eIF2B Complex Research Articles

Leukoencephalopathy with vanishing white matter disease: a case report study.

The authors report two cases of VWM disease. In the first case, an 8-month-old female child, brought to the pediatric clinic with seizure and loss of consciousness. The second case was a 24-month-old girl, presented with weakness, a disability to walk and swallow, and poor feeding. Her brain MRI demonstrated cystic changes (white matter rarefaction) in supratentorial peri-ventricular white matter and genetic testing result showed an EIF2B3 gene mutation. Leukoencephalopathy with VWM, also known as Cree encephalopathy is caused by mutations in the EIF2B gene. The disease is inherited in an autosomal recessive fashion. There are various agents leading to symptoms and signs of VWM disease. Physical stress like head trauma even in a mild degree, infections, and febrile diseases can be mentioned as causes of VWM. The eIF2B complex, plays a role as an important factor in the regulation of protein synthesis in cells under different conditions. As a conclusion, genetic counseling could be recommended to all individuals with VWM disease and their family members for next pregnancies and possible precautions for consanguineous marriages.

Adult-onset leukodystrophy with vanishing white matter: a case series of 19 patients.

Leukodystrophy with vanishing white matter (LVWM) is an autosomal recessive disease with typical pediatric-onset caused by mutations in one of the five EIF2B genes. Adult-onset (AO) cases are rare. In this observational study, we reviewed clinical and laboratory information of the patients with AO-LVWM assessed at two referral centers in Italy and Portugal from Jan-2007 to Dec-2019. We identified 18 patients (13 females) with AO-LVWM caused by EIF2B5 or EIF2B3 mutations. Age of neurological onset ranged from 16 to 60years, with follow-ups occurring from 2 to 37years. Crucial symptoms were cognitive and motor decline. In three patients, stroke-like events were the first manifestation; in another, bladder dysfunction remained the main complaint across decades. Brain MRI showed white matter (WM) rarefaction in all cases, except two. Diffusion-weighted imaging documented focal hyperintensity in the acute stage of stroke-like events. 1H-spectroscopy primarily showed N-acetyl-aspartate reduction; 18fluorodeoxyglucose-PET revealed predominant frontoparietal hypometabolism; evoked potential studies demonstrated normal-to-reduced amplitudes; neuro-ophthalmological assessment showed neuroretinal thinning, and b-wave reduction on full-field electroretinogram. Interestingly, we found an additional patient with LVWM-compatible phenotype and monoallelic variants in two distinct eIF2B genes, EIF2B1 and EIF2B2. AO-LVWM presents varying clinical manifestations at onset, including stroke-like events. WM rarefaction is the most consistent diagnostic clue even in the latest onset cases. Spectroscopy and electrophysiological features are compatible with axon, rather than myelin, damage. Cerebral glucose metabolic abnormalities and retinal alterations can be present. LVWM might also be caused by a digenic inheritance affecting the eIF2B complex.

Detection of anti-eIF2B autoantibodies in systemic sclerosis by immunoprecipitation-mass spectrometry.

Dear Editor, Anti-eukaryotic initiation factor 2B (eIF2B) autoantibodies are a recently described autoantibody specificity in SSc associated with diffuse cutaneous involvement, interstitial lung disease (ILD) and a cytoplasmic pattern on HEp-2 indirect immunofluorescence (HEp-2 IIF) [1–3]. Hitherto only 19 patients have been identified, all by radiolabelled protein immunoprecipitation [1–3]. Here we describe immunoprecipitation-mass spectrometry (IP-MS) as a detection method for anti-eIF2B autoantibodies and further characterize the precipitation pattern of the eIF2B complex as precipitated by various anti-eIF2B sera. We also further document the clinical associations of anti-eIF2B autoantibodies. The study was approved by the following Ethical Committees: University Hospitals Leuven (S60347/B32220071204) (Leuven) and Hospices civils de Lyon, Lyon (CT 69HCL21_0501) (Lyon). This was a retrospective study using left-over material of samples submitted to the clinical laboratory (secondary use) for which an informed consent waiver was authorized by the Ethical Committee of the University Hospitals Leuven, Belgium, and Hospices civils de Lyon, France. Patients were notified of the content and aim of the study. The patient data have been anonymized.

EP198: EIF3F compound heterozygous genotype-phenotype association

Phenotypes for newly described gene-disease associations often undergo rapid evolution as additional cases are described. The expansion of exome and genome sequencing has contributed to this phenomenon by allowing ascertainment of diseases based on genotype rather than phenotype. We describe an example of phenotype expansion for the EIF3F gene, which encodes an essential subunit of the mammalian eukaryotic initiation factor (eIF2B) complex. EIF2F is important for translation regulation and is involved in cell proliferation and growth, cell cycle control, differentiation, and apoptosis.

A C‐term truncated EIF2Bγ protein encoded by an intronically polyadenylated isoform introduces unfavorable EIF2Bγ–EIF2γ interactions

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

Vanishing white matter: Eukaryotic initiation factor 2B model and the impact of missense mutations.

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

De Novo Mutations in EIF2B1 Affecting eIF2 Signaling Cause Neonatal/Early-Onset Diabetes and Transient Hepatic Dysfunction.

Permanent neonatal diabetes mellitus (PNDM) is caused by reduced β-cell number or impaired β-cell function. Understanding of the genetic basis of this disorder highlights fundamental β-cell mechanisms. We performed trio genome sequencing for 44 patients with PNDM and their unaffected parents to identify causative de novo variants. Replication studies were performed in 188 patients diagnosed with diabetes before 2 years of age without a genetic diagnosis. EIF2B1 (encoding the eIF2B complex α subunit) was the only gene with novel de novo variants (all missense) in at least three patients. Replication studies identified two further patients with de novo EIF2B1 variants. In addition to having diabetes, four of five patients had hepatitis-like episodes in childhood. The EIF2B1 de novo mutations were found to map to the same protein surface. We propose that these variants render the eIF2B complex insensitive to eIF2 phosphorylation, which occurs under stress conditions and triggers expression of stress response genes. Failure of eIF2B to sense eIF2 phosphorylation likely leads to unregulated unfolded protein response and cell death. Our results establish de novo EIF2B1 mutations as a novel cause of permanent diabetes and liver dysfunction. These findings confirm the importance of cell stress regulation for β-cells and highlight EIF2B1's fundamental role within this pathway.

Integrated stress response on the brain

Structural Biology During translation, regulation of protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is a common consequence of diverse stress stimuli, which leads to reprogramming of gene expression. This process, known as the integrated stress response, is one of the most fundamental mechanisms of translational control conserved throughout eukaryotes. It is also a promising therapeutic target in neurodegenerative diseases and traumatic brain injury. Kashiwagi et al. report the cryo–electron microscopy and crystal structures and Kenner et al. report the cryo–electron microscopy structure of the guanine nucleotide exchange factor eIF2B in complex with eIF2 or phosphorylated eIF2. The structures of the eIF2•eIF2B complex reveal that the single phosphorylation modification on eIF2 changes how eIF2 binds to eIF2B and locks this enzyme into an inhibited complex. Science , this issue p. [495][1], p. [491][2] [1]: /lookup/doi/10.1126/science.aaw4104 [2]: /lookup/doi/10.1126/science.aaw2922

EIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response.

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

EIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Analysis of eIF2B bodies and their relationships with stress granules and P-bodies

Eukaryotic cells respond to stress and changes in the environment in part by repressing translation and forming cytoplasmic assemblies called stress granules and P-bodies, which harbor non-translating mRNAs and proteins. A third, but poorly understood, assembly called the eIF2B body can form and contains the eIF2B complex, an essential guanine exchange factor for the translation initiation factor eIF2. Hypomorphic EIF2B alleles can lead to Vanishing White Matter Disease (VWMD), a leukodystrophy that causes progressive white matter loss. An unexplored question is how eIF2B body formation is controlled and whether VWMD alleles in EIF2B alter the formation of eIF2B bodies, stress granules, or P-bodies. To examine these issues, we assessed eIF2B body, stress granule, and P-body induction in wild-type yeast cells and cells carrying VWMD alleles in the EIF2B2 (GCD7) and EIF2B5 (GCD6) subunits of eIF2B. We demonstrate eIF2B bodies are rapidly and reversibly formed independently of stress granules during acute glucose deprivation. VWMD mutations had diverse effects on stress-induced assemblies with some alleles altering eIF2B bodies, and others leading to increased P-body formation. Moreover, some VWMD-causing mutations in GCD7 caused hyper-sensitivity to chronic GCN2 activation, consistent with VWMD mutations causing hyper-sensitivity to eIF2α phosphorylation and thereby impacting VWMD pathogenesis.

Evolutionary engineering improves tolerance for medium-chain alcohols in Saccharomyces cerevisiae

BackgroundYeast-based chemical production is an environmentally friendly alternative to petroleum-based production or processes that involve harsh chemicals. However, many potential alcohol biofuels, such as n-butanol, isobutanol and n-hexanol, are toxic to production organisms, lowering the efficiency and cost-effectiveness of these processes. We set out to improve the tolerance of Saccharomyces cerevisiae toward these alcohols.ResultsWe evolved the laboratory strain of S. cerevisiae BY4741 to be more tolerant toward n-hexanol and show that the mutations which confer tolerance occur in proteins of the translation initiation complex. We found that n-hexanol inhibits initiation of translation and evolved mutations in the α subunit of eIF2 and the γ subunit of its guanine exchange factor eIF2B rescue this inhibition. We further demonstrate that translation initiation is affected by other alcohols such as n-pentanol and n-heptanol, and that mutations in the eIF2 and eIF2B complexes greatly improve tolerance to these medium-chain alcohols.ConclusionsWe successfully generated S. cerevisiae strains that have improved tolerance toward medium-chain alcohols and have demonstrated that the causative mutations overcome inhibition of translation initiation by these alcohols.

Abstract 733: Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5

Abstract Solid tumors exhibit a range of oxygen concentrations, in part due to abnormal vasculature and limited diffusion of oxygen which lead to hypoxia. Tumor hypoxia is associated with resistance to treatment, metastasis and poor patient outcome for many types of cancer. Assessing the impact of hypoxia in the tumor microenvironment and understanding how it contributes to tumorigenesis and survival is a challenging research goal for several reasons; a chief obstacle has been developing methods to analyze the transcriptome within precise regions of the tumor microenvironment in vivo. To address this task, we used Laser-Capture-Microdissection (LCM) of tumors labeled with the hypoxia probe, EF5, to specifically isolate normoxic and hypoxic regions of tumors from a murine model of head and neck cancer. We then carried out deep RNA-sequencing of the in vivo samples and corresponding in vitro samples grown in 0.5% oxygen conditions. Here we present the first described study of the hypoxic transcriptome in tumors at base-pair resolution. Our goal is to use these data to evaluate the potential of previously unstudied hypoxia-mediated pathways, such as the regulation of mRNA splicing, to be targeted therapeutically. We identified global changes in several classes of splicing in hypoxic compared to normoxic head and neck cancer cells, including changes in expression of last exons, retained introns and 3’ untranslated regions. Many of the alternatively spliced isoforms are in pathways central to hypoxic adaptation, such as glycolysis, cell cycle and translation. Among these genes, we uncovered a previously undescribed splicing event in the master regulator of translation, EIF2B5. The full-length protein encoded by EIF2B5 (eIF2Bå), is a necessary catalytic component of the eIF2B complex, which binds eIF2á and exchanges GDP for GTP to initiate translation. Strikingly, hypoxia induces retention of intron 12 in EIF2B5, which leads to insertion of an early stop codon in frame with the coding sequence; the resulting 65kDa protein isoform lacks a functional guanine nucleotide exchange (GEF) domain compared to the full-length isoform, but retains the region known to bind eIF2B complex subunits. Thus, we predict that the 65kDa isoform of eIF2Bå, may act in opposition to full-length eIF2Bå and inhibit translation initiation in conditions of oxygen deprivation. We are investigating EIF2B5, and other select hypoxia-regulated isoforms, to evaluate how alternative splicing impacts cell growth and survival in hypoxic tumors. Citation Format: Lauren K. Brady, Rohil Shekher, Vladimir Popov, Mircea Ivan, Milan Radovich, Constantinos Koumenis. Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 733.

Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.

Stoichiometry of the eIF2B complex is maintained by mutual stabilization of subunits.

The eukaryotic translation initiation factor eIF2B is a multi-subunit complex with a crucial role in the regulation of global protein synthesis in the cell. The complex comprises five subunits, termed α through ε in order of increasing size, arranged as a heterodecamer with two copies of each subunit. Regulation of the co-stoichiometric expression of the eIF2B subunits is crucial for the proper function and regulation of the eIF2B complex in cells. We have investigated the control of stoichiometric eIF2B complexes through mutual stabilization of eIF2B subunits. Our data show that the stable expression of the catalytic eIF2Bε subunit in human cells requires co-expression of eIF2Bγ. Similarly, stable expression of eIF2Bδ requires both eIF2Bβ and eIF2Bγ+ε. The expression of these subunits decreases despite there being no change in either the levels or the translation of their mRNAs. Instead, these subunits are targeted for degradation by the ubiquitin-proteasome system. The data allow us to propose a model for the formation of stoichiometric eIF2B complexes which can ensure their stoichiometric incorporation into the holocomplex.

Crystal structure of eukaryotic translation initiation factor 2B.

Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, β- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, β-, γ-, δ- and ε-subunits; its α-, β- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2β2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved 'NF motif', which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the 'nonproductive' eIF2-eIF2B complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.

EIF2B: recent structural and functional insights into a key regulator of translation.

The eukaryotic translation initiation factor (eIF) eIF2B is a key regulator of mRNA translation, being the guanine nt exchange factor (GEF) responsible for the recycling of the heterotrimeric G-protein, eIF2, which is required to allow translation initiation to occur. Unusually for a GEF, eIF2B is a multi-subunit protein, comprising five different subunits termed α through ε in order of increasing size. eIF2B is subject to tight regulation in the cell and may also serve additional functions. Here we review recent insights into the subunit organization of the mammalian eIF2B complex, gained both from structural studies of the complex and from studies of mutations of eIF2B that result in the neurological disorder leukoencephalopathy with vanishing white matter (VWM). We will also discuss recent data from yeast demonstrating a novel function of the eIF2B complex key for translational regulation.

No evidence for a role of Ile587Val polymorphism of EIF2B5 gene in multiple sclerosis in Kashmir Valley of India

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.

Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2.

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α–ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2BRSC). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)2 complex. Combined with mutational and biochemical data, we show that eIF2BRSC exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα2 homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2BRSC and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn−, Gcd− and VWM/CACH mutations and the evolutionary history of the eIF2B complex.

Biochemical effects of mutations in the gene encoding the alpha subunit of eukaryotic initiation factor (eIF) 2B associated with Vanishing White Matter disease.

BackgroundLeukoencephalopathy with Vanishing White Matter (VWM) is an autosomal recessive disorder caused by germline mutations in the genes EIF2B1-5, which encode the 5 subunits of the eukaryotic translation initiation factor eIF2B. To date, analysis of the biochemical effects of mutations in the EIF2B2-5 genes has been carried out, but no study has been performed on mutations in the EIF2B1 gene. This gene encodes eIF2Bα, the smallest subunit in eIF2B which has an important role in both the structure and regulation of the eIF2B complex.MethodseIF2B subunits were overexpressed in HEK293 cells and isolated from the resulting cell lysates by affinity chromatography. Formation of the eIF2B complex and binding of its substrate, eIF2, was assessed by western blot. Assays of the guanine nucleotide exchange (GEF) activity were also carried out.ResultsOf the 5 eIF2Bα mutations studied, we found 3 that showed loss or reduction of binding of eIF2Bα to the rest of the complex, one with increased GEF activity, and one where no effects on activity or complex formation were observed.ConclusionsThis is the first study on eIF2Bα VWM mutations. We show that some mutations cause expected decreases in GEF activity or complex formation, similar to a majority of observed VWM mutations. However, we also observe some unexpected changes which hint at other effects of these mutations on as yet undescribed functions of eIF2B.