Eukaryotic Translation Initiation Factor 2B Research Articles

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.

Transcriptional Changes and Preservation of Muscle Protein Content in Hibernating Black Bears

Physical inactivity decreases mechanical load on the skeleton, which, when prolonged, leads to loss of muscle mass and strength in most mammalian species. In contrast, hibernating bears demonstrate limited loss of muscle mass and protein content over the prolonged periods (5-6 months) of inactivity and fasting during winter. This suggests that bears have natural adaptive mechanisms preserving muscle mass and functionality. To identify transcriptional changes that underlie molecular mechanisms attenuating muscle loss, we conducted a large-scale gene expression profiling (14,194 genes) in quadriceps muscle of adult black bear males, comparing hibernating animals (n=5) sampled in March, about 1 month before expected emergence, and summer active animals (n=5) using next generation sequencing (RNA-seq) of the transcriptome. In total, 336 differentially expressed (FDR<0.05) genes were up-regulated and 677 genes were down-regulated in muscle of hibernating bears. Gene set enrichment analysis with Reactome database showed significantly elevated proportion of overexpressed genes involved in eukaryotic translation initiation (FDR<0.0001) and elongation (FDR<0.0001) as well as in the mTORC1 mediated signaling pathway (FDR=0.021). In contrast, down-regulated genes were overrepresented among genes involved in catabolism of branched chain amino acid (BCAA: leucine, isoleucine and valine, FDR<0.0001) that suggests preservation of BCAA. These findings imply induction of protein biosynthesis through the mTORC1 signaling positively activated by availability of leucine in muscle during hibernation. Support for this conclusion comes from significant overexpression of Ras-related GTP-binding protein D (RRAGD, FC=2.12, FDR<0.0001), crucial regulator of the mTORC1 response to leucine availability, and up-regulation of eukaryotic translation initiation factor 4B (EIF4B, FC=1.95, FDR=0.012), downstream target of the mTORC1 mediated signaling. In addition to regulation of protein biosynthesis, activation of the mTOR signaling suppresses autophagy-dependent protein degradation. Consistent with reduction of autophagy, microtubule associated protein 1 light chain (MAP1LC3A, FC=-2.46, FDR<0.0001) and unc-51 like autophagy activating kinase (ULK1, FC=-1.69, FDR=0.052) were down-regulated in hibernating muscle. No expression differences were detected for two muscle atrophy markers, key members of the ubiquitin proteasome degradation, FBXO32 (Atrogin-1, MAFBX, FDR=0.85) and TRIM63 (MURF-1, FDR=0.63). The induction of protein biosynthesis and decrease in protein catabolism through the mTORC1 mediated signaling as response to BCAA availability likely contribute to the maintenance of muscle protein content through prolonged periods of immobility and fasting during hibernation. The follow up studies need to include metabolomic quantification of BCAA, phospho-proteomic assessment of key genes (RPS6KB1, RPS6) involved in the mTORC1 signaling and identification of upstream negative regulators suppressing BCAA catabolism in hibernating muscle. The work was supported by NIH COBRE under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of Eukaryotic Translation Initiation Factor 4B as a Novel Candidate Gene for Congenital Hypothyroidism.

Congenital hypothyroidism (CH) is the most common endocrine disorder in neonates, but its etiology is still poorly understood. We performed whole exome sequencing to identify novel causative gene for CH and functional studies to validate its role in the occurrence of CH. Whole exome sequencing in 98 CH patients not harboring known CH candidate genes and bioinformatic analysis were performed. Functional analysis was performed using morpholino, a synthetic short antisense oligonucleotide that contains 25 DNA bases on a methylene morpholine backbone, in zebrafish and CRISPR‒Cas9-mediated gene knockout in mice. Eukaryotic translation initiation factor 4B (EIF4B) was identified as the most promising candidate gene. The EIF4B gene was inherited in an autosomal recessive model, and one patient with thyroid dysgenesis carried EIF4B biallelic variants (p.S430F/p.P328L). In zebrafish, the knockdown of eif4ba/b expression caused thyroid dysgenesis and growth retardation. Thyroid hormone levels were significantly decreased in morphants compared with controls. Thyroxine treatment in morphants partially rescued growth retardation. In mice, the homozygous conceptuses of Eif4b+/- parents did not survive. Eif4b knockout embryos showed severe growth retardation, including thyroid dysgenesis and embryonic lethality before E18.5. These experimental data supported a role for EIF4B function in the pathogenesis of the hypothyroid phenotype seen in CH patients. Our work indicated that EIF4B was identified as a novel candidate gene in CH. EIF4B is essential for animal survival, but further studies are needed to validate its role in the pathogenesis of CH.

Discovery of DNL343: A Potent, Selective, and Brain-Penetrant eIF2B Activator Designed for the Treatment of Neurodegenerative Diseases.

Eukaryotic translation initiation factor 2B (eIF2B) is a key component of the integrated stress response (ISR), which regulates protein synthesis and stress granule formation in response to cellular insult. Modulation of the ISR has been proposed as a therapeutic strategy for treatment of neurodegenerative diseases such as vanishing white matter (VWM) disease and amyotrophic lateral sclerosis (ALS) based on its ability to improve cellular homeostasis and prevent neuronal degeneration. Herein, we report the small-molecule discovery campaign that identified potent, selective, and CNS-penetrant eIF2B activators using both structure- and ligand-based drug design. These discovery efforts culminated in the identification of DNL343, which demonstrated a desirable preclinical drug profile, including a long half-life and high oral bioavailability across preclinical species. DNL343 was progressed into clinical studies and is currently undergoing evaluation in late-stage clinical trials for ALS.

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.

265 Interactive Effects of Dietary Isoleucine and Valine Ratios to Lysine in Response to Varying Leucine to Lysine Ratios on Pectoralis Major Muscle Protein Expression in Commercial Broilers

Abstract Optimizing the dietary concentrations and ratios of branched-chain amino acids (BCAA) is necessary to optimize broiler chicken growth performance and carcass meat yield. A central composite design (CCD) study was conducted to understand the impact of dietary BCAA concentrations and ratios on Pectoralis major (PM) muscle protein expression involved in mTOR and related protein synthesis pathways as well as and investigate the mechanisms behind how BCAA ratios affect broiler growth and muscle protein accretion. A total of 2,592 d-old Ross 344 × 708 male broilers were randomly placed in 144 floor pens (n = 18 birds per pen). Before the initiation of the experimental finisher phase, all pens were standardized to contain 15 birds per pen. Each pen received 1 of 15 dietary treatments in the 23 CCD with 6 center points from 20 to 35 d of age, varying in digestible ratios of valine:lysine (Val:Lys; 64 to 87), leucine:lysine (Leu:Lys; 110 to 185), and isoleucine:lysine (Ile:Lys; 52 to 75). On d 35, 1 bird per pen was randomly selected for PM protein extraction and proteomic analysis via data independent acquisition protein sequencing with a timsTOF Pro 2 LC/MS/MS and Spectronaut 15 software. Ribosomal protein S6 kinase (RPS6KA3), eukaryotic elongation factor 2 (EEF2), eukaryotic translation initiation factor 4B (EIF4B), eukaryotic translation initiation factor 4E (EIF4E), CAP-Glycine domain containing linker protein 1 (CLIP1), mitogen-activated protein kinase 3 (MAPK3), Protein kinase domain-containing protein (MAP2K1), Dual-specificity mitogen-activated protein kinase kinase 2 (MAP2K2), paxillin (PXN), and regulator complex protein (LAMTOR3) were identified, quantified, and analyzed as a CCD using the RSREG procedure of SAS ver. 9.4 with significance set at P ≤ 0.10. The surface response models for RPS6KA3, EEF2, EIF4B, EIF4E, MAPK3, MAP2K1, MAP2K2, CLIP1, PXN, and LAMTOR3 were not significant (P ≥ 0.926; R2 values ≤ 0.1). No linear (P ≥ 0.5223), quadratic (P ≥ 0.0.634) or cross product (P ≥ 0.6238) regression effects were observed for any of the proteins analyzed. A minimum stationary point was observed for RPS6KA3, EEF2, MAPK3, and LAMTOR3. However, optimal values for the other proteins could not be obtained due to stationary saddle points. Overall, the results in this study indicate that varying concentrations of dietary BCAA may not impact expression of proteins related to broiler skeletal muscle growth on d 35 following the 15-d treatment period, despite the influence on live performance. This is a limitation of this study and suggests that if the mTOR pathway was involved it was not still differentially up- or down-regulated at this time point. Further investigation and new studies with multiple time points will be required to better understand the mechanisms behind how BCAA impact broiler growth and muscle development.

SP1-Induced Upregulation of LncRNA AFAP1-AS1 Promotes Tumor Progression in Triple-Negative Breast Cancer by Regulating mTOR Pathway.

The long non-coding RNA (lncRNA) actin fiber-associated protein-1 antisense RNA 1 (AFAP1-AS1) exerted oncogenic activity in triple-negative breast cancer (TNBC). We designed this study and conducted it to investigate the upstream regulation mechanism of AFAP1-AS1 in TNBC tumorigenesis. In this work, we proved the localization of AFAP1-AS1 in the cytoplasm. We elucidated the mechanism by which the transcription factor specificity protein 1 (SP1) modulated AFAP1-AS1 in TNBC progression, which has yet to be thoroughly studied. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay revealed a strong affinity of SP1 toward the promoter regions P3 of AFAP1-AS1, proving the gene expression regulation of AFAP1-AS1 via SP1 in TNBC. Additionally, SP1 could facilitate the tumorigenesis of TNBC cells in vitro and in vivo by regulating the AFAP1-AS1 expression. Furthermore, silenced AFAP1-AS1 suppressed the expression of genes in the mTOR pathway, such as eukaryotic translation initiation factor 4B (EIF4B), mitogen-activated protein kinase-associated protein 1 (MAPKAP1), SEH1-like nucleoporin (SEH1L), serum/glucocorticoid regulated kinase 1 (SGK1), and its target NEDD4-like E3 ubiquitin protein ligase (NEDD4L), and promoted the gene expression of s-phase kinase-associated protein 2 (SKP2). Overall, this study emphasized the oncogenic role of SP1 and AFAP1-AS1 in TNBC and illustrated the AFAP1-AS1 upstream interaction with SP1 and the downstream modulatory of mTOR signaling, thus offering insights into the tumorigenesis mechanism in TNBC.

Nicotinamide mononucleotide (NMN) alleviates the poly(I:C)-induced inflammatory response in human primary cell cultures

NMN is the direct precursor of nicotinamide adenine dinucleotide (NAD+) and is considered as a key factor for increasing NAD+ levels and mitochondrial activity in cells. In this study, based on transcriptome analysis, we showed that NMN alleviates the poly(I:C)-induced inflammatory response in cultures of two types of human primary cells, human pulmonary microvascular endothelial cells (HPMECs) and human coronary artery endothelial cells (HCAECs). Major inflammatory mediators, including IL6 and PARP family members, were grouped into coexpressed gene modules and significantly downregulated under NMN exposure in poly(I:C)-activated conditions in both cell types. The Bayesian network analysis of module hub genes predicted common genes, including eukaryotic translation initiation factor 4B (EIF4B), and distinct genes, such as platelet-derived growth factor binding molecules, in HCAECs, which potentially regulate the identified inflammation modules. These results suggest a robust regulatory mechanism by which NMN alleviates inflammatory pathway activation, which may open up the possibility of a new role for NMN replenishment in the treatment of chronic or acute inflammation.

Backbone resonance assignments of the C-terminal region of human translation initiation factor eIF4B.

Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5' untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B. Analysis of the chemical shift values identifies one main helical region in the area previously linked to RNA binding, and confirms that the overall C-terminal region is intrinsically disordered.

Clinical and Neurophysiological Features of Epilepsy in Pediatric Patients with Vanishing White Matter Disease-A single institute experience and literature review (P12-9.006)

<h3>Objective:</h3> Refractory epilepsy is a common presentation in Vanishing white matter disease (VWMD) patients. However, there is little literature describing the nature and evolution of seizures. We aim to study the electroencephalograpic findings in patients with a genetically confirmed diagnosis of VWMD, and their correlation with MRI progression and clinical status longitudinally. <h3>Background:</h3> Vanishing White Matter Disease (VWMD) is one of the most prevalent leukodystrophies in pediatric patients with unique clinical, pathological, and molecular features. It mostly affects children, but may develop at all ages, from birth to senescence. Incidence in one study is reported to be 1 in 80,000 live births. The molecular basis of the disease has been shown to be due to mutations in subunits of Eukaryotic Translation Initiation Factor 2B (eIF2B) ie eIF2B 1–5. <h3>Design/Methods:</h3> An IRB approved retrospective chart review was conducted at Children’s Medical Center, Dallas TX. The initial search pulled up 42 patients that had an associated diagnosis of leukodystrophy. Four of these 42 patients were found to have a genetically confirmed diagnosis of VWMD. <h3>Results:</h3> In our cohort, there were 3males and 1female. Age of diagnosis ranged between 2–10 years and mutations in EIF2B5 was the most common. The earliest seizure onset was noted at 2 years of age at the time of diagnosis. Patients that presented with intractable seizures were noted to have significant MRI changes. Average time of seizure onset from time of diagnosis was 1.6 years. Our patient that was diagnosed with VWMD at 10 years of age did not go on to develop seizures and his EEG did not have any epileptiform discharges. His MRI did not show significant white matter changes and clinically patient had mild to moderate deficits. <h3>Conclusions:</h3> We observe that there is a wide range of severity of epilepsy in these patients &amp; not all patients with a diagnosis of VWMD go on to develop seizures. <b>Disclosure:</b> Dr. Laheji has nothing to disclose. Dr. Lowden has nothing to disclose.

Human-induced pluripotent stem cell-derived cerebral organoid of leukoencephalopathy with vanishing white matter.

Leukoencephalopathy with vanishing white matter (VWM) is a rare autosomal recessive leukoencephalopathy resulting from mutations in EIF2B1-5, which encode subunits of eukaryotic translation initiation factor 2B (eIF2B). Studies have found that eIF2B mutation has a certain influence on embryonic brain development. So far, the effect of the eIF2B mutations on the dynamic process of brain development is not fully understood yet. Three-dimensional brain organoid technology has promoted the study of human nervous system developmental diseases in recent years, providing a potential platform for elucidating the pathological mechanism of neurodevelopmental diseases. In this study, we aimed to investigate the effects of eIF2B mutation on the differentiation and development of different nerve cells during dynamic brain development process using 3D brain organoids. We constructed eIF2B mutant and wild-type brain organoid model with induced pluripotent stem cell (iPSC). Compared with the wild type, the mutant brain organoids were significantly smaller, accompanied by increase in apoptosis, which might be resulted from overactivation of unfolded protein response (UPR). Neuronal development was delayed in early stage, but with normal superficial neuronal differentiation in later stage. eIF2B mutations resulted in immature astrocytes with increased expression of GFAPδ, nestin, and αB-crystallin, and there were increased oligodendrocyte progenitor cells, decreased mature oligodendrocytes, and sparse myelin in mutant cerebral organoids in the later stage. we constructed the first eIF2B mutant cerebral organoids to explore the dynamic brain development process, which provides a platform for further research on the specific pathogenesis of VWM.

Exosomal miR-30a-5p promoted intrahepatic cholangiocarcinoma progression by increasing angiogenesis and vascular permeability in PDCD10 dependent manner.

Tumor-associated angiogenesis positively associates with malignant metastasis of intrahepatic cholangiocarcinoma (ICCA). Cancer cell-derived exosomes carrying microRNAs involves in tumor microenvironment (TME) regulation. We aimed to evaluate exosomal miR-30a-5p in ICCA development. Our data showed that increased miR-30a-5p level was correlated with higher microvascular density (MVD) and worse prognosis. Augmented miR-30a-5p expression was induced by hypoxia induced factor 1α (HIF-1α) in ICCA cell. Further exploration revealed that ICCA-derived miR-30a-5p could be transferred to endothelial and increased endothelial cells recruitment and proliferation, induced angiogenesis and vascular permeability in exosome dependent manner. In addition, circulating exosomal miR-30a-5p was higher in ICCA patients, and correlated with ICCA tissues-expressing miR-30a-5p. Hypoxic stress enhanced the effects of exosomal miR-30a-5p on endothelial-associated phenotypes. Rescued experiments showed that exosomal miR-30a-5p modulated endothelial-associated phenotypes in a way relied on programmed cell death 10 (PDCD10). Moreover, we revealed that the packing of miR-30a-5p into ICCA cells-derived exosomes was mediated by eukaryotic translation initiation factor 4B (EIF4B). More importantly, the combined application of targeting miR-30a-5p and apatinib could synergistically improve antiangiogenic efficacy in ICCA. Combined, ICCA-derived exosomal miR-30a-5p could be an excellent therapeutic and monitoring indicator for ICCA patients.

Effect of eIF4B knockout on apoptosis of mouse fetal liver cells

Eukaryotic translation initiation factor 4B (eIF4B) plays an important role in mRNA translation initiation, cell survival and proliferation in vitro, but the in vivo function is poorly understood. In this study, via various experimental techniques such as hematoxylin-eosin (HE) staining, flow cytometry, Western blotting, and immunohistochemistry, we investigated the role of eIF4B in mouse embryo development using an eIF4B knockout (KO) mouse model and explored the mechanism. We found that the livers, but not lungs, brain, stomach, or pancreas, derived from eIF4B KO mouse embryos displayed severe pathological changes characterized by enhanced apoptosis and necrosis. Accordingly, high expression of cleaved-caspase 3, and excessive activation of mTOR signaling as evidenced by increased expression and phosphorylation of p70S6K and enhanced phosphorylation of 4EBP1, were observed in mouse embryonic fibroblasts and fetal livers from eIF4B KO mice. These results uncover a critical role of eIF4B in mouse embryo development and provide important insights into the biological functions of eIF4B in vivo.

Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay.

Guanine nucleotide-exchange factors (GEFs) activate the function of guanine nucleotide-binding proteins (G-proteins) by promoting the exchange of GDP for GTP on the latter. Here, we describe a protocol for in vitro measurements of the GEF activity of eukaryotic translation initiation factor 2B, eIF2B, toward its substrate eIF2. This protocol provides a relatively simple method for determining the eIF2B's GEF activity in crude cell extracts. The eIF2 heterotrimeric substrate, with phosphorylated or unphosphorylated eIF2α, is prepared by immunoprecipitation, following subsequent loading of a fluorescent BODIPY-FL dye-attached GDP. The exchange of the bound fluorescent GDP molecule for an unlabeled one on eIF2 promoted by eIF2B is monitored kinetically using a fluorescence microplate reader.

Flaccidoxide Induces Apoptosis Through Down-regulation of PI3K/AKT/mTOR/p70S6K Signaling in Human Bladder Cancer Cells.

Urothelial carcinoma (UC) is the most common type of genitourinary cancer with high incidence and mortality rates in men. In this study, we used the BFTC-905 and T24 bladder cancer cell lines as in vitro models to investigate the pathways involved in flaccidoxide-induced apoptosis. We utilized MTT assays, colony assays, wound-healing assays and fluorescence with TUNEL to confirm the cytotoxicity of flaccidoxide in bladder cancer cell lines. Potential proliferative and apoptotic molecular mechanisms were evaluated by western blotting. The expression of anti-apoptotic proteins Bcl-2 and phosphorylated Bad (p-Bad) was attenuated with an increasing flaccidoxide concentration, while the expression of proapoptotic proteins Bax, Bad, cleaved caspase-3, cleaved caspase-9 and cleaved PARP-1 was found increased. Additionally, phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) in the PI3K/AKT/mTOR pathway was reduced, leading to a reduction in the phosphorylation of downstream 70-kDa ribosomal protein S6 kinase 1 (p70S6K), S6 ribosomal protein (S6) and eukaryotic translation initiation factor 4B (eIF4B). However, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein phosphorylation was increased due to attenuation of the upstream phosphorylation of mTOR protein. Flaccidoxide-induced apoptosis in BFTC-905 and T24 cells is mediated by mitochondrial dysfunction and down-regulation the PI3K/AKT/mTOR/p70S6K signaling pathway.

EIF4B Is a Novel Target of CAMK2G and Promotes Proliferation of Malignant Cells in Myelofibrosis

BackgroundMyelofibrosis (MF) is a myeloproliferative neoplasm which is associated with megakaryocytic atypia, fibrosis in the bone marrow (BM), and extramedullary hematopoiesis, followed by progressive hematopoietic failure and leukemic transformation. JAK1/JAK2 inhibitors are currently available and reduce spleen volume, improve symptoms related to MF and prolong the overall survival (OS). Although the benefits associated with JAK1/JAK2 inhibitors are well established, not all patients respond to these inhibitors, and long-term exposure to these inhibitors results in the emergence of resistant clones based on the reactivation of the JAK-STAT pathway. Therefore, new therapeutic strategies targeting other molecules can provide additional benefits to patients with MF. In the previous study, we identified Calcium/Calmodulin Dependent Protein Kinase II Gamma (CAMK2G) as a new therapeutic target for MF by performing compound screening. Furthermore, CAMK2G inhibition can overcome drug-resistance against JAK2 inhibitors. Therefore, it is important to investigate the mechanisms underlying the therapeutic effect of CAMK2G inhibition. In this study, to explore the mechanism underlying the therapeutic effect of CAMK2G inhibition, we performed the immunoprecipitation mass spectrometry to find out the protein that has the direct interaction with CAMK2G.MethodsQuantitative Proteomic Analysis of the Target Proteins of CAMK2GThe protein complexes with FLAG-tagged CAMK2G (FLAG-CAMK2G) were trapped by anti-FLAG antibody. The eluted assay mixtures were reduced, alkylated and digested into peptides. Each peptide solution and control mixture were labeled with differential stable isotope tags. The samples were quantitatively analyzed using a Q Exactive mass spectrometer (Thermo Fisher Scientific). The spectra were searched against the SWISS-PROT databases using SEQUEST on Proteome Discoverer software 2.2 (Thermo Scientific).ResultsTo reveal the , we conducted the immunoprecipitation assays by FLAG-CAMK2G or FLAG-tag alone, overexpressed in MF model cells. Because CAMK2G has kinase activity, we attempted to reveal the kinase-activity-dependent targets using unhydrolyzable ATP analog, AMP-PNP（5'-adenylyl-imidodiphosphate）that can maintain the strong-binding state of kinase with its target. The protein complexes with FLAG-CAMK2G were trapped by anti-FLAG antibody. After samples were digested into peptides and the candidate proteins were identified and quantitatively analyzed by mass spectrometry. As a result, we identified eukaryotic translation initiation factor 4B (eIF4B) as a protein that has a direct interaction with CAMK2G. eIF4B is a part of the complex involved in the initiation of translation. It has been reported that eIF4B plays a role in the translation of factors involved in anti-apoptosis (Bcl2, Bclxl, Mcl1), cell cycle (Cdc25c), and cell proliferation (c-Myc). We then checked whether knockdown of eIF4B decrease proliferation of MF model cell line. shRNA-mediated silencing of eIF4B decreased cell growth of these cells. Furthermore, the phosphorylation of eIF4B was increased by the ectopic expression of MPL W515L, one of the common mutations found in MF. Also, the phosphorylation of eIF4B was increased by the overexpression of CAMK2G. We then explored the proteins regulated by eIF4B in MF and identified that knockdown of eIF4B decreased the amount of Bcl2 protein.ConclusionIn our study, we performed immunoprecipitation mass spectrometry and identified eIF4B as a partner protein of CAMK2G. Since CAMK2G inhibition was shown to be effective against MF in vitro and in vivo, we focused on eIF4B as a potential effector in MF. We also showed that overexpression of MPL W515L and CAMK2G phosphorylates eIF4B and that knockdown of eIF4B inhibited proliferation of MF cells. Furthermore, Bcl2 can be one of the target proteins regulated by eIF4B. Based on these, eIF4B plays an important role in MF. We further perform ribosome profiling to comprehensively understand the regulation of translation by eIF4B. Our research not only elucidate the pathogenesis of MF but also identify new therapeutic targets for MF. DisclosuresMasamoto: MSD K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Kurokawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau.

Correlation Between Genotype and Age of Onset in Leukoencephalopathy With Vanishing White Matter.

Purpose: Leukoencephalopathy with vanishing white matter (VWM) is an autosomal recessive leukoencephalopathy caused by mutations in any of the five genes encoding the subunits of eukaryotic translation initiation factor 2B (eIF2B). The severity of the disease varies considerably, and its genotypic-phenotypic correlation is still unclear. Age of onset is the only independent clinical predictor for VWM severity. In this study, the correlation between genotype and age at onset of patients was investigated. Methods: Data were collected from patients with VWM in the available literature reports and from those diagnosed in Peking University First Hospital. The age of onset was divided into early-onset (≤4 years) and late-onset type (>4 years) for the analysis of the correlation between genotype and age of onset in patients with VWM. Results: A total of 341 patients were included, 281 were reported in 87 available articles and 60 were diagnosed in our center. A total of 180 different mutations were found, among which 86.1% were missense. The gene (EIF2B1-5) in which the mutation located, and the number of null alleles were not associated with age of onset in these patients. Certain mutations such as eIF2Bε[Arg195His] and eIF2Bε[Arg269Gln] that were predicted to have a serious influence on eIF2B structure were related to earlier age of onset. EIF2Bγ[Ala87Val] which was predicted to have a minimal influence on eIF2B structure, was related to later age of onset. Whereas eIF2Bβ[Glu213Gly], eIF2Bβ[Gly200Val] and eIF2Bε[Thr91Ala], also predicted having a small effect on the structure of eIF2B, did not show correlation with the age of onset. The onset age of patients with one or biallelic missense mutations located in the catalytic domain or other homologous domains in catalytic subunits (eIF2Bγ, ε) was earlier than that of patients with biallelic mutations located in the NT domain. Conclusion: The onset age of patients with different genotypes varied greatly. The degree of influence in protein structure of some missense mutations was correlated with phenotypic severity, but the results were not completely consistent. The combined effect of biallelic mutations, the role of regulatory genes, environmental stress and other potential factors on phenotypes need to be further explored.

Supplementing N-carbamoylglutamate in late gestation increases newborn calf weight by enhanced placental expression of mTOR and angiogenesis factor genes in dairy cows.

The objective of this study was to investigate whether supplementation with N-carbamoylglutamate (NCG) to cows during late gestation alters uteroplacental tissue nutrient transporters, calf metabolism and newborn weight. Thirty multiparous Chinese Holstein cows were used in a randomized complete block design experiment. During the last 28 d of pregnancy, cows were fed a diet without (CON) or with NCG (20 g/d per cow). The body weight of calves was weighed immediately after birth. Placentome samples were collected at parturition and used to assess mRNA expression of genes involved in transport of arginine, glucose, fatty acid and angiogenesis factors, as well as the mammalian target of rapamycin (mTOR) pathway. Blood samples of calves before colostrum consumption were also collected for the detection of plasma parameters, amino acids (AA) and metabolomics analysis. The newborn weight (P = 0.02) and plasma Arg concentration of NCG-calves was significantly higher (P = 0.05) than that of CON-calves, and the plasma concentrations of urea nitrogen tended to be lower (P = 0.10) in the NCG group. The mRNA abundance of genes involved in glucose transport (solute carrier family 2 member 3 [SLC2A3], P < 0.01), angiogenesis (nitric oxide synthase 3 [NOS3], P = 0.02), and mTOR pathway (serine/threonine-protein kinase 1 [AKT1], P = 0.10; eukaryotic translation initiation factor 4B pseudogene 1 [EIF4BP1], P = 0.08; EIF4EBP2, P = 0.04; and E74-like factor 2 [ELF2], P = 0.03) was upregulated in the placentome of NCG-supplemented cows. In addition, 17 metabolites were significantly different in the placentome of NCG-supplemented cows compared to non-supplemented cows, and these metabolites are mainly involved in arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and citrate cycle. In summary, the increased body weight of newborn calves from the NCG supplemented dairy cows may be attributed to the increased angiogenesis and uteroplacental nutrient transport and to the activated mTOR signal pathway, which may result in the increased nutrient supply to the fetus, and improved AA metabolism and urea cycle of the fetus.

Deficiency of eIF4B Increases Mouse Mortality and Impairs Antiviral Immunity.

Eukaryotic translation initiation factor 4B (eIF4B) plays an important role in mRNA translation initiation, cell survival and proliferation in vitro. However, its function in vivo is poorly understood. Here, we identified that eIF4B knockout (KO) in mice led to embryonic lethality, and the embryos displayed severe liver damage. Conditional KO (CKO) of eIF4B in adulthood profoundly increased the mortality of mice, characterized by severe pathological changes in several organs and reduced number of peripheral blood lymphocytes. Strikingly, eIF4B CKO mice were highly susceptible to viral infection with severe pulmonary inflammation. Selective deletion of eIF4B in lung epithelium also markedly promoted replication of influenza A virus (IAV) in the lung of infected animals. Furthermore, we observed that eIF4B deficiency significantly enhanced the expression of several important inflammation-associated factors and chemokines, including serum amyloid A1 (Saa1), Marco, Cxcr1, Ccl6, Ccl8, Ccl20, Cxcl2, Cxcl17 that are implicated in recruitment and activation of neutrophiles and macrophages. Moreover, the eIF4B-deficient mice exhibited impaired natural killer (NK) cell-mediated cytotoxicity during the IAV infection. Collectively, the results reveal that eIF4B is essential for mouse survival and host antiviral responses, and establish previously uncharacterized roles for eIF4B in regulating normal animal development and antiviral immunity in vivo.

Established and Emerging Regulatory Roles of Eukaryotic Translation Initiation Factor 5B (eIF5B).

Translational control (TC) is one the crucial steps that dictate gene expression and alter the outcome of physiological process like programmed cell death, metabolism, and proliferation in a eukaryotic cell. TC occurs mainly at the translation initiation stage. The initiation factor eIF5B tightly regulates global translation initiation and facilitates the expression of a subset of proteins involved in proliferation, inhibition of apoptosis, and immunosuppression under stress conditions. eIF5B enhances the expression of these survival proteins to allow cancer cells to metastasize and resist chemotherapy. Using eIF5B as a biomarker or drug target could help with diagnosis and improved prognosis, respectively. To achieve these goals, it is crucial to understand the role of eIF5B in translational regulation. This review recapitulates eIF5B’s regulatory roles in the translation initiation of viral mRNA as well as the cellular mRNAs in cancer and stressed eukaryotic cells.