsible for its binding capability.

Background: eIF2B is a critical translation factor and regulator of protein synthesis that is implicated in human disease. Results: Protein-protein interactions within the five-subunit eIF2B complex are identified. Conclusion: eIF2B complex formation requires extensive intersubunit domain interactions. Significance: The first experimental model is proposed for eIF2Bγ and -ϵ subunit interactions., In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α–ϵ) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ϵ that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bϵ for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bϵ and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγϵ subcomplex and propose a model for domain interactions between eIF2B subunits.

Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2α. eIF2α phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2α phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2Bδ variant 1 (V1). Replacement of total eIF2Bδ with V1 renders cells insensitive to eIF2α phosphorylation; specifically, they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2Bδ in ER stress response-deficient cells restores the stress response. Our data suggest that V1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2α phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases that result in ER stress.

Translation initiation is down-regulated in eukaryotes by phosphorylation of the alpha-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2alpha interacts with a subcomplex of eIF2B formed by the three regulatory subunits alpha/GCN3, beta/GCD7, and delta/GCD2, blocking the GDP-GTP exchange activity of the catalytic epsilon-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO(2) fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the alpha, beta, and delta subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the alpha-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the alpha-subunit of yeast eIF2 in vitro and to interact with eIF2Balpha/GCN3 in vivo in yeast. The aIF2B-eIF2alpha interaction was however independent of eIF2alpha phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2alpha, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2Bvarepsilon, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2alpha.

This paper presents the analysis of initiation control model of protein synthesis via eukaryotic initiation factor (eIF)-2 unit, introduced by [N.S. Bar, D.R. Morris, Dynamic model of the process of protein synthesis in eukaryoric cells, Bulletin of Mathematical Biology 69 (2007) 361–393, doi:10.1007/s11538-006-9128-2.] and propose methods to control it. Linearization of the model is presented as a measure to simplify the analysis and control application. The properties of the linear model were investigated and compared to the non-linear model using simulations. It was shown that the linear model is (marginally) stable and the states converge to a finite value. Linear optimal control theory can then be applied to the model under the value range where the linearized model is accurate. The effect of the input signals GCN2·tRNA and eIF-2 on the non-linear system was investigated. A few characteristics known from in vitro experiments of the initiation process were proven from a mathematical aspect and some conclusions about the function of the initiation complexes such as eIF2B and the ternary complex were derived. Consistent with published experiments, it was shown that overexpression of eIF-2 increases the concentration of 48S initiation complex and promote initiation rate. A state feedback control was applied in order to manipulate the initiation rate and it was proven that the 48S initiation complex can be driven to a desired value by calculating an input control law using measurement techniques available today. If this strategy can be implemented de facto , then a genuine control on protein synthesis process can be obtained.

In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).

Eukaryotic initiation factor 2 (eIF2) is a GDP-binding protein with three subunits: alpha, beta, and gamma. It delivers initiator tRNA (Met-tRNAi) to 40S ribosomes in a GTP-dependent manner. The factor regulates the translation of messenger RNAs through the phosphorylation of serine 51 residue in the small or alpha-subunit of eIF2 (eIF2alpha) and modulation of its interaction with a rate-limiting heteropentameric protein eIF2B. To understand the structural, functional, and regulatory roles of each of these subunits in the various activities of phosphorylated and unphosphorylated eIF2, such, as its ability to interact with GTP, Met-tRNAi, 40S ribosomes and with various proteins, we have for the first time over expressed all the three subunits of human eIF2 independently, and, also together in Sf9 cells using pFast Bac HT vector of baculovirus expression system. The expression of all subunits increased with increase in infection time up to 72 h. We have also over expressed three mutant forms of eIF2alpha viz, S51A, S51D, and S48A in which the serine at 51 or 48 position is replaced by an alanine or aspartic acid with 6x histidine tag at the N-terminus. Further, any of the two subunits or all the three subunits of eIF2 were coexpressed by multiple infection of cells with recombinant viruses. Purified alpha (wt and mutants) and beta subunits were found suitable to serve as substrates for different kinases. The recombinant subunits of eIF2alpha and beta-subunits were also phosphorylated in cultured insect cells. Phosphorylation of eIF2alpha in vitro was not significantly different in the presence and absence of the other subunits.

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.

Leukoencephalopathy with vanishing white matter (VWM) is an inherited childhood white matter disorder, caused by mutations in the genes encoding eukaryotic initiation factor 2B (eIF2B). The present study showed that, while the eIF2B activity was reduced in VWM lymphoblasts, the expression levels of the eIF2B subunits were similar to control lymphoblast lines. The mutations in eIF2B did not affect the interaction with eIF2. Strikingly, no apparent differences for the regulation of protein synthesis, measured by [ 35S]-methionine incorporation, were found between control and VWM lymphoblasts. Western blotting showed that, in some VWM cells, exposure to heat shock caused a decrease in the expression of specific eIF2B subunits. Most importantly, the increase in phosphorylation of eIF2α in response to heat shock was lower in VWM lymphoblasts than in control cells. These findings could form part of the explanation for the episodes of rapid and severe deterioration in VWM patients that are precipitated by febrile infections.

The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the alpha- and beta-subunits interact with each other. Consistent with this observation, the beta-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2alpha in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2alpha (S51D). These findings together with other observations raise the possibility that the beta-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2alpha kinase, is found to interact with wt and mutants of eIF2alpha in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2alpha. The dissociation constant of alpha-subunit-PERK complex (Kd alpha-subunit) is 0.74 microM and the interaction is stoichiometric.

Eukaryotic initiation factor eIF2 and its 'exchange factor' eIF2B play a key role in the regulation of protein synthesis in eukaryotes from yeast to mammals. Phosphorylation of eIF2 inhibits eIF2B and thus translation initiation. Four eIF2 kinases are now known in mammalian cells and these are activated in response to specific stress conditions. While phosphorylation of eIF2 serves to impair general protein synthesis, it causes upregulation of the translation of certain specific mRNAs that encode transcription factors. It can, therefore, exert effects on gene expression at multiple levels. The importance of correct control of eIF2 and eIF2B for normal physiology is exemplified by data from transgenic mice carrying knock-in or knock-out mutations and by the fact that mutations in the genes for the eIF2 kinase PERK or for eIF2B give rise to serious human diseases.

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of alpha- subunits. The alpha, beta, and delta subunits form a regulatory subcomplex, while the gamma and form a catalytic subcomplex. Archaea possess homologues of alpha, beta, and delta subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Balpha) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2A resolution. aIF2Balpha consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long alpha-helix (alpha5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the alpha/beta structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Balpha in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Balpha; the interaction is primarily mediated by the long alpha-helix at the N-domains. This structure suggests an architecture of the three subunits, alpha, beta, and delta, in the regulatory subcomplex within eIF2B.

Several neurodegenerative disorders are known to predominantly affect the white matter of the brain including vanishing white matter disease (VWMD), an autosomal recessive disorder characterized by leukodystrophy of varying severity in addition to variable systemic involvement. We report a consanguineous Arab family with three affected children, all of whom presented with severe neonatal epilepsy and profound neurodegenerative disease characterized by marked leukodystrophy with white matter cavitation mimicking VWMD. We combined autozygome and exome analysis to identify a novel variant in the gene encoding a member of the eIF2B-related family of proteins (MRI1). This is a poorly understood family of proteins of unclear function. Our results represent the first link between a variant in a member of this family and a human disease, and suggest that it converges with the highly homologous eIF2B, known to be mutated in VWMD, on the molecular pathogenesis of neurodegeneration.

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

There are conflicting reports over the question of whether the displacement by GTP of GDP bound to eIF2 catalyzed by eIF2B follows a substituted enzyme mechanism, as is believed to be the case for other guanine nucleotide exchange factors, or is a sequential mechanism. Analysis of data recently provided by Williams et al. (J. Biol. Chem. 276, 24697–24703, 2001) showing displacement by eIF2B of GDP bound to eIF2 in the absence of displacing nucleotide appears to offer a way of resolving the dispute and suggests that both mechanisms may be operative.

The effects of neurotrophic factors on translational activation were investigated in cortical neurons. Brain-derived neurotrophic factor (BDNF) increased protein synthesis within 30 min, whereas insulin produced a weaker enhancement of protein synthesis. BDNF-triggered protein synthesis was inhibited by LY294002, PD98059, and rapamycin, whereas the effect of insulin was unaffected by PD98059. To explore the mechanisms underlying this effect, the protein phosphorylation cascades that lead to the activation of translation initiation in neurons were examined. BDNF induced the phosphorylation of both eukaryote initiation factor (eIF) 4E and its binding protein (eIF4E-binding protein-1). The former reaction was inhibited by PD98059, whereas the latter was inhibited by LY294002 or rapamycin. In agreement, BDNF induced the phosphorylation of mammalian TOR (target of rapamycin) and enhanced its kinase activity toward eIF4E-binding protein-1. In contrast, insulin failed to activate MAPK and did not induce the phosphorylation of eIF4E. Since BDNF and insulin increased the activity of eIF2B and eIF2, the only difference between them was eIF4E phosphorylation. Thus, this may explain the lower activity of insulin in potentiating neuronal protein synthesis. These results suggest strongly that BDNF simultaneously activates multiple signaling cascades consisting of phosphatidylinositol 3-kinase, mammalian TOR, and MAPK to enhance translation initiation in neurons.

The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.

Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants. In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system. Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway. However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days. Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4. The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells. These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway.

eIF2 plays a central role in the maintenance of what is generally considered a rate-limiting step in mRNA translation. In this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S ribosomal subunit. At the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP and the eIF2.GDP complex is released from the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and is mediated by a second initiation factor, eIF2B. In what is probably the best-characterized mechanism for the regulation of mRNA translation, phosphorylation of eIF2 on its smallest, or alpha-, subunit converts eIF2 from a substrate of eIF2B into a competitive inhibitor. Thus, phosphorylation of eIF2 alpha effectively prevents formation of the eIF2.GTP.Met-tRNAi complex and inhibits global protein synthesis. Phosphorylation of eIF2 alpha occurs under a variety of conditions including viral infection, apoptosis, nutrient deprivation, heme-deprivation, and certain stresses.

The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i. e. eIF2, is phosphorylated on Ser51 of its alpha-subunit. Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.

Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the alpha-subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the alpha-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the delta- and epsilon-subunits of eIF2B, and eIF2B was shown to bind only to the beta-subunit of eIF2 by far-Western blot analysis. The binding site on eIF2beta for either the eIF2B holoprotein, or the isolated delta- or epsilon-subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the alpha-subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the beta-subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.

Protein synthesis is altered in both skeletal muscle and liver in response to nutritional status with food deprivation being associated with an inhibition of mRNA translation. In the present study, the effect of food-intake on the initiation of mRNA translation was examined in rats fasted for 18-h and then refed a complete diet. Fasting and refeeding caused alterations in translation initiation in both skeletal muscle and liver that were not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2 alpha. Instead, alterations in initiation were associated with changes in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G as well as the eIF4E binding protein, 4E-BP1. In muscle from fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was increased 5-fold compared to freely fed control animals. One hour after refeeding a complete diet, the amount of 4E-BP1 bound to eIF4E was reduced to freely fed control values. Reduced association of the two proteins was the result of increased phosphorylation of 4E-BP1. Refeeding a complete diet also stimulated the binding of eIF4E to eIF4G to form the active eIF4F complex. In liver, the amount of eIF4E associated with eIF4G, but not the amount of eIF4E associated with 4E-BP1, was altered by fasting and refeeding. Furthermore, in liver, but not in skeletal muscle, fasting and refeeding resulted in modulation of the phosphorylation state of eIF4E. Overall, the results suggest that protein synthesis may be differentially regulated in muscle and liver in response to fasting and refeeding. In muscle, protein synthesis is regulated through modulation of the binding of eIF4E to eIF4G and in liver through modulation of both phosphorylation of eIF4E as well as binding of eIF4E to eIF4G.

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.

Phosphorylation of serine 51 residue in the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) impairs the guanine nucleotide exchange (GNE) activity of eIF2B protein and thereby inhibits protein synthesis in mammalian systems, insects and yeast. It is not known if phosphorylation of plant eIF2 can inhibit an eIF2B-like activity. Interestingly purified wheat germ eIF2 (WG x eIF2) can exchange guanine nucleotides in vitro without the addition of any protein factor like eIF2B. It is not clear if this is due to a contaminant eIF2B-like activity associated with WG x eIF2 or because the affinity of WG x eIF2 for GDP and GTP is not markedly different. Our observations here indicate that the GNE activity of WG x eIF2 is not inhibited upon phosphorylation of the p41-42 doublet subunit in WG x eIF2 by reticulocyte eIF2alpha kinases, or in the presence of reticulocyte eIF2(alphaP) in which serine 51 residue is phosphorylated. Further, addition of WG x eIF2 reduces the inhibition in eIF2B activity, protein synthesis, and also the formation of 15S complex that occurs between reticulocyte eIF2(alphaP) and eIF2B protein in heme-deficient or poly(IC)-treated reticulocyte lysates, presumably by a mechanism of competition between wheat germ and reticulocyte eIF2 for phosphorylation. Unlike reticulocyte eIF2(alphaP), phosphorylated WG x eIF2 is unable to interact with reticulocyte eIF2B to form a 15S complex. The ability of WG x eIF2 to exchange guanine nucleotides independent of an eIF2B like protein and the inability of phosphorylated WG x eIF2 to interact with reticulocyte eIF2B suggests that WG x eIF2 is different from mammalian eIF2 and these differences may have occurred in evolution probably due to some changes in the amino acid sequences around the phosphorylation site in eIF2alpha.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the epsilon-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the alpha-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B alpha-subunit. The results demonstrate that eIF-2Balpha functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bepsilon is necessary and is perhaps sufficient for GEF activity in vitro.

The process of protein synthesis (mRNA translation) is essential for the growth and survival of all living organisms. Protein synthesis places heavy demands upon the cell—it requires a supply of amino acids as precursors and a great deal of energy. For these reasons, overall protein synthesis is under tight control. Furthermore, the translation of specific mRNAs is also subject to sophisticated control mechanisms, allowing the cell to modulate the production of certain proteins. Regulating translation rather than earlier stages in gene expression (transcription, splicing) confers important advantages—especially rapidity (by avoiding the requirement for de novo transcription and mRNA processing) and spatial control, e.g., in early development. Great strides have now been made in understanding how protein synthesis works and the functions and structures of the components involved. The chapters in this volume broadly concern two different areas of translational control. The first four primarily concern the regulation of the protein factors involved in the process of translation. This regulation generally involves alterations in their states of phosphorylation which influence their activity and interactions. The chapter by Proud describes the control of eukaryotic initiation factor 2. Its control has been intensively studied as it was a very early example of regulation of translation by protein phosphorylation. It is a target for four mammalian protein kinases which play important roles in regulating translation in response to diverse stress conditions. Recent data, using knock-in or knock-out mouse models, have clearly shown the importance of the kinases for normal physiology. It is also now clear that inherited mutations either in one of these kinases or in an ancillary factor for eIF2 (eIF2B) can lead to severe human diseases, as also discussed by Proud. Clemens discusses the mechanisms by which viruses seek to subvert the cellular protein synthesis machinery to favour expression of their own genetic information and the ways in which host cells strive to prevent this. A key player here is an eIF2 kinase termed PKR. Many of the mechanisms are also relevant in uninfected cells in response to stresses or early in apoptosis. As precursors for protein synthesis, amino acids exert important regulatory effects on the translational machinery. Some are exerted through eIF2, others through a key signaling pathway involving a protein termed the mammalian target of rapamycin, mTOR. Kimball focuses on recent studies that employ intact animals, rather than isolated cells, to explore the roles of amino acids in regulating translation factor activity. The mTOR pathway is currently the focus of intense attention. This reflects both its key roles in cellular and organismal physiology, and the links between this pathway and a range of human diseases including certain types of cancer and a condition termed tuberous sclerosis. Tee and Blenis discuss how studies on tuberous sclerosis have led to an improved understanding of mTOR signaling, and the roles of mTOR in health and disease. The second group of articles concern features of mRNA molecules themselves that are important for the control of their translation. In many cases, these features lie in the 5?- or 3?-untranslated regions (UTRs) of the message. Pickering and Willis review the roles of elements in the 5?-UTRs of certain mRNAs in mediating or controlling their translation. These include internal ribosome entry sites, which were initially found in viral mRNAs and which allow these mRNAs to be efficiently translated in the host cell. The articles by de Moor and colleagues and by Espel concern features found in the 3?-UTRs of specific mRNAs. The first chapter discusses 3?-UTR elements that are important in controlling the translation of specific mRNAs during early development and cell differentiation and during the cell cycle. The resulting translational control processes play critical roles in a diverse array of biological processes. A range of such regulatory elements have been identified, and these work by interacting with specific RNA-binding proteins. This is also true of the distinct 3?-UTR elements described by Espel: these AU-rich elements (AREs) play important roles in regulating the stability and translation of mRNAs encoding pro-inflammatory proteins and tumour-related proteins. As discussed by Espel, particular attention has focused on the role of AREs and ARE-binding proteins in inflammation, and on the signaling pathways involved. As the reader will appreciate from this set of articles, it is now abundantly clear that translational control is crucial for normal development and growth, and that dysregulation of these mechanisms leads to a range of serious human diseases.

Guanine nucleotide exchange factor (GEF) is a multisubunit protein involved in the initiation of translation. Although numerous models have been proposed for its mechanism of action, none have been definitive. An assay dependent on GEF activity was developed using highly purified eukaryotic initiation factor 2 (eIF-2) and GEF from Ehrlich cells. GEF was considered in terms of an enzyme whose catalytic function was the exchange of eIF-2-bound [alpha-32P]GDP for unlabeled nucleotide. The turnover number of GEF at 37 degrees C, calculated on the basis of enzyme kinetic methods is 0.027 s, which is consistent with in vivo rates of protein synthesis. Moreover, kinetic data support an enzyme-substituted mechanism as the mode of GEF function. This mechanism proposes the existence of a GEF.eIF-2.GDP complex and excludes the possibility of two guanine nucleotide binding sites on eIF-2. An analogous mechanism has been recently reported for elongation factor Ts, suggesting the importance of this mechanism to protein synthesis. The mechanism of inhibition of GEF function by eIF-2 alpha phosphorylation has also been investigated. It has been generally assumed that the mechanism by which eIF-2(P) traps GEF is an excessively stable complex, from which GEF is released very slowly. Data presented here, however, reveal that eIF-2(P).GDP is a competitive inhibitor of GEF (rather than an irreversible inhibitor) competing with eIF-2.GDP for binding to GEF. Even though the eIF-2(P).GDP.GEF complex dissociates too rapidly to measure, GEF is trapped because it has at least 150-fold greater affinity for eIF-2(P).GDP than for eIF-2.GDP. The implications of competitive inhibition with respect to the mechanism of reversal of inhibition by an eIF-2(P) phosphatase are discussed.