ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

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.

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring.

A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.

The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen-deuterium exchange-mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this 'switch-helix' controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.

eIF2Bδ blocks the integrated stress response and maintains eIF2B activity and cancer metastasis by overexpression in breast cancer stem cells.

Breast cancer (BC) metastasis involves cancer stem cells (CSCs) and their regulation by micro-RNAs (miRs), but miR targeting of the translation machinery in CSCs is poorly explored. We therefore screened miR expression levels in a range of BC cell lines, comparing non-CSCs to CSCs, and focused on miRs that target translation and protein synthesis factors. We describe a unique translation regulatory axis enacted by reduced expression of miR-183 in breast CSCs, which we show targets the eIF2Bδ subunit of guanine nucleotide exchange factor eIF2B, a regulator of protein synthesis and the integrated stress response (ISR) pathway. We report that reduced expression of miR-183 greatly increases eIF2Bδ protein levels, preventing strong induction of the ISR and eIF2α phosphorylation, by preferential interaction with P-eIF2α. eIF2Bδ overexpression is essential for BC cell invasion, metastasis, maintenance of metastases, and breast CSC expansion in animal models. Increased expression of eIF2Bδ, a site of action of the drug ISRIB that also prevents ISR signaling, is essential for breast CSC maintenance and metastatic capacity.

Evaluation of safety and early efficacy of AAV gene therapy in mouse models of vanishing white matter disease.

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.

Proteomic dissection of vanishing white matter pathogenesis.

Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5ho mouse model. We first mapped the temporal proteome in the cerebellum, corpus callosum, cortex, and brainstem of 2b5ho and wild-type (WT) mice. Protein changes observed in 2b5ho mice were then cross-referenced with published proteomic datasets from VWM patient brain tissue to define alterations relevant to the human disease. By comparing 2b5ho mice with their region- and age-matched WT counterparts, we showed that the proteome in the cerebellum and cortex of 2b5ho mice was already dysregulated prior to pathology development, whereas proteome changes in the corpus callosum only occurred after pathology onset. Remarkably, protein changes in the brainstem were transient, indicating that a compensatory mechanism might occur in this region. Importantly, 2b5ho mouse brain proteome changes reflect features well-known in VWM. Comparison of the 2b5ho mouse and VWM patient brain proteomes revealed shared changes. These could represent changes that contribute to the disease or even drive its progression in patients. Taken together, we show that the 2b5ho mouse brain proteome is affected in a region- and time-dependent manner. We found that the 2b5ho mouse model partly replicates the human disease at the protein level, providing a resource to study aspects of VWM pathogenesis by highlighting alterations from early to late disease stages, and those that possibly drive disease progression.

Protection of eIF2B from inhibitory phosphorylated eIF2: A viral strategy to maintain mRNA translation during the PKR-triggered integrated stress response.

The integrated stress response (ISR) protects cells from a variety of insults. Once elicited (e.g., by virus infections), it eventually leads to the block of mRNA translation. Central to the ISR are the interactions between translation initiation factors eIF2 and eIF2B. Under normal conditions, eIF2 drives the initiation of protein synthesis through hydrolysis of GTP, which becomes replenished by the guanine nucleotide exchange factor eIF2B. The antiviral branch of the ISR is activated by the RNA-activated kinase PKR which phosphorylates eIF2, thereby converting it into an eIF2B inhibitor. Here, we describe the recently solved structures of eIF2B in complex with eIF2 and a novel escape strategy used by viruses. While unphosphorylated eIF2 interacts with eIF2B in its "productive" conformation, phosphorylated eIF2 [eIF2(αP)] engages a different binding cavity on eIF2B and forces it into the "nonproductive" conformation that prohibits guanine nucleotide exchange factor activity. It is well established that viruses express so-called PKR antagonists that interfere with double-strand RNA, PKR itself, or eIF2. However recently, three taxonomically unrelated viruses were reported to encode antagonists targeting eIF2B instead. For one antagonist, the S segment nonstructural protein of Sandfly fever Sicilian virus, atomic structures showed that it occupies the eIF2(αP)-binding cavity on eIF2B without imposing a switch to the nonproductive conformation. S segment nonstructural protein thus antagonizes the activity of PKR by protecting eIF2B from inhibition by eIF2(αP). As the ISR and specifically eIF2B are central to neuroprotection and a wide range of genetic and age-related diseases, these developments may open new possibilities for treatments.

Adult-onset leukoencephalopathy with vanishing white matter with compound heterozygous EIF2B3 gene variants.

BACKGROUND: Leukoencephalopathy with vanishing white matter (VWM) is an autosomal recessive disorder affecting the white matter of the brain. It typically manifests during childhood, with clinical features including sudden and severe neurological deterioration triggered by stressors such as febrile illness, minor head trauma, or stressful events. Adult-onset cases of VWM are exceptionally uncommon. CASE PRESENTATION: In this case, we present an adult patient who exhibited late-onset progressive VWM characterized by ataxia, postural instability, cognitive impairment, and emotional disturbances. Comprehensive screening for endocrine, metabolic, tumor, and immunologic disorders yielded normal or negative results. Brain imaging revealed diffuse and confluent hyperintensity in the white matter on T2-weighted images, along with periventricular cavitations. Genetic testing confirmed the diagnosis of VWM, identifying two heterozygous variants in the eukaryotic translation initiation factor 2B subunit γ (EIF2B3) gene: a pathogenic variant, c.1037 T > C (p.I346T), and a variant of undetermined significance, c.22A > T (p.M8L). Upon a 2-year follow-up, the patient's symptoms deteriorated rapidly following a COVID-19 infection. CONCLUSIONS: In conclusion, we have presented a case of classical adult-onset VWM. Since there are no cures or definitive treatments for the disease, it's extremely important to focus on early diagnosis and the prevention of stressors to avoid acute deterioration.

Vanishing white matter disease: imaging, clinical and molecular correlation in Brazilian families.

PURPOSE: To characterize Vanishing White Matter Disease (VWM) cases from a Brazilian University Tertiary hospital, focusing on brain magnetic resonance image (MRI) aspects, clinical and molecular data. METHODS: Medical records and brain MRI of 13 genetically confirmed VWM patients were reviewed. Epidemiological data such as age at symptom onset, gender and main symptoms were analyzed, along with genetic mutations and MRI characteristics, such as the distribution of white matter lesions and atrophy. RESULTS: The majority of patients were female, with the age of symptom onset ranging from 1 year and 6 months to 40 years. All mutations were identified in the EIF2B5 gene, the most prevalent being c.338G > A (p.Arg113His), and a novel mutation related to the disease was discovered, c.1051G > A (p.Gly351Ser). Trauma or infection were significant triggers. The most frequent symptoms were ataxia and limb spasticity. All MRI scans displayed deep white matter involvement, cystic degeneration, with U-fibers relatively spared and a predilection for the frontoparietal region. Lesions in the corpus callosum and posterior fossa were present in all patients. Follow-up exams revealed the evolution of white matter lesions and cerebral atrophy, which correlated with clinical deterioration. CONCLUSIONS: VWM affects various age groups, with a significant clinical and genetic variability. A novel mutation associated with the disease is highlighted. MRI reveals a typical pattern of white matter involvement, characterized by diffuse lesions in the periventricular and deep regions, with subsequent extension to the subcortical areas, accompanied by cystic degeneration, and plays a crucial role in diagnosis and follow-up.

[Vanishing white matter disease, a rare leukodystrophy with mutation in the EIF2B5 gene]. / Eltuno fehérállomány-betegség, a leukodystrophiák ritka altípusa az EIF2B5 gén mutációjával.

Background - Leukodystrophies, a hete­ro­­ge­neous group of brain and spinal cord dis­orders, often pose challenges in es­tab­li­shing molecular etiology. Vanishing White Matter Disease (VWMD) is a rare sub­type of leu­ko­dys­trophies presenting with characteristic clinical and MRI features, ne­ver­theless, achieving diag­nostic certainty requires genetic studies.

Case presentation - Our patient is a nine year old girl, who developed progressive gait difficulties at around 3-4 years of age. Her brain MRI showed confluent lesions with in­­creased signal intensity in the cerebral and cerebellar white matter on T2/FLAIR se­quen­ces, within which hypointense regions ap­peared with signal intensity resembling that of the cerebrospinal fluid on T1 sequences. Whole exome sequencing identified a homozygous likely pathogenic variant within the EIF2B5 gene in the proband, which was present in a heterozygous state in both asymptomatic parents. Having the clinical and molecular genetic diagnosis established, we explored therapeutic possibilities for the patient.

Conclusion - VWMD is a severe form of leukodystrophies with little or no disease modifying therapy available until recently. A better understanding of its molecular pathogenesis offers some hope for new inventive therapies. 

Epithelial Endoplasmic Reticulum Stress Enhances the Risk of Muc5b-associated Lung Fibrosis.

The gain-of-function minor allele of the MUC5B (mucin 5B, oligomeric mucus/gel-forming) promoter (rs35705950) is the strongest risk factor for idiopathic pulmonary fibrosis (IPF), a devastating fibrotic lung disease that leads to progressive respiratory failure in adults. We have previously demonstrated that Muc5b overexpression in mice worsens lung fibrosis after bleomycin exposure and have hypothesized that excess Muc5b promotes endoplasmic reticulum (ER) stress and apoptosis, stimulating fibrotic lung injury. Here, we report that ER stress pathway members ATF4 (activating transcription factor 4) and ATF6 coexpress with MUC5B in epithelia of the distal IPF airway and honeycomb cyst and that this is more pronounced in carriers of the gain-of-function MUC5B promoter variant. Similarly, in mice exposed to bleomycin, Muc5b expression is temporally associated with markers of ER stress. Using bulk and single-cell RNA sequencing in bleomycin-exposed mice, we found that pathologic ER stress-associated transcripts Atf4 and Ddit3 (DNA damage inducible transcript 3) were elevated in alveolar epithelia of SFTPC-Muc5b transgenic (SFTPC-Muc5bTg) mice relative to wild-type (WT) mice. Activation of the ER stress response inhibits protein translation for most genes by phosphorylation of Eif2α (eukaryotic translation initiation factor 2 alpha), which prevents guanine exchange by Eif2B and facilitates translation of Atf4. The integrated stress response inhibitor (ISRIB) facilitates interaction of phosphorylated Eif2α with Eif2B, overcoming translation inhibition associated with ER stress and reducing Atf4. We found that a single dose of ISRIB diminished Atf4 translation in SFTPC-Muc5bTg mice after bleomycin injury. Moreover, ISRIB resolved the exaggerated fibrotic response of SFTPC-Muc5bTg mice to bleomycin. In summary, we demonstrate that MUC5B and Muc5b expression is associated with pathologic ER stress and that restoration of normal translation with a single dose of ISRIB promotes lung repair in bleomycin-injured Muc5b-overexpressing mice.

Stepwise assembly of the eukaryotic translation initiation factor 2 complex.

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2ß subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2ß with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease.

Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish.

Leukodystrophy with vanishing white matter (VWM), also called Childhood Ataxia with Central Nervous System Hypomyelination, is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5. However, little is known regarding the underlying pathogenetic mechanisms, and there is no curative treatment for VWM. In this study, we established the first EIF2B3 animal model for VWM disease in vertebrates by CRISPR mutagenesis of the highly conserved zebrafish ortholog eif2b3. Using CRISPR, we generated two mutant alleles in zebrafish eif2b3, 10- and 16-bp deletions, respectively. The eif2b3 mutants showed defects in myelin development and glial cell differentiation, and increased expression of genes in the induced stress response pathway. Interestingly, we also found ectopic angiogenesis and increased VEGF expression. Ectopic angiogenesis in the eif2b3 mutants was reduced by the administration of VEGF receptor inhibitor SU5416. Using the eif2b3 mutant zebrafish model together with in silico protein modeling analysis, we demonstrated the pathogenicity of 18 reported mutations in EIF2B3, as well as of a novel variant identified in a 19-month-old female patient: c.503 T > C (p.Leu168Pro). In summary, our zebrafish mutant model of eif2b3 provides novel insights into VWM pathogenesis and offers rapid functional analysis of human EIF2B3 gene variants.

The role of eIF2 phosphorylation in cell and organismal physiology: new roles for well-known actors.

Control of protein synthesis (mRNA translation) plays key roles in shaping the proteome and in many physiological, including homeostatic, responses. One long-known translational control mechanism involves phosphorylation of initiation factor, eIF2, which is catalysed by any one of four protein kinases, which are generally activated in response to stresses. They form a key arm of the integrated stress response (ISR). Phosphorylated eIF2 inhibits eIF2B (the protein that promotes exchange of eIF2-bound GDP for GTP) and thus impairs general protein synthesis. However, this mechanism actually promotes translation of certain mRNAs by virtue of specific features they possess. Recent work has uncovered many previously unknown features of this regulatory system. Several studies have yielded crucial insights into the structure and control of eIF2, including that eIF2B is regulated by several metabolites. Recent studies also reveal that control of eIF2 and the ISR helps determine organismal lifespan and surprising roles in sensing mitochondrial stresses and in controlling the mammalian target of rapamycin (mTOR). The latter effect involves an unexpected role for one of the eIF2 kinases, HRI. Phosphoproteomic analysis identified new substrates for another eIF2 kinase, Gcn2, which senses the availability of amino acids. Several genetic disorders arise from mutations in genes for eIF2α kinases or eIF2B (i.e. vanishing white matter disease, VWM and microcephaly, epileptic seizures, microcephaly, hypogenitalism, diabetes and obesity, MEHMO). Furthermore, the eIF2-mediated ISR plays roles in cognitive decline associated with Alzheimer's disease. New findings suggest potential therapeutic value in interfering with the ISR in certain settings, including VWM, for example by using compounds that promote eIF2B activity.

Phosphorylation of a reinitiation supporting protein, RISP, determines its function in translation reinitiation.

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2ß and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2ß, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.

CircAHNAK upregulates EIF2B5 expression to inhibit the progression of ovarian cancer by modulating the JAK2/STAT3 signaling pathway.

Recent studies highlighted non-coding RNAs as potential therapeutic targets in ovarian cancer. We aimed to investigate the roles of circAHNAK in ovarian cancer pathogenesis. Here, RNA immunoprecipitation, dual-luciferase reporter assay and RNA fluorescence in situ hybridization were adopted to determine circAHNAK, miR-28 or EIF2B5 interaction. CCK-8 assay was used to detect cell proliferation. Wound healing and Transwell assays were employed to assess cell migration and invasion, respectively. Flow cytometry was performed to measure cell apoptosis. The roles of circAHNAK on tumor growth in vivo were evaluated using subcutaneous xenograft model. The expression levels of circAHNAK, miR-28, EIF2B5, markers of EMT and JAK2/STAT3 pathway were measured by qRT-PCR, western blotting or immunohistochemistry staining. We reported that circAHNAK was decreased in ovarian cancer tissues. Forced expression of circAHNAK promoted apoptosis and inhibited cell proliferation, migration, invasion, EMT and JAK2/STAT3 signaling pathway. Mechanistically, circAHNAK acted as a miR-28 sponge. CircAHNAK deficiency resulted in the amassing of miR-28, which was elevated in ovarian cancer and promoted cancer cell malignancy. MiR-28 in turn inhibited EIF2B5 expression. Silence of EIF2B5 abolished the anticancer effects of miR-28 inhibitor. CircAHNAK overexpression retarded tumor growth in vivo, along with the decreased miR-28 and increased EIF2B, as well as EMT inhibition. In conclusion, circAHNAK targets miR-28 to upregulate EIF2B5 expression, thus inhibits progression of ovarian cancer by suppressing JAK2/STAT3 signaling pathway.

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

Eukaryotic translation initiates upon recruitment of the EIF2-GTP·Met-tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, ß, 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.

LncRNA KCNQ1OT1 promotes the metastasis of ovarian cancer by increasing the methylation of EIF2B5 promoter.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as regulators of human malignancies, including ovarian cancer (OC). LncRNA KCNQ1OT1 could promote OC progression, and EIF2B5 was associated with development of several tumors. This project was aimed to explore the role of lncRNA KCNQ1OT1 in OC development, as well as the involving action mechanism. METHODS: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) or Western blotting was employed to determine the expression levels of KCNQ1OT1 and EIF2B5. OC cell proliferation was evaluated by MTT and colony formation assays, and wound healing and Transwell assays were implemented to monitor cell migration and invasion, respectively. The methylation status of EIF2B5 promoter was examined by MS-PCR, to clarify whether the expression of EIF2B5 was decreased. The binding activity of KCNQ1OT1 to methyltransferases DNMT1, DNMT3A and DNMT3B was determined by dual luciferase reporter assay or RIP assay, to explore the potential of KCNQ1OT1 alters the expression of its downstream gene. ChIP assay was carried out to verify the combination between EIF2B5 promoter and above three methyltransferases. RESULTS: Expression of lncRNA KCNQ1OT1 was increased in OC tissues and cells. EIF2B5 expression was downregulated in OC, which was inversely correlated with KCNQ1OT1. Knockdown of KCNQ1OT1 inhibited OC cell proliferation and metastasis. KCNQ1OT1 could downregulate EIF2B5 expression by recruiting DNA methyltransferases into EIF2B5 promoter. Furthermore, interference of EIF2B5 expression rescued KCNQ1OT1 depletion-induced inhibitory impact on OC cell proliferation and metastasis. CONCLUSION: Our findings evidenced that lncRNA KCNQ1OT1 aggravated ovarian cancer metastasis by decreasing EIF2B5 expression level, and provided a novel therapeutic strategy for OC.

Secretomics Alterations and Astrocyte Dysfunction in Human iPSC of Leukoencephalopathy with Vanishing White Matter.

Leukoencephalopathy with vanishing white matter (VWM) is an inherited leukoencephalopathy characterized by progressive rarefaction of cerebral white matter. Dysfunction of patient astrocyte plays a central role in the pathogenesis, while the immaturity of oligodendrocyte is probably secondary. How eIF2B mutant astrocytes affect the maturation and myelination of oligodendrocyte precursor cells (OPCs) is unclear yet. We used induced pluripotent stem cells (iPSCs) derived from our patient with EIF2B5 mutations to differentiate into astrocytes (AS) and OPCs, and aimed to verify that patient astrocytes inhibited the differentiation of OPCs by abnormalities of secreted proteins. eIF2B mutant astrocytes and astrocyte-conditioned medium (ACM) both inhibited the maturation of OPCs. It was revealed that 13 promising proteins exhibited a similar up- or downregulation by the PRM method correlated well with TMT results. eIF2B mutant astrocytes may secrete abnormal extracellular matrix (HA, LAMA4, BGN, FBN1, VASN, PCOLCE, MFAP4), cytokines (IL-6, CRABP1, ISG15), growth factors (PDGF-AA, CNTF, IGF-II, sFRP1, SERPINF1) and increased FABP7, which might lead to the differentiation and maturation disorder of OPCs. We analyzed the astrocyte-conditioned medium to find the key secretory molecules affecting the differentiation and maturation of OPCs, which provides potential clues for further research on the mechanism of VWM.