Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Inhibitors of eIF4G1-eIF1 uncover its regulatory role of ER/UPR stress-response genes independent of eIF2α-phosphorylation.

During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation in vitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.

Inhibition of eIF6 Activity Reduces Hepatocellular Carcinoma Growth: An In Vivo and In Vitro Study.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.

Inhibitors of Eukaryotic Translational Machinery as Therapeutic Agents.

Inhibiting eukaryotic protein translation with small molecules is emerging as a powerful therapeutic strategy. The advantage of targeting cellular translational machinery is that it is required for the highly proliferative state of many neoplastic cells, replication of certain viruses, and ultimately the expression of a wide variety of protein targets. Although, this approach has been exploited to develop clinical agents, such as homoharringtonine (HHT, 1), used to treat chronic myeloid leukemia (CML), inhibiting components of the translational machinery is often associated with cytotoxic phenotypes. However, recent studies have demonstrated that certain small molecules can inhibit the translation of specific subsets of proteins, leading to lower cytotoxicity, and opening-up therapeutic opportunities for translation inhibitors to be deployed in indications beyond oncology and infectious disease. This review summarizes efforts to develop inhibitors of the eukaryotic translational machinery as therapeutic agents and highlights emerging opportunities for translation inhibitors in the future.

A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines.

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

Berberine Represses ß-Catenin Translation Involving 4E-BPs in Hepatocellular Carcinoma Cells.

Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of ß-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.

miR-331-3p Inhibits Proliferation and Promotes Apoptosis of Nasopharyngeal Carcinoma Cells by Targeting elf4B-PI3K-AKT Pathway.

BACKGROUND: The incidence of nasopharyngeal carcinoma is increasing gradually, but the pathogenesis is not completely clear. MicroRNA, a highly conserved endogenous noncoding small molecule RNA, plays an essential role in the regulation of gene expression and is a hotspot in cancer research worldwide. OBJECTIVES: Although previous studies have confirmed that the abnormal expression of microRNAs is closely related to the progression of nasopharyngeal carcinoma, the role of miRNA-331-3p in nasopharyngeal carcinoma has not been studied. The purpose of this study was to explore the role and mechanism of miRNA-331-3p in the progression of nasopharyngeal carcinoma. MATERIALS AND METHODS: Real-time quantitative reverse transcription polymerase chain reaction was performed to detect the expression of miRNA-331-3p in nasopharyngeal carcinoma clinical samples and cell lines (CNE-1 and 5-8F cells). After overexpression of miRNA-331-3p in CNE-1 cells, cell proliferation was measured by Cell Counting Kit-8 assay, cell invasion was detected by Transwell assay, and apoptosis was tested by flow cytometry. In addition, the dual-luciferase reporter assay was used to identify the target gene of miRNA-331-3p and Western blotting was performed to measure the relative protein expression. RESULTS: The expression of miRNA-331-3p in nasopharyngeal carcinoma clinical samples and cells was decreased significantly. Overexpression of miRNA-331-3p markedly inhibited the proliferation and invasion of CNE-1 cells and promoted cell apoptosis. Moreover, overexpression of miRNA-331-3p reduced the expression of target gene elF4B, leading to inhibition of the phosphorylation of Phosphoinositide 3-kinase (PI3K) and Serine/ threonine kinase (AKT). CONCLUSION: miRNA-331-3p inhibited cell proliferation and induced cell apoptosis in nasopharyngeal carcinoma by targeting elF4B gene and then blocked the PI3K-AKT signaling pathway. SIGNIFICANCE: The role of miRNA-331-3p in the development of NPC and its mechanism provide new ideas for the treatment of nasopharyngeal carcinoma.

Isolation and Culture of Primary Mouse Retinal Pigment Epithelial (RPE) Cells with Rho-Kinase and TGFßR-1/ALK5 Inhibitor.

BACKGROUND Primary RPE cells could be a reliable model for representing in vivo status of RPE compared with cell lines. We present a protocol for in vitro isolation and culture of primary RPE cells from C57BL mice. MATERIAL AND METHODS We used C57BL mice ages 7 days to 4 months. The RPE layer was separated from the neural retina layer by digestion with 2% Dispase for 45 min and scraped off from the choroid after 25-min incubation in 37°C. Collected RPE sheets were gently pipetted up into smaller sheets. RPE sheets were transferred into well plates and cultured in vitro for 2 weeks. To inhibit epithelial-mesenchymal transition (EMT) of RPE cells, we used Y27632 and Repsox to treat cultured primary RPE cells. RESULTS RPE cells isolated from C57BL mice maintained pigmented and hexagonal morphology in culture. However, long-term in vitro culture lead to the periphery cells of a RPE sheet becoming mesenchymal-like cells. In contrast to the control group, Y27632 and Repsox, which are inhibitors of Rho-kinase or TGFßR-1/ALK5, promoted primary RPE cells to maintain epithelial-like morphology and eventually become confluent. CONCLUSIONS RPE cells isolated from C57BL mice could be a powerful cell model to study the biological function of RPE. Especially, C57BL mice with different defective genetic background resulting in ocular diseases, would expand the genome type of RPE cells. The method presented here could be an efficient and applicable technique to obtain large numbers of primary RPE cells that maintain some characteristics of in vivo RPE.

Synthesis facilitates an understanding of the structural basis for translation inhibition by the lissoclimides.

The lissoclimides are unusual succinimide-containing labdane diterpenoids that were reported to be potent cytotoxins. Our short semisynthesis and analogue-oriented synthesis approaches provide a series of lissoclimide natural products and analogues that expand the structure-activity relationships (SARs) in this family. The semisynthesis approach yielded significant quantities of chlorolissoclimide (CL) to permit an evaluation against the National Cancer Institute's 60-cell line panel and allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. Although it shares a binding site with other imide-based natural product translation inhibitors, CL engages in a particularly interesting and novel face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis provides many more lissoclimide compounds, which were tested against aggressive human cancer cell lines and for protein synthesis inhibitory activity. Finally, computational modelling was used to explain the SARs of certain key compounds and set the stage for the structure-guided design of better translation inhibitors.

eIF1 modulates the recognition of suboptimal translation initiation sites and steers gene expression via uORFs.

Alternative translation initiation mechanisms such as leaky scanning and reinitiation potentiate the polycistronic nature of human transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by the eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4000 proteins and 10 000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. Here, the stringency of start codon selection and preference for an optimal nucleotide context were largely diminished leading to translational upregulation of uORFs with suboptimal start. Interestingly, genes affected by eIF1 deprivation were implicated in energy production and sensing of metabolic stress.

Overexpression of miR-519d in lung adenocarcinoma inhibits cell proliferation and invasion via the association of eIF4H.

Lung cancer is one of the deadliest types of cancer worldwide due to its high mortality rate. Adenocarcinoma constitutes 20%-30% of all lung cancers. In recent years, studies on the mechanisms of lung tumorigenesis and development have in part focused on the microRNAs for their crucial role in the progress of different cancers. As for our study, we demonstrated that miR-519d was differently downregulated and eIF4H was significantly overexpressed in lung adenocarcinoma via the detection of quantitative real-time polymerase chain reaction compared with the adjacent normal tissues. Furthermore, Cell Counting Kit-8 assay, colony formation assay, xenograft tumor experiment, Ki67 immunohistochemistry assay and transwell assay were performed to explain that the upregulated miR-519d could inhibit the proliferation and invasion of A549 and H1299 cells. To further advance our understanding of the mechanisms of miR-519d, we performed the bioinformatics analysis and the luciferase report assay. The results from these procedures revealed eIF4H to be one of the targets of miR-519d. Downregulated eIF4H was analogous to the overexpressed miR-519d obtained from miR-519d agomir and si-eIF4H transfection. In summary, it can be concluded that miR-519d targets eIF4H in lung adenocarcinoma to inhibit cell proliferation and invasion. This mechanism may offer new insights into the tumorigenesis and development of lung adenocarcinoma.

Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival.

The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK-eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK-eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival.

Crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis.

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.

4E-BPs Control Fat Storage by Regulating the Expression of Egr1 and ATGL.

Early growth response transcription factor Egr1 controls multiple aspects of cell physiology and metabolism. In particular, Egr1 suppresses lipolysis and promotes fat accumulation in adipocytes by inhibiting the expression of adipose triglyceride lipase. According to current dogma, regulation of the Egr1 expression takes place primarily at the level of transcription. Correspondingly, treatment of cultured adipocytes with insulin stimulates expression of Egr1 mRNA and protein. Unexpectedly, the MEK inhibitor PD98059 completely blocks insulin-stimulated increase in the Egr1 mRNA but has only a moderate effect on the Egr1 protein. At the same time, mTORC1 inhibitors rapamycin and PP242 suppress expression of the Egr1 protein and have an opposite effect on the Egr1 mRNA. Mouse embryonic fibroblasts with genetic ablations of TSC2 or 4E-BP1/2 express less Egr1 mRNA but more Egr1 protein than wild type controls. (35)S-labeling has confirmed that translation of the Egr1 mRNA is much more effective in 4E-BP1/2-null cells than in control. A selective agonist of the CB1 receptors, ACEA, up-regulates Egr1 mRNA, but does not activate mTORC1 and does not increase Egr1 protein in adipocytes. These data suggest that although insulin activates both the Erk and the mTORC1 signaling pathways in adipocytes, regulation of the Egr1 expression takes place predominantly via the mTORC1/4E-BP-mediated axis. In confirmation of this model, we show that 4E-BP1/2-null MEFs express less ATGL and accumulate more fat than control cells, while knock down of Egr1 in 4E-BP1/2-null MEFs increases ATGL expression and decreases fat storage.

Deficiency in either 4E-BP1 or 4E-BP2 augments innate antiviral immune responses.

Genetic deletion of both 4E-BP1 and 4E-BP2 was found to protect cells against viral infections. Here we demonstrate that the individual loss of either 4E-BP1 or 4E-BP2 in mouse embryonic fibroblasts (MEFs) is sufficient to confer viral resistance. shRNA-mediated silencing of 4E-BP1 or 4E-BP2 renders MEFs resistant to viruses, and compared to wild type cells, MEFs knockout for either 4E-BP1 or 4E-BP2 exhibit enhanced translation of Irf-7 and consequently increased innate immune response to viruses. Accordingly, the replication of vesicular stomatitis virus, encephalomyocarditis virus, influenza virus and Sindbis virus is markedly suppressed in these cells. Importantly, expression of either 4E-BP1 or 4E-BP2 in double knockout or respective single knockout cells diminishes their resistance to viral infection. Our data show that loss of 4E-BP1 or 4E-BP2 potentiates innate antiviral immunity. These results provide further evidence for translational control of innate immunity and support targeting translational effectors as an antiviral strategy.

Small RNA expression from the human macrosatellite DXZ4.

Small noncoding RNAs play several roles in regulating gene expression. In the nucleus, small RNA-Argonaute complexes recruit epigenetic modifying activities to genomic sites. This pathway has been described in mammals primarily for the germline; however, its role in somatic cells is less characterized. Here, we describe in human somatic cells a potential link between the expression of small RNAs from the macrosatellite DXZ4 and Argonaute-dependent DNA methylation of this locus. DXZ4 was found to express a wide range of small RNAs potentially representing several classes of small RNAs. A subpopulation of these RNAs is bound by Argonaute. Moreover, we show AGO association with DXZ4 and that the Argonaute proteins AGO-1 and PIWIL4 may play a role in DNA methylation of DXZ4. We hypothesize that the RNAs are involved in Argonaute-dependent methylation of DXZ4 DNA.

7-Ketocholesterol inhibits isocitrate dehydrogenase 2 expression and impairs endothelial function via microRNA-144.

Oxysterol is associated with the induction of endothelial oxidative stress and impaired endothelial function. Mitochondria play a central role in oxidative energy metabolism and the maintenance of proper redox status. The purpose of this study was to determine the effects and mechanisms of 7-ketocholesterol (7-KC) on isocitrate dehydrogenase 2 (IDH2) and its impact on endothelial function in both human aortic endothelial cells (HAECs) and C57BL/6J mice. HAECs treated with 7-KC showed significant reductions of IDH2 mRNA and protein levels and enzyme activity, leading to decreased NADPH concentration and an increased ratio of reduced-to-oxidized glutathione in the mitochondria. 7-KC induced the expression of a specific microRNA, miR-144, which in turn targets and downregulates IDH2. In silico analysis predicted that miR-144 could bind to the 3'-untranslated region of IDH2 mRNA. Overexpression of miR-144 decreased the expression of IDH2 and the levels of NADPH. A complementary finding is that a miR-144 inhibitor increased the mRNA and protein expression levels of IDH2. Furthermore, miR-144 level was elevated in HAECs in response to 7-KC. Anti-Ago1/2 immunoprecipitation coupled with a real-time polymerase chain reaction assay revealed that 7-KC increased the functional targeting of miR-144/IDH2 mRNA in HAECs. Infusion of 7-KC in vivo decreased vascular IDH2 expression and impaired vascular reactivity via miR-144. 7-KC controls miR-144 expression, which in turn decreases IDH2 expression and attenuates NO bioavailability to impair endothelial homeostasis. The newly identified 7-KC-miR-144-IDH2 pathway may contribute to atherosclerosis progression and provides new insight into 7-KC function and microRNA biology in cardiovascular disease.

mTORC1 targets the translational repressor 4E-BP2, but not S6 kinase 1/2, to regulate neural stem cell self-renewal in vivo.

The mammalian target of rapamycin complex 1 (mTORC1) integrates signals important for cell growth, and its dysregulation in neural stem cells (NSCs) is implicated in several neurological disorders associated with abnormal neurogenesis and brain size. However, the function of mTORC1 on NSC self-renewal and the downstream regulatory mechanisms are ill defined. Here, we found that genetically decreasing mTORC1 activity in neonatal NSCs prevented their differentiation, resulting in reduced lineage expansion and aborted neuron production. Constitutive activation of the translational repressor 4E-BP1, which blocked cap-dependent translation, had similar effects and prevented hyperactive mTORC1 induction of NSC differentiation and promoted self-renewal. Although 4E-BP2 knockdown promoted NSC differentiation, p70 S6 kinase 1 and 2 (S6K1/S6K2) knockdown did not affect NSC differentiation but reduced NSC soma size and prevented hyperactive mTORC1-induced increase in soma size. These data demonstrate a crucial role of mTORC1 and 4E-BP for switching on and off cap-dependent translation in NSC differentiation.

Synthesis of eukaryotic translation elongation inhibitor lactimidomycin via Zn(II)-mediated Horner-Wadsworth-Emmons macrocyclization.

An enantioselective synthesis of potent eukaryotic translation elongation inhibitor lactimidomycin has been accomplished in 21 linear steps. This synthesis features a Zn(II)-mediated Horner-Wadsworth-Emmons reaction that could be executed on a large scale to provide the highly strained 12-membered lactimidomycin macrolactone.

UVA, UVB and UVC induce differential response signaling pathways converged on the eIF2α phosphorylation.

It is clear that solar UV irradiation is a crucial environmental factor resulting in skin diseases partially through activation of cell signaling toward altered gene expression and reprogrammed protein translation. Such a key translational control mechanism is executed by the eukaryotic initiation factor 2α subunit (eIF2α) and the downstream events provoked by phosphorylation of eIF2α at Ser(51) are clearly understood, but the upstream signaling mechanisms on the eIF2α-Ser(51) phosphorylation responses to different types of UV irradiations, namely UVA, UVB and UVC, are still not well elucidated. Herein, our evidence reveals that UVA, UVB and UVC all induce a dose- and time-dependent phosphorylation of eIF2α-Ser(51) through distinct signaling mechanisms. UVA-induced eIF2α phosphorylation occurs through MAPKs, including ERKs, JNKs and p38 kinase, and phosphatidylinositol (PI)-3 kinase. By contrast, UVB-induced eIF2α phosphorylation is through JNKs and p38 kinase, but not ERKs or PI-3 kinase, whereas UVC-stimulated response to eIF2α phosphorylation is via JNKs alone. Furthermore, we have revealed that ATM is involved in induction of the intracellular responses to UVA and UVB, rather than UVC. These findings demonstrate that wavelength-specific UV irradiations activate differential response signaling pathways converged on the eIF2α phosphorylation. Importantly, we also show evidence that a direct eIF2α kinase PKR is activated though phosphorylation by either RSK1 or MSK1, two downstream kinases of MAPKs/PI-3 kinase-mediated signaling pathways.