The human eIF4E:4E-BP2 complex structure for studying hyperphosphorylation.

The cap-dependent mRNA translation is dysregulated in many kinds of cancers. The interaction between eIF4E and eIF4G through a canonical eIF4E-binding motif (CEBM) determines the efficacy of the cap-dependent mRNA translation. eIF4E-binding proteins (4E-BPs) share the CEBM and compete with eIF4G for the same binding surface of eIF4E and then inhibit the mRNA translation. 4E-BPs function as tumor repressors in nature. Hyperphosphorylation of 4E-BPs regulates the structure folding and causes the dissociation of 4E-BPs from eIF4E. However, until now, there has been no structure of the full-length 4E-BPs in complex with eIF4E. The regulation mechanism of phosphorylation is still unclear. In this work, we first investigate the interactions of human eIF4E with the CEBM and an auxiliary eIF4E-binding motif (AEBM) in eIF4G and 4E-BPs. The results unravel that the structure and interactions of the CEBM are highly conserved between eIF4G and 4E-BPs. However, the extended CEBM (ECEBM) in 4E-BPs forms a longer helix than that in eIF4G. The residue R62 in the ECEBM of 4E-BP2 forms salt bridges with E32 and E70 of eIF4E. The residue R63 of 4E-BP2 forms two special hydrogen bonds with N77 of eIF4E. Both of these interactions are missing in eIF4G. The AEBM of 4E-BPs folds into a ß-sheet conformation, which protects V81 inside a hydrophobic core in 4E-BP2. In eIF4G, the AEBM exists in a random coil state. The hydrophilic residues S637 and D638 of eIF4G open the hydrophobic core for solvents. The results show that the ECEBM and AEBM may be responsible for the competing advantage of 4E-BP2. Finally, based on our previous work (J. Zeng, F. Jiang and Y. D. Wu, J. Chem. Theory Comput., 2017, 13, 320), the human eIF4E:4E-BP2 complex (eIF4E:BP2P18-I88) including all reported phosphorylation sites is predicted. The eIF4E:BP2P18-I88 complex is different from the existing experimental eIF4E:eIF4G complex and provides an important structure for further studying the regulation mechanism of phosphorylation in 4E-BPs.

miR-483-5p orchestrates the initiation of protein synthesis by facilitating the decrease in phosphorylated Ser209eIF4E and 4E-BP1 levels.

Eukaryotic initiation factor 4E (eIF4E) is a pivotal protein involved in the regulatory mechanism for global protein synthesis in both physiological and pathological conditions. MicroRNAs (miRNAs) play a significant role in regulating gene expression by targeting mRNA. However, the ability of miRNAs to regulate eIF4E and its phosphorylation remains relatively unknown. In this study, we predicted and experimentally verified targets for miR-483-5p, including eukaryotic translation initiation factor eIF4E and its binding proteins, 4E-BPs, that regulate protein synthesis. Using the Web of Science database, we identified 28 experimentally verified miR-483-5p targets, and by the TargetScan database, we found 1818 predicted mRNA targets, including EIF4E, EIF4EBP1, and EIF4EBP2. We verified that miR-483-5p significantly reduced ERK1 and MKNK1 mRNA levels in HEK293 cells. Furthermore, we discovered that miR-483-5p suppressed EIF4EBP1 and EIF4EBP2, but not EIF4E. Finally, we found that miR-483-5p reduced the level of phosphorylated eIF4E (pSer209eIF4E) but not total eIF4E. In conclusion, our study suggests that miR-483-5p's multi-targeting effect on the ERK1/ MKNK1 axis modulates the phosphorylation state of eIF4E. Unlike siRNA, miRNA can have multiple targets in the pathway, and thereby exploring the role of miR-483-5p in various cancer models may uncover therapeutic options.

A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Computational inference of eIF4F complex function and structure in human cancers.

The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.

eIF4E plays the role of a pathogenic gene in psoriasis, and the inhibition of eIF4E phosphorylation ameliorates psoriasis-like skin damage.

Psoriasis is a complex inflammatory skin disease with uncertain pathogenesis. eIF4E (eukaryotic translation initiation factor 4E) and its phosphorylation state p-eIF4E are highly expressed in psoriatic tissues. However, the role eIF4E played in psoriasis is still unclear. To investigate the function of eIF4E and p-eIF4E in psoriasis and to figure out whether eFT-508 (Tomivosertib, eIF4E phosphorylation inhibitor) can relieve the disease severity and become a promising candidate for the psoriasis treatment. We first verified the expression of eIF4E and p-eIF4E in psoriasis patients' lesional skin. Then, we demonstrated the effect of eIF4E and p-eIF4E on the abnormal proliferation and inflammatory state of keratinocytes by using eIF4E-specific small interfering RNA (si-eIF4E) and eFT-508. In this study, all cell experiments were performed under the psoriasis-model condition. Moreover, the external application of eFT-508 on imiquimod (IMQ)-induced psoriasis mice was performed to explore its potential clinical value. Results showed that eIF4E and p-eIF4E were significantly overexpressed in skin lesions of psoriasis patients. Knocking down eIF4E or adding eFT-508 can relieve the abnormal proliferation and the excessive inflammatory state of keratinocytes by reducing the expression of cyclin D1, IL-1ß, CXCL10, IL23, Wnt 5a, NBS1 and p-AKT from mRNA or protein levels. Furthermore, these results were consistent with those obtained from the in vitro experiments. Then, we conclude that eIF4E plays the role of the pathogenic gene in psoriasis, and eFT-508 may be a promising candidate for anti-prosoriasis drugs.

eIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

Decoding the Heterogeneity and Specialized Function of Translation Machinery Through Ribosome Profiling in Yeast Mutants of Initiation Factors.

The nuanced heterogeneity and specialized functions of translation machinery are increasingly recognized as crucial for precise translational regulation. Here, high-throughput ribosomal profiling (ribo-seq) is used to analyze the specialized roles of eukaryotic initiation factors (eIFs) in the budding yeast. By examining changes in ribosomal distribution across the genome resulting from knockouts of eIF4A, eIF4B, eIF4G1, CAF20, or EAP1, or knockdowns of eIF1, eIF1A, eIF4E, or PAB1, two distinct initiation-factor groups, the "looping" and "scanning" groups are discerned, based on similarities in the ribosomal landscapes their perturbation induced. The study delves into the cis-regulatory sequence features of genes influenced predominantly by each group, revealing that genes more dependent on the looping-group factors generally have shorter transcripts and poly(A) tails. In contrast, genes more dependent on the scanning-group factors often possess upstream open reading frames and exhibit a higher GC content in their 5' untranslated regions. From the ribosomal RNA fragments identified in the ribo-seq data, ribosomal heterogeneity associated with perturbation of specific initiation factors is further identified, suggesting their potential roles in regulating ribosomal components. Collectively, the study illuminates the complexity of translational regulation driven by heterogeneity and specialized functions of translation machinery, presenting potential approaches for targeted gene translation manipulation.

The eukaryotic translation initiation factor eIF4E unexpectedly acts in splicing thereby coupling mRNA processing with translation: eIF4E induces widescale splicing reprogramming providing system-wide connectivity between splicing, nuclear mRNA export and translation.

Recent findings position the eukaryotic translation initiation factor eIF4E as a novel modulator of mRNA splicing, a process that impacts the form and function of resultant proteins. eIF4E physically interacts with the spliceosome and with some intron-containing transcripts implying a direct role in some splicing events. Moreover, eIF4E drives the production of key components of the splicing machinery underpinning larger scale impacts on splicing. These drive eIF4E-dependent reprogramming of the splicing signature. This work completes a series of studies demonstrating eIF4E acts in all the major mRNA maturation steps whereby eIF4E drives production of the RNA processing machinery and escorts some transcripts through various maturation steps. In this way, eIF4E couples the mRNA processing-export-translation axis linking nuclear mRNA processing to cytoplasmic translation. eIF4E elevation is linked to worse outcomes in acute myeloid leukemia patients where these activities are dysregulated. Understanding these effects provides new insight into post-transcriptional control and eIF4E-driven cancers.

Eukaryotic Initiation Factor 4E (eIF4E) as a Target of Anti-Psoriatic Treatment.

Eukaryotic initiation factor 4E (eIF4E) has been known to play a critical role in the regulation of gene expression and essential cellular processes, such as proliferation, apoptosis and differentiation. In this study, we explored its role in the pathophysiology of psoriasis. The inhibition of eIF4E by small interfering RNA or briciclib, an eIF4E small molecule inhibitor, downregulated the expression of eIF4E itself and its two complex partners eIF4A and G, as well as other eIFs (eg, eIF1A, eIF2α, eIF3A, eIF3B, eIF5, and eIF6). This inhibition also abolished psoriatic inflammation in both the imiquimod and TGFß mouse model, as well as in a human 3 dimensional-psoriasis tissue model. Downregulation of eIF4E and the other eIFs by application of briciclib (particularly when given topically) was linked to the normalization of cellular proliferation, epidermal hyperplasia, levels of proinflammatory cytokines (eg, TNFα, IL-1b, IL-17, and IL-22), and keratinocyte differentiation markers (eg, KRT16 and FLG). These results demonstrate translational imbalance and underline the crucial role played by eIF4E and other eIFs in the pathophysiology of psoriasis. This work opens up avenues for the development of novel topical antipsoriatic treatment strategies by targeting eIF4E.

Variable eIF4E-binding sites and their synergistic effect on cap-independent translation in a novel IRES of wheat yellow mosaic virus RNA2 isolates.

Some viral proteins are translated cap-independently via the internal ribosome entry site (IRES), which maintains conservative characteristic among different isolates of the same virus species. However, IRES activity showed a 7-fold variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates in this study. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and the hepta-nucleotide top loop of H2 in the LYJN isolate synergistically ensured higher IRES activity than that in the HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. WYMV RNA2 5'-UTR specifically interacted with the wheat eIF4E, accomplished by the top loop of H1 in the HC isolate or the top loop of H1 and H2 in the LYJN isolate. The high IRES activity of the WYMV RNA2 LYJN isolate was regulated by two eIF4E-binding sites, which showed a synergistic effect mediated by the proximity of the H1 and H2 top loops owing to the flexibility of the middle stem in H1. This report presents a novel evolution pattern of IRES, which altered the number of eIF4E-binding sites to regulate IRES activity.

Molecular dynamics simulations revealed topological frustration in the binding-wrapping process of eIF4G with eIF4E.

Interaction between the cap-binding protein eIF4E and the scaffolding protein eIF4G is essential for the cap-dependent translation initiation in eukaryotes. In the Saccharomyces cerevisiae eIF4G/eIF4E complex, the intrinsically disordered eIF4E-binding domain of eIF4G folds into a bracelet-like structure upon binding to eIF4E. Aiming to unveil the molecular mechanism underlying the binding-wrapping process of eIF4G with eIF4E, we performed extensive coarse-grained molecular dynamics simulations and transition path analysis in this work. The major transition pathway revealed from our simulations showed that docking of the eIF4E-binding motif of eIF4G to the folded core of eIF4E initiates the binding process and then the disordered eIF4G wraps around the N-terminal tail of eIF4E. Additionally, we identified a minor transition pathway which indicates the involvement of topological frustration in the binding process. By manipulating the interaction strength of the wrapping contacts and the latching contacts, we further dissected factors affecting the formation of topological frustration and the binding transition kinetics. Our findings provide new clues for experimental studies on the binding mechanism of eIF4G to eIF4E in the future and exemplify the involvement of topological frustration in the binding process of intrinsically disordered proteins.

FMRP Enhances the Translation of 4EBP2 mRNA during Neuronal Differentiation.

FMRP is a multifunctional protein encoded by the Fragile X Messenger Ribonucleoprotein 1 gene (FMR1). The inactivation of the FMR1 gene results in fragile X syndrome (FXS), a serious neurodevelopmental disorder. FMRP deficiency causes abnormal neurite outgrowth, which is likely to lead to abnormal learning and memory capabilities. However, the mechanism of FMRP in modulating neuronal development remains unknown. We found that FMRP enhances the translation of 4EBP2, a neuron-specific form of 4EBPs that inactivates eIF4E by inhibiting the interaction between eIF4E and eIF4G. Depletion of 4EBP2 results in abnormal neurite outgrowth. Moreover, the impairment of neurite outgrowth upon FMRP depletion was overcome by the ectopic expression of 4EBP2. These results suggest that FMRP controls neuronal development by enhancing 4EBP2 expression at the translational level. In addition, treatment with 4EGI-1, a chemical that blocks eIF4E activity, restored neurite length in FMRP-depleted and 4EBP2-depleted cells. In conclusion, we discovered that 4EBP2 functions as a key downstream regulator of FMRP activity in neuronal development and that FMRP represses eIF4E activity by enhancing 4EBP2 translation.

Arabidopsis eIF4E1 protects the translational machinery during TuMV infection and restricts virus accumulation.

Successful subversion of translation initiation factors eIF4E determines the infection success of potyviruses, the largest group of viruses affecting plants. In the natural variability of many plant species, resistance to potyvirus infection is provided by polymorphisms at eIF4E that renders them inadequate for virus hijacking but still functional in translation initiation. In crops where such natural resistance alleles are limited, the genetic inactivation of eIF4E has been proposed for the engineering of potyvirus resistance. However, recent findings indicate that knockout eIF4E alleles may be deleterious for plant health and could jeopardize resistance efficiency in comparison to functional resistance proteins. Here, we explored the cause of these adverse effects by studying the role of the Arabidopsis eIF4E1, whose inactivation was previously reported as conferring resistance to the potyvirus clover yellow vein virus (ClYVV) while also promoting susceptibility to another potyvirus turnip mosaic virus (TuMV). We report that eIF4E1 is required to maintain global plant translation and to restrict TuMV accumulation during infection, and its absence is associated with a favoured virus multiplication over host translation. Furthermore, our findings show that, in the absence of eIF4E1, infection with TuMV results in the production of a truncated eIFiso4G1 protein. Finally, we demonstrate a role for eIFiso4G1 in TuMV accumulation and in supporting plant fitness during infection. These findings suggest that eIF4E1 counteracts the hijacking of the plant translational apparatus during TuMV infection and underscore the importance of preserving the functionality of translation initiation factors eIF4E when implementing potyvirus resistance strategies.

C8ORF88: A Novel eIF4E-Binding Protein.

Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent protein synthesis. Binding of eIF4E to translational repressors (eIF4E-binding proteins [4E-BPs]) suppresses translation and is used by cells to link extra- and intracellular cues to protein synthetic rates. The best studied of these interactions involves repression of translation by 4E-BP1 upon inhibition of the PI3K/mTOR signaling pathway. Herein, we characterize a novel 4E-BP, C8ORF88, whose expression is predominantly restricted to early spermatids. C8ORF88:eIF4E interaction is dependent on the canonical eIF4E binding motif (4E-BM) present in other 4E-BPs. Whereas 4E-BP1:eIF4E interaction is dependent on the phosphorylation of 4E-BP1, these sites are not conserved in C8ORF88 indicating a different mode of regulation.

An intramolecular disulphide bond in human 4E-T affects its binding to eIF4E1a protein.

The cap at the 5'terminus of mRNA is a key determinant of gene expression in eukaryotic cells, which among others is required for cap dependent translation and protects mRNA from degradation. These properties of cap are mediated by several proteins. One of them is 4E-Transporter (4E-T), which plays an important role in translational repression, mRNA decay and P-bodies formation. 4E-T is also one of several proteins that interact with eukaryotic initiation factor 4E (eIF4E), a cap binding protein which is a key component of the translation initiation machinery. The molecular mechanisms underlying the interactions of these two proteins are crucial for mRNA processing. Studying the interactions between human eIF4E1a and the N-terminal fragment of 4E-T that possesses unstructured 4E-binding motifs under non-reducing conditions, we observed that 4E-T preferentially forms an intramolecular disulphide bond. This "disulphide loop" reduces affinity of 4E-T for eIF4E1a by about 300-fold. Considering that only human 4E-T possesses two cysteines located between the 4E binding motifs, we proposed that the disulphide bond may act as a switch to regulate interactions between the two proteins.

Allele mining of eukaryotic translation initiation factor genes in Prunus for the identification of new sources of resistance to sharka.

Members of the eukaryotic translation initiation complex are co-opted in viral infection, leading to susceptibility in many crop species, including stone fruit trees (Prunus spp.). Therefore, modification of one of those eukaryotic translation initiation factors or changes in their gene expression may result in resistance. We searched the crop and wild Prunus germplasm from the Armeniaca and Amygdalus taxonomic sections for allelic variants in the eIF4E and eIFiso4E genes, to identify alleles potentially linked to resistance to Plum pox virus (PPV). Over one thousand stone fruit accessions (1397) were screened for variation in eIF4E and eIFiso4E transcript sequences which are in single copy within the diploid Prunus genome. We identified new alleles for both genes differing from haplotypes associated with PPV susceptible accessions. Overall, analyses showed that eIFiso4E is genetically more constrained since it displayed less polymorphism than eIF4E. We also demonstrated more variations at both loci in the related wild species than in crop species. As the eIFiso4E translation initiation factor was identified as indispensable for PPV infection, a selection of ten different eIFiso4E haplotypes along 13 accessions were tested by infection with PPV and eight of them displayed a range of reduced susceptibility to resistance, indicating new potential sources of resistance to sharka.

Inhibition of Eukaryotic Initiating Factor eIF4E Overcomes Abemaciclib Resistance in Gastric Cancer.

OBJECTIVE: Aberrant activating mutations in cyclin-dependent kinases 4 and 6 (CDK4/6) are common in various cancers, including gastroesophageal malignancies. Although CDK4/6 inhibitors, such as abemaciclib and palbociclib, have been approved for breast cancer treatment, their effectiveness as a monotherapy remains limited for gastroesophageal tumors. The present study explored the underlying mechanism of abemaciclib resistance. METHODS: Abemaciclib-resistant gastric cancer cell lines were generated, and the phospho-eukaryotic translation initiation factor 4E (p-eIF4E) and eIF4E expression was compared between resistant and parental cell lines. In order to analyze the role of eIF4E in cell resistance, siRNA knockdown was employed. The effectiveness of ribavirin alone and its combination with abemaciclib was evaluated in the gastric cancer xenograft mouse model. RESULTS: The upregulation of eIF4E was a common feature in gastric cancer cells exposed to prolonged abemaciclib treatment. Gastric cancer cells with increased eIF4E levels exhibited a better response to eIF4E inhibition, especially those that were resistant to abemaciclib. Ribavirin, which is an approved anti-viral drug, significantly improved the efficacy of abemaciclib, both in vitro and in vivo, by inhibiting eIF4E. Importantly, ribavirin effectively suppressed the abemaciclib-resistant gastric cancer growth in mice without causing toxicity. CONCLUSION: These findings suggest that targeting eIF4E can enhance the abemaciclib treatment for gastric cancer, proposing the potential combination therapy of CDK4/6 inhibitors with ribavirin for advanced gastric cancer.

Lost in translation: a neglected mTOR target for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a cystic lung disease of women resulting from mutations in tuberous sclerosis complex (TSC) genes that suppress the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 activation enhances a plethora of anabolic cellular functions, mainly via the activation of mRNA translation through stimulation of ribosomal protein S6 kinase (S6K1)/ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic translation initiation factor 4E (eIF4E). Rapamycin (sirolimus), an allosteric inhibitor of mTORC1, stabilises lung function in many but not all LAM patients and, upon cessation of the drug, disease progression resumes. At clinically tolerable concentrations, rapamycin potently inhibits the ribosomal S6K1/S6 translation ribosome biogenesis and elongation axis, but not the translation 4E-BP1/eIF4E initiation axis. In this mini-review, we propose that inhibition of mTORC1-driven translation initiation is an obvious but underappreciated therapeutic strategy in LAM, TSC and other mTORC1-driven diseases.

Genetic Evidence Supporting a Causal Association Between mTOR-Dependent EIF-4E Circulating Protein Level and Osteoporosis.

INTRODUCTION: The mechanistic target of rapamycin (mTOR) regulates bone homeostasis, a crucial factor in osteoporosis (OP) development. However, most research is based on observational studies, and the causality remains uncertain. Therefore, we analyzed two samples of mendelian randomization (MR) to determine whether there is a causal relationship between mTOR-dependent circulating proteins and OP. METHODS: Mendelian weighting (weighted median [WM], inverse variance weighting [IVW], and MR-Egger regression) were applied to analyze the causality between bone phenotypes (bone mineral density [BMD] in forearm, femoral neck, lumbar spine, and heel) and mTOR-dependent circulating proteins (RP-S6K, 4EBP, EIF-4E, EIF-4A, and EIF-4G). Horizontal pleiotropy and heterogeneities were detected using Cochran's Q test, MR-Pleiotropy RE-Sidual Sum and Outlier (MR-PRESSO), and "leave-one-out" analysis. The proteomics-GWAS INTERVAL study was used to select the instrumental variables (IVs) for mTOR proteins. RESULTS: As phenotypes for OP, estimations of BMD were taken in four different sites: forearm (FA) (n = 8143), femoral neck (FN) (n = 32,735), lumbar spine (LS) (n = 28,498), and heel (eBMD) (n = 426,824). Based on IVW analysis, EIF4E is causally related to FA-BMD (OR = 0.938, 95% CI 0.887, 0.991, p = 0.024) but not to BMD elsewhere. CONCLUSION: MR analysis revealed a causal relationship between EIF-4E and FA-BMD, which may provide new insights into the underlying pathogenesis of OP and a new therapeutic target for OP.

P53 regulates CCAAT/Enhancer binding protein ß gene expression.

BACKGROUND: The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) is implicated in diverse processes and diseases. Its two isoforms, namely liver-enriched activator protein (LAP) and liver-enriched inhibitor protein (LIP) are translated from the same mRNA. They share the same C-terminal DNA binding domain except LAP has an extra N-terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, LIP can inhibit LAP transcriptional activity even at substoichiometric levels. However, the regulatory mechanism of C/EBPß gene expression and the LAP: LIP ratio is unclear. METHODS: In this study, the C/EBPß promoter sequence was scanned for conserved P53 response element (P53RE), and binding of P53 to the C/EBPß promoter was tested by Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation assay. P53 over-expression and dominant negative P53 expression plasmids were transfected into rat lung fibroblasts and tested for C/EBPß gene transcription and expression. Western blot analysis was used to test the regulation of C/EBPß LAP and LIP isoforms. Constructs containing the LAP 5'untranslated region (5'UTR) or the LIP 5'UTR region were used to test the importance of 5'UTR in the control of C/EBPß LAP and LIP translation. RESULTS: The C/EBPß promoter sequence was found to contain a conserved P53 response element (P53RE), which binds P53 as demonstrated by Electrophoresis Mobility Shift Assay and chromatin immunoprecipitation assays. P53 over-expression suppressed while dominant negative P53 stimulated C/EBPß gene transcription and expression. Western blot analysis showed that P53 differentially regulated the translation of the C/EBPß LAP and LIP isoforms through the regulation of eIF4E and eIF4E-BP1. Further studies with constructs containing the LAP 5'untranslated region (5'UTR) or the LIP 5'UTR region showed that the 5'UTR is important in differential control of C/EBPß LAP and LIP translation. CONCLUSION: Analysis of the effects of P53 on C/EBPß expression revealed a novel mechanism by which P53 could antagonize the effects of C/EBPß on its target gene expression. For the first time, P53 is shown to be a repressor of C/EBPß gene expression at both transcriptional and translational levels, with a differential effect in the magnitude of the effect on LAP vs. LIP isoforms.