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Sample records for 4e eif4e-binding protein

  1. eIF4E binding protein 1 expression is associated with clinical survival outcomes in colorectal cancer

    PubMed Central

    Chao, Min-Wu; Wang, Li-Ting; Lai, Chin-Yu; Yang, Xiao-Ming; Cheng, Ya-Wen; Lee, Kuo-Hsiung

    2015-01-01

    eIF4E binding protein 1 (4E-BP1), is critical for cap-dependent and cap-independent translation. This study is the first to demonstrate that 4E-BP1 expression correlates with colorectal cancer (CRC) progression. Compared to its expression in normal colon epithelial cells, 4E-BP1 was upregulated in CRC cell lines and was detected in patient tumor tissues. Furthermore, high 4E-BP1 expression was statistically associated with poor prognosis. Hypoxia has been considered as an obstacle for cancer therapeutics. Our previous data showed that YXM110, a cryptopleurine derivative, exhibited anticancer activity via 4E-BP1 depletion. Here, we investigated whether YXM110 could inhibit protein synthesis under hypoxia. 4E-BP1 expression was notably decreased by YXM110 under hypoxic conditions, implying that cap-independent translation could be suppressed by YXM110. Moreover, YXM110 repressed hypoxia-inducible factor 1α (HIF-1α) expression, which resulted in decreased downstream vascular endothelial growth factor (VEGF) expression. These observations highlight 4E-BP1 as a useful biomarker and therapeutic target, indicating that YXM110 could be a potent CRC therapeutic drug. PMID:26204490

  2. Embryonic-stage-dependent changes in the level of eIF4E-binding proteins during early development of sea urchin embryos.

    PubMed

    Salaün, Patrick; Boulben, Sandrine; Mulner-Lorillon, Odile; Bellé, Robert; Sonenberg, Nahum; Morales, Julia; Cormier, Patrick

    2005-04-01

    The eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) inhibit translation initiation by binding eIF4E and preventing recruitment of the translation machinery to mRNA. We have previously shown that fertilization of sea urchin eggs triggers eIF4E-4E-BP complex dissociation and 4E-BP degradation. Here, we show that microinjection of eIF4E-binding motif peptide into unfertilized eggs delays the onset of the first mitosis triggered by fertilization, demonstrating that dissociation of the eIF4E-4E-BP complex is functionally important for the first mitotic division in sea urchin embryos. We also show by gel filtration analyses that eIF4E is present in unfertilized eggs as an 80 kDa molecular mass complex containing 4E-BP and a new 4E-BP of 40 kDa. Fertilization triggers the dissociation of eIF4E from these two 4E-BPs and triggers the rapid recruitment of eIF4E into a high-molecular-mass complex. Release of eIF4E from the two 4E-BPs is correlated with a decrease in the total level of both 4E-BPs following fertilization. Abundance of the two 4E-BPs has been monitored during embryonic development. The level of the two proteins remains very low during the rapid cleavage stage of early development and increases 8 hours after fertilization. These results demonstrate that these two 4E-BPs are down- and upregulated during the embryonic development of sea urchins. Consequently, these data suggest that eIF4E availability to other partners represents an important determinant of the early development of sea urchin embryos.

  3. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    PubMed

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  4. Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins

    PubMed Central

    Cridge, Andrew G.; Castelli, Lydia M.; Smirnova, Julia B.; Selley, Julian N.; Rowe, William; Hubbard, Simon J.; McCarthy, John E.G.; Ashe, Mark P.; Grant, Christopher M.; Pavitt, Graham D.

    2010-01-01

    eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3′-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought. PMID:20705650

  5. Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

    SciTech Connect

    Groppo, Rachel; Brown, Bradley A.; Palmenberg, Ann C.

    2011-02-05

    Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C{sup pro}. Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

  6. eIF4E-binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs.

    PubMed

    Oulhen, Nathalie; Mulner-Lorillon, Odile; Cormier, Patrick

    2010-01-01

    Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap-binding protein eIF4E, which is released from its repressor 4E-BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E-BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E-BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E-BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E-BP and eIF4G.

  7. Dietary choline modulates immune responses, and gene expressions of TOR and eIF4E-binding protein2 in immune organs of juvenile Jian carp (Cyprinus carpio var. Jian).

    PubMed

    Wu, Pei; Jiang, Jun; Liu, Yang; Hu, Kai; Jiang, Wei-Dan; Li, Shu-Hong; Feng, Lin; Zhou, Xiao-Qiu

    2013-09-01

    The present work evaluates the effects of various levels of dietary choline on immune parameters, immune-related gene expression and protection against Aeromonas hydrophila (AH) in juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed with six different experimental diets containing graded levels of choline at 165 (choline-deficient control), 310, 607, 896, 1167 and 1820 mg kg(-1) diet for 65 days. At the end of the feeding trail, Fish were challenged with AH and mortalities were recorded over 17 days. Dietary choline significantly enhanced spleen and head kidney weights, spleen index, red blood cell and white blood cell counts, and intestinal Lactobacillus counts of juvenile Jian carp; whereas, intestinal Escherichia coli and A. hydrophila counts decreased. Moreover, the post-challenge survival rate, leucocyte phagocytic capacity, serum lysozyme and acid phosphatase activities, hemagglutination titer, complement 3 and 4 contents, immunoglobulin M content, and anti-AH antibody titer were significantly enhanced by choline and the lowest in choline-deficient group, while serum total iron-binding capacity was the highest in choline-deficient group. The relative gene expressions of interleukin 10 in spleen and head kidney, target of rapamycin (TOR) in spleen and eIF4E-binding protein2 (4E-BP2) in head kidney significantly increased with increasing of dietary choline up to a certain point. However, the relative gene expressions of interleukin 1β, tumor necrosis factor α and transforming growth factor β2 in spleen and head kidney, TOR in head kidney and 4E-BP2 in spleen significantly decreased. In conclusion, dietary choline improved disease resistance, enhanced the immune function, and regulated immune-related gene expression of juvenile Jian carp.

  8. Effects of simian virus 40 large and small tumor antigens on mammalian target of rapamycin signaling: small tumor antigen mediates hypophosphorylation of eIF4E-binding protein 1 late in infection.

    PubMed

    Yu, Yongjun; Kudchodkar, Sagar B; Alwine, James C

    2005-06-01

    We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.

  9. Effects of Simian Virus 40 Large and Small Tumor Antigens on Mammalian Target of Rapamycin Signaling: Small Tumor Antigen Mediates Hypophosphorylation of eIF4E-Binding Protein 1 Late in Infection

    PubMed Central

    Yu, Yongjun; Kudchodkar, Sagar B.; Alwine, James C.

    2005-01-01

    We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen. PMID:15890927

  10. Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

    PubMed

    Steiner, Jennifer L; Pruznak, Anne M; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R; Lang, Charles H

    2014-01-01

    Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

  11. Structural insights into parasite eIF4E binding specificity for m7G and m2,2,7G mRNA caps.

    PubMed

    Liu, Weizhi; Zhao, Rui; McFarland, Craig; Kieft, Jeffrey; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Stepinski, Janusz; Darzynkiewicz, Edward; Jones, David N M; Davis, Richard E

    2009-11-06

    The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m(7)G) or a trimethylguanosine (m(2,2,7)G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m(7)G and m(2,2,7)G caps. The eIF4E.m(7)GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m(7)GpppG and m(2,2,7)GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m(7)G versus m(2,2,7)G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m(2,2,7)G cap.

  12. Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress

    PubMed Central

    Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

    2017-01-01

    Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress. DOI: http://dx.doi.org/10.7554/eLife.21907.001 PMID:28177284

  13. Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

    PubMed

    Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

    2017-02-08

    Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

  14. Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity.

    PubMed

    Coleman, L J; Peter, M B; Teall, T J; Brannan, R A; Hanby, A M; Honarpisheh, H; Shaaban, A M; Smith, L; Speirs, V; Verghese, E T; McElwaine, J N; Hughes, T A

    2009-05-05

    Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

  15. Interaction of the eukaryotic initiation factor 4E with 4E-BP2 at a dynamic bipartite interface.

    PubMed

    Lukhele, Sabelo; Bah, Alaji; Lin, Hong; Sonenberg, Nahum; Forman-Kay, Julie D

    2013-12-03

    Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). Whereas the binding of 4E-BP peptides containing the eIF4E-binding ⁵⁴YXXXXLΦ⁶⁰ motif has been studied, atomic-level characterization of the interaction of eIF4E with full-length 4E-BPs has been lacking. Here, we use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to characterize the dynamic, structural and binding properties of 4E-BP2. Although disordered, 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical ⁵⁴YXXXXLΦ⁶⁰ and ⁷⁸IPGVT⁸² sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. This dynamic interaction facilitates exposure of regulatory phosphorylation sites within the complex. The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.

  16. Glycogen synthase kinase-3β positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1.

    PubMed

    Shin, S; Wolgamott, L; Tcherkezian, J; Vallabhapurapu, S; Yu, Y; Roux, P P; Yoon, S-O

    2014-03-27

    Protein synthesis has a key role in the control of cell proliferation, and its deregulation is associated with pathological conditions, notably cancer. Rapamycin, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), was known to inhibit protein synthesis. However, it does not substantially inhibit protein synthesis and cell proliferation in many cancer types. We were interested in finding a novel target in rapamycin-resistant cancer. The rate-limiting factor for translation is eukaryotic translation initiation factor 4E (eIF4E), which is negatively regulated by eIF4E-binding protein 1 (4E-BP1). Here, we provide evidence that glycogen synthase kinase (GSK)-3β promotes cell proliferation through positive regulation of protein synthesis. We found that GSK-3β phosphorylates and inactivates 4E-BP1, thereby increasing eIF4E-dependent protein synthesis. Considering the clinical relevance of pathways regulating protein synthesis, our study provides a promising new strategy and target for cancer therapy.

  17. Distinct Features of Cap Binding by eIF4E1b Proteins

    PubMed Central

    Kubacka, Dorota; Miguel, Ricardo Núñez; Minshall, Nicola; Darzynkiewicz, Edward; Standart, Nancy; Zuberek, Joanna

    2015-01-01

    eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m7GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α + β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N7 of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N7 position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding. PMID:25463438

  18. After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E.

    PubMed

    Oulhen, Nathalie; Salaün, Patrick; Cosson, Bertrand; Cormier, Patrick; Morales, Julia

    2007-02-01

    Release of eukaryotic initiation factor 4E (eIF4E) from its translational repressor eIF4E-binding protein (4E-BP) is a crucial event for the first mitotic division following fertilization of sea urchin eggs. Finding partners of eIF4E following fertilization is crucial to understand how eIF4E functions during this physiological process. The isolation and characterization of cDNA encoding Sphaerechinus granularis eIF4G (SgIF4G) are reported. mRNA of SgIF4G is present as a single 8.5-kb transcript in unfertilized eggs, suggesting that only one ortholog exists in echinoderms. The longest open reading frame predicts a sequence of 5235 nucleotides encoding a deduced polypeptide of 1745 amino acids with a predicted molecular mass of 192 kDa. Among highly conserved domains, SgIF4G protein possesses motifs that correspond to the poly(A) binding protein and eIF4E protein-binding sites. A specific polyclonal antibody was produced and used to characterize the SgIF4G protein in unfertilized and fertilized eggs by SDS-PAGE and western blotting. Multiple differentially migrating bands representing isoforms of sea urchin eIF4G are present in unfertilized eggs. Fertilization triggers modifications of the SgIF4G isoforms and rapid formation of the SgIF4G-eIF4E complex. Whereas rapamycin inhibits the formation of the SgIF4G-eIF4E complex, modification of these SgIF4G isoforms occurs independently from the rapamycin-sensitive pathway. Microinjection of a peptide corresponding to the eIF4E-binding site derived from the sequence of SgIF4G into unfertilized eggs affects the first mitotic division of sea urchin embryos. Association of SgIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization, suggesting that cap-dependent translation is highly regulated during this process. This hypothesis is strengthened by the evidence that microinjection of the cap analog m(7)GDP into unfertilized eggs inhibits the first mitotic division.

  19. Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP.

    PubMed

    Ruoff, Rachel; Katsara, Olga; Kolupaeva, Victoria

    2016-07-05

    Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

  20. eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo.

    PubMed

    Salaün, Patrick; Pyronnet, Stéphane; Morales, Julia; Mulner-Lorillon, Odile; Bellé, Robert; Sonenberg, Nahum; Cormier, Patrick

    2003-03-15

    The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.

  1. Eap1p, a Novel Eukaryotic Translation Initiation Factor 4E-Associated Protein in Saccharomyces cerevisiae

    PubMed Central

    Cosentino, Gregory P.; Schmelzle, Tobias; Haghighat, Ashkan; Helliwell, Stephen B.; Hall, Michael N.; Sonenberg, Nahum

    2000-01-01

    Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m7G(5′)ppp(5′)N, where N is any nucleotide] present at the 5′ termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae. PMID:10848587

  2. Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP

    PubMed Central

    Ruoff, Rachel; Katsara, Olga; Kolupaeva, Victoria

    2016-01-01

    Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type–dependent manner, with 4E-BP1 being a key player. PMID:27313212

  3. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3

    PubMed Central

    Tsukumo, Yoshinori; Alain, Tommy; Fonseca, Bruno D.; Nadon, Robert; Sonenberg, Nahum

    2016-01-01

    Targeting mTORC1 is a highly promising strategy in cancer therapy. Suppression of mTORC1 activity leads to rapid dephosphorylation of eIF4E-binding proteins (4E-BP1–3) and subsequent inhibition of mRNA translation. However, how the different 4E-BPs affect translation during prolonged use of mTOR inhibitors is not known. Here we show that the expression of 4E-BP3, but not that of 4E-BP1 or 4E-BP2, is transcriptionally induced during prolonged mTORC1 inhibition in vitro and in vivo. Mechanistically, our data reveal that 4E-BP3 expression is controlled by the transcription factor TFE3 through a cis-regulatory element in the EIF4EBP3 gene promoter. CRISPR/Cas9-mediated EIF4EBP3 gene disruption in human cancer cells mitigated the inhibition of translation and proliferation caused by prolonged treatment with mTOR inhibitors. Our findings show that 4E-BP3 is an important effector of mTORC1 and a robust predictive biomarker of therapeutic response to prolonged treatment with mTOR-targeting drugs in cancer. PMID:27319316

  4. eIF4E Is an Important Determinant of Adhesion and Pseudohyphal Growth of the Yeast S. cerevisiae

    PubMed Central

    Altmann, Michael

    2012-01-01

    eIF4E, the cytoplasmatic cap-binding protein, is required for efficient cap-dependent translation. We have studied the influence of mutations that alter the activity and/or expression level of eIF4E on haploid and diploid cells in the yeast S. cerevisiae. Temperature-sensitive eIF4E mutants with reduced levels of expression and reduced cap-binding affinity clearly show a loss in haploid adhesion and diploid pseudohyphenation upon starvation for nitrogen. Some of these mutations affect the interaction of the cap-structure of mRNAs with the cap-binding groove of eIF4E. The observed reduction in adhesive and pseudohyphenating properties is less evident for an eIF4E mutant that shows reduced interaction with p20 (an eIF4E-binding protein) or for a p20-knockout mutant. Loss of adhesive and pseudohyphenating properties was not only observed for eIF4E mutants but also for knockout mutants of components of eIF4F such as eIF4B and eIF4G1. We conclude from these experiments that mutations that affect components of the eIF4F-complex loose properties such as adhesion and pseudohyphal differentiation, most likely due to less effective translation of required mRNAs for such processes. PMID:23226381

  5. RNAi of the translation inhibition gene 4E-BP identified from the hard tick, Haemaphysalis longicornis, affects lipid storage during the off-host starvation period of ticks.

    PubMed

    Kume, Aiko; Boldbaatar, Damdinsuren; Takazawa, Yuko; Umemiya-Shirafuji, Rika; Tanaka, Tetsuya; Fujisaki, Kozo

    2012-08-01

    4E-BP, an eIF4E-binding protein, is well known as a cap-dependent translation inhibitor. Here, the 4E-BP homolog, Hl4E-BP, was isolated and identified from the hard tick Haemaphysalis longicornis. Hl4E-BP transcripts were ubiquitously expressed in the active stages, including the larvae, nymphs, and female adults, and the transcription levels were found to be higher in unfed than engorged ticks. In contrast, the expression levels of non-phosphorylated Hl4E-BP, which is a 13.4-kDa protein detected by the anti-recombinant Hl4E-BP antibody, were the highest in engorged ticks and significantly decreased progressively during the unfed starvation period of ticks. The functional role of Hl4E-BP as a metabolic brake was verified by histochemical observations on the lipid storage in midguts and fat bodies during the starvation period using ticks injected with dsHl4E-BP. The results indicate that Hl4E-BP is highly relevant to the lipid storage of ticks during the non-feeding starvation period. Our results suggest, for the first time, that Hl4E-BP may have a crucial role in the starvation resistance of ticks in an off-host condition via lipid metabolism control, although it was unclear whether Hl4E-BP might be involved in lipid synthesis regulation and/or lipid consumption inhibition.

  6. Insulin signaling controls neurotransmission via the 4eBP-dependent modification of the exocytotic machinery

    PubMed Central

    Mahoney, Rebekah Elizabeth; Azpurua, Jorge; Eaton, Benjamin A

    2016-01-01

    Altered insulin signaling has been linked to widespread nervous system dysfunction including cognitive dysfunction, neuropathy and susceptibility to neurodegenerative disease. However, knowledge of the cellular mechanisms underlying the effects of insulin on neuronal function is incomplete. Here, we show that cell autonomous insulin signaling within the Drosophila CM9 motor neuron regulates the release of neurotransmitter via alteration of the synaptic vesicle fusion machinery. This effect of insulin utilizes the FOXO-dependent regulation of the thor gene, which encodes the Drosophila homologue of the eif-4e binding protein (4eBP). A critical target of this regulatory mechanism is Complexin, a synaptic protein known to regulate synaptic vesicle exocytosis. We find that the amounts of Complexin protein observed at the synapse is regulated by insulin and genetic manipulations of Complexin levels support the model that increased synaptic Complexin reduces neurotransmission in response to insulin signaling. DOI: http://dx.doi.org/10.7554/eLife.16807.001 PMID:27525480

  7. The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    PubMed Central

    Castelli, Lydia M.; Talavera, David; Kershaw, Christopher J.; Mohammad-Qureshi, Sarah S.; Costello, Joseph L.; Rowe, William; Sims, Paul F. G.; Grant, Christopher M.; Hubbard, Simon J.; Ashe, Mark P.; Pavitt, Graham D.

    2015-01-01

    Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5’cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3’ UTR motif. Caf20p binds all tested motif-containing 3’ UTRs. Caf20p and the 3’UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3’UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the

  8. Polycomb silencing of the Drosophila 4E-BP gene regulates imaginal disc cell growth

    PubMed Central

    Mason-Suares, Heather; Tie, Feng; Yan, Christopher; Harte, Peter J.

    2015-01-01

    Polycomb group (PcG) proteins are best known for their role in maintaining stable, mitotically heritable silencing of the homeotic (HOX) genes during development. In addition to loss of homeotic gene silencing, some PcG mutants also have small imaginal discs. These include mutations in E(z), Su(z)12, esc and escl, which encode Polycomb Repressive Complex 2 (PRC2) subunits. The cause of this phenotype is not known, but the human homologs of PRC2 subunits have been shown to play a role in cell proliferation, are over-expressed in many tumors, and appear to be required for tumor proliferation. Here we show that the small imaginal disc phenotype arises, at least in part, from a cell growth defect. In homozygous E(z) mutants, imaginal disc cells are smaller than cells in normally proliferating discs. We show that the Thor gene, which encodes eIF4E-Binding Protein (4E-BP), the evolutionarily conserved inhibitor of cap-dependent translation and potent inhibitor of cell growth, is involved in the development of this phenotype. The Thor promoter region contains DNA binding motifs for transcription factors found in well-characterized Polycomb Response Elements (PREs), including PHO/PHOL, GAGA Factor, and others, suggesting that Thor may be a direct target of Polycomb silencing. We present chromatin immunoprecipitation evidence that PcG proteins are bound to the Thor 5’ region in vivo. The Thor gene is normally repressed in imaginal discs, but Thor mRNA and 4E-BP protein levels are elevated in imaginal discs of PRC2 subunit mutant larvae. Deletion of the Thor gene in E(z) mutants partially restores imaginal disc size toward wild-type and results in an increase in the fraction of larvae that pupariate. These results thus suggest that PcG proteins can directly modulate cell growth in Drosophila, in part by regulating Thor expression. PMID:23523430

  9. Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

    SciTech Connect

    Sekiyama, Naotaka; Arthanari, Haribabu; Papadopoulos, Evangelos; Rodriguez-Mias, Ricard A.; Wagner, Gerhard; Léger-Abraham, Mélissa

    2015-07-13

    The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54YXXXXLΦ60 (motif 1) but lack known phosphorylation sites. We report in this paper a 2.1-Å crystal structure of mouse eIF4E in complex with m7GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150–84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1–mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. Finally, the binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

  10. Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

    DOE PAGES

    Sekiyama, Naotaka; Arthanari, Haribabu; Papadopoulos, Evangelos; ...

    2015-07-13

    The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest formore » cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54YXXXXLΦ60 (motif 1) but lack known phosphorylation sites. We report in this paper a 2.1-Å crystal structure of mouse eIF4E in complex with m7GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150–84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1–mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. Finally, the binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.« less

  11. 4EGI-1 induces apoptosis and enhances radiotherapy sensitivity in nasopharyngeal carcinoma cells via DR5 induction on 4E-BP1 dephosphorylation

    PubMed Central

    Wen, Qiuyuan; Luo, Jiadi; Chu, Shuzhou; Chen, Lingjiao; Qing, Zhenzhen; Xie, Guiyuan; Xu, Lina; Alnemah, Mohannad Ma; Li, Meirong; Fan, Songqing; Zhang, Hongbo

    2016-01-01

    The eIF4F complex regulated by a various group of eIF4E-binding proteins (4E-BPs) can initial the protein synthesis. Small molecule compound 4EGI-1, an inhibitor of the cap-dependent translation initiation through disturbing the interaction between eIF4E and eIF4G which are main elements of the eIF4E complex, has been reported to suppress cell proliferation by inducing apoptosis in many types of cancer. And death receptor 5 (DR5) is a major component in the extrinsic apoptotic pathway. However, the correlation among 4EGI-1, DR5 and 4E-BPs have not been discovered in NPC now. Therefore, we intend to find out the effect of 4EGI-1 on the apoptosis process of NPC and the relationship among 4EGI-1, DR5 and 4E-BPs. Our results revealed a significant down regulation of DR5 expression in NPC tissues, which inversely correlated with lymph node metastasis status and clinical stages. Depressed DR5 expression was an independent biomarker for poor prognosis in NPC, and elevated DR5 expression showed longer overall survival time in 174 NPC patients. Besides, 4EGI-1 induced apoptosis in NPC cells through the DR5-caspase-8 axis on 4E-BP1 and eIF4E dephosphorylation exerting positive influence on their anti-tumor activities. The induction of DR5 also sensitized NPC cells to radiotherapy, and the SER was 1.195. These results establish the death receptor pathway as a novel anticancer mechanism of eIF4E/eIF4G interaction inhibitor in NPC. PMID:26942880

  12. 4EGI-1 induces apoptosis and enhances radiotherapy sensitivity in nasopharyngeal carcinoma cells via DR5 induction on 4E-BP1 dephosphorylation.

    PubMed

    Wang, Weiyuan; Li, Jiao; Wen, Qiuyuan; Luo, Jiadi; Chu, Shuzhou; Chen, Lingjiao; Qing, Zhenzhen; Xie, Guiyuan; Xu, Lina; Alnemah, Mohannad Ma; Li, Meirong; Fan, Songqing; Zhang, Hongbo

    2016-04-19

    The eIF4F complex regulated by a various group of eIF4E-binding proteins (4E-BPs) can initial the protein synthesis. Small molecule compound 4EGI-1, an inhibitor of the cap-dependent translation initiation through disturbing the interaction between eIF4E and eIF4G which are main elements of the eIF4E complex, has been reported to suppress cell proliferation by inducing apoptosis in many types of cancer. And death receptor 5 (DR5) is a major component in the extrinsic apoptotic pathway. However, the correlation among 4EGI-1, DR5 and 4E-BPs have not been discovered in NPC now. Therefore, we intend to find out the effect of 4EGI-1 on the apoptosis process of NPC and the relationship among 4EGI-1, DR5 and 4E-BPs. Our results revealed a significant down regulation of DR5 expression in NPC tissues, which inversely correlated with lymph node metastasis status and clinical stages. Depressed DR5 expression was an independent biomarker for poor prognosis in NPC, and elevated DR5 expression showed longer overall survival time in 174 NPC patients. Besides, 4EGI-1 induced apoptosis in NPC cells through the DR5-caspase-8 axis on 4E-BP1 and eIF4E dephosphorylation exerting positive influence on their anti-tumor activities. The induction of DR5 also sensitized NPC cells to radiotherapy, and the SER was 1.195. These results establish the death receptor pathway as a novel anticancer mechanism of eIF4E/eIF4G interaction inhibitor in NPC.

  13. PRMT5 is essential for the eIF4E-mediated 5′-cap dependent translation

    SciTech Connect

    Lim, Ji-Hong; Lee, Yoon-Mi; Lee, Gibok; Choi, Yong-Joon; Lim, Beong-Ou; Kim, Young Jun; Choi, Dong-Kug; Park, Jong-Wan

    2014-10-03

    Highlights: • PRMT5 participates in syntheses of HIF-1α, c-Myc and cyclin D1 proteins. • PRMT5 promotes the 5′-cap dependent translation. • PRMT5 is required for eIF4E binding to mRNA 5′-cap. • PRMT5 is essential for eIF4E-dependent cell proliferation. - Abstract: It is becoming clear that PRMT5 plays essential roles in cell cycle progression, survival, and responses to external stresses. However, the precise mechanisms underlying such roles of PRMT5 have not been clearly understood. Previously, we have demonstrated that PRMT5 participates in cellular adaptation to hypoxia by ensuring 5′-cap dependent translation of HIF-1α. Given that c-Myc and cyclin D1 expressions are also tightly regulated in 5′-cap dependent manner, we here tested the possibility that PRMT5 promotes cell proliferation by increasing de novo syntheses of the oncoproteins. c-Myc and cyclin D1 were found to be noticeably downregulated by PRMT5 knock-down. A RNA immunoprecipitation analysis, which can identify RNA–protein interactions, showed that PRMT5 is required for the interaction among eIF4E and 5′-UTRs of HIF-1α, c-Myc and cyclin D1 mRNAs. In addition, PRMT5 knock-down inhibited cell proliferation by inducing cell cycle arrest at the G1 phase. More importantly, ectopic expression of eIF4E significantly rescued the cell cycle progression and cell proliferation even in PRMT5-deficeint condition. Based on these results, we propose that PRMT5 determines cell fate by regulating 5′-cap dependent translation of proteins essential for proliferation and survival.

  14. Pin1 and PKMζ Sequentially Control Dendritic Protein Synthesis

    PubMed Central

    Westmark, Pamela R.; Westmark, Cara J.; Wang, SuQing; Levenson, Jonathan; O’Riordan, Kenneth J.; Burger, Corinna; Malter, James S.

    2010-01-01

    Some forms of learning and memory, and their electrophysiologic correlate, long-term potentiation (LTP), require dendritic translation. We demonstrate that Pin1, a peptidyl-prolyl isomerase, is present in dendritic spines and shafts and inhibits protein synthesis induced by glutamatergic signaling. Pin1 suppression increased dendritic translation, possibly through eIF4E binding proteins 1 and 2 (4E-BP1/2) and eukaryotic translation initiation factor 4E (eIF4E). Consistent with increased protein synthesis, hippocampal slices from Pin−/− mice had normal early LTP (E-LTP) but significantly enhanced late LTP (L-LTP) compared to wild-type controls. Protein kinase C ζ (PKCζ) and protein kinase M ζ (PKMζ) were increased in Pin1−/− mouse brain and their activity was required to maintain dendritic translation. PKMζ interacted with and inhibited Pin1 by phosphorylating Ser16. Therefore, glutamate-induced, dendritic protein synthesis is sequentially regulated by Pin1 and PKMζ signaling. PMID:20215645

  15. Ribavirin Inhibits the Activity of mTOR/eIF4E, ERK/Mnk1/eIF4E Signaling Pathway and Synergizes with Tyrosine Kinase Inhibitor Imatinib to Impair Bcr-Abl Mediated Proliferation and Apoptosis in Ph+ Leukemia

    PubMed Central

    Gong, Yuping; Shi, Rui; Yang, Xi; Naren, Duolan; Yan, Tianyou

    2015-01-01

    The eukaryotic translation initiation factor 4E (eIF4E), which is the main composition factor of eIF4F translation initiation complex, influences the growth of tumor through modulating cap-dependent protein translation. Previous studies reported that ribavirin could suppress eIF4E-controlled translation and reduce the synthesis of onco-proteins. Here, we investigated the anti-leukemic effects of ribavirin alone or in combination with tyrosine kinase inhibitor imatinib in Philadelphia chromosome positive (Ph+) leukemia cell lines SUP-B15 (Ph+ acute lymphoblastic leukemia cell line, Ph+ ALL) and K562 (chronic myelogenous leukemia cell line, CML). Our results showed that ribavirin had anti-proliferation effect; it down-regulated the phosphorylation levels of Akt, mTOR, 4EBP1, and eIF4E proteins in the mTOR/eIF4E signaling pathway, and MEK, ERK, Mnk1 and eIF4E proteins in ERK/Mnk1/eIF4E signaling pathway; reduced the expression of Mcl-1 (a translation substrates of eIF4F translation initiation complex) at protein synthesis level not mRNA transcriptional level; and induced cell apoptosis in both SUP-B15 and K562. 7-Methyl-guanosine cap affinity assay further demonstrated that ribavirin remarkably increased the eIF4E binding to 4EBP1 and decreased the combination of eIF4E with eIF4G, consequently resulting in a major inhibition of eIF4F complex assembly. The combination of ribavirin with imatinib enhanced antileukemic effects mentioned above, indicating that two drugs have synergistic anti-leukemic effect. Consistent with the cell lines, similar results were observed in Ph+ acute lymphoblastic primary leukemic blasts; however, the anti-proliferative role of ribavirin in other types of acute primary leukemic blasts was not obvious, which indicated that the anti-leukemic effect of ribavirin was different in cell lineages. PMID:26317515

  16. Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

    PubMed

    Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

    2013-08-09

    Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

  17. Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells.

    PubMed

    Yao, Kang; Yin, Yulong; Li, Xilong; Xi, Pengbin; Wang, Junjun; Lei, Jian; Hou, Yongqing; Wu, Guoyao

    2012-06-01

    α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.

  18. Intermittent bolus feeding has a greater stimulatory effect on protein synthesis in skeletal muscle than continuous feeding in neonatal pigs.

    PubMed

    Gazzaneo, María C; Suryawan, Agus; Orellana, Renán A; Torrazza, Roberto Murgas; El-Kadi, Samer W; Wilson, Fiona A; Kimball, Scot R; Srivastava, Neeraj; Nguyen, Hanh V; Fiorotto, Marta L; Davis, Teresa A

    2011-12-01

    Orogastric tube feeding, using either continuous or intermittent bolus delivery, is common in infants for whom normal feeding is contraindicated. To compare the impact of different feeding strategies on muscle protein synthesis, after withholding food overnight, neonatal pigs received a complete formula orally as a bolus feed every 4 h or were continuously fed. Protein synthesis rate and translational mechanisms in skeletal muscle were examined after 0, 24, and 25.5 h. Plasma amino acid and insulin concentrations increased minimally and remained constant in continuously fed compared to feed-deprived pigs; however, the pulsatile meal feeding pattern was mimicked in bolus-fed pigs. Muscle protein synthesis was stimulated by feeding and the greatest response occurred after a bolus meal. Bolus but not continuous feeds increased polysome aggregation, the phosphorylation of protein kinase B, tuberous sclerosis complex 2, proline-rich Akt substrate of 40 kDa, eukaryotic initiation factor (eIF) 4E binding protein (4EBP1), and rp S6 kinase and enhanced dissociation of the 4EBP1 ·eIF4E complex and formation of the eIF4E ·eIF4G complex compared to feed deprivation (P < 0.05). Activation of insulin receptor substrate-1, regulatory associated protein of mammalian target of rapamycin, AMP-activated protein kinase, eukaryotic elongation factor 2, and eIF2α phosphorylation were unaffected by either feeding modality. These results suggest that in neonates, intermittent bolus feeding enhances muscle protein synthesis to a greater extent than continuous feeding by eliciting a pulsatile pattern of amino acid- and insulin-induced translation initiation.

  19. In vivo modulation of 4E binding protein 1 (4E-BP1) phosphorylation by watercress: a pilot study.

    PubMed

    Syed Alwi, Sharifah S; Cavell, Breeze E; Telang, Urvi; Morris, Marilyn E; Parry, Barbara M; Packham, Graham

    2010-11-01

    Dietary intake of isothiocyanates (ITC) has been associated with reduced cancer risk. The dietary phenethyl ITC (PEITC) has previously been shown to decrease the phosphorylation of the translation regulator 4E binding protein 1 (4E-BP1). Decreased 4E-BP1 phosphorylation has been linked to the inhibition of cancer cell survival and decreased activity of the transcription factor hypoxia-inducible factor (HIF), a key positive regulator of angiogenesis, and may therefore contribute to potential anti-cancer effects of PEITC. In the present study, we have investigated the in vitro and in vivo effects of watercress, which is a rich source of PEITC. We first demonstrated that, similar to PEITC, crude watercress extracts inhibited cancer cell growth and HIF activity in vitro. To examine the effects of dietary intake of watercress, we obtained plasma and peripheral blood mononuclear cells following the ingestion of an 80 g portion of watercress from healthy participants who had previously been treated for breast cancer. Analysis of PEITC in plasma samples from nine participants demonstrated a mean maximum plasma concentration of 297 nm following the ingestion of watercress. Flow cytometric analysis of 4E-BP1 phosphorylation in peripheral blood cells from four participants demonstrated significantly reduced 4E-BP1 phosphorylation at 6 and 8 h following the ingestion of watercress. Although further investigations with larger numbers of participants are required to confirm these findings, this pilot study suggests that flow cytometry may be a suitable approach to measure changes in 4E-BP1 phosphorylation following the ingestion of watercress, and that dietary intake of watercress may be sufficient to modulate this potential anti-cancer pathway.

  20. Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

    PubMed

    Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

    2012-12-14

    Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

  1. Lysine suppresses protein degradation through autophagic-lysosomal system in C2C12 myotubes.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nedachi, Taku; Nagasawa, Takashi

    2014-06-01

    Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic-lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0-3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic-lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic-lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic-lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic-lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.

  2. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.

    PubMed

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S

    2015-05-12

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

  3. An Analog-sensitive Version of the Protein Kinase Slt2 Allows Identification of Novel Targets of the Yeast Cell Wall Integrity Pathway*

    PubMed Central

    Alonso-Rodríguez, Esmeralda; Fernández-Piñar, Pablo; Sacristán-Reviriego, Almudena; Molina, María; Martín, Humberto

    2016-01-01

    The yeast cell wall integrity MAPK Slt2 mediates the transcriptional response to cell wall alterations through phosphorylation of transcription factors Rlm1 and SBF. However, the variety of cellular functions regulated by Slt2 suggests the existence of a significant number of still unknown substrates for this kinase. To identify novel Slt2 targets, we generated and characterized an analog-sensitive mutant of Slt2 (Slt2-as) that can be specifically inhibited by bulky kinase inhibitor analogs. We demonstrated that Slt2-as is able to use adenosine 5′-[γ-thio]triphosphate analogs to thiophosphorylate its substrates in yeast cell extracts as well as when produced as recombinant proteins in Escherichia coli. Taking advantage of this chemical-genetic approach, we found that Slt2 phosphorylates the MAPK phosphatase Msg5 both in the N-terminal regulatory and C-terminal catalytic domains. Moreover, we identified the calcineurin regulator Rcn2, the 4E-BP (translation initiation factor eIF4E-binding protein) translation repressor protein Caf20, and the Golgi-associated adaptor Gga1 as novel targets for Slt2. The Slt2 phosphorylation sites on Rcn2 and Caf20 were determined. We also demonstrated that, in the absence of SLT2, the GGA1 paralog GGA2 is essential for cells to survive under cell wall stress and for proper protein sorting through the carboxypeptidase Y pathway. Therefore, Slt2-as provides a powerful tool that can expand our knowledge of the outputs of the cell wall integrity MAPK pathway. PMID:26786099

  4. Impaired translation initiation activation and reduced protein synthesis in weaned piglets fed a low-protein diet.

    PubMed

    Deng, Dun; Yao, Kang; Chu, Wuying; Li, Tiejun; Huang, Ruiling; Yin, Yulong; Liu, Zhiqiang; Zhang, Jianshe; Wu, Guoyao

    2009-07-01

    Weanling mammals (including infants) often experience intestinal dysfunction when fed a high-protein diet. Recent work with the piglet (an animal model for studying human infant nutrition) shows that reducing protein intake can improve gut function during weaning but compromises the provision of essential amino acids (EAA) for muscle growth. The present study was conducted with weaned pigs to test the hypothesis that supplementing deficient EAA (Lys, Met, Thr, Trp, Leu, Ile and Val) to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in tissues. Pigs were weaned at 21 days of age and fed diets containing 20.7, 16.7 or 12.7% crude protein (CP), with the low-CP diets supplemented with EAA to achieve the levels in the high-CP diet. On Day 14 of the trial, tissue protein synthesis was determined using the phenylalanine flooding dose method. Reducing dietary CP levels decreased protein synthesis in pancreas, liver, kidney and longissimus muscle. A low-CP diet reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) in skeletal muscle and liver while increasing the formation of an inactive eIF4E.4E-BP1 complex in muscle. Dietary protein deficiency also decreased the phosphorylation of mammalian target of rapamycin (mTOR) and the formation of an active eIF4E.eIF4G complex in liver. These results demonstrate for the first time that chronic feeding of a low-CP diet suppresses protein synthesis in animals partly by inhibiting mTOR signaling. Additionally, our findings indicate that supplementing deficient EAA to low-protein diets is not highly effective in restoring protein synthesis or whole-body growth in piglets. We suggest that conditionally essential amino acids (e.g., glutamine and arginine) may be required to maintain the activation of translation initiation factors and optimal protein synthesis in neonates.

  5. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  6. Feeding a DHA-enriched diet increases skeletal muscle protein synthesis in growing pigs: association with increased skeletal muscle insulin action and local mRNA expression of insulin-like growth factor 1.

    PubMed

    Wei, Hong-Kui; Zhou, Yuanfei; Jiang, Shuzhong; Tao, Ya-Xiong; Sun, Haiqing; Peng, Jian; Jiang, Siwen

    2013-08-01

    Dietary n-3 PUFA have been demonstrated to promote muscle growth in growing animals. In the present study, fractional protein synthesis rates (FSR) in the skeletal muscle of growing pigs fed a DHA-enriched (DE) diet (DE treatment) or a soyabean oil (SO) diet (SO treatment) were evaluated in the fed and feed-deprived states. Feeding-induced increases in muscle FSR, as well as the activation of the mammalian target of rapamycin and protein kinase B, were higher in the DE treatment as indicated by the positive interaction between diet and feeding. In the fed state, the activation of eIF4E-binding protein 1 in the skeletal muscle of pigs on the DE diet was higher than that in pigs on the SO diet (P<0·05). Feeding the DE diet increased muscle insulin-like growth factor 1 (IGF-1) expression (P<0·05) and insulin action (as demonstrated by increased insulin receptor (IR) phosphorylation, P<0·05), resulting in increased IR substrate 1 activation in the fed state. However, no difference in plasma IGF-1 concentration or hepatic IGF-1 expression between the two treatments was associated. The increased IGF-1 expression in the DE treatment was associated with increased mRNA expression of the signal transducer and activator of transcription 5A and decreased mRNA expression of protein tyrosine phosphatase, non-receptor type 3 in skeletal muscle. Moreover, mRNA expression of protein tyrosine phosphatase, non-receptor type 1 (PTPN1), the activation of PTPN1 and the activation of NF-κB in muscle were significantly lower in the DE treatment (P<0·05). The results of the present study suggest that feeding a DE diet increased feeding-induced muscle protein synthesis in growing pigs, and muscle IGF-1 expression and insulin action were involved in this action.

  7. Translational control of nociception via 4E-binding protein 1

    PubMed Central

    Khoutorsky, Arkady; Bonin, Robert P; Sorge, Robert E; Gkogkas, Christos G; Pawlowski, Sophie Anne; Jafarnejad, Seyed Mehdi; Pitcher, Mark H; Alain, Tommy; Perez-Sanchez, Jimena; Salter, Eric W; Martin, Loren; Ribeiro-da-Silva, Alfredo; De Koninck, Yves; Cervero, Fernando; Mogil, Jeffrey S; Sonenberg, Nahum

    2015-01-01

    Activation of the mechanistic/mammalian target of rapamycin (mTOR) kinase in models of acute and chronic pain is strongly implicated in mediating enhanced translation and hyperalgesia. However, the molecular mechanisms by which mTOR regulates nociception remain unclear. Here we show that deletion of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a major mTOR downstream effector, which represses eIF4E activity and cap-dependent translation, leads to mechanical, but not thermal pain hypersensitivity. Mice lacking 4E-BP1 exhibit enhanced spinal cord expression of neuroligin 1, a cell-adhesion postsynaptic protein regulating excitatory synapse function, and show increased excitatory synaptic input into spinal neurons, and a lowered threshold for induction of synaptic potentiation. Pharmacological inhibition of eIF4E or genetic reduction of neuroligin 1 levels normalizes the increased excitatory synaptic activity and reverses mechanical hypersensitivity. Thus, translational control by 4E-BP1 downstream of mTOR effects the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, and thereby contributes to enhanced mechanical nociception. DOI: http://dx.doi.org/10.7554/eLife.12002.001 PMID:26678009

  8. Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex

    ERIC Educational Resources Information Center

    Sui, Li; Wang, Jing; Li, Bao-Ming

    2008-01-01

    Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

  9. Hypoxia and DNA-damaging agent bleomycin both increase the cellular level of the protein 4E-BP.

    PubMed

    Le Bouffant, Ronan; Cormier, Patrick; Mulner-Lorillon, Odile; Bellé, Robert

    2006-09-01

    The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.

  10. An Isoform of Eukaryotic Initiation Factor 4E from Chrysanthemum morifolium Interacts with Chrysanthemum Virus B Coat Protein

    PubMed Central

    Chen, Sumei; Sun, Zuxia; Guan, Zhiyong; Fang, Weimin; Teng, Nianjun; Chen, Fadi

    2013-01-01

    Background Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in plant virus infection as well as the regulation of gene translation. Methodology/Principal Findings Here, we describe the isolation of a cDNA encoding CmeIF(iso)4E (GenBank accession no. JQ904592), an isoform of eIF4E from chrysanthemum, using RACE PCR. We used the CmeIF(iso)4E cDNA for expression profiling and to analyze the interaction between CmeIF(iso)4E and the Chrysanthemum virus B coat protein (CVBCP). Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity of CmeIF(iso)4E with other reported plant eIF(iso)4E sequences varied between 69.12% and 89.18%, indicating that CmeIF(iso)4E belongs to the eIF(iso)4E subfamily of the eIF4E family. CmeIF(iso)4E was present in all chrysanthemum organs, but was particularly abundant in the roots and flowers. Confocal microscopy showed that a transiently transfected CmeIF(iso)4E-GFP fusion protein distributed throughout the whole cell in onion epidermis cells. A yeast two hybrid assay showed CVBCP interacted with CmeIF(iso)4E but not with CmeIF4E. BiFC assay further demonstrated the interaction between CmeIF(iso)4E and CVBCP. Luminescence assay showed that CVBCP increased the RLU of Luc-CVB, suggesting CVBCP might participate in the translation of viral proteins. Conclusions/Significance These results inferred that CmeIF(iso)4E as the cap-binding subunit eIF(iso)4F may be involved in Chrysanthemum Virus B infection in chrysanthemum through its interaction with CVBCP in spatial. PMID:23505421

  11. Optimal dietary protein level improved growth, disease resistance, intestinal immune and physical barrier function of young grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, Jing; Wu, Pei; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-08-01

    This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis

  12. mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

    PubMed Central

    Jossé, Lyne; Xie, Jianling; Proud, Christopher G.; Smales, C. Mark

    2016-01-01

    Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. PMID:27760840

  13. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

    PubMed

    Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

    2015-09-15

    Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1.

  14. Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive

    PubMed Central

    Laurent, Sébastien; Richard, Adrien; Mulner-Lorillon, Odile; Morales, Julia; Flament, Didier; Glippa, Virginie; Bourdon, Jérémie; Gosselin, Pauline; Siegel, Anne; Cormier, Patrick; Bellé, Robert

    2014-01-01

    Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR. PMID:24834072

  15. Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive.

    PubMed

    Laurent, Sébastien; Richard, Adrien; Mulner-Lorillon, Odile; Morales, Julia; Flament, Didier; Glippa, Virginie; Bourdon, Jérémie; Gosselin, Pauline; Siegel, Anne; Cormier, Patrick; Bellé, Robert

    2014-01-01

    Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR.

  16. Effects of dietary protein levels on the disease resistance, immune function and physical barrier function in the gill of grass carp (Ctenopharyngodon idella) after challenged with Flavobacterium columnare.

    PubMed

    Xu, Jing; Feng, Lin; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2016-10-01

    The effects of dietary protein levels on the disease resistance, gill immune function and physical barrier function of grass carp (Ctenopharyngodon idella) were investigated in this study. A total of 540 grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that optimal levels of dietary protein had the following effects: (1) the production of antibacterial components increased, and anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels were up-regulated, whereas mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, IκB kinase (IKK) α, IKKβ, IKKγ, eIF4E-binding proteins (4E-BP) 1 and 4E-BP2 were down-regulated in the gills of grass carp (P < 0.05), indicating that fish gill immune function was enhanced at an optimal level of dietary protein; (2) the activities and mRNA levels of antioxidant enzymes and glutathione content increased, the contents of reactive oxygen species, malondialdehyde and protein carbonyl (PC) decreased, and NF-E2-related factor 2, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and tight junction complexes mRNA levels were up-regulated, whereas Kelch-like-ECH-associated protein (Keap) 1a, Keap1b, cysteinyl aspartic acid-protease 3, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, c-Jun N-terminal protein kinase, myosin light chain kinase and p38 mitogen-activated protein kinase mRNA levels were down-regulated in the gills of grass carp (P < 0.05), indicating that the fish gill physical barrier function improved at an optimal level of dietary protein. Finally, based on the gill rot morbidity, ACP activity and PC

  17. Expression, Purification and Characterization of Recombinant Mouse Translation Initiation factor eIF-4E as a Dihydrofolate Reductase (DHFR) Fusion Protein

    PubMed Central

    Ghosh, Phalguni; Cheng, Jilin; Chou, Tsui-Fen; Jia, Yan; Avdulov, Svetlana; Bitterman, Peter B.; Polunovsky, Vitaly A.; Wagner, Carston R.

    2008-01-01

    One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR-eIF4E protein by using affinity and anion-exchange chromatography. Fluorescence quenching experiments indicated the cap analogue, 7MeGTP, bound to DHFR-eIF4E and eIF4E with a dissociation constant (Kd) of 6±5 and 10±3 nM, respectively. Recombinant eIF4E and DHFR-eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 uM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 uM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR-eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs. PMID:18479935

  18. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

  19. Fas-activated Ser/Thr phosphoprotein (FAST) is a eukaryotic initiation factor 4E-binding protein that regulates mRNA stability and cell survival

    PubMed Central

    Li, Wei; Ivanov, Pavel; Anderson, Paul

    2013-01-01

    The recognition of T cell intracellular antigen-1 (TIA-1) by Fas-activated Ser/Thr phosphoprotein (FAST) results in prolonged cell survival by inducing the expression of inhibitors of apoptosis. Here we show that the functional effects of FAST are dependent on its interactions with eukaryotic translation initiation factor 4E (eIF4E) which is the major cytosolic cap binding protein in cells. FAST binds to eIF4E via a consensus motif (428YXXXXLL433) that is also found in eIF4G, 4E-BP1/2/3, 4E-T, and cup. A point mutation within this motif at Y428 dampens the ability of FAST to recognize eIF4E. Wild-type (WT) FAST, but not its Y428G mutant, increases the expression of co-transfected cellular inhibitor of apoptosis-1 (cIAP-1) and β-gal mRNA and protein, but inhibits the Fas-induced activation of caspase-3. Increased expression of the co-transfected proteins results, in part, from stabilization of mRNA, suggesting that FAST:eIF4E interactions can inhibit mRNA decay. We propose that eIF4E:FAST:TIA-1 complexes regulate the translation and stability of specific mRNAs that encode proteins important for cell survival. PMID:26824015

  20. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs

    PubMed Central

    Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

    2015-01-01

    of rapamycin (mTOR), eIF-4E binding protein 1, and ribosomal protein S6 kinase 1 (P<0.001). There were no interactions between dietary protein levels and CS supplementation for all traits. In conclusion, dietary protein levels and CS supplementation influenced growth and protein metabolism through independent mechanisms in pigs. In addition, LP diets supplemented with EAA did not affect growth performance and other traits except the concentrations of SS and PUN probably through maintenance of protein synthesis and degradation signaling. Moreover, CS supplementation improved growth performance by increasing plasma IGF-1 concentrations possibly through alterations of mTOR and Akt/FOXO signaling pathways in skeletal muscle of finishing pigs. PMID:26422009

  1. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    PubMed

    Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

    2015-01-01

    of rapamycin (mTOR), eIF-4E binding protein 1, and ribosomal protein S6 kinase 1 (P<0.001). There were no interactions between dietary protein levels and CS supplementation for all traits. In conclusion, dietary protein levels and CS supplementation influenced growth and protein metabolism through independent mechanisms in pigs. In addition, LP diets supplemented with EAA did not affect growth performance and other traits except the concentrations of SS and PUN probably through maintenance of protein synthesis and degradation signaling. Moreover, CS supplementation improved growth performance by increasing plasma IGF-1 concentrations possibly through alterations of mTOR and Akt/FOXO signaling pathways in skeletal muscle of finishing pigs.

  2. Rapid dephosphorylation of eIF4E by dietary protein in the skeletal muscle and liver of food-deprived rats.

    PubMed

    Yoshizawa, F; Kido, T; Nagasawa, T

    2001-04-01

    The effect of dietary protein on eIF4E phosphorylation was examined in rats starved for 18 h and then fed on either a 20% casein diet (20C) or a protein-free diet (0C). Refeeding with the 20C diet, but not the 0C diet, resulted in partial dephosphorylation of eIF4E in both the skeletal muscle and liver. The results suggest that the dephosphorylation of eIF4E in response to food intake was regulated by the increase in plasma amino acid concentration that occurred after feeding with the 20C diet.

  3. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    PubMed Central

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-01-01

    Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function. PMID:27402756

  4. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    PubMed

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

  5. Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein

    PubMed Central

    Lyabin, D. N.; Ovchinnikov, L. P.

    2016-01-01

    The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors. PMID:26931209

  6. Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

    PubMed

    Lyabin, D N; Ovchinnikov, L P

    2016-03-02

    The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

  7. Barley Yellow Mosaic Virus VPg Is the Determinant Protein for Breaking eIF4E-Mediated Recessive Resistance in Barley Plants

    PubMed Central

    Li, Huangai; Kondo, Hideki; Kühne, Thomas; Shirako, Yukio

    2016-01-01

    In this study, we investigated the barley yellow mosaic virus (BaYMV, genus Bymovirus) factor(s) responsible for breaking eIF4E-mediated recessive resistance genes (rym4/5/6) in barley. Genome mapping analysis using chimeric infectious cDNA clones between rym5-breaking (JT10) and rym5-non-breaking (JK05) isolates indicated that genome-linked viral protein (VPg) is the determinant protein for breaking the rym5 resistance. Likewise, VPg is also responsible for overcoming the resistances of rym4 and rym6 alleles. Mutational analysis identified that amino acids Ser-118, Thr-120, and His-142 in JT10 VPg are the most critical residues for overcoming rym5 resistance in protoplasts. Moreover, the rym5-non-breaking JK05 could accumulate in the rym5 protoplasts when eIF4E derived from a susceptible barley cultivar was expressed from the viral genome. Thus, the compatibility between VPg and host eIF4E determines the ability of BaYMV to infect barley plants. PMID:27746794

  8. Regulation of sperm-specific proteins by IFE-1, a germline-specific homolog of eIF4E, in C. elegans.

    PubMed

    Kawasaki, Ichiro; Jeong, Myung-Hwan; Shim, Yhong-Hee

    2011-02-01

    ABSTEACT: IFE-1 is one of the five C. elegans homologs of eIF4E, which is the mRNA 5' cap-binding component of the translation initiation complex eIF4F. Depletion of IFE-1 causes defects in sperm, suggesting that IFE-1 regulates a subset of genes required for sperm functions. To further understand the molecular function of IFE-1, proteomic analysis was performed to search for sperm proteins that are downregulated in ife-1(ok1978); fem-3(q20) mutants relative to the fem-3(q20) control. The fem-3(q20) mutant background was used because it only produces sperm at restrictive temperature. Total worm proteins were subjected to 2D-DIGE, and differentially expressed protein spots were further identified by MALDI-TOF mass spectrometry. Among the identified proteins, GSP-3 and Major Sperm Proteins (MSPs) were found to be significantly down-regulated in the ife-1(ok1978) mutant. Moreover, RNAi of gsp-3 caused an ife-1-like phenotype. These results suggest that IFE-1 is required for efficient expression of some sperm-specific proteins, and the fertilization defect of ife-1 mutant is caused mainly by a reduced level of GSP-3.

  9. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

    PubMed

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L

    2015-08-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

  10. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

    PubMed Central

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

    2015-01-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

  11. Loss of PINK1 attenuates HIF-1α induction by preventing 4E-BP1-dependent switch in protein translation under hypoxia.

    PubMed

    Lin, William; Wadlington, Natasha L; Chen, Linan; Zhuang, Xiaoxi; Brorson, James R; Kang, Un Jung

    2014-02-19

    Parkinson's disease (PD) has multiple proposed etiologies with implication of abnormalities in cellular homeostasis ranging from proteostasis to mitochondrial dynamics to energy metabolism. PINK1 mutations are associated with familial PD and here we discover a novel PINK1 mechanism in cellular stress response. Using hypoxia as a physiological trigger of oxidative stress and disruption in energy metabolism, we demonstrate that PINK1(-/-) mouse cells exhibited significantly reduced induction of HIF-1α protein, HIF-1α transcriptional activity, and hypoxia-responsive gene upregulation. Loss of PINK1 impairs both hypoxia-induced 4E-BP1 dephosphorylation and increase in the ratio of internal ribosomal entry site (IRES)-dependent to cap-dependent translation. These data suggest that PINK1 mediates adaptive responses by activating IRES-dependent translation, and the impairments in translation and the HIF-1α pathway may contribute to PINK1-associated PD pathogenesis that manifests under cellular stress.

  12. Alteration of the major phosphorylation site of eukaryotic protein synthesis initiation factor 4E prevents its association with the 48 S initiation complex.

    PubMed

    Joshi-Barve, S; Rychlik, W; Rhoads, R E

    1990-02-15

    Site-directed mutagenesis was used to replace the serine residue at the primary phosphorylation site of human eukaryotic initiation factor (eIF) 4E with an alanine residue. The mutated cDNA was transcribed in vitro, and the transcript was used to direct protein synthesis in a reticulocyte lysate system. The variant protein (eIF-4EAla) was retained on a 7-methylguanosine 5'-triphosphate (m7GTP)-Sepharose affinity column and was specifically eluted by m7GTP. Examination of eIF-4EAla by isoelectric focusing revealed two species which had the same pI values as the phosphorylated and nonphosphorylated forms of unaltered eIF-4E (here designated eIF-4ESer). However, conversion of unphosphorylated eIF-4EAla to the putative phosphorylated eIF-4EAla in the reticulocyte lysate system was slower than the corresponding conversion of eIF-4ESer. The possibility that the more acidic form of eIF-4EAla was due to NH2-terminal acetylation was ruled out by an experiment in which the acetyl-CoA pool of the reticulocyte lysate system was depleted with oxaloacetate and citrate synthase. The more acidic form of eIF-4EAla was, however, eliminated by treatment with calf intestine alkaline phosphatase, suggesting that it results from a second-site phosphorylation. When translation reaction mixtures were resolved on sucrose density gradients, the 35S-labeled eIF-4ESer was found on the 48 S initiation complex in the presence of guanylyl imidodiphosphate, as reported earlier (Hiremath, L.S., Hiremath, S.T., Rychlik, W., Joshi, S., Domier, L.L., and Rhoads, R.E. (1989) J. Biol. Chem. 264, 1132-1138). eIF-4EAla, by contrast, was not found on the 48 S complex, suggesting that phosphorylation of eIF-4E is necessary for it to carry out its role of transferring mRNA to the 48 S complex. Supporting this interpretation was the finding that eIF-4ESer isolated from 48 S initiation complexes consisted predominantly of the phosphorylated form.

  13. Elevated levels of p-Mnk1, p-eIF4E and p-p70S6K proteins are associated with tumor recurrence and poor prognosis in astrocytomas.

    PubMed

    Fan, Weibing; Wang, Weiyuan; Mao, Xinfa; Chu, Shuzhou; Feng, Juan; Xiao, Desheng; Zhou, Jianhua; Fan, Songqing

    2017-02-01

    Malignant astrocytomas are able to invade neighboring and distant areas of the normal brain. Signaling pathway alterations play important role in the development of astrocytomas. Deregulation of eukaryotic translation initiation factor 4E (eIF4E) by MAP kinase-interacting kinases (Mnk) on Ser-209 directly or PI3K/mTOR/S6K pathway indirectly has a critical effect on promoting cellular proliferation, malignant transformation and metastasis. We examined and analyzed the correlation between expression of p-Mnk1, p-eIF4E and p-p70S6K proteins and clinicopathological features in 103 astrocytomas and 54 non-tumorous brain tissues. The results indicated that positive percentage of overexpression of p-Mnk1 and p-eIF4E proteins in astrocytomas were significantly higher than that of in the non-tumorous brain tissues (P < 0.05). Elevated p-Mnk1 and p-eIF4E and co-overexpressed three proteins were associated with tumor recurrence (P = 0.003, P = 0.006, P = 0.007, respectively). Overexpressed p-eIF4E significantly correlated with the tumor size (P = 0.019). In addition, overexpression of p-eIF4E and three proteins common expression were related to the WHO grade of astrocytomas (P = 0.001, P = 0.044 respectively). Spearman's rank correlation test further showed that the expression of p-Mnk1 was strongly positive correlated with the expression of p-eIF4E in astrocytomas (r = 0.294, P = 0.003). Besides, overexpression of p-eIF4E and co-expression of p-Mnk1, p-eIF4E and p-p70S6K proteins were inversely correlated with overall survival rates of astrocytomas. Multivariate Cox regression analysis further identified that the elevated p-eIF4E expression, three proteins common expression were correlated with unfavorable prognosis of astrocytomas regardless of ages and WHO grades. Taken together, overexpression of p-eIF4E and co-expression of p-Mnk1, p-eIF4E and p-p70S6K proteins could be used as novel independent poor prognostic biomarkers for patients

  14. HIV-1 Gag Blocks Selenite-Induced Stress Granule Assembly by Altering the mRNA Cap-Binding Complex

    PubMed Central

    Cinti, Alessandro; Le Sage, Valerie; Ghanem, Marwan

    2016-01-01

    ABSTRACT Stress granules (SGs) are dynamic accumulations of stalled preinitiation complexes and translational machinery that assemble under stressful conditions. Sodium selenite (Se) induces the assembly of noncanonical type II SGs that differ in morphology, composition, and mechanism of assembly from canonical SGs. Se inhibits translation initiation by altering the cap-binding activity of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). In this work, we show that human immunodeficiency virus type 1 (HIV-1) Gag is able to block the assembly of type II noncanonical SGs to facilitate continued Gag protein synthesis. We demonstrate that expression of Gag reduces the amount of hypophosphorylated 4EBP1 associated with the 5′ cap potentially through an interaction with its target, eIF4E. These results suggest that the assembly of SGs is an important host antiviral defense that HIV-1 has evolved for inhibition through several distinct mechanisms. PMID:27025252

  15. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    SciTech Connect

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-07-11

    mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

  16. Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in Planta.

    PubMed

    Beauchemin, Chantal; Boutet, Nathalie; Laliberté, Jean-François

    2007-01-01

    The RNA genome of Turnip mosaic virus is covalently linked at its 5' end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.

  17. Deficiency of the eIF4E isoform nCBP limits the cell-to-cell movement of a plant virus encoding triple-gene-block proteins in Arabidopsis thaliana

    PubMed Central

    Keima, Takuya; Hagiwara-Komoda, Yuka; Hashimoto, Masayoshi; Neriya, Yutaro; Koinuma, Hiroaki; Iwabuchi, Nozomu; Nishida, Shuko; Yamaji, Yasuyuki; Namba, Shigetou

    2017-01-01

    One of the important antiviral genetic strategies used in crop breeding is recessive resistance. Two eukaryotic translation initiation factor 4E family genes, eIF4E and eIFiso4E, are the most common recessive resistance genes whose absence inhibits infection by plant viruses in Potyviridae, Carmovirus, and Cucumovirus. Here, we show that another eIF4E family gene, nCBP, acts as a novel recessive resistance gene in Arabidopsis thaliana toward plant viruses in Alpha- and Betaflexiviridae. We found that infection by Plantago asiatica mosaic virus (PlAMV), a potexvirus, was delayed in ncbp mutants of A. thaliana. Virus replication efficiency did not differ between an ncbp mutant and a wild type plant in single cells, but viral cell-to-cell movement was significantly delayed in the ncbp mutant. Furthermore, the accumulation of triple-gene-block protein 2 (TGB2) and TGB3, the movement proteins of potexviruses, decreased in the ncbp mutant. Inoculation experiments with several viruses showed that the accumulation of viruses encoding TGBs in their genomes decreased in the ncbp mutant. These results indicate that nCBP is a novel member of the eIF4E family recessive resistance genes whose loss impairs viral cell-to-cell movement by inhibiting the efficient accumulation of TGB2 and TGB3. PMID:28059075

  18. 4EBP1 Is Dephosphorylated by Respiratory Syncytial Virus Infection.

    PubMed

    Pérez-Gil, Gustavo; Landa-Cardeña, Adriana; Coutiño, Rocío; García-Román, Rebeca; Sampieri, Clara L; Mora, Silvia I; Montero, Hilda

    2015-01-01

    Respiratory syncytial virus (RSV) requires protein biosynthesis machinery to generate progeny. There is evidence that RSV might alter some translation components since stress granules are formed in their host cells. Consistent with these observations, we found that RSV induces dephosphorylation of 4EBP1 (eIF4E-binding protein), an important cellular translation factor. Our results show no correlation between the 4EBP1 dephosphorylation time and the decrease in the global rate of protein synthesis. Interestingly, treatment with rapamycin stimulates virus generation. The results suggest that RSV is a virus that still contains unknown mechanisms involved in the translation of their mRNAs through the alteration or modification of some translation factors, such as 4EBP1, possibly to favor its replicative cycle.

  19. RPPA-based protein profiling reveals eIF4G overexpression and 4E-BP1 serine 65 phosphorylation as molecular events that correspond with a pro-survival phenotype in chronic lymphocytic leukemia

    PubMed Central

    Shull, Austin Y.; Noonepalle, Satish K.; Awan, Farrukh T.; Liu, Jimei; Pei, Lirong; Bollag, Roni J.; Salman, Huda; Ding, Zhiyong; Shi, Huidong

    2015-01-01

    Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival. PMID:25999352

  20. eIF4E association with 4E-BP decreases rapidly following fertilization in sea urchin.

    PubMed

    Cormier, P; Pyronnet, S; Morales, J; Mulner-Lorillon, O; Sonenberg, N; Bellé, R

    2001-04-15

    The eukaryotic translation initiation factor (eIF) 4F facilitates the recruitment of ribosomes to the mRNA 5' end. The 4E-BPs are small proteins with hypophosphorylated forms that interact with the cap binding protein eIF4E, preventing its interaction with eIF4G, thereby preventing ribosome interaction with mRNA. In sea urchin, fertilization triggers a rapid rise in protein synthesis. Here, we demonstrate that a 4E-BP homologue exists and is associated with eIF4E in unfertilized eggs. We also show that 4E-BP/eIF4E association diminishes a few minutes following fertilization. This decrease is correlated with a decrease in the total amount of 4E-BP in combination with an increase in the phosphorylation of the protein. We propose that 4E-BP acts as a repressor of protein synthesis in unfertilized sea urchin eggs and that 4E-BP/eIF4E dissociation plays an important role in the rise in protein synthesis that occurs shortly following fertilization.

  1. Transcriptional repressor E4-binding protein 4 (E4BP4) regulates metabolic hormone fibroblast growth factor 21 (FGF21) during circadian cycles and feeding.

    PubMed

    Tong, Xin; Muchnik, Marina; Chen, Zheng; Patel, Manish; Wu, Nan; Joshi, Shree; Rui, Liangyou; Lazar, Mitchell A; Yin, Lei

    2010-11-19

    Fibroblast growth factor 21 (FGF21) is a potent antidiabetic and triglyceride-lowering hormone whose hepatic expression is highly responsive to food intake. FGF21 induction in the adaptive response to fasting has been well studied, but the molecular mechanism responsible for feeding-induced repression remains unknown. In this study, we demonstrate a novel link between FGF21 and a key circadian output protein, E4BP4. Expression of Fgf21 displays a circadian rhythm, which peaks during the fasting phase and is anti-phase to E4bp4, which is elevated during feeding periods. E4BP4 strongly suppresses Fgf21 transcription by binding to a D-box element in the distal promoter region. Depletion of E4BP4 in synchronized Hepa1c1c-7 liver cells augments the amplitude of Fgf21 expression, and overexpression of E4BP4 represses FGF21 secretion from primary mouse hepatocytes. Mimicking feeding effects, insulin significantly increases E4BP4 expression and binding to the Fgf21 promoter through AKT activation. Thus, E4BP4 is a novel insulin-responsive repressor of FGF21 expression during circadian cycles and feeding.

  2. Translation Initiation Factor eIF4E and eIFiso4E Are Both Required for Peanut stripe virus Infection in Peanut (Arachis hypogaea L.).

    PubMed

    Xu, Manlin; Xie, Hongfeng; Wu, Juxiang; Xie, Lianhui; Yang, Jinguang; Chi, Yucheng

    2017-01-01

    Peanut stripe virus (PStV) belongs to the genus Potyvirus and is the most important viral pathogen of cultivated peanut (Arachis hypogaea L.). The eukaryotic translation initiation factor, eIF4E, and its isoform, eIF(iso)4E, play key roles during virus infection in plants, particularly Potyvirus. In the present study, we cloned the eIF4E and eIF(iso)4E homologs in peanut and named these as PeaeIF4E and PeaeIF(iso)4E, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed that these two genes were expressed during all growth periods and in all peanut organs, but were especially abundant in young leaves and roots. These also had similar expression levels. Yeast two-hybrid analysis showed that PStV multifunctional helper component proteinase (HC-Pro) and viral protein genome-linked (VPg) both interacted with PeaeIF4E and PeaeIF(iso)4E. Bimolecular fluorescence complementation assay showed that there was an interaction between HC-Pro and PeaeIF4E/PeaeIF(iso)4E in the cytoplasm and between VPg and PeaeIF4E/PeaeIF(iso)4E in the nucleus. Silencing either PeaeIF4E or PeaeIF(iso)4E using a virus-induced gene silencing system did not significantly affect PStV accumulation. However, silencing both PeaeIF4E and PeaeIF(iso)4E genes significantly weakened PStV accumulation. The findings of the present study suggest that PeaeIF4E and PeaeIF(iso)4E play important roles in the PStV infection cycle and may potentially contribute to PStV resistance.

  3. Translation Initiation Factor eIF4E and eIFiso4E Are Both Required for Peanut stripe virus Infection in Peanut (Arachis hypogaea L.)

    PubMed Central

    Xu, Manlin; Xie, Hongfeng; Wu, Juxiang; Xie, Lianhui; Yang, Jinguang; Chi, Yucheng

    2017-01-01

    Peanut stripe virus (PStV) belongs to the genus Potyvirus and is the most important viral pathogen of cultivated peanut (Arachis hypogaea L.). The eukaryotic translation initiation factor, eIF4E, and its isoform, eIF(iso)4E, play key roles during virus infection in plants, particularly Potyvirus. In the present study, we cloned the eIF4E and eIF(iso)4E homologs in peanut and named these as PeaeIF4E and PeaeIF(iso)4E, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed that these two genes were expressed during all growth periods and in all peanut organs, but were especially abundant in young leaves and roots. These also had similar expression levels. Yeast two-hybrid analysis showed that PStV multifunctional helper component proteinase (HC-Pro) and viral protein genome-linked (VPg) both interacted with PeaeIF4E and PeaeIF(iso)4E. Bimolecular fluorescence complementation assay showed that there was an interaction between HC-Pro and PeaeIF4E/PeaeIF(iso)4E in the cytoplasm and between VPg and PeaeIF4E/PeaeIF(iso)4E in the nucleus. Silencing either PeaeIF4E or PeaeIF(iso)4E using a virus-induced gene silencing system did not significantly affect PStV accumulation. However, silencing both PeaeIF4E and PeaeIF(iso)4E genes significantly weakened PStV accumulation. The findings of the present study suggest that PeaeIF4E and PeaeIF(iso)4E play important roles in the PStV infection cycle and may potentially contribute to PStV resistance. PMID:28344571

  4. The mechanism of insulin-stimulated 4E-BP protein binding to mammalian target of rapamycin (mTOR) complex 1 and its contribution to mTOR complex 1 signaling.

    PubMed

    Rapley, Joseph; Oshiro, Noriko; Ortiz-Vega, Sara; Avruch, Joseph

    2011-11-04

    Insulin activation of mTOR complex 1 is accompanied by enhanced binding of substrates. We examined the mechanism and contribution of this enhancement to insulin activation of mTORC1 signaling in 293E and HeLa cells. In 293E, insulin increased the amount of mTORC1 retrieved by the transiently expressed nonphosphorylatable 4E-BP[5A] to an extent that varied inversely with the amount of PRAS40 bound to mTORC1. RNAi depletion of PRAS40 enhanced 4E-BP[5A] binding to ∼70% the extent of maximal insulin, and PRAS40 RNAi and insulin together did not increase 4E-BP[5A] binding beyond insulin alone, suggesting that removal of PRAS40 from mTORC1 is the predominant mechanism of an insulin-induced increase in substrate access. As regards the role of increased substrate access in mTORC1 signaling, RNAi depletion of PRAS40, although increasing 4E-BP[5A] binding, did not stimulate phosphorylation of endogenous mTORC1 substrates S6K1(Thr(389)) or 4E-BP (Thr(37)/Thr(46)), the latter already ∼70% of maximal in amino acid replete, serum-deprived 293E cells. In HeLa cells, insulin and PRAS40 RNAi also both enhanced the binding of 4E-BP[5A] to raptor but only insulin stimulated S6K1 and 4E-BP phosphorylation. Furthermore, Rheb overexpression in 293E activated mTORC1 signaling completely without causing PRAS40 release. In the presence of Rheb and insulin, PRAS40 release is abolished by Akt inhibition without diminishing mTORC1 signaling. In conclusion, dissociation of PRAS40 from mTORC1 and enhanced mTORC1 substrate binding results from Akt and mTORC1 activation and makes little or no contribution to mTORC1 signaling, which rather is determined by Rheb activation of mTOR catalytic activity, through mechanisms that remain to be fully elucidated.

  5. MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

    PubMed Central

    Landon, Ari L.; Muniandy, Parameswary A.; Shetty, Amol C.; Lehrmann, Elin; Volpon, Laurent; Houng, Simone; Zhang, Yongqing; Dai, Bojie; Peroutka, Raymond; Mazan-Mamczarz, Krystyna; Steinhardt, James; Mahurkar, Anup; Becker, Kevin G.; Borden, Katherine L.; Gartenhaus, Ronald B.

    2014-01-01

    The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL. PMID:25403230

  6. The Translational Repressor 4E-BP1 Contributes to Diabetes-Induced Visual Dysfunction

    PubMed Central

    Miller, William P.; Mihailescu, Maria L.; Yang, Chen; Barber, Alistair J.; Kimball, Scot R.; Jefferson, Leonard S.; Dennis, Michael D.

    2016-01-01

    Purpose The translational repressor 4E-BP1 interacts with the mRNA cap-binding protein eIF4E and thereby promotes cap-independent translation of mRNAs encoding proteins that contribute to diabetic retinopathy. Interaction of 4E-BP1 with eIF4E is enhanced in the retina of diabetic rodents, at least in part, as a result of elevated 4E-BP1 protein expression. In the present study, we examined the role of 4E-BP1 in diabetes-induced visual dysfunction, as well as the mechanism whereby hyperglycemia promotes 4E-BP1 expression. Methods Nondiabetic and diabetic wild-type and 4E-BP1/2 knockout mice were evaluated for visual function using a virtual optomotor test (Optomotry). Retinas were harvested from nondiabetic and type 1 diabetic mice and analyzed for protein abundance and posttranslational modifications. Similar analyses were performed on cells in culture exposed to hyperglycemic conditions or an O-GlcNAcase inhibitor (Thiamet G [TMG]). Results Diabetes-induced visual dysfunction was delayed in mice deficient of 4E-BP1/2 as compared to controls. 4E-BP1 protein expression was enhanced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in retinal cells in culture. A similar elevation in 4E-BP1 expression was observed with TMG. The rate of 4E-BP1 degradation was significantly prolonged by either hyperglycemic conditions or TMG. A PEST motif in the C-terminus of 4E-BP1 regulated polyubiquitination, turnover, and binding of an E3 ubiquitin ligase complex containing CUL3. Conclusions The findings support a model whereby elevated 4E-BP1 expression observed in the retina of diabetic rodents is the result of O-GlcNAcylation of 4E-BP1 within its PEST motif. PMID:26998719

  7. Alpha-lipoic acid supplementation reduces mTORC1 signaling in skeletal muscle from high fat fed, obese Zucker rats.

    PubMed

    Li, Zhuyun; Dungan, Cory M; Carrier, Bradley; Rideout, Todd C; Williamson, David L

    2014-12-01

    The mammalian target of rapamycin (mTOR) signaling pathway is hyperactive in liver, adipose and skeletal muscle tissues of obese rodents. Alpha-lipoic acid (αLA) has been well accepted as a weight-loss treatment, though there are limited studies on its effect on mTOR signaling in high-fat fed, obese rodents. Therefore, the goal of this study was to determine mTOR signaling and oxidative protein alterations in skeletal muscle of high-fat fed, obese rats after αLA supplementation. Phosphorylation of the mTOR substrate, eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and eIF4B were significantly reduced (p < 0.05) in muscle from αLA supplemented rats. Activation of AMP-activated protein kinase (AMPK), an mTOR inhibitory kinase, was higher (p < 0.05) in the αLA group. Protein expression of markers of oxidative metabolism, acetyl CoA carboxylase (ACC), cytochrome c oxidase IV (COX IV), peroxisome proliferator-activated receptor (PPAR), and PPAR gamma coactivator 1-alpha (PGC-1α) were significantly higher (p < 0.05) after αLA supplementation compared to non-supplemented group. Our findings show that αLA supplementation limits the negative ramifications of consuming a high fat diet on skeletal muscle markers of oxidative metabolism and mTORC1 signaling.

  8. Two zebrafish eIF4E family members are differentially expressed and functionally divergent.

    PubMed

    Robalino, Javier; Joshi, Bhavesh; Fahrenkrug, Scott C; Jagus, Rosemary

    2004-03-12

    Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role

  9. The Extracellular-Regulated Kinase Effector Lk6 is Required for Glutamate Receptor Localization at the Drosophila Neuromuscular Junction

    PubMed Central

    Hussein, Nizar A.; Delaney, Taylor L.; Tounsel, Brittany L.; Liebl, Faith L.W.

    2016-01-01

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). We used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. Our data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. We also find that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. PMID:27199570

  10. The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

    PubMed Central

    Soto-Rifo, Ricardo; Rubilar, Paulina S.; Ohlmann, Théophile

    2013-01-01

    Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m7GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m7GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled. PMID:23630313

  11. The Role of eIF4E Activity in Breast Cancer

    DTIC Science & Technology

    2010-08-01

    estimates of eIF4E activity in tumours, would act as predictive markers for cellular responses to eIF4E directed therapies. 2 Body Tissue micro-arrays...2352–2358 Armengol G, Rojo F, Castellvi J, Iglesias C, Cuatrecasas M, Pons B, Baselga J, Ramon y Cajal S (2007) 4E-binding protein 1: a key...Baselga J, Ramon y Cajal S (2006) Phosphorylated 4E binding protein 1: a hallmark of cell signaling that correlates with survival in ovarian cancer. Cancer

  12. Characterization of the cloned full-length and a truncated human target of rapamycin: Activity, specificity, and enzyme inhibition as studied by a high capacity assay

    SciTech Connect

    Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig; LaRocque, James; Gibbons, James; Yu, Ker . E-mail: yuk@wyeth.com

    2005-06-24

    The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (K {sub m}) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 {mu}M, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors.

  13. Modulation of gurken translation by insulin and TOR signaling in Drosophila

    PubMed Central

    Ferguson, Scott B.; Blundon, Malachi A.; Klovstad, Martha S.; Schüpbach, Trudi

    2012-01-01

    Localized Gurken (Grk) translation specifies the anterior–posterior and dorsal–ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, we identified a mutation in the lnk gene. We show that lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS–TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). We hypothesize that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed. PMID:22328499

  14. Ubiquilin-mediated Small Molecule Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling*

    PubMed Central

    Coffey, Rory T.; Shi, Yuntao; Long, Marcus J. C.; Marr, Michael T.; Hedstrom, Lizbeth

    2016-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism, growth, and proliferation. mTORC1 has been implicated in many diseases such as cancer, diabetes, and neurodegeneration, and is a target to prolong lifespan. Here we report a small molecule inhibitor (Cbz-B3A) of mTORC1 signaling. Cbz-B3A inhibits the phosphorylation of eIF4E-binding protein 1 (4EBP1) and blocks 68% of translation. In contrast, rapamycin preferentially inhibits the phosphorylation of p70S6k and blocks 35% of translation. Cbz-B3A does not appear to bind directly to mTORC1, but instead binds to ubiquilins 1, 2, and 4. Knockdown of ubiquilin 2, but not ubiquilins 1 and 4, decreases the phosphorylation of 4EBP1, suggesting that ubiquilin 2 activates mTORC1. The knockdown of ubiquilins 2 and 4 decreases the effect of Cbz-B3A on 4EBP1 phosphorylation. Cbz-B3A slows cellular growth of some human leukemia cell lines, but is not cytotoxic. Thus Cbz-B3A exemplifies a novel strategy to inhibit mTORC1 signaling that might be exploited for treating many human diseases. We propose that Cbz-B3A reveals a previously unappreciated regulatory pathway coordinating cytosolic protein quality control and mTORC1 signaling. PMID:26740621

  15. Gcn4 is required for the response to peroxide stress in the yeast Saccharomyces cerevisiae.

    PubMed

    Mascarenhas, Claire; Edwards-Ingram, Laura C; Zeef, Leo; Shenton, Daniel; Ashe, Mark P; Grant, Chris M

    2008-07-01

    An oxidative stress occurs when reactive oxygen species overwhelm the cellular antioxidant defenses. We have examined the regulation of protein synthesis in Saccharomyces cerevisiae in response to oxidative stress induced by exposure to hydroperoxides (hydrogen peroxide, and cumene hydroperoxide), a thiol oxidant (diamide), and a heavy metal (cadmium). Examination of translational activity indicates that these oxidants inhibit translation at the initiation and postinitiation phases. Inhibition of translation initiation in response to hydroperoxides is entirely dependent on phosphorylation of the alpha subunit of eukaryotic initiation factor (eIF)2 by the Gcn2 kinase. Activation of Gcn2 is mediated by uncharged tRNA because mutation of its HisRS domain abolishes regulation in response to hydroperoxides. Furthermore, Gcn4 is translationally up-regulated in response to H(2)O(2), and it is required for hydroperoxide resistance. We used transcriptional profiling to identify a wide range of genes that mediate this response as part of the Gcn4-dependent H(2)O(2)-regulon. In contrast to hydroperoxides, regulation of translation initiation in response to cadmium and diamide depends on both Gcn2 and the eIF4E binding protein Eap1. Thus, the response to oxidative stress is mediated by oxidant-specific regulation of translation initiation, and we suggest that this is an important mechanism underlying the ability of cells to adapt to different oxidants.

  16. Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver.

    PubMed

    Goggin, Melissa M; Nelsen, Christopher J; Kimball, Scot R; Jefferson, Leonard S; Morley, Simon J; Albrecht, Jeffrey H

    2004-09-01

    Following acute injuries that diminish functional liver mass, the remaining hepatocytes substantially increase overall protein synthesis to meet increased metabolic demands and to allow for compensatory liver growth. Previous studies have not clearly defined the mechanisms that promote protein synthesis in the regenerating liver. In the current study, we examined the regulation of key proteins involved in translation initiation following 70% partial hepatectomy (PH) in mice. PH promoted the assembly of eukaryotic initiation factor (eIF) 4F complexes consisting of eIF4E, eIF4G, eIF4A1, and poly-A binding protein. eIF4F complex formation after PH occurred without detectable changes in eIF4E-binding protein 1 (4E-BP1) phosphorylation or its binding eIF4E. The amount of serine 1108-phosphorylated eIF4G (but not Ser209-phosphorylated eIF4E) was induced following PH. These effects were antagonized by treatment with rapamycin, indicating that target of rapamycin (TOR) activity is required for eIF4F assembly in the regenerating liver. Rapamycin inhibited the induction of cyclin D1, a known eIF4F-sensitive gene, at the level of protein expression but not messenger RNA (mRNA) expression. In conclusion, increased translation initiation mediated by the mRNA cap-binding complex eIF4F contributes to the induction of protein synthesis during compensatory liver growth. Further study of factors that regulate translation initiation may provide insight into mechanisms that govern metabolic homeostasis and regeneration in response to liver injury.

  17. eIF4E Resistance: Natural Variation Should Guide Gene Editing.

    PubMed

    Bastet, Anna; Robaglia, Christophe; Gallois, Jean-Luc

    2017-02-28

    eIF4E translation initiation factors have emerged as major susceptibility factors for RNA viruses. Natural eIF4E-based resistance alleles are found in many species and are mostly variants that maintain the translation function of the protein. eIF4E genes represent major targets for engineering viral resistance, and gene-editing technologies can be used to make up for the lack of natural resistance alleles in some crops, often by knocking out eIF4E susceptibility factors. However, we report here how redundancy among eIF4E genes can restrict the efficient use of knockout alleles in breeding. We therefore discuss how gene-editing technologies can be used to design de novo functional alleles, using knowledge about the natural evolution of eIF4E genes in different species, to drive resistance to viruses without affecting plant physiology.

  18. Cation-dependent folding of 3' cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G.

    PubMed

    Kraft, Jelena J; Treder, Krzysztof; Peterson, Mariko S; Miller, W Allen

    2013-03-01

    The 3'-untranslated regions of many plant viral RNAs contain cap-independent translation elements (CITEs) that drive translation initiation at the 5'-end of the mRNA. The barley yellow dwarf virus-like CITE (BTE) stimulates translation by binding the eIF4G subunit of translation initiation factor eIF4F with high affinity. To understand this interaction, we characterized the dynamic structural properties of the BTE, mapped the eIF4G-binding sites on the BTE and identified a region of eIF4G that is crucial for BTE binding. BTE folding involves cooperative uptake of magnesium ions and is driven primarily by charge neutralization. Footprinting experiments revealed that functional eIF4G fragments protect the highly conserved stem-loop I and a downstream bulge. The BTE forms a functional structure in the absence of protein, and the loop that base pairs the 5'-untranslated region (5'-UTR) remains solvent-accessible at high eIF4G concentrations. The region in eIF4G between the eIF4E-binding site and the MIF4G region is required for BTE binding and translation. The data support the model in which the eIF4F complex binds directly to the BTE which base pairs simultaneously to the 5'-UTR, allowing eIF4F to recruit the 40S ribosomal subunit to the 5'-end.

  19. Effect of dietary arginine on growth, intestinal enzyme activities and gene expression in muscle, hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

    PubMed

    Chen, Gangfu; Feng, Lin; Kuang, Shengyao; Liu, Yang; Jiang, Jun; Hu, Kai; Jiang, Weidan; Li, Shuhong; Tang, Ling; Zhou, Xiaoqiu

    2012-07-01

    The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.

  20. Methionine, leucine, isoleucine, or threonine effects on mammary cell signaling and pup growth in lactating mice.

    PubMed

    Liu, G M; Hanigan, M D; Lin, X Y; Zhao, K; Jiang, F G; White, R R; Wang, Y; Hu, Z Y; Wang, Z H

    2017-02-22

    Two studies were undertaken to assess the effects of individual essential AA supplementation of a protein-deficient diet on lactational performance in mice using litter growth rates as a response variable. The first study was designed to establish a dietary protein response curve, and the second to determine the effects of Leu, Ile, Met, and Thr supplementation of a protein-deficient diet on lactational performance. In both studies, dams were fed test diets from parturition through d 17 of lactation, when the studies ended. Mammary tissue was collected on d 17 from mice on the second experiment and analyzed for mammalian target of rapamycin (mTOR) pathway signaling. Supplementation with Ile, Leu, or Met independently increased litter weight gain by 11, 9, and 10%, respectively, as compared with the protein-deficient diet. These responses were supported by independent phosphorylation responses for mTOR and eIF4E binding protein 1 (4eBP1). Supplementation of Ile, Leu, and Met increased phosphorylation of mTOR by 55, 34, and 47%, respectively, as compared with the protein-deficient diet. Phosphorylation of 4eBP1 increased in response to Ile and Met supplementation by 60 and 40%, respectively. Supplementation of Ile and Met increased phosphorylation of Akt/protein kinase B (Akt) by 41 and 59%, respectively. This work demonstrated that milk production responds nonlinearly to protein supply, and milk production and the mTOR pathway responded independently to supplementation of individual AA. The former demonstrates that a linear breakpoint model is an inappropriate description of the responses, and the latter demonstrates that no single factor limits AA for lactation. Incorporation of a multiple-limiting AA concept and nonlinear responses into milk protein response models will help improve milk yield predictions and allow derivation of diets that will increase postabsorptive N efficiency and reduce N excretion by lactating animals.

  1. Emx2 homeodomain transcription factor interacts with eukaryotic translation initiation factor 4E (eIF4E) in the axons of olfactory sensory neurons.

    PubMed

    Nédélec, Stéphane; Foucher, Isabelle; Brunet, Isabelle; Bouillot, Colette; Prochiantz, Alain; Trembleau, Alain

    2004-07-20

    We report that Emx2 homeogene is expressed at the mRNA and protein levels in the adult mouse olfactory neuroepithelium. As expected for a transcription factor, Emx2 is present in the nucleus of immature and mature olfactory sensory neurons. However, the protein is also detected in the axonal compartment of these neurons, both in the olfactory mucosa axon bundles and in axon terminals within the olfactory bulb. Emx2 axonal staining is heterogeneous, suggesting an association with particles. Subcellular fractionations of olfactory bulb synaptosomes, combined with chemical lesions of olfactory neurons, confirm the presence of Emx2 in axon terminals. Significant amounts of Emx2 protein cosediment with high density synaptosomal subfractions containing eukaryotic translation initiation factor 4E (eIF4E). Nonionic detergents and RNase treatments failed to detach eIF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and eIF4E can be coimmunoprecipitated from olfactory mucosa and bulb extracts and interact directly, as demonstrated in pull-down experiments. Emx2 axonal localization, association with high-density particles and interaction with eIF4E strongly suggest that this transcription factor has new nonnuclear functions most probably related to the local control of protein translation in the olfactory sensory neuron axons. Finally, we show that two other brain-expressed homeoproteins, Otx2 and Engrailed 2, also bind eIF4E, indicating that several homeoproteins may modulate eIF4E functions in the developing and adult nervous system.

  2. microRNA-558 facilitates the expression of hypoxia-inducible factor 2 alpha through binding to 5′-untranslated region in neuroblastoma

    PubMed Central

    Song, Huajie; Jiao, Wanju; Li, Dan; Fang, Erhu; Wang, Xiaojing; Mei, Hong; Pu, Jiarui; Huang, Kai; Tong, Qiangsong

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Our previous studies have shown that hypoxia-inducible factor 2 alpha (HIF-2α), one member of the bHLH-PAS transcription factor family, facilitates the progression of NB under non-hypoxic conditions. However, the mechanisms underlying HIF-2α expression in NB still remain largely unknown. Herein, through analyzing the computational algorithm programs, we identified microRNA-558 (miR-558) as a crucial regulator of HIF-2α expression in NB. We demonstrated that miR-558 promoted the expression of HIF-2α at translational levels in NB cells through recruiting Argonaute 2 (AGO2). Mechanistically, miR-558 directly bound with its complementary site within 5′-untranslated region (5′-UTR) to facilitate the binding of AGO2 to eukaryotic translation initiation factor 4E (eIF4E) binding protein 1, resulting in increased eIF4E enrichment and HIF-2α translation. In addition, miR-558 promoted the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo, and these biological features were rescued by knockdown of AGO2, eIF4E, or HIF-2α. In clinical NB specimens, miR-558, AGO2, and eIF4E were highly expressed and positively correlated with HIF-2α expression. Patients with high miR-558, HIF-2α, AGO2, or eIF4E levels had lower survival probability. Taken together, these results demonstrate that miR-558 facilitates the expression of HIF-2α through bindingto its 5′-UTR, thus promoting the tumorigenesis and aggressiveness of NB. PMID:27276678

  3. Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies

    PubMed Central

    Lipstein, Mark R.; Scotto, Luigi; Jirau Serrano, Xavier O.; Mangone, Michael A.; Li, Shirong; Vendome, Jeremie; Hao, Yun; Xu, Xiaoming; Deng, Shi-Xian; Realubit, Ronald B.; Tatonetti, Nicholas P.; Karan, Charles; Lentzsch, Suzanne; Fruman, David A.; Honig, Barry; Landry, Donald W.; O’Connor, Owen A.

    2017-01-01

    Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc–dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. PMID:27784673

  4. A Carma1/MALT1-dependent, Bcl10-independent, pathway regulates antigen receptor-mediated mTOR signaling in T cells

    PubMed Central

    Hamilton, Kristia S.; Phong, Binh; Corey, Catherine; Cheng, Jing; Gorentla, Balachandra; Zhong, Xiaoping; Shiva, Sruti; Kane, Lawrence P.

    2015-01-01

    Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. These effects are mediated in part by the mTOR targets S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Most models place Akt upstream of the best-studied mTOR complex, mTORC1; however, studies have called into question whether Akt is necessary for this pathway, at least in T cells. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1 (Carma1)] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the coreceptor CD28. However, another common binding partner of Carma1 and MALT1, Bcl10, was not required for TCR-dependent activation of the mTOR pathway. Consistent with these findings, MALT1 activity was required for the proliferation of CD4+ T cells, but not early TCR-dependent activation events. Also consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR. PMID:24917592

  5. [eIF4E and developmental decisions: when translation drives the development].

    PubMed

    Oulhen, Nathalie; Cormier, Patrick

    2006-05-01

    Regulation of mRNA translation is an important regulatory step in gene expression. During embryonic development, mRNA translation is tightly regulated to produce the protein at the right place, at the right time. The eukaryotic initiation factor 4E (eIF4E) is a major target for the regulation of cap-dependent translation, that plays a key role during embryogenesis including gametogenesis, fertilization and establishment of embryonic axes. In this review, we describe recent advances illustrating the importance of the translational regulator eIF4E and its partners in developmental decisions. double dagger.

  6. Allele mining in the pepper gene pool provided new complementation effects between pvr2-eIF4E and pvr6-eIF(iso)4E alleles for resistance to pepper veinal mottle virus.

    PubMed

    Rubio, Manuel; Nicolaï, Maryse; Caranta, Carole; Palloix, Alain

    2009-11-01

    Molecular cloning of recessive resistance genes to potyviruses in a large range of host species identified the eukaryotic translation initiation factor 4E (eIF4E) as an essential determinant in the outcome of potyvirus infection. Resistance results from a few amino acid changes in the eIF4E protein encoded by the recessive resistance allele that disrupt the direct interaction with the potyviral protein VPg. In plants, several loci encode two protein subfamilies, eIF4E and eIF(iso)4E. While most eIF4E-mediated resistance to potyviruses depends on mutations in a single eIF4E protein, simultaneous mutations in eIF4E (corresponding to the pvr2 locus) and eIF(iso)4E (corresponding to the pvr6 locus) are required to prevent pepper veinal mottle virus (PVMV) infection in pepper. We used this model to look for additional alleles at the pvr2-eIF4E locus that result in resistance when combined with the pvr6-eIF(iso)4E resistant allele. Among the 12 pvr2-eIF4E resistance alleles sequenced in the pepper gene pool, three were shown to have a complementary effect with pvr6-eIF(iso)4E for resistance. Two amino acid changes were exclusively shared by these three alleles and were systematically associated with a second amino acid change, suggesting that these substitutions are associated with resistance expression. The availability of new resistant allele combinations increases the possibility for the durable deployment of resistance against this pepper virus which is prevalent in Africa.

  7. Disruption of Parallel and Converging Signaling Pathways Contributes to the Synergistic Antitumor Effects of Simultaneous mTOR and EGFR Inhibition in GBM Cells1

    PubMed Central

    Rao, Ravi D; Mladek, Ann C; Lamont, Jeffrey D; Goble, Jennie M; Erlichman, Charles; James, C David; Sarkaria, Jann N

    2005-01-01

    Abstract Elevated epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) signaling are known to contribute to the malignant properties of glioblastoma multiforme (GBM), which include uncontrolled cell proliferation and evasion of apoptosis. Small molecule inhibitors that target these protein kinases have been evaluated in multiple clinical trials for cancer patients, including those with GBM. Here we have examined the cellular and molecular effects of a combined kinase inhibition of mTOR (rapamycin) and EGFR (EKI-785) in U87 and U251 GBM cells. Simultaneous treatment with rapamycin and EKI-785 results in synergistic antiproliferative as well as proapoptotic effects. At a molecular level, rapamycin alone significantly decreases S6 phosphorylation, whereas EKI-785 alone promotes substantially reduced signal transducer and activator of transcription (STAT3) phosphorylation. Treatment with rapamycin alone also increases Akt phosphorylation on Ser-473, but this effect is blocked by a simultaneous administration of EKI-785. Individually, EKI-785 diminishes while rapamycin promotes the binding of the translation inhibitor eukaryotic initiation factor 4E binding protein (4EBP1) to the eukaryotic translation initiation factor 4E (eIF4E). In spite of these opposing effects, the highest level of 4EBP1-eIF4E binding occurs with the combination of the two inhibitors. These results indicate that the inhibition of EGFR and mTOR has distinct as well as common signaling consequences and provides a molecular rationale for the synergistic antitumor effects of EKI-785 and rapamycin administration. PMID:16242075

  8. Deficiency in Either 4E-BP1 or 4E-BP2 Augments Innate Antiviral Immune Responses

    PubMed Central

    Nehdi, Atef; Sean, Polen; Linares, Izzar; Colina, Rodney; Jaramillo, Maritza; Alain, Tommy

    2014-01-01

    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. PMID:25531441

  9. The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond

    SciTech Connect

    Monzingo,A.; Dhaliwal, S.; Dutt-Chaudhuri, A.; Lyon, A.; Sadow, J.; Hoffman, D.; Robertus, J.; Browning, K.

    2007-01-01

    Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m{sup 7} guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight {beta}-strands, three {alpha}-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m{sup 7}GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m{sup 7}GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m{sup 7}GTP in a similar and labile manner, with dissociation rates in the range of 20

  10. The DDX6–4E-T interaction mediates translational repression and P-body assembly

    PubMed Central

    Kamenska, Anastasiia; Simpson, Clare; Vindry, Caroline; Broomhead, Helen; Bénard, Marianne; Ernoult-Lange, Michèle; Lee, Benjamin P.; Harries, Lorna W.; Weil, Dominique; Standart, Nancy

    2016-01-01

    4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis. PMID:27342281

  11. The Distribution of eIF4E-Family Members across Insecta

    PubMed Central

    Tettweiler, Gritta; Kowanda, Michelle; Lasko, Paul; Sonenberg, Nahum; Hernández, Greco

    2012-01-01

    Insects are part of the earliest faunas that invaded terrestrial environments and are the first organisms that evolved controlled flight. Nowadays, insects are the most diverse animal group on the planet and comprise the majority of extant animal species described. Moreover, they have a huge impact in the biosphere as well as in all aspects of human life and economy; therefore understanding all aspects of insect biology is of great importance. In insects, as in all cells, translation is a fundamental process for gene expression. However, translation in insects has been mostly studied only in the model organism Drosophila melanogaster. We used all publicly available genomic sequences to investigate in insects the distribution of the genes encoding the cap-binding protein eIF4E, a protein that plays a crucial role in eukaryotic translation. We found that there is a diversity of multiple ortholog genes encoding eIF4E isoforms within the genus Drosophila. In striking contrast, insects outside this genus contain only a single eIF4E gene, related to D. melanogaster eIF4E-1. We also found that all insect species here analyzed contain only one Class II gene, termed 4E-HP. We discuss the possible evolutionary causes originating the multiplicity of eIF4E genes within the genus Drosophila. PMID:22745595

  12. Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise.

    PubMed

    Moberg, Marcus; Apró, William; Ekblom, Björn; van Hall, Gerrit; Holmberg, Hans-Christer; Blomstrand, Eva

    2016-06-01

    Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (Placebo4E (eIF4E)-binding protein 1 (4E-BP1) at Thr(37/46) was unaffected by supplementation, while that of Thr(46) alone exhibited a pattern similar to that of S6K1, being 18% higher with EAA than BCAA. However, after 180 min of recovery this difference between EAA and BCAA had disappeared, although with both these supplements the increases were still higher than with leucine (40%, P < 0.05) and placebo (100%, P < 0.05). In summary, EAA ingestion appears to stimulate translation initiation more effectively than the other supplements, although the results also suggest that this effect is primarily attributable to the BCAA.

  13. Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity

    PubMed Central

    Ling, Jun; Lopez-Dee, Zenaida P.; Cottell, Colby; Wolfe, Laura; Nye, Derek

    2016-01-01

    Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities. PMID:27992464

  14. Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

    PubMed

    Ling, Jun; Lopez-Dee, Zenaida P; Cottell, Colby; Wolfe, Laura; Nye, Derek

    2016-01-01

    Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

  15. Coordinated Regulation of Cap-Dependent Translation and MicroRNA Function by Convergent Signaling Pathways

    PubMed Central

    La Rocca, Gaspare; Radler, Megan R.; Egan, Shawn M.; Xiang, Qing; Garippa, Ralph

    2016-01-01

    Cell growth and proliferation require the coordinated activation of many cellular processes, including cap-dependent mRNA translation. MicroRNAs oppose cap-dependent translation and set thresholds for expression of target proteins. Emerging data suggest that microRNA function is enhanced by cellular activation due in part to induction of the RNA-induced silencing complex (RISC) scaffold protein GW182. In the current study, we demonstrate that increased expression of GW182 in activated or transformed immune cells results from effects of phosphoinositol 3-kinase–Akt–mechanistic target of rapamycin (PI3K-Akt-mTOR) and Jak-Stat-Pim signaling on the translation of GW182 mRNA. Both signaling pathways enhanced polysome occupancy and eukaryotic initiation factor 4E (eIF4E) binding to the 5′ 7mG cap of GW182 mRNA. The effect of Jak-Stat-Pim signaling on polysome occupancy and expression of GW182 protein was greater than that of PI3K-Akt-mTOR signaling, likely resulting from enhanced eIF4A-dependent unwinding of G-quadruplexes in the 5′ untranslated region of GW182 mRNA. Consistent with this, GW182 expression and microRNA function were reduced by inhibition of mTOR or Pim kinases, translation initiation complex assembly, or eIF4A function. Taken together, these data provide a mechanistic link between microRNA function and cap-dependent translation that allows activated immune cells to maintain microRNA-mediated repression of targets despite enhanced rates of protein synthesis. PMID:27354062

  16. zif-1 translational repression defines a second, mutually exclusive OMA function in germline transcriptional repression.

    PubMed

    Guven-Ozkan, Tugba; Robertson, Scott M; Nishi, Yuichi; Lin, Rueyling

    2010-10-01

    Specification of primordial germ cells requires global repression of transcription. In C. elegans, primordial germ cells are generated through four rounds of asymmetric divisions, starting from the zygote P0, each producing a transcriptionally repressed germline blastomere (P1-P4). Repression in P2-P4 requires PIE-1, which is provided maternally in oocytes and segregated to all germline blastomeres. We have shown previously that OMA-1 and OMA-2 repress global transcription in P0 and P1 by sequestering TAF-4, an essential component of TFIID. Soon after the first mitotic cycle, OMA proteins undergo developmentally regulated degradation. Here, we show that OMA proteins also repress transcription in P2-P4 indirectly, through a completely different mechanism that operates in oocytes. OMA proteins bind to both the 3' UTR of the zif-1 transcript and the eIF4E-binding protein, SPN-2, repressing translation of zif-1 mRNA in oocytes. zif-1 encodes the substrate-binding subunit of the E3 ligase for PIE-1 degradation. Inhibition of zif-1 translation in oocytes ensures high PIE-1 levels in oocytes and germline blastomeres. The two OMA protein functions are strictly regulated in both space and time by MBK-2, a kinase activated following fertilization. Phosphorylation by MBK-2 facilitates the binding of OMA proteins to TAF-4 and simultaneously inactivates their function in repressing zif-1 translation. Phosphorylation of OMA proteins displaces SPN-2 from the zif-1 3' UTR, releasing translational repression. We propose that MBK-2 phosphorylation serves as a developmental switch, converting OMA proteins from specific translational repressors in oocytes to global transcriptional repressors in embryos, together effectively repressing transcription in all germline blastomeres.

  17. Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

    PubMed Central

    Finton, Kathryn A. K.; Larimore, Kevin; Larman, H. Benjamin; Friend, Della; Correnti, Colin; Rupert, Peter B.; Elledge, Stephen J.; Greenberg, Philip D.; Strong, Roland K.

    2013-01-01

    The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3

  18. Knockdown of eIF4E suppresses cell proliferation, invasion and enhances cisplatin cytotoxicity in human ovarian cancer cells.

    PubMed

    Wan, Jing; Shi, Fang; Xu, Zhanzhan; Zhao, Min

    2015-12-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in cap-dependent translation. The overexpression of eIF4E gene has been found in a variety of human malignancies. In this study, we attempted to identify the potential effects of eIF4E and explore the possibility of eIF4E as a therapeutic target for the treatment of human ovarian cancer. First the activation of eIF4E protein was detected with m7-GTP cap binding assays in ovarian cancer and control cells. Next, the eIF4E-shRNA expression plasmids were used to specifically inhibit eIF4E activity in ovarian cancer cells line A2780 and C200. The effects of knockdown eIF4E gene on cell proliferation, migration and invasion were investigated in vitro. Moreover, the changes of cell cycle and apoptosis of ovarian cancer cells were detected by flow cytometry. Finally, we investigated the effect of knockdown of eIF4E on the chemosensitivity of ovarian cancer cells to cisplatin in vitro. Our results show there is elevated activation of eIF4E in ovarian cancer cells compared with normal human ovarian epithelial cell line. The results of BrdU incorporation and FCM assay indicate that knockdown of eIF4E efficiently suppressed cell growth and induce cell cycle arrest in G1 phase and subsequent apoptosis in ovarian cancer cells. From Transwell assay analysis, knockdown eIF4E significantly decrease cellular migration and invasion of ovarian cancer cells. We also confirmed that knockdown eIF4E could synergistically enhance the cytotoxicity effects of cisplatin to cancer cells and sensitized cisplatin-resistant C200 cells in vitro. This study demonstrates that the activation of eIF4E gene is an essential component of the malignant phenotype in ovarian cancer, and aberration of eIF4E expression is associated with proliferation, migration, invasion and chemosensitivity to cisplatin in ovarian cancer cells. Knockdown eIF4E gene can be used as a potential therapeutic target for the treatment of human ovarian cancer.

  19. The Role of elF4E Activity in Breast Cancer

    DTIC Science & Technology

    2011-08-01

    Page Introduction…………………………………………………………….………..….. 1 Body ………………………………………………………………………………….. 2 Key Research Accomplishments...responses to eIF4E directed therapies. Hughes TA; W81XWH-08-1-0108; The role of eIF4E activity in breast cancer 2 2. Body 2.1 Examination of archival...Cancer 112: 2352 – 2358 Armengol G, Rojo F, Castellvi J, Iglesias C, Cuatrecasas M, Pons B, Baselga J, Ramon y Cajal S (2007) 4E-binding protein 1: a

  20. Drosophila Longevity Assurance Conferred by Reduced Insulin Receptor Substrate Chico Partially Requires d4eBP

    PubMed Central

    Bai, Hua; Post, Stephanie; Kang, Ping; Tatar, Marc

    2015-01-01

    Mutations of the insulin/IGF signaling (IIS) pathway extend Drosophila lifespan. Based on genetic epistasis analyses, this longevity assurance is attributed to downstream effects of the FOXO transcription factor. However, as reported FOXO accounts for only a portion of the observed longevity benefit, suggesting there are additional outputs of IIS to mediate aging. One candidate is target of rapamycin complex 1 (TORC1). Reduced TORC1 activity is reported to slow aging, whereas reduced IIS is reported to repress TORC1 activity. The eukaryotic translation initiation factor 4E binding protein (4E-BP) is repressed by TORC1, and activated 4E-BP is reported to increase Drosophila lifespan. Here we use genetic epistasis analyses to test whether longevity assurance mutants of chico, the Drosophila insulin receptor substrate homolog, require Drosophila d4eBP to slow aging. In chico heterozygotes, which are robustly long-lived, d4eBP is required but not sufficient to slow aging. Remarkably, d4eBP is not required or sufficient for chico homozygotes to extend longevity. Likewise, chico heterozygote females partially require d4eBP to preserve age-dependent locomotion, and both chico genotypes require d4eBP to improve stress-resistance. Reproduction and most measures of growth affected by either chico genotype are always independent of d4eBP. In females, chico heterozygotes paradoxically produce more rather than less phosphorylated 4E-BP (p4E-BP). Altered IRS function within the IIS pathway of Drosophila appears to have partial, conditional capacity to regulate aging through an unconventional interaction with 4E-BP. PMID:26252766

  1. Drosophila Longevity Assurance Conferred by Reduced Insulin Receptor Substrate Chico Partially Requires d4eBP.

    PubMed

    Bai, Hua; Post, Stephanie; Kang, Ping; Tatar, Marc

    2015-01-01

    Mutations of the insulin/IGF signaling (IIS) pathway extend Drosophila lifespan. Based on genetic epistasis analyses, this longevity assurance is attributed to downstream effects of the FOXO transcription factor. However, as reported FOXO accounts for only a portion of the observed longevity benefit, suggesting there are additional outputs of IIS to mediate aging. One candidate is target of rapamycin complex 1 (TORC1). Reduced TORC1 activity is reported to slow aging, whereas reduced IIS is reported to repress TORC1 activity. The eukaryotic translation initiation factor 4E binding protein (4E-BP) is repressed by TORC1, and activated 4E-BP is reported to increase Drosophila lifespan. Here we use genetic epistasis analyses to test whether longevity assurance mutants of chico, the Drosophila insulin receptor substrate homolog, require Drosophila d4eBP to slow aging. In chico heterozygotes, which are robustly long-lived, d4eBP is required but not sufficient to slow aging. Remarkably, d4eBP is not required or sufficient for chico homozygotes to extend longevity. Likewise, chico heterozygote females partially require d4eBP to preserve age-dependent locomotion, and both chico genotypes require d4eBP to improve stress-resistance. Reproduction and most measures of growth affected by either chico genotype are always independent of d4eBP. In females, chico heterozygotes paradoxically produce more rather than less phosphorylated 4E-BP (p4E-BP). Altered IRS function within the IIS pathway of Drosophila appears to have partial, conditional capacity to regulate aging through an unconventional interaction with 4E-BP.

  2. Translation initiation factor (iso) 4E interacts with BTF3, the beta subunit of the nascent polypeptide-associated complex.

    PubMed

    Freire, Miguel Angel

    2005-01-31

    A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-binding proteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4E binding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.

  3. REDD1 Is a Major Target of Testosterone Action in Preventing Dexamethasone-Induced Muscle Loss

    PubMed Central

    Wu, Yong; Zhao, Weidong; Zhao, Jingbo; Zhang, Yuanfei; Qin, Weiping; Pan, Jiangping; Bauman, William A.; Blitzer, Robert D.; Cardozo, Christopher

    2010-01-01

    Glucocorticoids are a well-recognized and common cause of muscle atrophy that can be prevented by testosterone. However, the molecular mechanisms underlying such protection have not been described. Thus, the global effects of testosterone on dexamethasone-induced changes in gene expression were evaluated in rat gastrocnemius muscle using DNA microarrays. Gene expression was analyzed after 7-d administration of dexamethasone, dexamethasone plus testosterone, or vehicle. Dexamethasone changed expression of 876 probe sets by at least 2-fold. Among these, 474 probe sets were changed by at least 2-fold in the opposite direction in the dexamethasone plus testosterone group (genes in opposition). Major biological themes represented by genes in opposition included IGF-I signaling, myogenesis and muscle development, and cell cycle progression. Testosterone completely prevented the 22-fold increase in expression of the mammalian target of rapamycin (mTOR) inhibitor regulated in development and DNA damage responses 1 (REDD1), and attenuated dexamethasone induced increased expression of eIF4E binding protein 1, Forkhead box O1, and the p85 regulatory subunit of the IGF-I receptor but prevented decreased expression of IRS-1. Testosterone attenuated increases in REDD1 protein in skeletal muscle and L6 myoblasts and prevented dephosphorylation of p70S6 kinase at the mTOR-dependent site Thr389 in L6 myoblast cells. Effects of testosterone on REDD1 mRNA levels occurred within 1 h, required the androgen receptor, were blocked by bicalutamide, and were due to inhibition of transcriptional activation of REDD1 by dexamethasone. These data suggest that testosterone blocks dexamethasone-induced changes in expression of REDD1 and other genes that collectively would otherwise down-regulate mTOR activity and hence also down-regulate protein synthesis. PMID:20032058

  4. Multi-Functional Regulation of 4E-BP Gene Expression by the Ccr4-Not Complex

    PubMed Central

    Okada, Hirokazu; Schittenhelm, Ralf B.; Straessle, Anna; Hafen, Ernst

    2015-01-01

    The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3’UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression. PMID:25793896

  5. Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.

    PubMed

    Coffman, Kimberly; Yang, Bing; Lu, Jie; Tetlow, Ashley L; Pelliccio, Emelia; Lu, Shan; Guo, Da-Chuan; Tang, Chun; Dong, Meng-Qiu; Tamanoi, Fuyuhiko

    2014-02-21

    mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.

  6. eIF4E promotes tumorigenesis and modulates chemosensitivity to cisplatin in esophageal squamous cell carcinoma

    PubMed Central

    Zhao, Hui; Deng, Juan; Long, Ying; Shuai, Meng-ting; Li, Qian; Gu, Huan; Chen, Ya-qi; Leng, Ai-min

    2016-01-01

    Patients with esophageal squamous cell cancer are often diagnosed with advanced diseases that respond poorly to chemotherapy. Overexpression of eIF4E leads to enhance the translation of key malignancy-related proteins and enabling tumor growth and chemoresistance in a variety of human malignancies, but whether it has a role in ESCC remains obscure. We hypothesized that eIF4E promoted ESCC tumorigenesis and facilitated the development of acquired resistance to the cisplatin-based chemotherapy. In this study, we showed that eIF4E expression was increased significantly in clinical ESCC tissues and and ESCC cell lines and its expression level was correlated with lymph node metastasis, TNM stage, as well as overall and disease-free survival of ESCC. We also showed here that knockdown of eIF4E in EC9706 would dramatically reduced cell proliferation, colony formation, migration and invasion, apoptosis in vitro as well as in vivo, and vice versa. Moreover, “weak mRNAs” were demonstrated to be regulated by eIF4E in ESCC, which might interpret the above function. Overexpression of eIF4E decreased the efficacy of cisplatin-induced cell growth inhibition in ESCC cell line and xenograft model (P < 0.05). eIF4E knockdown by shRNA increased cisplatin-induced cytotoxicity in ESCC cell lines, and enhanced chemosensitivity to cisplatin in xenograft tumor models. Furthermore, we found that the PI3K/AKT pathway and Bcl-2/Bax ratio might be responsible for the eIF4E-induced cisplatin resistance in ESCC. Our data collectively show association of eIF4E expression with chemotherapeutic response in ESCC, and suggest that therapeutically targeting eIF4E may be a viable means of improving chemotherapy response in ESCC. PMID:27588477

  7. Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.

    PubMed

    Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2014-08-01

    The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.

  8. Inactivation of the mTORC1-Eukaryotic Translation Initiation Factor 4E Pathway Alters Stress Granule Formation

    PubMed Central

    Fournier, Marie-Josée; Coudert, Laetitia; Mellaoui, Samia; Adjibade, Pauline; Gareau, Cristina; Côté, Marie-France; Sonenberg, Nahum; Gaudreault, René C.

    2013-01-01

    Stress granules (SG) are cytoplasmic multimeric RNA bodies that form under stress conditions known to inhibit cap-dependent translation. SG contain translation initiation factors, RNA binding proteins, and signaling molecules. SG are known to inhibit apoptotic pathways, thus contributing to chemo- and radioresistance in tumor cells. However, whether stress granule formation involves oncogenic signaling pathways is currently unknown. Here, we report a novel role of the mTORC1-eukaryotic translation initiation factor 4E (eIF4E) pathway, a key regulator of cap-dependent translation initiation of oncogenic factors, in SG formation. mTORC1 specifically drives the eIF4E-mediated formation of SG through the phosphorylation of 4E-BP1, a key factor known to inhibit formation of the mTORC1-dependent eIF4E-eIF4GI interactions. Disrupting formation of SG by inactivation of mTOR with its specific inhibitor pp242 or by depletion of eIF4E or eIF4GI blocks the SG-associated antiapoptotic p21 pathway. Finally, pp242 sensitizes cancer cells to death in vitro and inhibits the growth of chemoresistant tumors in vivo. This work therefore highlights a novel role of the oncogenic mTORC1-eIF4E pathway, namely, the promotion of formation of antiapoptotic SG. PMID:23547259

  9. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4).

    PubMed

    Hirai, Takao; Tanaka, Kenjiro; Togari, Akifumi

    2014-06-13

    Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts.

  10. The Role of eIF4E Activity in Breast Cancer

    DTIC Science & Technology

    2010-08-01

    Page Introduction…………………………………………………………….………..….. 1 Body ………………………………………………………………………………….. 2 Key Research...therapies. Hughes TA; W81XWH-08-1-0108; The role of eIF4E activity in breast cancer 2 2. Body 2.1 Examination of archival breast tumours (Statement...112: 2352 – 2358 Armengol G, Rojo F, Castellvi J, Iglesias C, Cuatrecasas M, Pons B, Baselga J, Ramon y Cajal S (2007) 4E-binding protein 1: a key

  11. Signaling involved in PTTH-stimulated 4E-BP phosphorylation in prothoracic gland cells of Bombyx mori.

    PubMed

    Gu, Shi-Hong; Hsieh, Yun-Chih; Lin, Pei-Ling

    2017-01-01

    Our previous studies showed that adenosine 5'-monophosphate-activated protein kinase (AMPK)/the target of rapamycin (TOR) signaling is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the signaling involved in PTTH-stimulated phosphorylation of 4E-BP. We found that 4E-BP phosphorylation stimulated by PTTH was partially reduced in Ca(2+)-free medium, indicating the involvement of Ca(2+). In addition, we found that a potent and specific inhibitor of phospholipase C (PLC), U73122, greatly inhibited 4E-BP phosphorylation. However, PTTH-stimulated 4E-BP phosphorylation was not attenuated by a protein kinase C (PKC) inhibitor (chelerythrine C). These results indicate that PLC, but not PKC, is involved in PTTH-stimulated 4E-BP phosphorylation. When PGs were treated with agents that directly elevate the intracellular Ca(2+) concentration (either A23187 or thapsigargin), a great increase in 4E-BP phosphorylation was observed. A23187-stimulated phosphorylation of 4E-BP was blocked by a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), but not by U0126, indicating involvement of AMPK and PI3K. Determination of AMPK phosphorylation showed that treatment with either A23187 or thapsigargin inhibited AMPK phosphorylation. Moreover, PTTH appeared to inhibit AMPK phosphorylation in a Ca(2+)-dependent manner. Altogether, these results indicate interconnections among Ca(2+) signaling, AMPK, and 4E-BP phosphorylation in PTTH-activated PGs of B. mori.

  12. (4E)-dehydrocitrals [(2E,4E)- and (2Z,4E )-3,7-dimethyl-2,4,6-octatrienals] from acarid mite Histiogaster sp. A096 (Acari: Acaridae).

    PubMed

    Hiraoka, H; Mori, N; Nishida, R; Kuwahara, Y

    2001-12-01

    A mixture of two monoterpenes was obtained as the opisthonotal gland secretion from unidentified Histiogaster sp. A096 (Acari: Acaridae), and their structures were elucidated to be (4E)-dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals] by GC/MS, GC/FT-IR, UV and 1H-NMR spectra. Both isomers of (4E)-dehydrocitral prepared by syntheses in 4 steps from 3-methyl-2-butenal with 34.2% yields (based on the ylide) were separated by column chromatography into the (2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. Mass spectra together with GC retention times of the purified natural (4E)-dehydrocitrals were identical with those of synthetic (2E,4E)-3,7-dimethyl-2,4,6-octatrienal and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. The geometry at the 2-C position of both synthetic (4E)-dehydrocitrals was confirmed by NOESY analyses. This is the first identification of (4E)-dehydrocitrals from the animal kingdom.

  13. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

    SciTech Connect

    Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan; Li, Qiuping; Yang, Zhiyuan; Wu, Guoqiang; Sun, Shuhui; Gu, Jianxin; Wei, Yuanyan; Jiang, Jianhai

    2010-07-09

    Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

  14. Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3

    PubMed Central

    Robichaud, Nathaniel; del Rincon, Sonia V.; Huor, Bonnie; Alain, Tommy; Petruccelli, Andy; Hearnden, Jaclyn; Goncalves, Christophe; Grotegut, Stefan; Spruck, Charles H.; Furic, Luc; Larsson, Ola; Miller, Wilson H.; Sonenberg, Nahum

    2016-01-01

    The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The mRNA cap-binding protein eIF4E is an oncoprotein that plays an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eIF4E cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that TGFβ induces eIF4E phosphorylation to promote translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFβ requires translational activation via the non-canonical TGFβ signaling branch acting through eIF4E phosphorylation. PMID:24909168

  15. Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle.

    PubMed

    Liu, Zhenqi; Li, Guolian; Kimball, Scot R; Jahn, Linda A; Barrett, Eugene J

    2004-08-01

    Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence (n = 8) or presence (n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70(S6K)), and eIF2alpha and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 (P < 0.04) and p70(S6K) (P < 0.001) and the dephosphorylation of eIF2alpha (P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70(S6K), or eIF2alpha; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 (P = 0.31) without affecting amino acid-induced phosphorylation of p70(S6K) (P = 0.002) or dephosphorylation of eIF2alpha (P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70(S6K) and glucocorticoid excess blocks the former action in vivo in human skeletal muscle.

  16. 4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging.

    PubMed

    Kang, Min-Ji; Vasudevan, Deepika; Kang, Kwonyoon; Kim, Kyunggon; Park, Jung-Eun; Zhang, Nan; Zeng, Xiaomei; Neubert, Thomas A; Marr, Michael T; Ryoo, Hyung Don

    2017-01-02

    Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

  17. eIF4F is a nexus of resistance to anti-BRAF and anti-MEK cancer therapies.

    PubMed

    Boussemart, Lise; Malka-Mahieu, Hélène; Girault, Isabelle; Allard, Delphine; Hemmingsson, Oskar; Tomasic, Gorana; Thomas, Marina; Basmadjian, Christine; Ribeiro, Nigel; Thuaud, Frédéric; Mateus, Christina; Routier, Emilie; Kamsu-Kom, Nyam; Agoussi, Sandrine; Eggermont, Alexander M; Désaubry, Laurent; Robert, Caroline; Vagner, Stéphan

    2014-09-04

    In BRAF(V600)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway, on activation of the alternative, PI(3)K-AKT-mTOR, pathway (which is ERK independent) or on modulation of the caspase-dependent apoptotic cascade. All three pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex, which binds to the 7-methylguanylate cap (m(7)G) at the 5' end of messenger RNA, thereby modulating the translation of specific mRNAs. Here we show that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF, anti-MEK and anti-BRAF plus anti-MEK drug combinations in BRAF(V600)-mutant melanoma, colon and thyroid cancer cell lines. Resistance to treatment and maintenance of eIF4F complex formation is associated with one of three mechanisms: reactivation of MAPK signalling, persistent ERK-independent phosphorylation of the inhibitory eIF4E-binding protein 4EBP1 or increased pro-apoptotic BCL-2-modifying factor (BMF)-dependent degradation of eIF4G. The development of an in situ method to detect the eIF4E-eIF4G interactions shows that eIF4F complex formation is decreased in tumours that respond to anti-BRAF therapy and increased in resistant metastases compared to tumours before treatment. Strikingly, inhibiting the eIF4F complex, either by blocking the eIF4E-eIF4G interaction or by targeting eIF4A, synergizes with inhibiting BRAF(V600) to kill the cancer cells. eIF4F not only appears to be an indicator of both innate and acquired resistance but also is a promising therapeutic target. Combinations of drugs targeting BRAF (and/or MEK) and eIF4F may overcome most of the resistance mechanisms arising in BRAF(V600)-mutant cancers.

  18. Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E

    PubMed Central

    Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Osborne, Michael J.; Ramteke, Anup; Sun, Qingxiang; Niesman, Ashley; Chook, Yuh Min; Borden, Katherine L. B.

    2016-01-01

    Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m7G)–capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients’ responses. During clinical responses to the m7G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m7G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8–eIF4E complex as a novel therapeutic target. PMID:27114554

  19. The absence of eukaryotic initiation factor eIF(iso)4E affects the systemic spread of a Tobacco etch virus isolate in Arabidopsis thaliana.

    PubMed

    Contreras-Paredes, Carlos A; Silva-Rosales, Laura; Daròs, José-Antonio; Alejandri-Ramírez, Naholi D; Dinkova, Tzvetanka D

    2013-04-01

    Translation initiation factor eIF4E exerts an important role during infection of viral species in the family Potyviridae. Particularly, a eIF(iso)4E family member is required for Arabidopsis thaliana susceptibility to Turnip mosaic virus, Lettuce mosaic virus, and Tobacco etch virus (TEV). In addition, a resistance mechanism named restriction of TEV movement (RTM) in A. thaliana controls the systemic spread of TEV in Col-0 ecotype. Here, we describe that TEV-TAMPS, a Mexican isolate, overcomes the RTM resistance mechanism reported for TEV-7DA infection of the Col-0 ecotype but depends on eIF(iso)4E for its systemic spread. To understand at which level eIF(iso)4E participates in A. thaliana TEV-TAMPS infection, the viral RNA replication and translation were measured. The absence or overexpression of eIF(iso)4E did not affect viral translation, and replication was still observed in the absence of eIF(iso)4E. However, the TEV-TAMPS systemic spread was completely abolished in the null mutant. The viral protein genome-linked (VPg) precursor NIa was found in coimmunoprecipitated complexes with both, eIF(iso)4E and eIF4E. However, the viral coat protein (CP) was only present in the eIF(iso)4E complexes. Since both the VPg and the CP proteins are needed for systemic spread, we propose a role of A. thaliana eIF(iso)4E in the movement of TEV-TAMPS within this host.

  20. Ectopic expression of eIF4E-transporter triggers the movement of eIF4E into P-bodies, inhibiting steady-state translation but not the pioneer round of translation

    SciTech Connect

    Lee, Hyung Chul; Cho, Hana; Kim, Yoon Ki

    2008-05-16

    Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism; this process removes faulty mRNAs harboring premature termination codons (PTCs). NMD targets newly synthesized mRNAs bound by nuclear cap-binding proteins 80/20 (CBP80/20) and exon junction complex (EJC), the former of which is thought to recruit the ribosome to initiate the pioneer round of translation. After completion of the pioneer round of translation, CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E, which mediates steady-state translation in the cytoplasm. Here, we show that overexpression of eIF4E-T preferentially inhibits cap-dependent steady-state translation, but not the pioneer round of translation. We also demonstrate that overexpression of eIF4E-T or Dcp1a triggers the movement of eIF4E into the processing bodies. These results suggest that the pioneer round of translation differs from steady-state translation in terms of ribosome recruitment.

  1. Loss-of-function analysis reveals distinct requirements of the translation initiation factors eIF4E, eIF4E-3, eIF4G and eIF4G2 in Drosophila spermatogenesis.

    PubMed

    Ghosh, Sanjay; Lasko, Paul

    2015-01-01

    In eukaryotes, post-transcriptional regulation of gene expression has a key role in many cellular and developmental processes. Spermatogenesis involves a complex developmental program that includes changes in cell cycle dynamics and dramatic cellular remodeling. Translational control is critical for spermatogenesis in Drosophila as many mRNAs synthesized in the spermatocytes are translated only much later during spermatid differentiation. Testes-specific translation initiation factors eIF4E-3 and eIF4G2 are essential specifically for male fertility. However, details of their roles during different stages of spermatogenesis are unknown, and the role of canonical translation initiation factors in spermatogenesis remains unexplored. In this study, we addressed the functional role of eIF4E-1, eIF4E-3, eIF4G and eIF4G2 in testes development and formation of mature sperm. Using the UAS-Gal4 system and RNA interference, we systematically knocked down these four genes in different stages of germ cell development, and in the somatic cells. Our results show that eIF4E-1 function in early germ cells and the surrounding somatic cells is critical for spermatogenesis. Both eIF4E-1 and eIF4E-3 are required in spermatocytes for chromosome condensation and cytokinesis during the meiotic stages. Interestingly, we find that eIF4G knockdown did not affect male fertility while eIF4G2 has distinct functions during spermatogenesis; it is required in early germ cells for proper meiotic divisions and spermatid elongation while its abrogation in spermatocytes caused meiotic arrest. Double knockdown of eIF4G and eIF4G2 shows that these proteins act redundantly during the early stages of spermatogenesis. Taken together, our analysis reveals spatio-temporal roles of the canonical and testes-specific translation initiation factors in coordinating developmental programs during spermatogenesis.

  2. Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti-HIV mAb 4E10 from transgenic tobacco.

    PubMed

    Platis, Dimitris; Maltezos, Anastasios; Ma, Julian K-C; Labrou, Nikolaos E

    2009-01-01

    Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.

  3. Small‑molecule COH-SR4 inhibits adipocyte differentiation via AMPK activation.

    PubMed

    Figarola, James L; Rahbar, Samuel

    2013-05-01

    Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure. It is one of the principal causative factors involved in the development of metabolic syndrome and cancer. Inhibition of adipocyte differentiation has often been a target of anti-obesity strategies since obesity is caused not only by hypertrophy but also by adipocyte hyperplasia. In this study, we investigated the effects of COH-SR4, a novel compound with anticancer properties, on the adipogenesis in 3T3-L1 cells. Treatment with COH-SR4 significantly inhibited adipocyte differentiation in a dose-dependent manner. This inhibitory effect mainly occurred at the early phase of differentiation through inhibition of mitotic clonal expansion and cell cycle arrest at the G1/S phase transition. In differentiating adipocytes, COH-SR4 significantly reduced intracellular lipid accumulation and downregulated the expression of key adipogenesis-related transcription factors and lipogenic proteins. COH-SR4 exhibited no cytotoxic effects in 3T3-L1 cells, but indirectly activated AMP-activated protein kinase (AMPK). AMPK activation by COH-SR4 also resulted in the phosphorylation of raptor and tuberous sclerosis protein 2 (TSC2), two proteins involved in the mammalian target of rapamycin (mTOR) signaling pathways. Additionally, COH-SR4 decreased the phosphorylation of p70 kDa ribosomal protein S6 kinase (S6K) and initiation factor 4E (eIF4E) binding protein 1 (4EB‑P1), two downstream effectors of mTOR that regulate protein synthesis. Interestingly, knockdown of AMPKα1/α2 prevented the ability of COH-SR4 to inhibit cell cycle arrest and overall adipogenesis and lipid accumulation in the differentiating 3T3-L1 cells. Taken together, these results suggest that COH-SR4 inhibits 3T3-L1 adipogenesis via AMPK activation. COH-SR4 may be a promising compound for the treatment of obesity and related metabolic disorders.

  4. 49 CFR 178.68 - Specification 4E welded aluminum cylinders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 false Specification 4E welded aluminum cylinders. 178.68... PACKAGINGS Specifications for Cylinders § 178.68 Specification 4E welded aluminum cylinders. (a) Type, size and service pressure. A DOT 4E cylinder is a welded aluminum cylinder with a water capacity...

  5. 49 CFR 178.68 - Specification 4E welded aluminum cylinders.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false Specification 4E welded aluminum cylinders. 178.68... PACKAGINGS Specifications for Cylinders § 178.68 Specification 4E welded aluminum cylinders. (a) Type, size and service pressure. A DOT 4E cylinder is a welded aluminum cylinder with a water capacity...

  6. 49 CFR 178.68 - Specification 4E welded aluminum cylinders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false Specification 4E welded aluminum cylinders. 178.68... PACKAGINGS Specifications for Cylinders § 178.68 Specification 4E welded aluminum cylinders. (a) Type, size and service pressure. A DOT 4E cylinder is a welded aluminum cylinder with a water capacity...

  7. 49 CFR 178.68 - Specification 4E welded aluminum cylinders.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false Specification 4E welded aluminum cylinders. 178.68... PACKAGINGS Specifications for Cylinders § 178.68 Specification 4E welded aluminum cylinders. (a) Type, size and service pressure. A DOT 4E cylinder is a welded aluminum cylinder with a water capacity...

  8. Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

    PubMed Central

    Yángüez, Emilio; Rodriguez, Paloma; Goodfellow, Ian; Nieto, Amelia

    2012-01-01

    The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins are responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNAs translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTRs sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNAs translation. PMID:22112850

  9. Structure-Based Mutational Analysis of eIF4E in Relation to sbm1 Resistance to Pea Seed-Borne Mosaic Virus in Pea

    PubMed Central

    Ashby, Jamie A.; Stevenson, Clare E. M.; Jarvis, Gavin E.; Lawson, David M.; Maule, Andrew J.

    2011-01-01

    Background Pea encodes eukaryotic translation initiation factor eIF4E (eIF4ES), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4ER) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection. Methodology/Principal Findings To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4ES bound to m7GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m7GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4ER and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m7GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation. Conclusions/Significance The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional

  10. EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility

    PubMed Central

    Nieto, Cristina; Piron, Florence; Dalmais, Marion; Marco, Cristina F; Moriones, Enrique; Gómez-Guillamón, Ma Luisa; Truniger, Verónica; Gómez, Pedro; Garcia-Mas, Jordi; Aranda, Miguel A; Bendahmane, Abdelhafid

    2007-01-01

    Background Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method. Results A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV. Conclusion This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles. PMID:17584936

  11. Translation Initiation Factor AteIF(iso)4E Is Involved in Selective mRNA Translation in Arabidopsis Thaliana Seedlings

    PubMed Central

    Martínez-Silva, Ana Valeria; Aguirre-Martínez, César; Flores-Tinoco, Carlos E.; Alejandri-Ramírez, Naholi D.; Dinkova, Tzvetanka D.

    2012-01-01

    One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions. PMID:22363683

  12. Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

    PubMed

    Frei, Julia C; Kielian, Margaret; Lai, Jonathan R

    2015-11-01

    Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

  13. The Role of eIF4E Activity in Breast Cancer

    DTIC Science & Technology

    2009-08-01

    may provide a potentially powerful therapy predictive marker for therapies directed against the eIF4E pathway, such as sirolimus9, everolimus 10and 4E...C, Zoellner U, Tang P & Piccart M (2008) The oral mTOR inhibitor RAD001 ( everolimus ) in combination with letrozole in patients with advanced breast

  14. The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability

    PubMed Central

    Chung, Jacky; Bauer, Daniel E.; Ghamari, Alireza; Nizzi, Christopher P.; Deck, Kathryn M.; Kingsley, Paul D.; Yien, Yvette Y.; Huston, Nicholas C.; Chen, Caiyong; Schultz, Iman J.; Dalton, Arthur J.; Wittig, Johannes G.; Palis, James; Orkin, Stuart H.; Lodish, Harvey F.; Eisenstein, Richard S.; Cantor, Alan B.; Paw, Barry H.

    2015-01-01

    In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. Here, we found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mechanistic target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E-binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly L-leucine. PMID:25872869

  15. Identification of promoter motifs regulating ZmeIF4E expression level involved in maize rough dwarf disease resistance in maize (Zea Mays L.).

    PubMed

    Shi, Liyu; Weng, Jianfeng; Liu, Changlin; Song, Xinyuan; Miao, Hongqin; Hao, Zhuanfang; Xie, Chuanxiao; Li, Mingshun; Zhang, Degui; Bai, Li; Pan, Guangtang; Li, Xinhai; Zhang, Shihuang

    2013-04-01

    Maize rough dwarf disease (MRDD, a viral disease) results in significant grain yield losses, while genetic basis of which is largely unknown. Based on comparative genomics, eukaryotic translation initiation factor 4E (eIF4E) was considered as a candidate gene for MRDD resistance, validation of which will help to understand the possible genetic mechanism of this disease. ZmeIF4E (orthologs of eIF4E gene in maize) encodes a protein of 218 amino acids, harboring five exons and no variation in the cDNA sequence is identified between the resistant inbred line, X178 and susceptible one, Ye478. ZmeIF4E expression was different in the two lines plants treated with three plant hormones, ethylene, salicylic acid, and jasmonates at V3 developmental stage, suggesting that ZmeIF4E is more likely to be involved in the regulation of defense gene expression and induction of local and systemic resistance. Moreover, four cis-acting elements related to plant defense responses, including DOFCOREZM, EECCRCAH1, GT1GAMSCAM4, and GT1CONSENSUS were detected in ZmeIF4E promoter for harboring sequence variation in the two lines. Association analysis with 163 inbred lines revealed that one SNP in EECCRCAH1 is significantly associated with CSI of MRDD in two environments, which explained 3.33 and 9.04 % of phenotypic variation, respectively. Meanwhile, one SNP in GT-1 motif was found to affect MRDD resistance only in one of the two environments, which explained 5.17 % of phenotypic variation. Collectively, regulatory motifs respectively harboring the two significant SNPs in ZmeIF4E promoter could be involved in the defense process of maize after viral infection. These results contribute to understand maize defense mechanisms against maize rough dwarf virus.

  16. Isolation of the serotoninergic 5-HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6-glial and CHO cell lines

    PubMed Central

    Mialet, Jeanne; Berque-Bestel, Isabelle; Eftekhari, Pierre; Gastineau, Monique; Giner, Mireille; Dahmoune, Yamina; Donzeau-Gouge, Patrick; Hoebeke, Johan; Langlois, Michel; Sicsic, Sames; Fischmeister, Rodolphe; Lezoualc'h, Frank

    2000-01-01

    RT–PCR technique was used to clone the human 5-HT4(e) receptor (h5-HT4(e)) from heart atrium. We showed that this h5-HT4(e) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT4(e) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg−1 protein, respectively. Expression of h5-HT4(e) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [3H]-GR113808 of a number of 5-HT4 ligands, was consistent with that previously reported for other 5-HT4 receptor isoforms. Surprisingly, we found that the rank order of potencies (EC50) of 5-HT4 agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (Ki) obtained from binding assays. Furthermore, EC50 values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT4(e) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca2+ currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT4(e) receptor was observed in CHO cells in the absence of any 5-HT4 ligand and two 5-HT4 antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT4(e) receptor has a pharmacological profile which is close to the native h5-HT4 receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed. PMID:10683202

  17. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering

    PubMed Central

    Raza, M. Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M. Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S.; Drayna, Dennis

    2015-01-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  18. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    PubMed

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-05

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.

  19. Targeting of the MNK–eIF4E axis in blast crisis chronic myeloid leukemia inhibits leukemia stem cell function

    PubMed Central

    Lim, Sharon; Saw, Tzuen Yih; Zhang, Min; Janes, Matthew R.; Nacro, Kassoum; Hill, Jeffrey; Lim, An Qi; Chang, Chia-Tien; Fruman, David A.; Rizzieri, David A.; Tan, Soo Yong; Fan, Hung; Chuah, Charles T. H.; Ong, S. Tiong

    2013-01-01

    Chronic myeloid leukemia responds well to therapy targeting the oncogenic fusion protein BCR-ABL1 in chronic phase, but is resistant to treatment after it progresses to blast crisis (BC). BC is characterized by elevated β-catenin signaling in granulocyte macrophage progenitors (GMPs), which enables this population to function as leukemia stem cells (LSCs) and act as a reservoir for resistance. Because normal hematopoietic stem cells (HSCs) and LSCs depend on β-catenin signaling for self-renewal, strategies to specifically target BC will require identification of drugable factors capable of distinguishing between self-renewal in BC LSCs and normal HSCs. Here, we show that the MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in BC GMPs but not normal HSCs, and that MNK kinase-dependent eIF4E phosphorylation at serine 209 activates β-catenin signaling in BC GMPs. Mechanistically, eIF4E overexpression and phosphorylation leads to increased β-catenin protein synthesis, whereas MNK-dependent eIF4E phosphorylation is required for nuclear translocation and activation of β-catenin. Accordingly, we found that a panel of small molecule MNK kinase inhibitors prevented eIF4E phosphorylation, β-catenin activation, and BC LSC function in vitro and in vivo. Our findings identify the MNK–eIF4E axis as a specific and critical regulator of BC self-renewal, and suggest that pharmacologic inhibition of the MNK kinases may be therapeutically useful in BC chronic myeloid leukemia. PMID:23737503

  20. 4E-BP2/SH2B1/IRS2 Are Part of a Novel Feedback Loop That Controls β-Cell Mass.

    PubMed

    Blandino-Rosano, Manuel; Scheys, Joshua O; Jimenez-Palomares, Margarita; Barbaresso, Rebecca; Bender, Aaron S; Yanagiya, Akiko; Liu, Ming; Rui, Liangyou; Sonenberg, Nahum; Bernal-Mizrachi, Ernesto

    2016-08-01

    The mammalian target of rapamycin complex 1 (mTORC1) regulates several biological processes, although the key downstream mechanisms responsible for these effects are poorly defined. Using mice with deletion of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), we determine that this downstream target is a major regulator of glucose homeostasis and β-cell mass, proliferation, and survival by increasing insulin receptor substrate 2 (IRS2) levels and identify a novel feedback mechanism by which mTORC1 signaling increases IRS2 levels. In this feedback loop, we show that 4E-BP2 deletion induces translation of the adaptor protein SH2B1 and promotes the formation of a complex with IRS2 and Janus kinase 2, preventing IRS2 ubiquitination. The changes in IRS2 levels result in increases in cell cycle progression, cell survival, and β-cell mass by increasing Akt signaling and reducing p27 levels. Importantly, 4E-BP2 deletion confers resistance to cytokine treatment in vitro. Our data identify SH2B1 as a major regulator of IRS2 stability, demonstrate a novel feedback mechanism linking mTORC1 signaling with IRS2, and identify 4E-BP2 as a major regulator of proliferation and survival of β-cells.

  1. Translational control of entrainment and synchrony of the suprachiasmatic circadian clock by mTOR/4E-BP1 signaling.

    PubMed

    Cao, Ruifeng; Robinson, Barry; Xu, Haiyan; Gkogkas, Christos; Khoutorsky, Arkady; Alain, Tommy; Yanagiya, Akiko; Nevarko, Tatiana; Liu, Andrew C; Amir, Shimon; Sonenberg, Nahum

    2013-08-21

    Protein synthesis is critical for circadian clock function, but little is known of how translational regulation controls the master pacemaker in mammals, the suprachiasmatic nucleus (SCN). Here we demonstrate that the pivotal translational repressor, the eukaryotic translational initiation factor 4E binding protein 1 (4E-BP1), is rhythmically regulated via the mechanistic target of rapamycin (mTOR) signaling in the SCN and preferentially represses vasoactive intestinal peptide (Vip) mRNA translation. Knockout (KO) of Eif4ebp1 (gene encoding 4E-BP1) leads to upregulation of VIP and higher amplitude of molecular rhythms in the SCN. Consequently, the 4E-BP1 null mice exhibit accelerated re-entrainment to a shifted light/dark cycle and are more resistant to the rhythm-disruptive effects of constant light. Conversely, in Mtor(+/-) mice VIP expression is decreased and susceptibility to the effects of constant light is increased. These results reveal a key role for mTOR/4E-BP1-mediated translational control in regulating entrainment and synchrony of the master clock.

  2. The role and mechanism of CRL4 E3 ubiquitin ligase in cancer and its potential therapy implications

    PubMed Central

    Sang, Youzhou; Yan, Fan; Ren, Xiubao

    2015-01-01

    CRLs (Cullin-RING E3 ubiquitin ligases) are the largest E3 ligase family in eukaryotes, which ubiquitinate a wide range of substrates involved in cell cycle regulation, signal transduction, transcriptional regulation, DNA damage response, genomic integrity, tumor suppression and embryonic development. CRL4 E3 ubiquitin ligase, as one member of CRLs family, consists of a RING finger domain protein, cullin4 (CUL4) scaffold protein and DDB1–CUL4 associated substrate receptors. The CUL4 subfamily includes two members, CUL4A and CUL4B, which share extensively sequence identity and functional redundancy. Aberrant expression of CUL4 has been found in a majority of tumors. Given the significance of CUL4 in cancer, understanding its detailed aspects of pathogenesis of human malignancy would have significant value for the treatment of cancer. Here, the work provides an overview to address the role of CRL4 E3 ubiquitin ligase in cancer development and progression, and discuss the possible mechanisms of CRL4 ligase involving in many cellular processes associated with tumor. Finally, we discuss its potential value in cancer therapy. PMID:26460955

  3. CHINESE PLAT, 1919 (L19 19 4 E, SALT LAKE CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CHINESE PLAT, 1919 (L19 19 4 E, SALT LAKE CITY CEMETERY LOCATER), SALT LAKE CITY, UT. VIEW LOOKING NORTHEAST AT CHINESE PLAT MARKER AND BURNER. - Salt Lake City Cemetery, 200 N Street, Salt Lake City, Salt Lake County, UT

  4. Chemical bonding in electron-deficient boron oxide clusters: core boronyl groups, dual 3c-4e hypervalent bonds, and rhombic 4c-4e bonds.

    PubMed

    Chen, Qiang; Lu, Haigang; Zhai, Hua-Jin; Li, Si-Dian

    2014-04-28

    We explore the structural and bonding properties of the electron-deficient boron oxide clusters, using a series of B3On(-/0/+) (n = 2-4) clusters as examples. Global-minimum structures of these boron oxide clusters are identified via unbiased Coalescence Kick and Basin Hopping searches, which show a remarkable size and charge-state dependence. An array of new bonding elements are revealed: core boronyl groups, dual 3c-4e hypervalent bonds (ω-bonds), and rhombic 4c-4e bonds (o-bonds). In favorable cases, oxygen can exhaust all its 2s/2p electrons to facilitate the formation of B-O bonds. The current findings should help understand the bonding nature of low-dimensional boron oxide nanomaterials and bulk boron oxides.

  5. eIF4E and eIF4GI have distinct and differential imprints on multiple myeloma's proteome and signaling

    PubMed Central

    Attar-Schneider, Oshrat; Pasmanik-Chor, Metsada; Tartakover-Matalon, Shelly

    2015-01-01

    Accumulating data indicate translation plays a role in cancer biology, particularly its rate limiting stage of initiation. Despite this evolving recognition, the function and importance of specific translation initiation factors is unresolved. The eukaryotic translation initiation complex eIF4F consists of eIF4E and eIF4G at a 1:1 ratio. Although it is expected that they display interdependent functions, several publications suggest independent mechanisms. This study is the first to directly assess the relative contribution of eIF4F components to the expressed cellular proteome, transcription factors, microRNAs, and phenotype in a malignancy known for extensive protein synthesis-multiple myeloma (MM). Previously, we have shown that eIF4E/eIF4GI attenuation (siRNA/Avastin) deleteriously affected MM cells' fate and reduced levels of eIF4E/eIF4GI established targets. Here, we demonstrated that eIF4E/eIF4GI indeed have individual influences on cell proteome. We used an objective, high throughput assay of mRNA microarrays to examine the significance of eIF4E/eIF4GI silencing to several cellular facets such as transcription factors, microRNAs and phenotype. We showed different imprints for eIF4E and eIF4GI in all assayed aspects. These results promote our understanding of the relative contribution and importance of eIF4E and eIF4GI to the malignant phenotype and shed light on their function in eIF4F translation initiation complex. PMID:25717031

  6. A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance

    PubMed Central

    Terhzaz, Selim; Cabrero, Pablo; Brinzer, Robert A.; Halberg, Kenneth A.; Dow, Julian A.T.; Davies, Shireen-A.

    2015-01-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. PMID:26073628

  7. Unifying the 2e(-) and 4e(-) Reduction of Oxygen on Metal Surfaces.

    PubMed

    Viswanathan, Venkatasubramanian; Hansen, Heine Anton; Rossmeisl, Jan; Nørskov, Jens K

    2012-10-18

    Understanding trends in selectivity is of paramount importance for multi-electron electrochemical reactions. The goal of this work is to address the issue of 2e(-) versus 4e(-) reduction of oxygen on metal surfaces. Using a detailed thermodynamic analysis based on density functional theory calculations, we show that to a first approximation an activity descriptor, ΔGOH*, the free energy of adsorbed OH*, can be used to describe trends for the 2e(-) and 4e(-) reduction of oxygen. While the weak binding of OOH* on Au(111) makes it an unsuitable catalyst for the 4e(-) reduction, this weak binding is optimal for the 2e(-) reduction to H2O2. We find quite a remarkable agreement between the predictions of the model and experimental results spanning nearly 30 years.

  8. CHINESE PLAT, 1919 (L19 19 4 E, SALT LAKE CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CHINESE PLAT, 1919 (L19 19 4 E, SALT LAKE CITY CEMETERY LOCATER), SALT LAKE CITY, UT. VIEW LOOKING NORTHEAST AT CHINESE PLAT MARKER, BURNER & CHINESE GRAVES. - Salt Lake City Cemetery, 200 N Street, Salt Lake City, Salt Lake County, UT

  9. Acyclic sulfides, garlicnins L-1-L-4, E, and F, from Allium sativum.

    PubMed

    Nohara, Toshihiro; Fujiwara, Yukio; Ikeda, Tsuyoshi; Yamaguchi, Koki; Manabe, Hideyuki; Murakami, Kotaro; Ono, Masateru; Nakano, Daisuke; Kinjo, Junei

    2014-01-01

    Six novel acyclic sulfides, named garlicnins L-1-L-4 (1-4), E (5), and F (6), were isolated from the acetone extracts, with the ability to suppress M2 macrophage activation, of the bulbs of garlic (Allium sativum L.), and their chemical structures were characterized.

  10. Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection*

    PubMed Central

    Royall, Elizabeth; Doyle, Nicole; Abdul-Wahab, Azimah; Emmott, Ed; Morley, Simon J.; Goodfellow, Ian; Roberts, Lisa O.; Locker, Nicolas

    2015-01-01

    Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction. PMID:25561727

  11. Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

    PubMed

    Duffy, A G; Makarova-Rusher, O V; Ulahannan, S V; Rahma, O E; Fioravanti, S; Walker, M; Abdullah, S; Raffeld, M; Anderson, V; Abi-Jaoudeh, N; Levy, E; Wood, B J; Lee, S; Tomita, Y; Trepel, J B; Steinberg, S M; Revenko, A S; MacLeod, A R; Peer, C J; Figg, W D; Greten, T F

    2016-10-01

    The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis.

  12. Transforming Growth Factor-β1 Induced Epithelial Mesenchymal Transition is blocked by a chemical antagonist of translation factor eIF4E

    PubMed Central

    Smith, K. A.; Zhou, B.; Avdulov, S.; Benyumov, A.; Peterson, M.; Liu, Y.; Okon, A.; Hergert, P.; Braziunas, J.; Wagner, C. R.; Borok, Z.; Bitterman, P. B.

    2015-01-01

    The epithelial to mesenchymal transition (EMT) imparts disease-defining properties to epithelial cells in cancer and organ fibrosis. Prior studies identify EMT control points at the level of transcription and translation, and indicate that activation of translation initiation factor 4E (eIF4E) is involved in the mechanisms coordinating these two levels of control. Here we show that 4Ei-1, a specific chemical antagonist of the eIF4E-mRNA cap interaction, potently inhibits transforming growth factor beta 1 (TGF-β1) mediated EMT in lung epithelial cells. Upon treatment with TGF-β1, we observed a rapid recruitment of Snail1 mRNA into the actively translated polysome pool accompanied by accumulation of the EMT transcription factor Snail1 in the nucleus. 4Ei-1 blocks ribosome recruitment to the Snail1 transcript thereby preventing accumulation of the Snail1 protein in the nucleus. Our findings establish an obligatory role for upstream translational control of downstream Snail1-mediated transcriptional events in TGF-β1 induced EMT, and provide proof of concept for efforts to pharmacologically modulate the eIF4E-cap interaction as a means to inhibit pathological EMT in the setting of cancer and organ fibrosis. PMID:26678431

  13. Icariside II, a natural mTOR inhibitor, disrupts aberrant energy homeostasis via suppressing mTORC1-4E-BP1 axis in sarcoma cells

    PubMed Central

    Zhang, Chao; Yang, Lei; Geng, Ya-di; An, Fa-liang; Xia, Yuan-zheng; Guo, Chao; Luo, Jian-guang; Zhang, Lu-yong; Guo, Qing-long; Kong, Ling-yi

    2016-01-01

    The aberrant energy homeostasis that characterized by high rate of energy production (glycolysis) and energy consumption (mRNA translation) is associated with the development of cancer. As mammalian target of rapamycin (mTOR) is a critical regulator of aberrant energy homeostasis, it is an attractive target for anti-tumor intervention. The flavonoid compound Icariside II (IS) is a natural mTOR inhibitor derived from Epimedium. Koreanum. Herein, we evaluate the effect of IS on aberrant energy homeostasis. The reduction of glycolysis and mRNA translation in U2OS (osteosarcoma), S180 (fibrosarcoma) and SW1535 (chondrosarcoma) cells observed in our study, indicate that, IS inhibits aberrant energy homeostasis. This inhibition is found to be due to suppression of mammalian target of rapamycin complex 1 (mTORC1)-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) axis through blocking the assembly of mTORC1. Furthermore, IS inhibits the cap-dependent translation of c-myc through mTORC1-4E-BP1 axis which links the relationship between mRNA translation and glycolysis. Inhibition of aberrant energy homeostasis by IS, contributes to its in vitro and in vivo anti-proliferation activity. These data indicate that IS disrupts aberrant energy homeostasis of sarcoma cells through suppression of mTORC1-4E-BP1 axis, providing a novel mechanism of IS to inhibit cell proliferation in sarcoma cells. PMID:27056897

  14. Silencing of the host factor eIF(iso)4E gene confers plum pox virus resistance in plum.

    PubMed

    Wang, Xinhua; Kohalmi, Susanne E; Svircev, Antonet; Wang, Aiming; Sanfaçon, Hélène; Tian, Lining

    2013-01-01

    Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.

  15. A Novel IEEE 802.15.4e DSME MAC for Wireless Sensor Networks

    PubMed Central

    Sahoo, Prasan Kumar; Pattanaik, Sudhir Ranjan; Wu, Shih-Lin

    2017-01-01

    IEEE 802.15.4e standard proposes Deterministic and Synchronous Multichannel Extension (DSME) mode for wireless sensor networks (WSNs) to support industrial, commercial and health care applications. In this paper, a new channel access scheme and beacon scheduling schemes are designed for the IEEE 802.15.4e enabled WSNs in star topology to reduce the network discovery time and energy consumption. In addition, a new dynamic guaranteed retransmission slot allocation scheme is designed for devices with the failure Guaranteed Time Slot (GTS) transmission to reduce the retransmission delay. To evaluate our schemes, analytical models are designed to analyze the performance of WSNs in terms of reliability, delay, throughput and energy consumption. Our schemes are validated with simulation and analytical results and are observed that simulation results well match with the analytical one. The evaluated results of our designed schemes can improve the reliability, throughput, delay, and energy consumptions significantly. PMID:28275216

  16. A Novel IEEE 802.15.4e DSME MAC for Wireless Sensor Networks.

    PubMed

    Sahoo, Prasan Kumar; Pattanaik, Sudhir Ranjan; Wu, Shih-Lin

    2017-01-16

    IEEE 802.15.4e standard proposes Deterministic and Synchronous Multichannel Extension (DSME) mode for wireless sensor networks (WSNs) to support industrial, commercial and health care applications. In this paper, a new channel access scheme and beacon scheduling schemes are designed for the IEEE 802.15.4e enabled WSNs in star topology to reduce the network discovery time and energy consumption. In addition, a new dynamic guaranteed retransmission slot allocation scheme is designed for devices with the failure Guaranteed Time Slot (GTS) transmission to reduce the retransmission delay. To evaluate our schemes, analytical models are designed to analyze the performance of WSNs in terms of reliability, delay, throughput and energy consumption. Our schemes are validated with simulation and analytical results and are observed that simulation results well match with the analytical one. The evaluated results of our designed schemes can improve the reliability, throughput, delay, and energy consumptions significantly.

  17. Sonochemical synthesis of novel pyrano[3,4-e][1,3]oxazines: A green protocol.

    PubMed

    Saleh, Tamer S; Al-Bogami, Abdullah S; Mekky, Ahmed E M; Alkhathlan, Hamad Z

    2017-05-01

    The atom-efficient and green protocol for formation of pyrano[3,4-e][1,3]oxazines utilizing dimethyl carbonate under ultrasound irradiation in a presence of KF/basic alumina was reported. We provide a novel series of pyrano[3,4-e][1,3]oxazine derivatives interesting for biological screening tests. In general, it was found that ultrasound irradiations enable the reactions to occur which could not be carried out under silent conditions. These remarkable effects appeared in sonicated reactions can be reasonably interpreted in terms of acoustic cavitation phenomenon. Structures of the products were established on analytical and spectral data. This protocol offers several advantages attain many principles of green chemistry including, save energy, atom economy, clean reactions, inexpensive green reagent and use catalysts rather than stoichiometric reagents.

  18. Photoelectron spectroscopy of B4O4-: Dual 3c-4e π hyperbonds and rhombic 4c-4e o-bond in boron oxide clusters

    NASA Astrophysics Data System (ADS)

    Tian, Wen-Juan; Zhao, Li-Juan; Chen, Qiang; Ou, Ting; Xu, Hong-Guang; Zheng, Wei-Jun; Zhai, Hua-Jin; Li, Si-Dian

    2015-04-01

    Gas-phase anion photoelectron spectroscopy (PES) is combined with global structural searches and electronic structure calculations at the hybrid Becke 3-parameter exchange functional and Lee-Yang-Parr correlation functional (B3LYP) and single-point coupled-cluster with single, double, and perturbative triple excitations (CCSD(T)) levels to probe the structural and electronic properties and chemical bonding of the B4O40/- clusters. The measured PES spectra of B4O4- exhibit a major band with the adiabatic and vertical detachment energies (ADE and VDE) of 2.64 ± 0.10 and 2.81 ± 0.10 eV, respectively, as well as a weak peak with the ADE and VDE of 1.42 ± 0.08 and 1.48 ± 0.08 eV. The former band proves to correspond to the Y-shaped global minimum of Cs B4O4- (2A″), with the calculated ADE/VDE of 2.57/2.84 eV at the CCSD(T) level, whereas the weak band is associated with the second lowest-energy, rhombic isomer of D2h B4O4- (2B2g) with the predicted ADE/VDE of 1.43/1.49 eV. Both anion structures are planar, featuring a B atom or a B2O2 core bonded with terminal BO and/or BO2 groups. The same Y-shaped and rhombic structures are also located for the B4O4 neutral cluster, albeit with a reversed energy order. Bonding analyses reveal dual three-center four-electron (3c-4e) π hyperbonds in the Y-shaped B4O40/- clusters and a four-center four-electron (4c-4e) π bond, that is, the so-called o-bond in the rhombic B4O40/- clusters. This work is the first experimental study on a molecular system with an o-bond.

  19. The putative tumor suppressor microRNA-497 modulates gastric cancer cell proliferation and invasion by repressing eIF4E

    SciTech Connect

    Li, Weidong; Jin, Xuejun; Deng, Xubin; Zhang, Gong; Zhang, Bingqian; Ma, Lei

    2014-06-27

    Highlights: • MiR-497 expression was down-regulated in GC patients and GC cell lines. • MiR-497 inhibited cell proliferation and invasion of GC cells in vitro. • MiR-497 modulated eIF4E expression in GC cells. • Restoration of miR-497 decreased tumor growth and metastasis in vivo. - Abstract: Accumulating evidence has shown that microRNAs are involved in multiple processes in gastric cancer (GC) development and progression. Aberrant expression of miR-497 has been frequently reported in cancer studies; however, the role and mechanism of its function in GC remains unknown. Here, we reported that miR-497 was frequently downregulated in GC tissues and associated with aggressive clinicopathological features of GC patients. Further in vitro observations showed that the enforced expression of miR-497 inhibited cell proliferation by blocking the G1/S transition and decreased the invasion of GC cells, implying that miR-497 functions as a tumor suppressor in the progression of GC. In vivo study indicated that restoration of miR-497 inhibited tumor growth and metastasis. Luciferase assays revealed that miR-497 inhibited eIF4E expression by targeting the binding sites in the 3′-untranslated region of eIF4E mRNA. qRT-PCR and Western blot assays verified that miR-497 reduced eIF4E expression at both the mRNA and protein levels. A reverse correlation between miR-497 and eIF4E expression was noted in GC tissues. Taken together, our results identify a crucial tumor suppressive role of miR-497 in the progression of GC and suggest that miR-497 might be an anticancer therapeutic target for GC patients.

  20. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  1. Inhibition of 4E-BP1 Sensitizes U87 Glioblastoma Xenograft Tumors to Irradiation by Decreasing Hypoxia Tolerance

    SciTech Connect

    Dubois, Ludwig; Magagnin, Michael G.; Cleven, Arjen H.G.; Weppler, Sherry A.; Grenacher, Beat; Landuyt, Willy; Lieuwes, Natasja; Lambin, Philippe; Gorr, Thomas A.; Koritzinsky, Marianne

    2009-03-15

    Purpose: Eukaryotic initiation factor 4E (eIF4E) is an essential rate-limiting factor for cap-dependent translation in eukaryotic cells. Elevated eIF4E activity is common in many human tumors and is associated with disease progression. The growth-promoting effects of eIF4E are in turn negatively regulated by 4E-BP1. However, although 4E-BP1 harbors anti-growth activity, its expression is paradoxically elevated in some tumors. The aim of this study was to investigate the functional role of 4E-BP1 in the context of solid tumors. Methods and Materials: In vitro and in vivo growth properties, hypoxia tolerance, and response to radiation were assessed for HeLa and U87 cells, after stable expression of shRNA specific for 4E-BP1. Results: We found that loss of 4E-BP1 expression did not significantly alter in vitro growth but did accelerate the growth of U87 tumor xenografts, consistent with the growth-promoting function of deregulated eIF4E. However, cells lacking 4E-BP1 were significantly more sensitive to hypoxia-induced cell death in vitro. Furthermore, 4E-BP1 knockdown cells produced tumors more sensitive to radiation because of a reduction in the viable fraction of radioresistant hypoxic cells. Decreased hypoxia tolerance in the 4E-BP1 knockdown tumors was evident by increased cleaved caspase-3 levels and was associated with a reduction in adenosine triphosphate (ATP). Conclusions: Our results suggest that although tumors often demonstrate increases in cap-dependent translation, regulation of this activity is required to facilitate energy conservation, hypoxia tolerance, and tumor radioresistance. Furthermore, we suggest that targeting translational control may be an effective way to target hypoxic cells and radioresistance in metabolically hyperactive tumors.

  2. Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation.

    PubMed

    Soukarieh, Fadi; Nowicki, Matthew W; Bastide, Amandine; Pöyry, Tuija; Jones, Carolyn; Dudek, Kate; Patwardhan, Geetanjali; Meullenet, François; Oldham, Neil J; Walkinshaw, Malcolm D; Willis, Anne E; Fischer, Peter M

    2016-11-29

    Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5'-terminal mRNA cap structure (m(7)GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N(7)-substituent of m(7)GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N'-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m(7)GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5'-deoxy-5'-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N(7)-methyl-guanosine (4a), N(7)-3-chlorobenzyl-5'-deoxy-5'-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N(7)-benzyl-5'-deoxy-5'-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents.

  3. NEDD4 E3 ligase inhibits the activity of the Hippo pathway by targeting LATS1 for degradation.

    PubMed

    Salah, Zaidoun; Cohen, Sherri; Itzhaki, Ella; Aqeilan, Rami I

    2013-12-15

    Proper regulation of cell proliferation, cell apoptosis, and cell death are vital for the development and survival of living organisms. Failure or dysfunction of any of these processes can have devastating effects, including cancer. The Hippo pathway, first discovered in Drosophila, has been found to be a major growth-regulatory signaling pathway that controls these crucial processes and has been implicated in cell-progress regulation and organ size determination. Abnormal regulation of this pathway has been found in several cancer types. However, the mechanisms that regulate the pathway and its core members yet have to be elucidated. One of the main core components of this pathway is LATS1, a serine/threonine kinase. Therefore, understanding how LATS1 activity is regulated is expected to shed light on new mechanisms that regulate the Hippo pathway. In the current work, we identified several potential LATS1 regulators and proved that NEDD4 E3 ubiquitin ligase controls LATS1 stability. We demonstrate that NEDD4 directly interacts with LATS1, leading to ubiquitination and decreased levels of LATS1 and, thus, increased YAP localization in the nucleus, which subsequently increases the transcriptional activity of YAP. As such, we show that NEDD4 acts as an additional regulator of the Hippo pathway on the protein level via interactions between WW domain-containing and PPxY motif-containing proteins. These findings might be applied in the development of new therapeutic approaches through the activation of LATS1.

  4. NEDD4 E3 ligase inhibits the activity of the Hippo pathway by targeting LATS1 for degradation

    PubMed Central

    Salah, Zaidoun; Cohen, Sherri; Itzhaki, Ella; Aqeilan, Rami I

    2013-01-01

    Proper regulation of cell proliferation, cell apoptosis, and cell death are vital for the development and survival of living organisms. Failure or dysfunction of any of these processes can have devastating effects, including cancer. The Hippo pathway, first discovered in Drosophila, has been found to be a major growth-regulatory signaling pathway that controls these crucial processes and has been implicated in cell-progress regulation and organ size determination. Abnormal regulation of this pathway has been found in several cancer types. However, the mechanisms that regulate the pathway and its core members yet have to be elucidated. One of the main core components of this pathway is LATS1, a serine/threonine kinase. Therefore, understanding how LATS1 activity is regulated is expected to shed light on new mechanisms that regulate the Hippo pathway. In the current work, we identified several potential LATS1 regulators and proved that NEDD4 E3 ubiquitin ligase controls LATS1 stability. We demonstrate that NEDD4 directly interacts with LATS1, leading to ubiquitination and decreased levels of LATS1 and, thus, increased YAP localization in the nucleus, which subsequently increases the transcriptional activity of YAP. As such, we show that NEDD4 acts as an additional regulator of the Hippo pathway on the protein level via interactions between WW domain-containing and PPxY motif-containing proteins. These findings might be applied in the development of new therapeutic approaches through the activation of LATS1. PMID:24107629

  5. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  6. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  7. FUS-DDIT3 Prevents the Development of Adipocytic Precursors in Liposarcoma by Repressing PPARγ and C/EBPα and Activating eIF4E

    PubMed Central

    Pérez-Mancera, Pedro A.; Bermejo-Rodríguez, Camino; Sánchez-Martín, Manuel; Abollo-Jiménez, Fernando; Pintado, Belén; Sánchez-García, Isidro

    2008-01-01

    Background FUS-DDIT3 is a chimeric protein generated by the most common chromosomal translocation t(12;16)(q13;p11) linked to liposarcomas, which are characterized by the accumulation of early adipocytic precursors. Current studies indicate that FUS-DDIT3- liposarcoma develops from uncommitted progenitors. However, the precise mechanism whereby FUS-DDIT3 contributes to the differentiation arrest remains to be elucidated. Methodology/Principal Findings Here we have characterized the adipocyte regulatory protein network in liposarcomas of FUS-DITT3 transgenic mice and showed that PPARγ2 and C/EBPα expression was altered. Consistent with in vivo data, FUS-DDIT3 MEFs and human liposarcoma cell lines showed a similar downregulation of both PPARγ2 and C/EBPα expression. Complementation studies with PPARγ but not C/EBPα rescued the differentiation block in committed adipocytic precursors expressing FUS-DDIT3. Our results further show that FUS-DDIT3 interferes with the control of initiation of translation by upregulation of the eukaryotic translation initiation factors eIF2 and eIF4E both in FUS-DDIT3 mice and human liposarcomas cell lines, explaining the shift towards the truncated p30 isoform of C/EBPα in liposarcomas. Suppression of the FUS-DDIT3 transgene did rescue this adipocyte differentiation block. Moreover, eIF4E was also strongly upregulated in normal adipose tissue of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be a primary event in the initiation of liposarcomas. Reporter assays showed FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas and that both domains of the fusion protein are required for affecting eIF4E expression. Conclusions/Significance Taken together, this study provides evidence of the molecular mechanisms involve in the disruption of normal adipocyte differentiation program in liposarcoma harbouring the chimeric gene FUS-DDIT3. PMID:18596980

  8. In vivo study of breast carcinoma radiosensitization by targeting eIF4E

    SciTech Connect

    Yang, Hua; Li, Li-Wen; Shi, Mei; Wang, Jian-Hua; Xiao, Feng; Zhou, Bin; Diao, Li-Qiong; Long, Xiao-Li; Liu, Xiao-Li; Xu, Lin

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer eIF4E is associated with the formation and progression for breast cancer. Black-Right-Pointing-Pointer pSecX-t4EBP1 can downregulated the expression of eIF4E in direct binding. Black-Right-Pointing-Pointer We transfected pSecX-t4EBP1 into a mouse xenograft model. Black-Right-Pointing-Pointer It can significantly inhibit tumor growth and enhance the radiosensitivity. Black-Right-Pointing-Pointer The possible mechanism is downregulation of HIF-1{alpha} expression. -- Abstract: Background: Eukaryotic initiation factor eIF4E, an important regulator of translation, plays a crucial role in the malignant transformation, progression and radioresistance of many human solid tumors. The overexpression of this gene has been associated with tumor formation in a wide range of human malignancies, including breast cancer. In the present study, we attempted to explore the use of eIF4E as a therapeutic target to enhance radiosensitivity for breast carcinomas in a xenograft BALB/C mice model. Materials and methods: Ninety female BALB/C mice transfected with EMT-6 cells were randomly divided into six groups: control, irradiation (IR), pSecX-t4EBP1, pSecX-t4EBP1 + irradiation, pSecX and pSecX + irradiation. At the end of the experiments, all mice were sacrificed, the xenografts were harvested to measure the tumor volume and mass, and the tumor inhibition rates were calculated. Apoptosis was detected with a flow cytometric assay. Immunohistochemistry was used to detect the expression of HIF-1{alpha}. Results: The xenografts in pSecX-t4EBP1 mice showed a significantly delayed growth and smaller tumor volume, with a higher tumor inhibition rate compared with the control and pSecX groups. A similar result was obtained in the pSecX-t4EBP1 + IR group compared with IR alone and pSecX + irradiation. The expression of HIF-1{alpha} in the tumor cells was significantly decreased, while the apoptosis index was much higher. Conclusions: pSecX-t4EBP1 can

  9. The eukaryotic translation initiation factor eIF4E wears a “cap” for many occasions

    PubMed Central

    Borden, Katherine L. B.

    2016-01-01

    ABSTRACT The eukaryotic translation initiation factor eIF4E plays important roles in controlling the composition of the proteome. Indeed, dysregulation of eIF4E is associated with poor prognosis cancers. The traditional view has been that eIF4E acts solely in translation. However, over the last ∼25 years, eIF4E was found in the nucleus where it acts in mRNA export and in the last ∼10 years, eIF4E was found in cytoplasmic processing bodies (P-bodies) where it functions in mRNA sequestration and stability. The common biochemical thread for these activities is the ability of eIF4E to bind the 7-methylguanosine cap on the 5′ end of mRNAs. Recently, the possibility that eIF4E directly binds some mRNA elements independently of the cap has also been raised. Importantly, the effects of eIF4E are not genome-wide with a subset of transcripts targeted depending on the presence of specific mRNA elements and context-dependent regulatory factors. Indeed, eIF4E governs RNA regulons through co-regulating the expression of groups of transcripts acting in the same biochemical pathways. In addition, studies over the past ∼15 years indicate that there are multiple strategies that regulatory factors employ to modulate eIF4E activities in context-dependent manners. This perspective focuses on these new findings and incorporates them into a broader model for eIF4E function. PMID:28090419

  10. Semisolid forming of 42CrMo4E steel grade

    NASA Astrophysics Data System (ADS)

    Lozares, Jokin; Plata, Gorka; Azpilgain, Zigor; Álvarez, Gonzalo

    2016-10-01

    Reduction of production costs is the aim of many companies in order to become more competitive. In this field, one of the so called `near net shape' processes, the semisolid metal forming (SSF), has revealed a high potential in terms of raw material and energetic savings. The lack of materials that result in good mechanical properties after SSF makes it difficult to implement this technology into the industry. This fact requires further thixoformability investigations on different steels to be overcome. Therefore, this research work, which is the continuation of [1], focuses on analyzing the thixoformability of the commercially available 42CrMo4E steel grade by thixoforging of an automotive spindle. Microstructure and mechanical properties evaluation has been as well carried-out to conclude, first, the impossibility of estimating by quenching and quantitative metallography analysis the liquid fraction of 42CrMo4E, and second, the great accordance of the mechanical properties of SSF component with the requirements of hot forged part.

  11. mTOR downstream effectors, 4EBP1 and eIF4E, are overexpressed and associated with HPV status in precancerous lesions and carcinomas of the uterine cervix

    PubMed Central

    Asimomytis, Aristidis; Karanikou, Maria; Rodolakis, Alexander; Vaiopoulou, Anna; Tsetsa, Paraskevi; Creatsas, George; Stefos, Theodoros; Antsaklis, Aristidis; Patsouris, Efstratios; Rassidakis, George Z.

    2016-01-01

    The present study aims to investigate the expression levels of two critical mammalian target of rapamycin (mTOR) downstream effectors, 4E binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) proteins, in precancerous squamous intraepithelial lesions and cancer of the uterine cervix, and their association with human papilloma virus (HPV) infection status. Uterine cervical biopsies from 73 patients were obtained, including 40 fresh-frozen samples and 42 archival formalin-fixed, paraffin-embedded tissue specimens. Whole protein extracts were analyzed for the expression of 4EBP1 and eIF4E proteins using western blotting. In addition, distribution of 4EBP1 and eIF4E protein expression and 4EBP1 phosphorylation (P-4EBP1) were analyzed by immunohistochemistry in archival tissues and correlated with the degree of dysplasia. The presence of high-risk HPV (HR-HPV) types was assessed by polymerase chain reaction. Using western blot analysis, high expression levels of 4EBP1 and eIF4E were observed in all uterine cervical carcinomas, which significantly correlated with the degree of dysplasia. By immunohistochemistry, overexpression of 4EBP1 and eIF4E was detected in 20 of 21 (95%) and 17 of 21 (81%) samples, respectively, in patients with high-grade dysplasia and carcinomas, compared with 1 of 20 (5%) and 2 of 20 (10%) samples, respectively, in patients with low-grade lesions or normal histology. All 4EBP1-positive cases tested were also positive for P-4EBP1. Furthermore, overexpression of 4EBP1 and eIF4E significantly correlated with the presence of HR-HPV oncogenic types. The present study demonstrated that critical effectors of mTOR signaling, which control protein synthesis initiation, are overexpressed in cervical high-grade dysplasia and cancer, and their levels correlate with oncogenic HPV types. These findings may provide novel targets for investigational therapeutic approaches in patients with cancer of the uterine cervix. PMID:27899988

  12. Dual modulation of Ras-Mnk and PI3K-AKT-mTOR pathways: A Novel c-FLIP inhibitory mechanism of 3-AWA mediated translational attenuation through dephosphorylation of eIF4E

    PubMed Central

    ur Rasool, Reyaz; Rah, Bilal; Amin, Hina; Nayak, Debasis; Chakraborty, Souneek; Rawoof, Abdul; Mintoo, Mubashir Javed; Yousuf, Khalid; Mukherjee, Debaraj; Kumar, Lekha Dinesh; Mondhe, Dilip Manikaro; Goswami, Anindya

    2016-01-01

    The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development. PMID:26728896

  13. Dual modulation of Ras-Mnk and PI3K-AKT-mTOR pathways: A Novel c-FLIP inhibitory mechanism of 3-AWA mediated translational attenuation through dephosphorylation of eIF4E.

    PubMed

    ur Rasool, Reyaz; Rah, Bilal; Amin, Hina; Nayak, Debasis; Chakraborty, Souneek; Rawoof, Abdul; Mintoo, Mubashir Javed; Yousuf, Khalid; Mukherjee, Debaraj; Kumar, Lekha Dinesh; Mondhe, Dilip Manikaro; Goswami, Anindya

    2016-01-05

    The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development.

  14. Targeting the eIF4E/β-catenin axis sensitizes cervical carcinoma squamous cells to chemotherapy

    PubMed Central

    Xu, Hai; Wang, Zhiyin; Xu, Lang; Mo, Guoyan; Duan, Gangfeng; Wang, Yali; Sun, Zhengang; Chen, Hao

    2017-01-01

    Chemotherapy has improved the clinical outcomes of cervical cancer patients. However, patients develop chemoresistance, whose underlying mechanisms are not well understood. In this study, we investigated the phosphorylation levels of eukaryotic translation initiation factor 4E (eIF4E) in cervical cancer cells subjected to chemotherapy. Results showed that chemotherapeutic drugs significantly increased eIF4E phosphorylation at S209 in HeLa and SiHa cells. Upregulation of phosphorylated eIF4E (p-eIF4E) levels has also been shown in cisplatin-resistant HeLa cells and has been observed to be a common response of cervical cancer patients undergoing chemotherapy. We further showed that chemotherapeutic drugs increase β-catenin activity and mRNA levels of Wnt/β-catenin target genes in cervical cancer cells but not in eIF4E-depleted cells, suggesting that chemotherapeutic drugs activate Wnt/β-catenin signaling in an eIF4E-dependent manner. Inhibiting eIF4E via siRNA knockdown or Wnt/β-catenin using the Wnt inhibitor pyrvinium effectively enhanced the anti-proliferative and pro-apoptotic effects of cisplatin in cervical cancer cells both in vitro and in vivo. Our findings demonstrate that eIF4E/β-catenin signaling plays a positive regulatory role in the resistance of cervical cancer cell to chemotherapy and thus highlight the therapeutic value of eIF4E or β-catenin inhibition in overcoming chemoresistance. PMID:28386346

  15. [1,2]Oxazolo[5,4-e]isoindoles as promising tubulin polymerization inhibitors.

    PubMed

    Spanò, Virginia; Pennati, Marzia; Parrino, Barbara; Carbone, Anna; Montalbano, Alessandra; Lopergolo, Alessia; Zuco, Valentina; Cominetti, Denis; Diana, Patrizia; Cirrincione, Girolamo; Zaffaroni, Nadia; Barraja, Paola

    2016-11-29

    A series of [1,2]Oxazolo [5,4-e]isoindoles has been synthesized through a versatile and high yielding sequence. All the new structures showed in the (1)HNMR spectra, the typical signal in the 8.34-8.47 ppm attributable to the H-3 of the [1,2]oxazole moiety. Among all derivatives, methoxy benzyl substituents at positions 3 and 4 or/and 5 were very effective in reducing the growth of different tumor cell lines, including diffuse malignant peritoneal mesothelioma (DMPM), an uncommon and rapidly malignancy poorly responsive to available therapeutic options. The most active compound 6j was found to impair tubulin polymerization, cause cell cycle arrest at G2/M phase and induce apoptosis in DMPM cells, making it as a new lead for the discovery of new potent antimitotic drugs.

  16. 4-[(E)-2-(Pyridin-2-yl)ethen-yl]pyridine-terephthalic acid (2/1).

    PubMed

    Castro-Montes, Paola; Guerrero-Alvarez, Jorge A; Hopfl, Herbert; Campos-Gaxiola, Jose J; Cruz-Enriquez, Adriana

    2012-12-01

    The title 2:1 co-crystal, 2C12H10N2·C8H6O4, crystallizes with one mol-ecule of 4-[(E)-2-(pyridin-2-yl)ethen-yl]pyridine (A) and one half-mol-ecule of terephthalic acid (B) in the asymmetric unit. In the crystal, the components are linked through heterodimeric COOH⋯Npyridine synthons, forming linear aggregates of composition -A-B-A-B-. Further linkage through weak C-H⋯O and C-H⋯π inter-actions gives two-dimensional hydrogen-bonded undulating sheets propagating in the [100] and [010] directions. These layers are connected through additional weak C-H⋯O contacts, forming a three-dimensional structure.

  17. Identification of a CAPS marker in an eIF4E gene Linked to Zucchini yellow mosaic virus resistant locus in watermelon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes that encode eukaryotic initiation factors (eIF) 4E and iso(4E) have been associated with the recessively inherited resistance to potyviruses in a number of plant species. Using previously developed degenerate primers, partial eIF4E and eIF(iso)4E gene sequence regions were obtained through po...

  18. Immune tolerance negatively regulates B cells in knock-in mice expressing broadly neutralizing HIV antibody 4E10

    PubMed Central

    Cooper, Anthony B.; Ota, Takayuki; Skog, Patrick; Dawson, Phillip E.; Zwick, Michael B.; Schief, William R.; Burton, Dennis R.; Nemazee, David

    2013-01-01

    A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing antibodies (bNAbs). This has proved to be challenging, however. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated “knock-in” mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the accompanying study, 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms including receptor editing, clonal deletion and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1−/− background 4E10HL mice had virtually no serum immunoglobulins of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of an MPER-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly-described bNAb 10E8. PMID:23940276

  19. Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance.

    PubMed

    Rodríguez-Hernández, Ana M; Gosalvez, Blanca; Sempere, Raquel N; Burgos, Lorenzo; Aranda, Miguel A; Truniger, Verónica

    2012-09-01

    Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.

  20. Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10.

    PubMed

    Irimia, Adriana; Sarkar, Anita; Stanfield, Robyn L; Wilson, Ian A

    2016-01-19

    Numerous studies of the anti-HIV-1 envelope glycoprotein 41 (gp41) broadly neutralizing antibody 4E10 suggest that 4E10 also interacts with membrane lipids, but the antibody regions contacting lipids and its orientation with respect to the viral membrane are unknown. Vaccine immunogens capable of re-eliciting these membrane proximal external region (MPER)-like antibodies may require a lipid component to be successful. We performed a systematic crystallographic study of lipid binding to 4E10 to identify lipids bound by the antibody and the lipid-interacting regions. We identified phosphatidic acid, phosphatidylglycerol, and glycerol phosphate as specific ligands for 4E10 in the crystal structures. 4E10 used its CDRH1 loop to bind the lipid head groups, while its CDRH3 interacted with the hydrophobic lipid tails. Identification of the lipid binding sites on 4E10 may aid design of immunogens for vaccines that include a lipid component in addition to the MPER on gp41 for generation of broadly neutralizing antibodies.

  1. 4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

    PubMed Central

    Nakamura, Kyle J.; Gach, Johannes S.; Jones, Laura; Semrau, Katherine; Walter, Jan; Bibollet-Ruche, Frederic; Decker, Julie M.; Heath, Laura; Decker, William D.; Sinkala, Moses; Kankasa, Chipepo; Thea, Donald; Mullins, James; Kuhn, Louise; Zwick, Michael B.; Aldrovandi, Grace M.

    2010-01-01

    Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope. PMID:20352106

  2. Toxicity of abate® 4E (temephos) in mallard ducklings and the influence of cold

    USGS Publications Warehouse

    Fleming, W.J.; Heinz, G.H.; Franson, J.C.; Rattner, B.A.

    1985-01-01

    Diets mixed to contain 0, 0.1, 1.0, 10 and 100 ppm temephos (determined chemically to contain less than 0.5, less than 0.5, 0.89, 6.0 and 59 ppm temephos, respectively) in an Abate® 4E formulation, were fed to mallard (Anas platyrhynchos) ducklings for 7 d. During this period, half of the ducklings in each dietary treatment group were housed in a heated brooder (39 to 41 °C) and half were housed in an unheated brooder (10 to 18°C). Mortality in all dietary groups in the unheated brooder was higher than in the heated brooder. High temephos-related mortality occurred in the 100 ppm group in the unheated brooder but not in any other diet-temperature groups. Ingestion of the 100 ppm temephos diet inhibited plasma Cholinesterase (ChE) activity and elevated plasma corticosterone concentration and creatine phosphokinase activity, but other selected plasma chemistries were not affected in a dose-related manner. Brain ChE activity was depressed only in the 100 ppm dietary groups; maximum inhibition of brain ChE activity was 48%. These findings suggest that diets containing up to 10 ppm temephos do not directly affect duckling survival during the first week of life and that the toxicity of 100 ppm temephos is markedly enhanced by cold.

  3. Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists

    PubMed Central

    Jagus, Rosemary; Bachvaroff, Tsvetan R.; Joshi, Bhavesh; Place, Allen R.

    2012-01-01

    The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in “text-book” model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed. PMID:22778692

  4. Structural analysis of the eukaryotic initiation factor 4E gene controlling potyvirus resistance in pepper: exploitation of a BAC library.

    PubMed

    Ruffel, Sandrine; Caranta, Carole; Palloix, Alain; Lefebvre, Véronique; Caboche, Michel; Bendahmane, Abdelhafid

    2004-09-01

    The pvr2 locus in pepper codes for a eukaryotic translation initiation factor 4E (eIF4E) gene that confers resistance to viruses belonging to the potyvirus genus. In this work, we describe the isolation and characterisation of the genomic sequence carrying the pvr2 locus. A Bacterial Artificial Chromosome (BAC) library that consisted of 239,232 clones with an average insert size of 123 kilobases (kb) was constructed from a Capsicum annuum line with the pvr2(+) allele for susceptibility to potato virus Y (PVY) and tobacco etch virus (TEV). Based on a polymerase chain reaction (PCR) screen with single-copy markers, three to seven positive BAC clones per markers were identified, indicating that the BAC library is suitable for pepper genome analysis. To determine the genomic organization of the pepper eIF4E gene, the library was screened with primers designed from the cDNA sequence and four positive BAC clones carrying the pvr2 locus were identified. A 7-kb DNA fragment containing the complete eIF4E gene was sub-cloned from the positive BAC clones and analysed. The eIF4E gene is organised into five exons and four introns and showed a strictly conserved exon/intron structure with eIF4E genes from Arabidopsis thaliana and rice. Moreover, the splice sites between plant exons 1/2 and 2/3 are conserved among eukaryotes including human, Drosophila and yeast. Several potential binding sites for MADS box transcription factors within the 5' flanking region of eIF4E genes from the three plant species were also predicted.

  5. Spatial and temporal translational control of germ cell mRNAs mediated by the eIF4E isoform IFE-1.

    PubMed

    Friday, Andrew J; Henderson, Melissa A; Morrison, J Kaitlin; Hoffman, Jenna L; Keiper, Brett D

    2015-12-15

    Regulated mRNA translation is vital for germ cells to produce new proteins in the spatial and temporal patterns that drive gamete development. Translational control involves the de-repression of stored mRNAs and their recruitment by eukaryotic initiation factors (eIFs) to ribosomes. C. elegans expresses five eIF4Es (IFE-1-IFE-5); several have been shown to selectively recruit unique pools of mRNA. Individual IFE knockouts yield unique phenotypes due to inefficient translation of certain mRNAs. Here, we identified mRNAs preferentially translated through the germline-specific eIF4E isoform IFE-1. Differential polysome microarray analysis identified 77 mRNAs recruited by IFE-1. Among the IFE-1-dependent mRNAs are several required for late germ cell differentiation and maturation. Polysome association of gld-1, vab-1, vpr-1, rab-7 and rnp-3 mRNAs relies on IFE-1. Live animal imaging showed IFE-1-dependent selectivity in spatial and temporal translation of germline mRNAs. Altered MAPK activation in oocytes suggests dual roles for IFE-1, both promoting and suppressing oocyte maturation at different stages. This single eIF4E isoform exerts positive, selective translational control during germ cell differentiation.

  6. mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction on 4E-BP1 dephosphorylation.

    PubMed

    He, K; Zheng, X; Li, M; Zhang, L; Yu, J

    2016-01-14

    The mammalian target of rapamycin (mTOR) is commonly activated in colon cancer. mTOR complex 1 (mTORC1) is a major downstream target of the PI3K/ATK pathway and activates protein synthesis by phosphorylating key regulators of messenger RNA translation and ribosome synthesis. Rapamycin analogs Everolimus and Temsirolimus are non-ATP-competitive mTORC1 inhibitors, and suppress proliferation and tumor angiogenesis and invasion. We now show that apoptosis plays a key role in their anti-tumor activities in colon cancer cells and xenografts through the DR5, FADD and caspase-8 axis, and is strongly enhanced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and 5-fluorouracil. The induction of DR5 by rapalogs is mediated by the ER stress regulator and transcription factor CHOP, but not the tumor suppressor p53, on rapid and sustained inhibition of 4E-BP1 phosphorylation, and attenuated by eIF4E expression. ATP-competitive mTOR/PI3K inhibitors also promote DR5 induction and FADD-dependent apoptosis in colon cancer cells. These results establish activation of ER stress and the death receptor pathway as a novel anticancer mechanism of mTOR inhibitors.

  7. mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction upon 4E-BP1 dephosphorylation

    PubMed Central

    He, Kan; Zheng, Xingnan; Li, Mei; Zhang, Lin; Yu, Jian

    2015-01-01

    The mammalian target of rapamycin (mTOR) is commonly activated in colon cancer. mTOR complex 1 (mTORC1) is a major downstream target of the PI3K/ATK pathway and activates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. Rapamycin analogs Everolimus and Temsirolimus are non-ATP-competitive mTORC1 inhibitors, and suppress proliferation and tumor angiogenesis and invasion. We now show that apoptosis plays a key role in their anti-tumor activities in colon cancer cells and xenografts through the DR5, FADD and caspase-8 axis, and is strongly enhanced by TRAIL and 5-fluorouracil. The induction of DR5 by rapalogs is mediated by the ER stress regulator and transcription factor CHOP, but not the tumor suppressor p53, upon rapid and sustained inhibition of 4E-BP1 phosphorylation, and attenuated by eIF4E expression. ATP-competitive mTOR/PI3K inhibitors also promote DR5 induction and FADD-dependent apoptosis in colon cancer cells. These results establish activation of ER stress and the death receptor pathway as a novel anticancer mechanism of mTOR inhibitors. PMID:25867072

  8. The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase.

    PubMed Central

    Haghighat, A; Svitkin, Y; Novoa, I; Kuechler, E; Skern, T; Sonenberg, N

    1996-01-01

    The 2A proteinases (2Apro) of certain picornaviruses induce the cleavage of the eIF4G subunit of the cap-binding protein complex, eIF4F. Several reports have demonstrated that 2Apro of rhinovirus and coxsackievirus B4 cleave eIF4G directly. However, it was suggested that in poliovirus infection, the 2Apro induces the activation of a cellular proteinase which in turn cleaves eIF4G. Furthermore, it is not clear whether eIF4G is cleaved as part of the eIF4F complex or as an individual polypeptide. To address these issues, recombinant eIF4G was purified from Sf9 insect cells and tested for cleavage by purified rhinovirus 2Apro. Here we report that eIF4G alone is a relatively poor substrate for cleavage by the rhinovirus 2Apro. However, an eIF4G-eIF4E complex is cleaved efficiently by the 2Apro, suggesting that eIF4F is a preferred substrate for cleavage by rhinovirus 2Apro. Furthermore, 2Apr drastically reduced the translation of a capped mRNA. An eIF4G-eIF4E complex, but not eIF4G alone, was required to restore translation. PMID:8970966

  9. Acoustic measurements of F-4E aircraft operating in hush house, NSN 4920-02-070-2721

    NASA Astrophysics Data System (ADS)

    Miller, V. R.; Plzak, G. A.; Chinn, J. M.

    1981-09-01

    The primary purpose of this test program was to measure the acoustic environment in the hush house facility located at Kelly Air Force Base, Texas, during operation of the F-4E aircraft to ensure that aircraft structural acoustic design limits were not exceeded. The acoustic measurements showed that sonic fatigue problems are anticipated with the F-4E aircraft aft fuselage structure during operation in the hush house. The measured acoustic levels were less than those measured in an F-4E aircraft water cooled hush house at Hill AFB in the lower frequencies, but were increased over that measured during ground run up on some areas of the aircraft. It was recommended that the acoustic loads measured in this program should be specified in the structural design criteria for aircraft which will be subjected to hush house operation or defining requirements for associated equipment. Recommendations were also made to increase the fatigue life of the aft fuselage.

  10. Effects of Altosid and Abate-4E on deformities and survival in southern leopard frogs under semi-natural conditions

    USGS Publications Warehouse

    Sparling, D.W.; Kaiser, Hinrich; Casper, Gary S.; Bernstein, Neil P.

    2000-01-01

    Experimental wetlands were sprayed with Abate-4E (a.i. temephos) and Altosid (a.i. methoprene) through the summer following label directions. In late August and early Septemeber metamorphing tadpoles were captured and examined for deformities. Tadpoles captured from ponds sprayed with Altosid had a 15% deformity rate mostly involving total or partially missing hind limbs. Tadpoles from control ponds had a 5% rate of deformities. The difference was statistically significant. The relative abundance of tadpoles from ponds sprayed with Abate-4E was significantly lower than those from Altosid-sprayed or control wetlands.

  11. Temporal changes in ERK phosphorylation are harmonious with 4E-BP1, but not p70S6K, during clenbuterol-induced hypertrophy in the rat gastrocnemius.

    PubMed

    Sumi, Koichiro; Higashi, Seiichiro; Natsume, Midori; Kawahata, Keiko; Nakazato, Koichi

    2014-08-01

    Extracellular signal-regulated kinase (ERK) is required for clenbuterol (CB)-dependent fast-type myofibril enlargement; however, its contribution to translation control is unclear. ERK mediates translational regulation through mammalian target of rapamycin complex 1 (mTORC1) activation and (or) mTORC1-independent pathways. In this study, we aimed to investigate the role of ERK in translational control during CB-induced muscular hypertrophy by measuring time-dependent changes in the phosphorylation statuses of ERK, p70 ribosomal S6 kinase (p70S6K; an indicator of mTORC1 activity), 4E-binding protein 1 (4E-BP1), eukaryotic elongation factor 2 (eEF2), and other related signaling molecules in rat gastrocnemius muscles. Five-day administration of CB induced phenotypes associated with muscular hypertrophy (significant increases in wet weight and isometric ankle flexion torque in the gastrocnemius muscle), but was not accompanied by elevated ERK or p70S6K phosphorylation. One-day administration of CB caused significant increases in the phosphorylation of ERK, p70S6K, and 4E-BP1. In contrast, 3-day administration of CB caused significant increases in the phosphorylation of ERK and 4E-BP1, but not p70S6K. In addition, positive correlations were observed between ERK and 4E-BP1 on days 1 and 3, whereas a correlation between ERK and p70S6K was only observed on day 1. eEF2 phosphorylation was unchanged on both days 1 and 3. These findings suggest that ERK accelerates the initiation of translation, but does not support the involvement of ERK in translational elongation. Furthermore, ERK may play a major role in promoting translational initiation by mediating the phosphorylation of 4E-BP1, and may contribute to the initial activation of mTORC1 during CB administration.

  12. Phosphorylation of mTOR and S6RP predicts the efficacy of everolimus in patients with metastatic renal cell carcinoma

    PubMed Central

    2014-01-01

    Background The incidence of renal cell cancer (RCC) has been increasing for the past decade, and the 5-year survival for patients with metastatic RCC (mRCC) is rather low. Everolimus (RAD001), a new inhibitor for mammalian target of rapamycin (mTOR), is generally well tolerated, and demonstrates clinical benefit to patients with anti-VEGF-refractory mRCC. However, factors for selection of patients who may benefit from everolimus remain largely unknown. Here we aimed to explore potential molecular indicators for mRCC patients who may benefit from everolimus treatment. Methods Paraffin-embedded tumor tissue specimens derived from 18 mRCC patients before everolimus treatment, who participated the phase 1b trial of everolimus in VEGF receptor (VEGFR)-tyrosine kinase inhibitor (TKI)-refractory Chinese patients with mRCC (clinicaltrials.gov, NCT01152801), were examined for the expression levels of phosphorylated AKT, mTOR, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4EBP1) and 40S ribosomal protein S6 (S6RP) by immunohistochemistry. Clinical benefit rate (complete response [CR], partial response [PR], plus stable disease [SD] ≥ 6 months) and progression-free survival time (PFS) were correlated with expression levels of these mTOR-associated molecules. Results In these 18 patients, there were 1 PR, 15 SDs (including 9 SDs ≥ 6 months), and 2 progressive diseases (PD). The clinical benefit rate (CBR) was 55.6% (10/18), and the median PFS time was 8.4 months. Patients with positive expression of phospho-mTOR showed a better CBR (71.4% versus 0%, P = 0.023) and PFS time (11.3 versus 3.7 months, P = 0.001) than those patients with negative expression. The median PFS of patients with positive phospho-S6RP expression was longer (11.3 versus 3.7 months, P = 0.002) than that of patients negative for phospho-S6RP expression. However, expression levels of phospho-4EBP1 and phospho-AKT were unassociated to efficacy of everolimus treatment

  13. Experimental discrimination between charge 2e/3 top quark and charge 4e/3 exotic quark production scenarios.

    PubMed

    Abazov, V M; Abbott, B; Abolins, M; Acharya, B S; Adams, M; Adams, T; Agelou, M; Ahn, S H; Ahsan, M; Alexeev, G D; Alkhazov, G; Alton, A; Alverson, G; Alves, G A; Anastasoaie, M; Andeen, T; Anderson, S; Andrieu, B; Anzelc, M S; Arnoud, Y; Arov, M; Askew, A; Asman, B; Assis Jesus, A C S; Atramentov, O; Autermann, C; Avila, C; Ay, C; Badaud, F; Baden, A; Bagby, L; Baldin, B; Bandurin, D V; Banerjee, P; Banerjee, S; Barberis, E; Bargassa, P; Baringer, P; Barnes, C; Barreto, J; Bartlett, J F; Bassler, U; Bauer, D; Bean, A; Begalli, M; Begel, M; Belanger-Champagne, C; Bellantoni, L; Bellavance, A; Benitez, J A; Beri, S B; Bernardi, G; Bernhard, R; Berntzon, L; Bertram, I; Besançon, M; Beuselinck, R; Bezzubov, V A; Bhat, P C; Bhatnagar, V; Binder, M; Biscarat, C; Black, K M; Blackler, I; Blazey, G; Blekman, F; Blessing, S; Bloch, D; Bloom, K; Blumenschein, U; Boehnlein, A; Boeriu, O; Bolton, T A; Borissov, G; Bos, K; Bose, T; Brandt, A; Brock, R; Brooijmans, G; Bross, A; Brown, D; Buchanan, N J; Buchholz, D; Buehler, M; Buescher, V; Burdin, S; Burke, S; Burnett, T H; Busato, E; Buszello, C P; Butler, J M; Calfayan, P; Calvet, S; Cammin, J; Caron, S; Carvalho, W; Casey, B C K; Cason, N M; Castilla-Valdez, H; Chakraborty, D; Chan, K M; Chandra, A; Charles, F; Cheu, E; Chevallier, F; Cho, D K; Choi, S; Choudhary, B; Christofek, L; Claes, D; Clément, B; Clément, C; Coadou, Y; Cooke, M; Cooper, W E; Coppage, D; Corcoran, M; Cousinou, M-C; Cox, B; Crépé-Renaudin, S; Cutts, D; Cwiok, M; da Motta, H; Das, A; Das, M; Davies, B; Davies, G; Davis, G A; De, K; de Jong, P; de Jong, S J; De La Cruz-Burelo, E; De Oliveira Martins, C; Degenhardt, J D; Déliot, F; Demarteau, M; Demina, R; Demine, P; Denisov, D; Denisov, S P; Desai, S; Diehl, H T; Diesburg, M; Doidge, M; Dominguez, A; Dong, H; Dudko, L V; Duflot, L; Dugad, S R; Duggan, D; Duperrin, A; Dyer, J; Dyshkant, A; Eads, M; Edmunds, D; Edwards, T; Ellison, J; Elmsheuser, J; Elvira, V D; Eno, S; Ermolov, P; Evans, H; Evdokimov, A; Evdokimov, V N; Fatakia, S N; Feligioni, L; Ferapontov, A V; Ferbel, T; Fiedler, F; Filthaut, F; Fisher, W; Fisk, H E; Fleck, I; Ford, M; Fortner, M; Fox, H; Fu, S; Fuess, S; Gadfort, T; Galea, C F; Gallas, E; Galyaev, E; Garcia, C; Garcia-Bellido, A; Gardner, J; Gavrilov, V; Gay, A; Gay, P; Gelé, D; Gelhaus, R; Gerber, C E; Gershtein, Y; Gillberg, D; Ginther, G; Gollub, N; Gómez, B; Goussiou, A; Grannis, P D; Greenlee, H; Greenwood, Z D; Gregores, E M; Grenier, G; Gris, Ph; Grivaz, J-F; Grünendahl, S; Grünewald, M W; Guo, F; Guo, J; Gutierrez, G; Gutierrez, P; Haas, A; Hadley, N J; Haefner, P; Hagopian, S; Haley, J; Hall, I; Hall, R E; Han, L; Hanagaki, K; Hansson, P; Harder, K; Harel, A; Harrington, R; Hauptman, J M; Hauser, R; Hays, J; Hebbeker, T; Hedin, D; Hegeman, J G; Heinmiller, J M; Heinson, A P; Heintz, U; Hensel, C; Herner, K; Hesketh, G; Hildreth, M D; Hirosky, R; Hobbs, J D; Hoeneisen, B; Hoeth, H; Hohlfeld, M; Hong, S J; Hooper, R; Houben, P; Hu, Y; Hubacek, Z; Hynek, V; Iashvili, I; Illingworth, R; Ito, A S; Jabeen, S; Jaffré, M; Jain, S; Jakobs, K; Jarvis, C; Jenkins, A; Jesik, R; Johns, K; Johnson, C; Johnson, M; Jonckheere, A; Jonsson, P; Juste, A; Käfer, D; Kahn, S; Kajfasz, E; Kalinin, A M; Kalk, J M; Kalk, J R; Kappler, S; Karmanov, D; Kasper, J; Kasper, P; Katsanos, I; Kau, D; Kaur, R; Kehoe, R; Kermiche, S; Khalatyan, N; Khanov, A; Kharchilava, A; Kharzheev, Y M; Khatidze, D; Kim, H; Kim, T J; Kirby, M H; Klima, B; Kohli, J M; Konrath, J-P; Kopal, M; Korablev, V M; Kotcher, J; Kothari, B; Koubarovsky, A; Kozelov, A V; Kozminski, J; Krop, D; Kryemadhi, A; Kuhl, T; Kumar, A; Kunori, S; Kupco, A; Kurca, T; Kvita, J; Lammers, S; Landsberg, G; Lazoflores, J; Le Bihan, A-C; Lebrun, P; Lee, W M; Leflat, A; Lehner, F; Lesne, V; Leveque, J; Lewis, P; Li, J; Li, Q Z; Lima, J G R; Lincoln, D; Linnemann, J; Lipaev, V V; Lipton, R; Liu, Z; Lobo, L; Lobodenko, A; Lokajicek, M; Lounis, A; Love, P; Lubatti, H J; Lynker, M; Lyon, A L; Maciel, A K A; Madaras, R J; Mättig, P; Magass, C; Magerkurth, A; Magnan, A-M; Makovec, N; Mal, P K; Malbouisson, H B; Malik, S; Malyshev, V L; Mao, H S; Maravin, Y; Martens, M; McCarthy, R; Meder, D; Melnitchouk, A; Mendes, A; Mendoza, L; Merkin, M; Merritt, K W; Meyer, A; Meyer, J; Michaut, M; Miettinen, H; Millet, T; Mitrevski, J; Molina, J; Mondal, N K; Monk, J; Moore, R W; Moulik, T; Muanza, G S; Mulders, M; Mulhearn, M; Mundim, L; Mutaf, Y D; Nagy, E; Naimuddin, M; Narain, M; Naumann, N A; Neal, H A; Negret, J P; Neustroev, P; Noeding, C; Nomerotski, A; Novaes, S F; Nunnemann, T; O'dell, V; O'neil, D C; Obrant, G; Oguri, V; Oliveira, N; Oshima, N; Otec, R; Otero Y Garzón, G J; Owen, M; Padley, P; Parashar, N; Park, S-J; Park, S K; Parsons, J; Partridge, R; Parua, N; Patwa, A; Pawloski, G; Perea, P M; Perez, E; Peters, K; Pétroff, P; Petteni, M; Piegaia, R; Piper, J; Pleier, M-A; Podesta-Lerma, P L M; Podstavkov, V M; Pogorelov, Y; Pol, M-E; Pompos, A; Pope, B G; Popov, A V; Potter, C; Prado da Silva, W L; Prosper, H B; Protopopescu, S; Qian, J; Quadt, A; Quinn, B; Rangel, M S; Rani, K J; Ranjan, K; Ratoff, P N; Renkel, P; Reucroft, S; Rijssenbeek, M; Ripp-Baudot, I; Rizatdinova, F; Robinson, S; Rodrigues, R F; Royon, C; Rubinov, P; Ruchti, R; Rud, V I; Sajot, G; Sánchez-Hernández, A; Sanders, M P; Santoro, A; Savage, G; Sawyer, L; Scanlon, T; Schaile, D; Schamberger, R D; Scheglov, Y; Schellman, H; Schieferdecker, P; Schmitt, C; Schwanenberger, C; Schwartzman, A; Schwienhorst, R; Sekaric, J; Sengupta, S; Severini, H; Shabalina, E; Shamim, M; Shary, V; Shchukin, A A; Shephard, W D; Shivpuri, R K; Shpakov, D; Siccardi, V; Sidwell, R A; Simak, V; Sirotenko, V; Skubic, P; Slattery, P; Smith, R P; Snow, G R; Snow, J; Snyder, S; Söldner-Rembold, S; Song, X; Sonnenschein, L; Sopczak, A; Sosebee, M; Soustruznik, K; Souza, M; Spurlock, B; Stark, J; Steele, J; Stolin, V; Stone, A; Stoyanova, D A; Strandberg, J; Strandberg, S; Strang, M A; Strauss, M; Ströhmer, R; Strom, D; Strovink, M; Stutte, L; Sumowidagdo, S; Sznajder, A; Talby, M; Tamburello, P; Taylor, W; Telford, P; Temple, J; Tiller, B; Titov, M; Tokmenin, V V; Tomoto, M; Toole, T; Torchiani, I; Towers, S; Trefzger, T; Trincaz-Duvoid, S; Tsybychev, D; Tuchming, B; Tully, C; Turcot, A S; Tuts, P M; Unalan, R; Uvarov, L; Uvarov, S; Uzunyan, S; Vachon, B; van den Berg, P J; Van Kooten, R; van Leeuwen, W M; Varelas, N; Varnes, E W; Vartapetian, A; Vasilyev, I A; Vaupel, M; Verdier, P; Vertogradov, L S; Verzocchi, M; Villeneuve-Seguier, F; Vint, P; Vlimant, J-R; Von Toerne, E; Voutilainen, M; Vreeswijk, M; Wahl, H D; Wang, L; Wang, M H L S; Warchol, J; Watts, G; Wayne, M; Weber, M; Weerts, H; Wermes, N; Wetstein, M; White, A; Wicke, D; Wilson, G W; Wimpenny, S J; Wobisch, M; Womersley, J; Wood, D R; Wyatt, T R; Xie, Y; Xuan, N; Yacoob, S; Yamada, R; Yan, M; Yasuda, T; Yatsunenko, Y A; Yip, K; Yoo, H D; Youn, S W; Yu, C; Yu, J; Yurkewicz, A; Zatserklyaniy, A; Zeitnitz, C; Zhang, D; Zhao, T; Zhou, B; Zhu, J; Zielinski, M; Zieminska, D; Zieminski, A; Zutshi, V; Zverev, E G

    2007-01-26

    We present the first experimental discrimination between the 2e/3 and 4e/3 top quark electric charge scenarios, using top quark pairs (tt) produced in pp collisions at (square root) s = 1.96 TeV by the Fermilab Tevatron Collider. We use 370 pb;{-1} of data collected by the D0 experiment and select events with at least one high transverse momentum electron or muon, high transverse energy imbalance, and four or more jets. We discriminate between b- and b-quark jets by using the charge and momenta of tracks within the jet cones. The data are consistent with the expected electric charge, |q|=2e/3. We exclude, at the 92% C.L., that the sample is solely due to the production of exotic quark pairs QQ with |q|=4e/3. We place an upper limit on the fraction of QQ pairs rho<0.80 at the 90% C.L.

  14. Kalman Filter Design for the Long Range Intercept Function of the F-4E/G Fire Control System.

    DTIC Science & Technology

    1985-12-01

    Budget Studies ............ 107 Filter Tuning Using Measurement Noise . . . 110 Filter Adaptation to F-4E/G Roll Rate . . . 112 Filter Comparisons on...140 Appendix F: Filter Tuning Using Driving Noise . . . . 171 Appendix G: Error Budget Studies .. ........ . . 199 Appendix H...Airspeed a L i " x0 i""ix ...-... ... •....... ....-... .. ..- . ..........-.-...... .- .. . . . ., AFIT/GE/ENG/85D-20 Abstract 1 * This study examines

  15. Microstructural effects on the spall properties of ECAE and SWAP magnesium alloys: AZ31B-4E and AMX602

    NASA Astrophysics Data System (ADS)

    Williams, C. L.; Farbaniec, L.; Kecskes, L.; Bradley, J.

    2017-01-01

    The effects of microstructure on the spall properties of two magnesium alloys fabricated via Equal-Channel Angular Extrusion (ECAE) and Spinning Water Atomization Process (SWAP) were investigated. The Hugoniot Elastic Limit (HEL) for both AZ31B-4E and AMX602 magnesium alloys were found to be approximately 0.181±0.003 GPa and 0.187±0.012 GPa, respectively. The spall strengths extracted from the free surface velocity profiles were found to decrease by approximately 4% for AZ31B-4E between 1.7 GPa to 4.6 GPa shock stress. Although this reduction in spall strength may lie within the experimental error, the microstructure of the post-shocked magnesium alloy show that manganese intermetallic inclusions in the AZ31B-4E magnesium were perhaps responsible for the reduction in spall strength as a function of shock stress. On the contrary, the spall strength for AMX602 was found to be random for the same shock stress range studied. This random behavior of the AMX602 was likely due to the incomplete sintering during mechanical processing. The fracture surfaces of both materials were dominated by nanovoids and the AMX602 fracture surface was found to be striated. A more in-depth study is needed to better understand the spall behavior of both materials.

  16. Effects of temephos (Abate? 4E) on fiddler crabs (Uca pugnax and Uca minax) on a Delaware salt marsh

    USGS Publications Warehouse

    Pinkney, A.E.; McGowan, P.C.; Murphy, D.R.; Lowe, T.P.; Sparling, D.W.; Meredith, W.H.

    1999-01-01

    The non-target effects of temephos (as Abate 4E, 44.6% active ingredient) on fiddler crabs were examined on the salt marsh at Bombay Hook National Wildlife Refuge (NWR), near Dover, DE. Six 170 x 170 m plots were established; 3 were sprayed on 4 occasions at a rate of 1.5 fl oz/acre (0.054 kg active ingredient/ha) and 3 were controls. On each plot, marsh fiddler crab (Uca pugnax) populations were monitored by repeatedly counting the number of burrow holes in 2 counting areas marked out along tidal guts. One half of each counting area was covered with bird netting to evaluate sublethal toxic effects, which, if present, could result in increased susceptibility to bird predation. A statistically significant linear association was established between the number of holes and the number of crabs. No significant differences were found in the numbers of holes (or crabs) in the sprayed vs. control plots and in the covered vs. uncovered sections. However, survival of juvenile crabs in in situ bioassays was significantly reduced (16% lower) by the spraying. Median acetylcholinesterase activity in claw muscle of red-jointed fiddler crabs (U. minax) collected 2 days after an operational spray with Abate 4E was significantly reduced (28% lower) compared to unsprayed crabs. In view of the toxicity to juvenile crabs and the cholinesterase inhibition, we recommend continued monitoring and research for non-target impacts of Abate 4E on fiddler crabs to establish whether the reported level of cholinesterase inhibition results in acute or chronic toxicity.

  17. Architecture for an advanced biomedical collaboration domain for the European paediatric cancer research community (ABCD-4-E).

    PubMed

    Nitzlnader, Michael; Falgenhauer, Markus; Gossy, Christian; Schreier, Günter

    2015-01-01

    Today, progress in biomedical research often depends on large, interdisciplinary research projects and tailored information and communication technology (ICT) support. In the context of the European Network for Cancer Research in Children and Adolescents (ENCCA) project the exchange of data between data source (Source Domain) and data consumer (Consumer Domain) systems in a distributed computing environment needs to be facilitated. This work presents the requirements and the corresponding solution architecture of the Advanced Biomedical Collaboration Domain for Europe (ABCD-4-E). The proposed concept utilises public as well as private cloud systems, the Integrating the Healthcare Enterprise (IHE) framework and web-based applications to provide the core capabilities in accordance with privacy and security needs. The utility of crucial parts of the concept was evaluated by prototypic implementation. A discussion of the design indicates that the requirements of ENCCA are fully met. A whole system demonstration is currently being prepared to verify that ABCD-4-E has the potential to evolve into a domain-bridging collaboration platform in the future.

  18. Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to Zucchini yellow mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant wa...

  19. A germline-specific isoform of eIF4E (IFE-1) is required for efficient translation of stored mRNAs and maturation of both oocytes and sperm.

    PubMed

    Henderson, Melissa A; Cronland, Elizabeth; Dunkelbarger, Steve; Contreras, Vince; Strome, Susan; Keiper, Brett D

    2009-05-15

    Fertility and embryonic viability are measures of efficient germ cell growth and development. During oogenesis and spermatogenesis, new proteins are required for both mitotic expansion and differentiation. Qualitative and quantitative changes in protein synthesis occur by translational control of mRNAs, mediated in part by eIF4E, which binds the mRNAs 5' cap. IFE-1 is one of five eIF4E isoforms identified in C. elegans. IFE-1 is expressed primarily in the germ line and associates with P granules, large mRNPs that store mRNAs. We isolated a strain that lacks IFE-1 [ife-1(bn127)] and demonstrated that the translation of several maternal mRNAs (pos-1, pal-1, mex-1 and oma-1) was inefficient relative to that in wild-type worms. At 25 degrees C, ife-1(bn127) spermatocytes failed in cytokinesis, prematurely expressed the pro-apoptotic protein CED-4/Apaf-1, and accumulated as multinucleate cells unable to mature to spermatids. A modest defect in oocyte development was also observed. Oocytes progressed normally through mitosis and meiosis, but subsequent production of competent oocytes became limiting, even in the presence of wild-type sperm. Combined gametogenesis defects decreased worm fertility by 80% at 20 degrees C; ife-1 worms were completely sterile at 25 degrees C. Thus, IFE-1 plays independent roles in late oogenesis and spermatogenesis through selective translation of germline-specific mRNAs.

  20. Tamoxifen synergizes with 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, novel azaresveratrol analogs, in inhibiting the proliferation of breast cancer cells

    PubMed Central

    Ronghe, Amruta; Chatterjee, Anwesha; Bhat, Nimee K.; Padhye, Subhash; Bhat, Hari K.

    2016-01-01

    We have recently shown that 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD) and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), novel analogs of resveratrol (Res), selectively inhibited the proliferation of breast cancer cells. In the current study, we tested HPIMBD and TIMBD individually in combination with tamoxifen (Tam) for inhibition of growth of breast cancer cells. Tamoxifen was first tested on non-neoplastic breast epithelial cell lines and its dose that does not inhibit their growth was determined. A combination of this low dose of Tam with either of the Res analogs HPIMBD or TIMBD, resulted in synergistic inhibition of proliferation of breast cancer cells. Both estrogen receptor (ER)-positive and negative breast cancer cell lines responded to the combination. The combination resulted in a substantial decrease in IC50 values of Res analogs in all breast cancer cell lines tested. Mechanistic studies showed a synergistic increase in apoptosis and autophagy genes (beclin-1 and LC3BII/I) with the combination in ER-negative MDA-MB-231 cells. In ER-positive MCF-7 and T47D cells, the mechanism of synergy was found to be inhibition of expression of ERα and oncogene c-Myc. The combination treatment had a synergistic effect in inhibiting the colony forming and spheroid forming ability of cancer cells. Taken together, our findings indicate that a combination of Tam and Res analogs HPIMBD or TIMBD represents a novel approach to enhancing the use of Tam in therapy for breast cancers. Considering the urgent need for novel therapeutic strategies to treat ER-negative breast cancers and overcoming resistance in ER-positive cancers, this combinatorial approach is worthy of continued investigation. PMID:27351134

  1. Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells

    PubMed Central

    Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen

    2015-01-01

    Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity. PMID:25986608

  2. Tanshinone IIA Inhibits HIF-1α and VEGF Expression in Breast Cancer Cells via mTOR/p70S6K/RPS6/4E-BP1 Signaling Pathway

    PubMed Central

    Li, Guobing; Shan, Changyu; Liu, Lei; Zhou, Ting; Zhou, Jing; Hu, Xiaoye; Chen, Yibiao; Cui, Hongjuan; Gao, Ning

    2015-01-01

    Hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) play important roles in angiogenesis and tumor growth. Tanshinone IIA (T2A) is a novel antiangiogenic agent with promising antitumor effects; however, the molecular mechanism underlying the antiangiogenic effects of T2A remains unclear. In the present study, we provided evidence showing that T2A inhibited angiogenesis and breast cancer growth by down-regulating VEGF expression. Specifically, T2A repressed HIF-1α expression at the translational level and inhibited the transcriptional activity of HIF-1α, which led to the down-regulation of VEGF expression. Suppression of HIF-1α synthesis by T2A correlated with strong dephosphorylation of mammalian target of rapamycin (mTOR) and its effectors ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a pathway regulating HIF-1α expression at the translational level. In addition, we also found that T2A inhibited the angiogenesis and growth of human breast cancer xenografts in nude mice through suppression of HIF-1α and VEGF. Our study provides novel perspectives and potential targets for the treatment of human breast cancer. PMID:25659153

  3. A TILLING approach to generate broad-spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy.

    PubMed

    Gauffier, Camille; Lebaron, Caroline; Moretti, André; Constant, Carole; Moquet, Frédéric; Bonnet, Grégori; Caranta, Carole; Gallois, Jean-Luc

    2016-03-01

    Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING-induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh-eIF4E1(PI24)-pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re-establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss-of-function alleles to generate resistance may be counter-productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.

  4. Computational and experimental studies of 2-[(E)-hydrazinylidenemethyl]-6-methoxy-4-[(E)-phenyldiazenyl]phenol and its tautomers

    NASA Astrophysics Data System (ADS)

    Sayin, Koray; Kurtoglu, Nurcan; Kose, Muhammet; Karakas, Duran; Kurtoglu, Mukerrem

    2016-09-01

    A new azo-chromophore group containing a hydrazine-Schiff base compound, 2-[(E)-hydrazinylidenemethyl]-6-methoxy-4-[(E)-phenyldiazenyl]phenol, was synthesized and structurally characterized by single crystal X-ray diffraction study. The compound was found to crystallise in orthorhombic crystal system with Pca2(1) space group. In the structure, the molecule exhibits a phenol-imine intramolecular hydrogen bond and the sbnd NH2 group also involves in intermolecular hydrogen bonding with one of the nitrogen atom of the azo group (-Ndbnd N-) forming a 1D zigzag chain. Computational studies were performed on the titled compound and its tautomers. As computationally, this compound and its tautomers were optimized by using M062X/6-311G(d,p) level. According to thermodynamic parameters, the most stable tautomer was found to be azo-enol form. This result was then taken into account and spectral studies, which are IR, UV-Vis and NMR spectra, of this compound were performed and examined in detail. All calculations were performed at gas phase (ε = 1.000), 2-propanol (ε = 19.264), 1,2-ethanediol (ε = 40.245), water (ε = 78.355), formamide (ε = 108.940) and N-methylformamide-mixture (ε = 181.560).

  5. Preclinical Activity of New [1,2]Oxazolo[5,4-e]isoindole Derivatives in Diffuse Malignant Peritoneal Mesothelioma.

    PubMed

    Spanò, Virginia; Pennati, Marzia; Parrino, Barbara; Carbone, Anna; Montalbano, Alessandra; Cilibrasi, Vincenzo; Zuco, Valentina; Lopergolo, Alessia; Cominetti, Denis; Diana, Patrizia; Cirrincione, Girolamo; Barraja, Paola; Zaffaroni, Nadia

    2016-08-11

    A series of 22 derivatives of the [1,2]oxazolo[5,4-e]isoindole system were synthesized through an efficient and versatile procedure that involves the annelation of the [1,2]oxazole moiety to the isoindole ring, producing derivatives with a wide substitution pattern. The structure-activity relationship indicates that the N-4-methoxybenzyl group appears crucial for potent activity. In addition, the presence of a 6-phenyl moiety is important and the best activity is reached with a 3,4,5-trimethoxy substituent. The most active compound, bearing both the structural features, was able to inhibit tumor cell proliferation at nanomolar concentrations when tested against the full NCI human tumor cell line panel. Interestingly, this compound was effective in reducing in vitro and in vivo cell growth, impairing cell cycle progression and inducing apoptosis, as a consequence of the inhibition of tubulin polymerization, in experimental models of diffuse malignant peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional therapeutic strategies.

  6. RIS4E at Kilauea's December 1974 Flow: Assessing the Integration of Portable Infrared Multispectral Imaging into Planetary Surface Exploration

    NASA Astrophysics Data System (ADS)

    Ito, G.; Rogers, D.; Bleacher, J. E.; Young, K. E.; Edwards, C. S.; Glotch, T. D.

    2015-12-01

    Portable, hand-held geochemical and mineralogical instruments are potentially valuable tools to be used in sample collection and site documentation activities during future human missions to planetary bodies. The main purpose of these instruments is to allow fast in situ analyses of rocks and soils so that astronauts can quickly document sample characteristics and context, and make strategic decisions on sample selection in the context of predefined scientific objectives. As part of the Remote, In Situ, and Synchrotron Studies for Science and Exploration (RIS4E) investigation, we test the performance of candidate instruments and operational procedures through fieldwork expeditions that simulate lunar and asteroid environments on Earth. Our field site, Kilauea Volcano in Hawaii, is a lava field with landscape and mineralogy that represent a reasonable analog to the Moon and some differentiated asteroids. In this paper, we focus on one of the candidate instruments, the infrared multispectral imager. During field expeditions in 2014 and 2015, we explored the applicability of the multispectral imager in manned surface operations. From these expeditions, our instrument calibration techniques and data collection procedures matured. Current work focuses on assessment of data product usefulness, through comparison with detailed laboratory chemical and spectral measurements, and field descriptions of surface textures. Our field expeditions will continue in other analog locations to obtain improved understanding of the multispectral imager and its role in sampling workflow so that science return can be maximized in future human missions.

  7. Kepler Flares. IV. A Comprehensive Analysis of the Activity of the dM4e Star GJ 1243

    NASA Astrophysics Data System (ADS)

    Silverberg, Steven M.; Kowalski, Adam F.; Davenport, James R. A.; Wisniewski, John P.; Hawley, Suzanne L.; Hilton, Eric J.

    2016-10-01

    We present a comprehensive study of the active dM4e star GJ 1243. We use previous observations and ground-based echelle spectroscopy to determine that GJ 1243 is a member of the Argus association of field stars, suggesting it is ∼ 30{--}50 {{Myr}} old. We analyze 11 months of 1 minute cadence data from Kepler, presenting Kepler flare frequency distributions, as well as determining correlations between flare energy, amplitude, duration, and decay time. We find that the exponent α of the power-law flare energy distribution varies in time, primarily due to completeness of sample and the low frequency of high-energy flares. We also find a deviation from a single power law at high energy. We use ground-based spectroscopic observations that were simultaneous with the Kepler data to provide simultaneous photometric and spectroscopic analysis of three low-energy flares, the lowest-energy dMe flares with detailed spectral analysis to date on any star. The spectroscopic data from these flares extend constraints for radiative hydrodynamic flare models to a lower energy regime than has previously been studied. We use this simultaneous spectroscopy and Kepler photometry to develop approximate conversions from the Kepler bandpass to the traditional U and B bands. This conversion will be a critical factor in comparing any Kepler flare analyses to the canon of previous ground-based flare studies.

  8. 3c/4e σ-type long-bonding: a novel transitional motif toward the metallic delocalization limit.

    PubMed

    Landis, C R; Weinhold, F

    2013-05-06

    We describe a novel "long-bonding" motif that appears in the framework of natural bond orbital (NBO) analysis as a surprising form of 3-center, 4-electron (3c/4e) L···A···L' bonding with "inverted" electronegativity pattern Ξ(A) > Ξ(L), Ξ(L'). Such long-bonding (denoted L(^)L') underlies the predicted (meta)stability of exotic rare gas species with highly electronegative ligands (e.g., HeF2, NeF2) as well as the absolute stability of low-electronegativity metallic triads (e.g., BeLi2, ZnCu2, and related species) that are experimentally unknown but can be anticipated from simple valency and electronegativity trends. We focus particularly on the BeLi2 triad, whose Lewis-type Li(^)Li' long bond is of paradoxical antibonding phase pattern, denoted σ*(LiLi') to suggest its essential 2(-1/2)(s(Li) - s(Li')) orbital composition. We demonstrate how the long-bonded triad serves as a fundamental building-block for numerous 1-, 2-, and 3-d structures that are predicted to exhibit extraordinary calorimetric, vibrational, and electric polarizability properties, commonly associated with the delocalized metallic limit. Both thermodynamic and kinetic results support the NBO inference that σ/σ*-type long-bonding signals the transition to a fundamentally new regime of chemical association, separated by significant activation barriers from the covalent molecular domain and characterized by reversed perturbative precedence of Lewis-type vs resonance-type donor-acceptor contributions. Long-bond resonance therefore appears to be of central importance to a broadened conceptual picture of molecular and metallic interaction phenomena.

  9. Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

    SciTech Connect

    Li Jing; Chen Xi; Jiang Shibo Chen Yinghua

    2008-11-07

    The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformation as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.

  10. Structure of Antibody F425-B4e8 in Complex With a V3 Peptide Reveals a New Binding Mode for Hiv-1 Neutralization

    SciTech Connect

    Bell, C.H.; Pantophlet, R.; Schiefner, A.; Cavacini, L.A.; Stanfield, R.L.; Burton, D.R.; Wilson, I.A.

    2009-05-11

    F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 A resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue alpha-turn around the conserved GPGRA apex of the beta-hairpin loop. In agreement with previous mutagenesis analyses, the Fab interacts primarily with V3 through side-chain contacts with just two residues, Ile(P309) and Arg(P315), while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.

  11. Activating the translational repressor 4E-BP or reducing S6K-GSK3β activity prevents accelerated axon growth induced by hyperactive mTOR in vivo

    PubMed Central

    Gong, Xuan; Zhang, Longbo; Huang, Tianxiang; Lin, Tiffany V.; Miyares, Laura; Wen, John; Hsieh, Lawrence; Bordey, Angélique

    2015-01-01

    Abnormal axonal connectivity and hyperactive mTOR complex 1 (mTORC1) are shared features of several neurological disorders. Hyperactive mTORC1 alters axon length and polarity of hippocampal neurons in vitro, but the impact of hyperactive mTORC1 on axon growth in vivo and the mechanisms underlying those effects remain unclear. Using in utero electroporation during corticogenesis, we show that increasing mTORC1 activity accelerates axon growth without multiple axon formation. This was prevented by counteracting mTORC1 signaling through p70S6Ks (S6K1/2) or eukaryotic initiation factor 4E-binding protein (4E-BP1/2), which both regulate translation. In addition to regulating translational targets, S6K1 indirectly signals through GSK3β, a regulator of axogenesis. Although blocking GSK3β activity did not alter axon growth under physiological conditions in vivo, blocking it using a dominant-negative mutant or lithium chloride prevented mTORC1-induced accelerated axon growth. These data reveal the contribution of translational and non-translational downstream effectors such as GSK3β to abnormal axon growth in neurodevelopmental mTORopathies and open new therapeutic options for restoring long-range connectivity. PMID:26220974

  12. Two novel organic-inorganic hybrid materials from tetrachloridometallate(II) salts and 4-[(E)-2-(pyridin-1-ium-2-yl)ethenyl]pyridinium.

    PubMed

    Campos-Gaxiola, José J; Arredondo Rea, Susana P; Corral Higuera, Ramón; Höpfl, Herbert; Cruz Enríquez, Adriana

    2015-01-01

    Two organic-inorganic hybrid compounds have been prepared by the combination of the 4-[(E)-2-(pyridin-1-ium-2-yl)ethenyl]pyridinium cation with perhalometallate anions to give 4-[(E)-2-(pyridin-1-ium-2-yl)ethenyl]pyridinium tetrachloridocobaltate(II), (C12H12N2)[CoCl4], (I), and 4-[(E)-2-(pyridin-1-ium-2-yl)ethenyl]pyridinium tetrachloridozincate(II), (C12H12N2)[ZnCl4], (II). The compounds have been structurally characterized by single-crystal X-ray diffraction analysis, showing the formation of a three-dimensional network through X-H...ClnM(-) (X = C, N(+); n = 1, 2; M = Co(II), Zn(II)) hydrogen-bonding interactions and π-π stacking interactions. The title compounds were also characterized by FT-IR spectroscopy and thermogravimetric analysis (TGA).

  13. W4E HYDROPOWER DIRECT DRIVE IN-LINE HYDROTURBINE GENERATOR FULL SCALE PROTOTYPE VALIDATION TESTING REPORT MAY 2013 ALDEN LABORATORIES

    SciTech Connect

    Cox, Chad W

    2013-09-24

    The W4E is a patent-pending, direct-drive, variable force turbine/generator. The equipment generates electricity through the water dependent engagement of a ring of rotating magnets with coils mounted on a stator ring. Validation testing of the W4e was performed at Alden Laboratories in the Spring of 2013. The testing was independently observed and validated by GZA GeoEnvironmental, Inc. The observations made during testing and the results of the testing are included in the Test Summary Report

  14. An allele-specific SNP mutation in the eIF4E gene is associated with the Zucchini yellow mosaic virus resistance in watermelon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced an eIF4E gene in the ZYMV-resistant PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon c...

  15. Stabilization of p21 by mTORC1/4E-BP1 predicts clinical outcome of head and neck cancers

    PubMed Central

    Llanos, Susana; García-Pedrero, Juana M.; Morgado-Palacin, Lucia; Rodrigo, Juan P.; Serrano, Manuel

    2016-01-01

    The levels, regulation and prognostic value of p21 in head and neck squamous cell carcinomas (HNSCC) has been puzzling for years. Here, we report a new mechanism of regulation of p21 by the mTORC1/4E-BP1 pathway. We find that non-phosphorylated 4E-BP1 interacts with p21 and induces its degradation. Accordingly, hyper-activation of mTORC1 results in phosphorylation of 4E-BP1 and stabilization of p21. In HNSCC, p21 levels strongly correlate with mTORC1 activity but not with p53 status. Finally, clinical data indicate that HNSCC patients with p21 and phospho-S6-double-positive tumours present a better disease-specific survival. We conclude that over-activation of the mTORC1/4E-BP1/p21 pathway is a frequent and clinically relevant alteration in HNSCC. PMID:26832959

  16. 3-Substituted Indazoles as Configurationally Locked 4EGI-1 Mimetic and Inhibitors of eIF4E/eIF4G Interaction

    PubMed Central

    Yefidoff-Freedman, Revital; Chen, Ting; Sahoo, Rupam; Chen, Limo; Wagner, Gerhard; Halperin, Jose A.; Aktas, Bertal H.; Chorev, Michael

    2014-01-01

    4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small molecule libraries using a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 holds a potential for studying molecular mechanisms involved in human disorders characterized by loss of physiologic restrains on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the liability of the configurational instability in 4EGI-1, which stems from the (E)-to-(Z) isomerization of the hydrazone function. We identified compound 1a, in which the labile hydrazone was incorporated into a rigid indazole scaffold as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study aimed at probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidating the role of cap-dependent translation initiation in a host of pathophysiological states. PMID:24458973

  17. Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc-3 is associated with the homozygotic presence of a mutated eIF4E allele

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris, four recessive genes, bc-1, bc-2, bc-3...

  18. 3-substituted indazoles as configurationally locked 4EGI-1 mimetics and inhibitors of the eIF4E/eIF4G interaction.

    PubMed

    Yefidoff-Freedman, Revital; Chen, Ting; Sahoo, Rupam; Chen, Limo; Wagner, Gerhard; Halperin, Jose A; Aktas, Bertal H; Chorev, Michael

    2014-03-03

    4EGI-1, the prototypic inhibitor of eIF4E/eIF4G interaction, was identified in a high-throughput screening of small-molecule libraries with the aid of a fluorescence polarization assay that measures inhibition of binding of an eIF4G-derived peptide to recombinant eIF4E. As such, the molecular probe 4EGI-1 has potential for the study of molecular mechanisms involved in human disorders characterized by loss of physiological restraints on translation initiation. A hit-to-lead optimization campaign was carried out to overcome the configurational instability in 4EGI-1, which stems from the E-to-Z isomerization of the hydrazone function. We identified compound 1 a, in which the labile hydrazone was incorporated into a rigid indazole scaffold, as a promising rigidified 4EGI-1 mimetic lead. In a structure-activity relationship study directed towards probing the structural latitude of this new chemotype as an inhibitor of eIF4E/eIF4G interaction and translation initiation we identified 1 d, an indazole-based 4EGI-1 mimetic, as a new and improved lead inhibitor of eIF4E/eIF4G interaction and a promising molecular probe candidate for elucidation of the role of cap-dependent translation initiation in a host of pathophysiological states.

  19. Estimating the probability of polyreactive antibodies 4E10 and 2F5 disabling a gp41 trimer after T cell-HIV adhesion.

    PubMed

    Hu, Bin; Liao, Hua-Xin; Alam, S Munir; Goldstein, Byron

    2014-01-01

    A few broadly neutralizing antibodies, isolated from HIV-1 infected individuals, recognize epitopes in the membrane proximal external region (MPER) of gp41 that are transiently exposed during viral entry. The best characterized, 4E10 and 2F5, are polyreactive, binding to the viral membrane and their epitopes in the MPER. We present a model to calculate, for any antibody concentration, the probability that during the pre-hairpin intermediate, the transient period when the epitopes are first exposed, a bound antibody will disable a trivalent gp41 before fusion is complete. When 4E10 or 2F5 bind to the MPER, a conformational change is induced that results in a stably bound complex. The model predicts that for these antibodies to be effective at neutralization, the time to disable an epitope must be shorter than the time the antibody remains bound in this conformation, about five minutes or less for 4E10 and 2F5. We investigate the role of avidity in neutralization and show that 2F5 IgG, but not 4E10, is much more effective at neutralization than its Fab fragment. We attribute this to 2F5 interacting more stably than 4E10 with the viral membrane. We use the model to elucidate the parameters that determine the ability of these antibodies to disable epitopes and propose an extension of the model to analyze neutralization data. The extended model predicts the dependencies of IC50 for neutralization on the rate constants that characterize antibody binding, the rate of fusion of gp41, and the number of gp41 bridging the virus and target cell at the start of the pre-hairpin intermediate. Analysis of neutralization experiments indicate that only a small number of gp41 bridges must be disabled to prevent fusion. However, the model cannot determine the exact number from neutralization experiments alone.

  20. In Vitro Characterization of the Pharmacological Properties of the Anti-Cancer Chelator, Bp4eT, and Its Phase I Metabolites

    PubMed Central

    Potůčková, Eliška; Roh, Jaroslav; Macháček, Miloslav; Sahni, Sumit; Stariat, Ján; Šesták, Vít; Jansová, Hana; Hašková, Pavlína; Jirkovská, Anna; Vávrová, Kateřina; Kovaříková, Petra; Kalinowski, Danuta S.; Richardson, Des R.; Šimůnek, Tomáš

    2015-01-01

    Cancer cells have a high iron requirement and many experimental studies, as well as clinical trials, have demonstrated that iron chelators are potential anti-cancer agents. The ligand, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), demonstrates both potent anti-neoplastic and anti-retroviral properties. In this study, Bp4eT and its recently identified amidrazone and semicarbazone metabolites were examined and compared with respect to their anti-proliferative activity towards cancer cells (HL-60 human promyelocytic leukemia, MCF-7 human breast adenocarcinoma, HCT116 human colon carcinoma and A549 human lung adenocarcinoma), non-cancerous cells (H9c2 neonatal rat-derived cardiomyoblasts and 3T3 mouse embryo fibroblasts) and their interaction with intracellular iron pools. Bp4eT was demonstrated to be a highly potent and selective anti-neoplastic agent that induces S phase cell cycle arrest, mitochondrial depolarization and apoptosis in MCF-7 cells. Both semicarbazone and amidrazone metabolites showed at least a 300-fold decrease in cytotoxic activity than Bp4eT towards both cancer and normal cell lines. The metabolites also lost the ability to: (1) promote the redox cycling of iron; (2) bind and mobilize iron from labile intracellular pools; and (3) prevent 59Fe uptake from 59Fe-labeled transferrin by MCF-7 cells. Hence, this study demonstrates that the highly active ligand, Bp4eT, is metabolized to non-toxic and pharmacologically inactive analogs, which most likely contribute to its favorable pharmacological profile. These findings are important for the further development of this drug candidate and contribute to the understanding of the structure-activity relationships of these agents. PMID:26460540

  1. The kissing-loop T-shaped structure translational enhancer of Pea enation mosaic virus can bind simultaneously to ribosomes and a 5' proximal hairpin.

    PubMed

    Gao, Feng; Gulay, Suna P; Kasprzak, Wojciech; Dinman, Jonathan D; Shapiro, Bruce A; Simon, Anne E

    2013-11-01

    The Pea enation mosaic virus (PEMV) 3' translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5'-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5' and 3' PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3' translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5' hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5' end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation.

  2. Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to zucchini yellow mosaic virus.

    PubMed

    Ling, Kai-Shu; Harris, Karen R; Meyer, Jenelle D F; Levi, Amnon; Guner, Nihat; Wehner, Todd C; Bendahmane, Abdelhafid; Havey, Michael J

    2009-12-01

    Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar 'New Hampshire Midget' ('NHM') showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F(2) and 114 BC(1R) progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F(2) and BC(1R) populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.

  3. Experimental validation of CASMO-4E and CASMO-5M for radial fission rate distributions in a westinghouse SVEA-96 Optima2 BWR fuel assembly

    SciTech Connect

    Grimm, P.; Perret, G.

    2012-07-01

    Measured and calculated radial total fission rate distributions are compared for the three axial sections of a Westinghouse SVEA-96 Optima2 BWR fuel assembly, comprising 96, 92 and 84 fuel rods, respectively. The measurements were performed on a full-size fuel assembly in the PROTEUS zero-power experimental facility. The measured fission rates are compared to the results of the CASMO-4E and CASMO-5M fuel assembly codes. Detailed measured geometrical data were used in the models, and effects of the surrounding zones of the reactor were taken into account by correction factors derived from MCNPX calculations. The results of the calculations agree well with those of the experiments, with root-mean-square deviations between 1.2% and 1.5% and maximum deviations of 3-4%. The quality of the predictions by CASMO-4E and CASMO-5M is comparable. (authors)

  4. (2S,4E)-2-Hydroxy-4-octen-3-one, a Male-Produced Attractant Pheromone of the Cerambycid Beetle Tylonotus bimaculatus.

    PubMed

    Zou, Yunfan; Millar, Jocelyn G; Blackwood, J Scott; Van Duzor, Ryan; Hanks, Lawrence M; Mongold-Diers, Judith A; Wong, Joseph C H; Ray, Ann M

    2015-07-01

    We report the identification of a novel pheromone structure from males of the cerambycid beetle Tylonotus bimaculatus Haldeman (Cerambycinae: Hesperophanini), a species native to eastern North America. Volatiles collected from adult males contained (2S,4E)-2-hydroxyoct-4-en-3-one (71%), (3R,4E)-3-hydroxyoct-4-en-2-one (15%), (E)-4-octen-2,3-dione (13%), and 2,3-octanedione (1.5%). Four independent field bioassays with synthetic compounds confirmed that adults of both sexes were attracted by the racemate of the major component, (E)-2-hydroxyoct-4-en-3-one. No other cerambycid species were attracted in significant numbers. Attraction of both sexes is consistent with the male-produced pheromones of many other species in the subfamily Cerambycinae, but T. bimaculatus is unusual in having a pheromone chemistry that is so far unique among species in that subfamily.

  5. Exploring the Integration of Field Portable Instrumentation into Real-Time Surface Science Operations with the RIS4E SSERVI Team

    NASA Astrophysics Data System (ADS)

    Young, K. E.; Bleacher, J. E.; Rogers, D.; Garry, W. B.; McAdam, A.; Scheidt, S. P.; Carter, L. M.; Glotch, T. D.

    2015-12-01

    The Remote, In Situ, and Synchrotron Studies for Science (RIS4E) team represents one node of the Solar System Exploration Research Virtual Institute (SSERVI) program. While the RIS4E team consists of four themes, each dedicated to a different aspect of airless body exploration, this submission details the RIS4E work underway to maximize an astronaut's effectiveness while conducting surface science. The next generation of surface science operations will look quite different than the EVAs (extravehicular activities) conducted during Apollo. Astronauts will possess data of much higher resolution than the Apollo reconnaissance data, and the EVAs will thus be designed to answer targeted science questions. Additionally, technological advancements over the last several decades have made it possible to conduct in situ analyses of a caliber much greater than was achievable during Apollo. For example, lab techniques such as x-ray fluorescence, x-ray diffraction, and multi-spectral imaging are now available in field portable formats, meaning that astronauts can gain real-time geochemical awareness during sample collection. The integration of these instruments into EVA operations, however, has not been widely tested. While these instruments will provide the astronaut with a high-resolution look at regional geochemistry and structure, their implementation could prove costly to the already constrained astronaut EVA timeline. The RIS4E team, through fieldwork at the December 1974 lava flow at Kilauea Volcano, HI, investigates the incorporation of portable technologies into planetary surface exploration and explores the relationship between science value added from these instruments and the cost associated with integrating them into an EVA timeline. We also consider what an appropriate instrumentation suite would be for the exploration of a volcanic terrain using this ideal terrestrial analog (see Rogers et al., Young et al., Bleacher et al., and Yant et al., this meeting).

  6. Involvement of the P1 cistron in overcoming eIF4E-mediated recessive resistance against Clover yellow vein virus in pea.

    PubMed

    Nakahara, Kenji S; Shimada, Ryoko; Choi, Sun-Hee; Yamamoto, Haruko; Shao, Jun; Uyeda, Ichiro

    2010-11-01

    Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and wlm against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the P1 cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that P1 is involved in eIF4E-mediated recessive resistance.

  7. Probing the Repulsive Core of the Nucleon-Nucleon Interaction via the He4(e ,e'pN) Triple-Coincidence Reaction

    NASA Astrophysics Data System (ADS)

    Korover, I.; Muangma, N.; Hen, O.; Shneor, R.; Sulkosky, V.; Kelleher, A.; Gilad, S.; Higinbotham, D. W.; Piasetzky, E.; Watson, J. W.; Wood, S. A.; Aguilera, P.; Ahmed, Z.; Albataineh, H.; Allada, K.; Anderson, B.; Anez, D.; Aniol, K.; Annand, J.; Armstrong, W.; Arrington, J.; Averett, T.; Badman, T.; Baghdasaryan, H.; Bai, X.; Beck, A.; Beck, S.; Bellini, V.; Benmokhtar, F.; Bertozzi, W.; Bittner, J.; Boeglin, W.; Camsonne, A.; Chen, C.; Chen, J.-P.; Chirapatpimol, K.; Cisbani, E.; Dalton, M. M.; Daniel, A.; Day, D.; de Jager, C. W.; De Leo, R.; Deconinck, W.; Defurne, M.; Flay, D.; Fomin, N.; Friend, M.; Frullani, S.; Fuchey, E.; Garibaldi, F.; Gaskell, D.; Gilman, R.; Glamazdin, O.; Gu, C.; Gueye, P.; Hamilton, D.; Hanretty, C.; Hansen, J.-O.; Hashemi Shabestari, M.; Holmstrom, T.; Huang, M.; Iqbal, S.; Jin, G.; Kalantarians, N.; Kang, H.; Khandaker, M.; LeRose, J.; Leckey, J.; Lindgren, R.; Long, E.; Mammei, J.; Margaziotis, D. J.; Markowitz, P.; Marti Jimenez-Arguello, A.; Meekins, D.; Meziani, Z.; Michaels, R.; Mihovilovic, M.; Monaghan, P.; Munoz Camacho, C.; Norum, B.; Nuruzzaman, Pan, K.; Phillips, S.; Pomerantz, I.; Posik, M.; Punjabi, V.; Qian, X.; Qiang, Y.; Qiu, X.; Rakhman, A.; Reimer, P. E.; Riordan, S.; Ron, G.; Rondon-Aramayo, O.; Saha, A.; Schulte, E.; Selvy, L.; Shahinyan, A.; Sirca, S.; Sjoegren, J.; Slifer, K.; Solvignon, P.; Sparveris, N.; Subedi, R.; Tireman, W.; Wang, D.; Weinstein, L. B.; Wojtsekhowski, B.; Yan, W.; Yaron, I.; Ye, Z.; Zhan, X.; Zhang, J.; Zhang, Y.; Zhao, B.; Zhao, Z.; Zheng, X.; Zhu, P.; Zielinski, R.; Jefferson Lab Hall A Collaboration

    2014-07-01

    We studied simultaneously the He4(e ,e'p), He4(e ,e'pp), and He4(e ,e'pn) reactions at Q2=2(GeV/c)2 and xB>1, for an (e ,e'p) missing-momentum range of 400 to 830 MeV/c. The knocked-out proton was detected in coincidence with a proton or neutron recoiling almost back to back to the missing momentum, leaving the residual A =2 system at low excitation energy. These data were used to identify two-nucleon short-range correlated pairs and to deduce their isospin structure as a function of missing momentum, in a region where the nucleon-nucleon (NN) force is expected to change from predominantly tensor to repulsive. The abundance of neutron-proton pairs is reduced as the nucleon momentum increases beyond ˜500 MeV/c. The extracted fraction of proton-proton pairs is small and almost independent of the missing momentum. Our data are compared with calculations of two-nucleon momentum distributions in He4 and discussed in the context of probing the elusive repulsive component of the NN force.

  8. Synthesis and COX-2 Inhibitory Activity of 4-[(E)-2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethenyl]benzene-1-sulfonamideand Its Analogs

    PubMed Central

    Hayun; Hudiyono, Sumi; Hanafi, Muhammad; Yanuar, Arry

    2012-01-01

    Some novel 3-phenyl-2-[(E)-2-phenylethenyl]-3,4-dihydroquinazolin-4-one derivatives possessing para-sulfonamides groups on the phenyl ring of the 2-phenylethenyl moiety have been synthesized and their COX-2 inhibitory activity evaluated. The stuctures of the synthesized compounds were confirmed on the basis of FT-IR, 1H-NMR, 13C-NMR and mass spectral data. The COX-2 inhibition screening assay revealed that 4-[(E)-2-{3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide had a maximum COX-2 inhibition (47.1%), at a concentration of 20 μM. PMID:24281337

  9. MABGEL 1: First Phase 1 Trial of the Anti-HIV-1 Monoclonal Antibodies 2F5, 4E10 and 2G12 as a Vaginal Microbicide

    PubMed Central

    Morris, Georgina C.; Wiggins, Rebecca C.; Woodhall, Sarah C.; Bland, J. Martin; Taylor, Carol R.; Jespers, Vicky; Vcelar, Brigitta A.; Lacey, Charles J.

    2014-01-01

    Background Monoclonal antibodies (mAbs) which potently neutralize a broad range of HIV isolates are potential microbicide candidates. To date, topical application of mAbs in humans and their stability in vaginal secretions has not been studied. Objectives To assess the pharmacokinetics and safety of the mAbs 2F5, 4E10 and 2G12 when applied vaginally in women. Design A randomized, double-blind, placebo-controlled phase 1 trial. Methods Twenty-eight healthy, sexually abstinent women administered 2.5 g of gel daily for 12 days containing either 10 or 20 mg/g of each mAb (MABGEL) or placebo. Main clinical evaluations and sampling occurred at baseline, 1, 8, and 24 hours post-1st dose and 12 and 36 hours post-12th dose. Results After adjustment for dilution factors, median levels of 2F5, 4E10 and 2G12 in vaginal secretions at 1 hour post high-dose MABGEL were 7.74, 5.28 and 7.48 mg/ml respectively. Levels of 2F5 and 4E10 declined exponentially thereafter with similar estimated half-lives (4.6 and 4.3 hours). In contrast, 2G12 levels declined more rapidly in the first 8 hours, with an estimated half-life of 1.4 hours during this period. There was no evidence of systemic absorption. There were no significant differences in local or systemic adverse event rates or vaginal flora changes (by qPCR) between active and placebo gel arms. Whilst at least 1 adverse event was recorded in 96% of participants, 95% were mild and none were serious. Conclusions Vaginal application of 50 mg of each mAb daily was safe over a 12 day period. Median mAb concentrations detected at 8 hours post dose were potentially sufficient to block HIV transmission.2G12 exhibited more rapid elimination from the human vagina than 4E10 and 2F5, likely due to poor stability of 2G12 in acidic human vaginal secretions. Further research is needed to develop mAb-based vaginal microbicides and delivery systems. Trial Registration ISRCTN 64808733 UK CRN Portfolio 6470 PMID:25546420

  10. The Role of the p38-MNK-eIF4E Signaling Axis in TNF Production Downstream of the NOD1 Receptor.

    PubMed

    Pashenkov, Mikhail V; Balyasova, Lyudmila S; Dagil, Yulia A; Pinegin, Boris V

    2017-02-15

    Activation of nucleotide-binding oligomerization domain (NOD) 1 and NOD2 by muropeptides triggers a complex transcriptional program in innate immune cells. However, little is known about posttranscriptional regulation of NOD1- and NOD2-dependent responses. When stimulated with a prototypic NOD1 agonist, N-acetylglucosaminyl-N-acetylmuramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid (GM-triDAP), human monocyte-derived macrophages (MDM) produced an order of magnitude more TNF, IL-6, and pro-IL-1β than did monocyte-derived dendritic cells (MDDC), despite similar NOD1 expression, similar cytokine mRNA kinetics, and comparable responses to LPS. TNF production by GM-triDAP-activated MDM was independent of autocrine IL-1. However, GM-triDAP-activated MDM translated TNF mRNA more efficiently than did MDDC. As an underlying mechanism, NOD1 triggering in MDM caused a more potent and long-lasting activation of the signaling axis involving p38 MAPK, MAPK-interacting kinase (MNK), and eukaryotic translation initiation factor 4E, which is a critical regulator of translation. Furthermore, MNK controlled TNF mRNA abundance in MDDC and MDM upon NOD1 triggering. NOD1-dependent responses were more sensitive to MNK inhibition than were TLR4-dependent responses. These results demonstrate the importance of the p38-MNK-eukaryotic translation initiation factor 4E axis in TNF production downstream of NOD1.

  11. The study of bonding in pyramidanes [(Me3Si)4C4]E (E = Ge, Sn, Pb) by optical (Raman, UV-vis) spectroscopy and quantum-chemical methods

    NASA Astrophysics Data System (ADS)

    Leites, Larissa A.; Aysin, Rinat R.; Bukalov, Sergey S.; Lee, Vladimir Ya.; Sugasawa, Hakura; Sekiguchi, Akira

    2017-02-01

    The nature of the apex-base bonds in organometallics of a novel class - pyramidanes [(Me3Si)4C4]E (E = Ge, Sn, Pb) was shown to be covalent but with a high degree of polarity on the basis of the Raman data and the results of QTAIM analysis. NICS data suggest three-dimensional aromaticity of the C4E pyramid.

  12. Dodeca-2(E),4(E)-dienoic acid isobutylamide enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling.

    PubMed

    Choi, Kyeong-Mi; Kim, Wonkyun; Hong, Jin Tae; Yoo, Hwan-Soo

    2017-02-01

    Dodeca-2(E),4(E)-dienoic acid isobutylamide (DDI), an alkamide derived from the plant Echinacea purpurea, promotes adipocyte differentiation and activates peroxisome proliferator-activated receptor γ, which is associated with enhanced insulin sensitivity. In the present study, we investigated whether DDI may increase glucose uptake through activation of the insulin signaling pathway in 3T3-L1 adipocytes. DDI increased insulin-stimulated glucose uptake, and expression and translocation of glucose transporter 4 in adipocytes treated with sub-optimal levels of insulin. Additionally, DDI enhanced Akt phosphorylation, whereas phosphoinositide 3-kinase/Akt inhibitors suppressed DDI-induced glucose uptake. These results suggest that DDI may improve insulin sensitivity through the activation of Akt signaling, which leads to enhanced glucose uptake.

  13. Quantum reactive scattering of O(3P)+H2 at collision energies up to 4.4 eV.

    PubMed

    Gacesa, Marko; Kharchenko, Vasili

    2014-10-28

    We report the results of quantum scattering calculations for the O((3)P)+H2 reaction for a range of collision energies from 0.4 to 4.4 eV, important for astrophysical and atmospheric processes. The total and state-to-state reactive cross sections are calculated using a fully quantum time-independent coupled-channel approach on recent potential energy surfaces of (3)A' and (3)A″ symmetry. A larger basis set than in the previous studies was used to ensure single-surface convergence at higher energies. Our results agree well with the published data at lower energies and indicate the breakdown of reduced dimensionality approach at collision energies higher than 1.5 eV. Differential cross sections and momentum transfer cross sections are also reported.

  14. Quantum reactive scattering of O({sup 3}P)+H{sub 2} at collision energies up to 4.4 eV

    SciTech Connect

    Gacesa, Marko; Kharchenko, Vasili

    2014-10-28

    We report the results of quantum scattering calculations for the O({sup 3}P)+H{sub 2} reaction for a range of collision energies from 0.4 to 4.4 eV, important for astrophysical and atmospheric processes. The total and state-to-state reactive cross sections are calculated using a fully quantum time-independent coupled-channel approach on recent potential energy surfaces of {sup 3}A{sup ′} and {sup 3}A{sup ″} symmetry. A larger basis set than in the previous studies was used to ensure single-surface convergence at higher energies. Our results agree well with the published data at lower energies and indicate the breakdown of reduced dimensionality approach at collision energies higher than 1.5 eV. Differential cross sections and momentum transfer cross sections are also reported.

  15. X-ray structures of precursors of styrylpyridine-derivatives used to obtain 4-((E)-2-(pyridin-2-yl)vinyl)benzamido-TEMPO: synthesis and characterization.

    PubMed

    Soriano-Moro, Guillermo; Percino, María Judith; Sánchez, Ana Laura; Chapela, Víctor Manuel; Cerón, Margarita; Castro, María Eugenia

    2015-04-02

    The synthesis and characterization of the precursor isomers trans-4-(2-(pyridin-2-yl)vinylbenzaldehyde (I), trans-4-(2-(pyridin-4-yl)vinylbenzaldehyde (II), trans-4-(2-(pyridin-2-yl)vinylbenzoic acid (III) and (E)-4-(2-(pydridin-4-yl)vinylbenzoic acid (IV) are reported. These compounds were prepared in order to obtain trans-4-((E)-2-(pyridin-2-yl)vinyl)benzamide-TEMPO (V). Compounds I and II were obtained by using a Knoevenagel reaction in the absence of a condensing agent and solvent. Oxidation of the aldehyde group using the Jones reagent afforded the corresponding acid forms III and IV. A condensation reaction with 4-amino-TEMPO using oxalyl chloride/DMF/CH2Cl2 provided the 4-((E)-2-(pyridin-2-yl)vinyl)benzamide-TEMPO. Single crystals of compounds I, II and III were obtained and characterized by X-ray diffraction. Compound I belongs to space group P2(1)/c, a = 12.6674(19) Å, b = 7.2173(11) Å, c = 11.5877(14) Å, b = 97.203(13)° and the asymmetric unit was Z = 4, whereas compound II was in the space group P2(1), with a = 3.85728(9) Å, b = 10.62375(19) Å, c = 12.8625(2) Å, b = 91.722 (2)° and the asymmetric unit was Z = 2. Compound III crystallized as single colorless needle crystals, belonging to the monoclinic system with space group P2(1), with Z = 2, with a = 3.89359(7) Å, b = 17.7014(3) Å, c = 8.04530(12) Å, b = 94.4030 (16)°. All compounds were completely characterized by IR, (1)H-NMR, EI-MS and UV-Vis.

  16. Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

    PubMed

    Papadopoulos, Evangelos; Jenni, Simon; Kabha, Eihab; Takrouri, Khuloud J; Yi, Tingfang; Salvi, Nicola; Luna, Rafael E; Gavathiotis, Evripidis; Mahalingam, Poornachandran; Arthanari, Haribabu; Rodriguez-Mias, Ricard; Yefidoff-Freedman, Revital; Aktas, Bertal H; Chorev, Michael; Halperin, Jose A; Wagner, Gerhard

    2014-08-05

    The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

  17. Regulation of global protein translation and protein degradation in aerobic dormancy.

    PubMed

    Ramnanan, Christopher J; Allan, Marcus E; Groom, Amy G; Storey, Kenneth B

    2009-03-01

    We hypothesized that protein turnover would be substantially suppressed during estivation in the land snail, Otala lactea, as part of a wholesale move to conserve ATP in the hypometabolic state, and that decreased rates of protein synthesis and degradation would be mediated by altering the phosphorylation state of key proteins. Rates of protein translation, measured in vitro, decreased by approximately 80% in extracts of foot muscle and hepatopancreas after 2 days of estivation, and this reduction was associated with strong increases in the phosphorylation of ribosomal factors, eIF2 alpha and eEF2, as well as decreased phosphorylation of 4E-BP1. Reductions in levels of markers of ribosomal biogenesis and a tissue-specific reduction in the phosphorylation state of eIF4E and eIF4GI were also evident after 14 days of estivation. Activity of the 20S proteasome decreased by 60-80% after 2 days of estivation and this decrease was mediated by protein kinase G in vitro, whereas protein phosphatase 2A activated the proteasome. Levels of protein carbonyls did not change in snail tissues during estivation whereas the expression heat shock proteins increased, suggesting that protein resistance to damage is enhanced in estivation. In conclusion, protein synthesis and degradation rates were coordinately suppressed during estivation in O. lactea and this is associated with the phosphorylation of ribosomal initiation and elongation factors and the 20S proteasome.

  18. Synthesis, spectroscopic investigation and theoretical studies of 2-((E)-(2-(2-cyanoacetyl)hydrazono)methyl)-4-((E)-phenyldiazenyl)phenyl methyl carbonate

    NASA Astrophysics Data System (ADS)

    Arokiasamy, A.; Manikandan, G.; Thanikachalam, V.; Gokula Krishnan, K.

    2017-04-01

    Synthesis and computational optimization studies have been carried out by Hartree-Fock (HF) and Density Functional Theory (DFT-B3LYP) methods with 6-31+G(d, p) basis set for 2-((E)-(2-(2-cyanoacetyl)hydrazono)methyl)-4-((E)-phenyldiazenyl)phenyl methyl carbonate (CHPMC). The stable configuration of CHPMC was confirmed theoretically by potential energy surface scan analysis. The complete vibrational assignments were performed on the basis of total energy distribution (TED) analysis. The vibrational properties studied by IR and Raman spectroscopic data complemented by quantum chemical calculations support the formation of intramolecular hydrogen bond. Furthermore, the UV-Vis spectra are interpreted in terms of TD-DFT quantum chemical calculations. The shapes of the simulated absorption spectra are in good agreement with the experimental data. The comparison between the experimental and theoretical values of FT-IR, FT-Raman vibrational spectra, NMR (1H and 13C) and UV-Vis spectra have also been discussed.

  19. Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

    SciTech Connect

    Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu; Huang, Jingsong

    2015-06-03

    Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap is distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.

  20. Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

    DOE PAGES

    Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu; ...

    2015-06-03

    Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

  1. Common tolerance mechanisms, but distinct cross-reactivities associated with gp41 and lipids, limit production of HIV-1 broad neutralizing antibodies 2F5 and 4E10.

    PubMed

    Chen, Yao; Zhang, Jinsong; Hwang, Kwan-Ki; Bouton-Verville, Hilary; Xia, Shi-Mao; Newman, Amanda; Ouyang, Ying-Bin; Haynes, Barton F; Verkoczy, Laurent

    2013-08-01

    Developing an HIV-1 vaccine has been hampered by the inability of immunogens to induce broadly neutralizing Abs (BnAbs) that protect against infection. Previously, we used knockin (KI) mice expressing a prototypical gp41-specific BnAb, 2F5, to demonstrate that immunological tolerance triggered by self-reactivity of the 2F5 H chain impedes BnAb induction. In this study, we generate KI models expressing H chains from two other HIV-1 Abs, 4E10 (another self-/polyreactive, anti-gp41 BnAb) and 48d (an anti-CD4 inducible, nonpolyreactive Ab), and find a similar developmental blockade consistent with central B cell deletion in 4E10, but not in 48d VH KI mice. Furthermore, in KI strains expressing the complete 2F5 and 4E10 Abs as BCRs, we find that residual splenic B cells arrest at distinct developmental stages, yet exhibit uniformly low BCR densities, elevated basal activation, and profoundly muted responses to BCR ligation and, when captured as hybridoma mAb lines, maintain their dual (gp41/lipid) affinities and capacities to neutralize HIV-1, establishing a key role for anergy in suppressing residual 2F5- or 4E10-expressing B cells. Importantly, serum IgGs from naive 2F5 and 4E10 KI strains selectively eliminate gp41 and lipid binding, respectively, suggesting B cells expressing 2F5 or 4E10 as BCRs exhibit specificity for a distinct spectrum of host Ags, including selective interactions by 2F5 BCR(+) B cells (i.e., and not 4E10 BCR(+) B cells) with those mimicked by its gp41 neutralization epitope.

  2. RIS4E at Kilauea's December 1974 Flow: Chemical, mineralogical and spectral characteristics of Hawaiian basaltic alteration products measured with portable instruments

    NASA Astrophysics Data System (ADS)

    Young, K. E.; Rogers, D.; Dyar, M. D.; Ito, G.; Yant, M.; McAdam, A.; Bleacher, J. E.; Glotch, T. D.

    2015-12-01

    A major objective of the SSERVI RIS4E (Remote, In-situ, and Synchrotron Studies for Science and Exploration) investigation is to evaluate the performance of portable chemical and mineralogical instruments in a variety of planetary volcanic analog settings. To that end, we used a suite of true/proxy portable instruments (XRF, LIBS, XRD, near-IR and mid-IR spectrometers), to measure the chemical and spectral characteristics of young basaltic flows (erupted December 1974, or D1974) within the southwest rift zone of Kilauea, Hawaii. The D1974 lavas exhibit multiple flow morphologies and textures, and have undergone alteration by a variety of processes, including acid weathering, oxidation and devitrification. The mineralogy, chemistry and infrared spectral properties of select samples from these altered surfaces have been well characterized by previous groups using high resolution (e.g. SEM, TEM) and/or laboratory measurements (XRD, Mossbauer, infrared). Typical alteration products include coatings of Fe-Ti-oxide +/- an overlying silica-rich coating. Coatings are commonly discontinuous and vary in color. Oxidation fronts are also present, most visible as reddish brown discoloration along the edges of broken and uplifted flow crusts. The previous detailed characterizations provide the basis for evaluating instrument performance and also allow us to assess areas where portable instruments can contribute new information to current understanding. These areas include characterizing the spatial variability in alteration chemistry/mineralogy, relating chemical/mineralogical properties to texture and context, and comparing chemical/mineralogical variations with infrared spectral properties. Because infrared spectra are commonly used to assess compositional variations of a site remotely, either from the ground or from orbit, relating changes in chemistry and mineralogy to spectral variations is particularly important. Last, the D1974 site provides an excellent location to test

  3. RIS4E at Kilauea's December 1974 (D1974) Flow: In Situ Geochemical Analysis and Laboratory Spectral Characterization of Fumarolic Alteration.

    NASA Astrophysics Data System (ADS)

    Yant, M.; Rogers, D.; Young, K. E.; Ito, G.; Bleacher, J. E.; McAdam, A.; Evans, C. A.; Eigenbrode, J. L.; Dyar, M. D.; Glotch, T. D.; Scheidt, S. P.

    2015-12-01

    The December 1974 (D1974) flow in the SW rift zone at Kilauea Volcano, Hawaii, has been established as a Mars analog due to its physical and chemical properties as well as its interaction with the outgassing plume from the primary Kilauea caldera. The RIS4E (Remote, In Situ and Synchrotron Studies for Science and Exploration) node of the SSERVI (Solar System Exploration Research Virtual Institute) program has conducted two field campaigns to the D1974 flow in an effort to study both its morphology and emplacement history as well as its geochemical and mineralogical signature. Several field portable instruments were deployed at the field site, including handheld x-ray fluorescence, field portable x-ray diffraction, a multispectral imager, Light Detection and Ranging, Ground Penetrating Radar, and a kite capable of producing high-resolution images and Digital Terrain Model data products. This study specifically focuses on the combination of field data with laboratory infrared spectra acquired in the MIR and VNIR ranges. We focus on the solfatara site which consists of hydrothermally-altered basalt deposited in and around an actively degassing volcanic vent situated directly adjacent to the D1974 flow on its NW side. Preliminary laboratory results indicate that the samples exhibit IR signatures consistent with various sulfates (K, Ca, Mg, Na, Al, Fe+3, Cu, Zn, Sr), clay minerals, amorphous silica, Fe-oxides, and/or sulfur. This data will give us a rich understanding of the solfatara site and enable us to make inferences about the hydrothermal alteration products formed in similar Martian environments.

  4. Low energy (0-4 eV) electron impact to N{sub 2}O clusters: Dissociative electron attachment, ion-molecule reactions, and vibrational Feshbach resonances

    SciTech Connect

    Vizcaino, Violaine; Denifl, Stephan; Maerk, Tilmann D.; Scheier, Paul; Illenberger, Eugen

    2010-10-21

    Electron attachment to clusters of N{sub 2}O in the energy range of 0-4 eV yields the ionic complexes [(N{sub 2}O){sub n}O]{sup -}, [(N{sub 2}O){sub n}NO]{sup -}, and (N{sub 2}O){sub n}{sup -} . The shape of the ion yields of the three homologous series differs substantially reflecting the different formation mechanisms. While the generation of [(N{sub 2}O){sub n}O]{sup -} can be assigned to dissociative electron attachment (DEA) of an individual N{sub 2}O molecule in the target cluster, the formation of [(N{sub 2}O){sub n}NO]{sup -} is interpreted via a sequence of ion molecule reactions involving the formation of O{sup -} via DEA in the first step. The nondecomposed complexes (N{sub 2}O){sub n}{sup -} are preferentially formed at very low energies (below 0.5 eV) as a result of intramolecular stabilization of a diffuse molecular anion at low energy. The ion yields of [(N{sub 2}O){sub n}O]{sup -} and (N{sub 2}O){sub n}{sup -} versus electron energy show sharp peaks at the threshold region, which can be assigned to vibrational Feshbach resonances mediated by the diffuse anion state as already observed in an ultrahigh resolution electron attachment study of N{sub 2}O clusters [E. Leber, S. Barsotti, J. Boemmels, J. M. Weber, I. I. Fabrikant, M.-W. Ruf, and H. Hotop, Chem. Phys. Lett. 325, 345 (2000)].

  5. Differing requirements for CCR4, E-selectin, and α4β1 for the migration of memory CD4 and activated T cells to dermal inflammation.

    PubMed

    Gehad, Ahmed; Al-Banna, Nadia A; Vaci, Maria; Issekutz, Andrew C; Mohan, Karkada; Latta, Markus; Issekutz, Thomas B

    2012-07-01

    CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)β(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ∼75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ∼60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)β(1) was more important. Anti-α(4)β(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)β(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)β(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)β(1), and CCR4(-) cells preferentially use α(4)β(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.

  6. Synthesis, characterization and antimicrobial studies of 2-{(E)-[(2-hydroxy-5-methylphenyl)imino]methyl}-4-[(E)-phenyldiazenyl]phenol as a novel azo-azomethine dye

    NASA Astrophysics Data System (ADS)

    Köse, Muhammet; Kurtoglu, Nurcan; Gümüşsu, Özkan; Tutak, Mustafa; McKee, Vickie; Karakaş, Duran; Kurtoglu, Mukerrem

    2013-12-01

    A novel dye, 2-{(E)-[(2-hydroxy-5-methylphenyl)imino]methyl}-4-[(E)-phenyldiazenyl]phenol dye was synthesized by the condensation reaction of 2-hydroxy-5-[(E)-phenyldiazenyl]benzaldehyde with 2-amino-4-methylphenol in methanol. The title dye was characterized by its melting point, elemental analysis, FT-IR, 1H, 13C NMR and mass spectroscopic studies. Molecular structure of the title dye was determined by single crystal X-ray diffraction study. X-ray data showed that the dye crystallizes in the monoclinic space group P21/c with cell parameters a = 18.541(2) Å, b = 4.7091(5) Å, c = 20.586(2) Å, V = 1761.5(3) Å3 and Z = 4. The title dye adopts azo-enamine tautomer in the solid state. The molecules crystallises as dimers assembled by two molecules of methanol via intermolecular hydrogen bonding resulting in R64(18) hydrogen bonding motif. Additionally, there is an intramolecular keto-amine hydrogen bond (NH⋯O) with a distance of 2.6172(17) Å. Optimized structures of the three possible tautomers of the compound were obtained using B3LYP method with 6-311++G(d,p), 6-31G and 3-21G basis sets in the gas phase. Thermal properties of the prepared dye were examined by thermogravimetric analysis and results indicated that the framework of the dye is stable up to 172 °C. Furthermore, the pathogenic activities of the synthesized dye were tested in vitro against the sensitive organisms, Bacillus cereous (ATCC 33019) and Staphylococcus aureus (ATCC 25923) as gram positive bacteria, Escherichia coli (ATCC 11229), and Klebsiella pneumoniae (ATCC 13883) as gram negative bacteria and the results are discussed. The results indicated that the prepared dye had antibacterial activities against gram-positive bacteria (S. aureus and Bacillus cereuss), but it exhibited no activity against gram-negative bacteria (E. coli and K. pneumoniae).

  7. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  8. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  9. The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

    PubMed

    Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

    2010-04-01

    Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

  10. Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis.

    PubMed

    Coldwell, Mark J; Cowan, Joanne L; Vlasak, Markete; Mead, Abbie; Willett, Mark; Perry, Lisa S; Morley, Simon J

    2013-12-01

    Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

  11. Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest

    PubMed Central

    2014-01-01

    Background HIV-1 Vpr-mediated G2 cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G2 arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown. Methods Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response. Results We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX. Conclusions The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the

  12. Targeted Dual pH-Sensitive Lipid ECO/siRNA Self-Assembly Nanoparticles Facilitate In Vivo Cytosolic sieIF4E Delivery and Overcome Paclitaxel Resistance in Breast Cancer Therapy.

    PubMed

    Gujrati, Maneesh; Vaidya, Amita M; Mack, Margaret; Snyder, Dayton; Malamas, Anthony; Lu, Zheng-Rong

    2016-11-01

    RNAi-mediated knockdown of oncogenes associated with drug resistance can potentially enhance the efficacy of chemotherapy. Here, we have designed and developed targeted dual pH-sensitive lipid-siRNA self-assembly nanoparticles, RGD-PEG(HZ)-ECO/siRNA, which can efficiently silence the oncogene, eukaryotic translation initiation factor 4E (eIF4E), and consequently resensitize triple-negative breast tumors to paclitaxel. The dual pH-sensitive function of these nanoparticles facilitates effective cytosolic siRNA delivery in cancer cells, both in vitro and in vivo. Intravenous injection of RGD-PEG(HZ)-ECO/siRNA nanoparticles (1.0 mg-siRNA/kg) results in effective gene silencing for at least one week in MDA-MB-231 tumors. In addition, treatment of athymic nude mice with RGD-PEG(HZ)-ECO/sieIF4E every 6 days for 6 weeks down-regulates the overexpression of eIF4E and resensitizes paclitaxel-resistant MDA-MB-231 tumors to paclitaxel, resulting in significant tumor regression at a low dose, with negligible side effects. Moreover, repeated injections of the RGD-PEG(HZ)-ECO/siRNA nanoparticles in immunocompetent mice result in minimal immunogenicity, demonstrating their safety and low toxicity. These multifunctional lipid/siRNA nanoparticles constitute a versatile platform of delivery of therapeutic siRNA for treating cancer and other human diseases.

  13. Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region.

    PubMed

    Banerjee, Saikat; Shi, Heliang; Banasik, Marisa; Moon, Hojin; Lees, William; Qin, Yali; Harley, Andrew; Shepherd, Adrian; Cho, Michael W

    2017-02-23

    The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 ((671)NWFDITNWLWYIK(683)). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

  14. 75 FR 11740 - S-Abscisic Acid, (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-1-cyclohex-2-enyl)-3-methyl-penta-(2Z,4E...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... AGENCY 40 CFR Part 180 S-Abscisic Acid, (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-1- cyclohex-2-enyl)-3-methyl-penta-(2Z,4E)-dienoic Acid; Amendment to an Exemption from the Requirement of a Tolerance AGENCY... exemption from the requirement of a tolerance for residues of the biochemical pesticide S-Abscisic Acid,...

  15. Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis†

    PubMed Central

    Tudor, Jennifer C.; Davis, Emily J.; Peixoto, Lucia; Wimmer, Mathieu E.; van Tilborg, Erik; Park, Alan J.; Poplawski, Shane G.; Chung, Caroline W.; Havekes, Robbert; Huang, Jiayan; Gatti, Evelina; Pierre, Philippe; Abel, Ted

    2016-01-01

    Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eukaryotic initiation factor 4G (eIF4G) in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons prior to sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. PMID:27117251

  16. Le transfert de connaissances entre les mathematiques et les sciences. Une etude exploratoire aupres d'eleves de 4e secondaire

    NASA Astrophysics Data System (ADS)

    Samson, Ghislain

    2003-06-01

    Au moment ou dans plusieurs pays on travaille a refondre les programmes d'etudes, tant au primaire qu'au secondaire, l'interet pour le transfert renait. Un des concepts fondamentaux en apprentissage consiste en l'habilete a reutiliser de facon consciente et efficace un acquis d'une situation a une autre situation. Cette recherche emane de preoccupations professionnelles au moment ou le chercheur etait enseignant au secondaire. Au cours de ces annees, il lui a ete possible de constater que plusieurs eleves percevaient difficilement les liens presents entre les disciplines mathematiques et scientifiques. Des travaux en psychologie cognitive et plus particulierement selon une perspective du traitement de l'information ont servi de cadre de reference pour evaluer et analyser les capacites de transfert aupres d'eleves de 4e secondaire. Ce cadre de reference permet de formuler le principal objectif qui est de mieux comprendre le processus de transfert chez des eleves en situation de resolution de problemes scientifiques. Cette these s'interesse donc au transfert en tant que phenomene important du processus d'apprentissage au sens de l'integration. La methode de recherche choisie, de nature qualitative, est principalement axee sur l'evaluation de la capacite a transferer des connaissances lors d'une epreuve et d'un entretien. Pour evaluer ce potentiel de transfert, nous avons elabore deux outils: une epreuve en mathematiques et en sciences et un guide d'entretien. Pour la passation de l'epreuve, le chercheur a pu compter sur la collaboration de 130 sujets provenant de deux ecoles. L'entretien complete la prise de donnees avec 13 sujets ayant accepte de poursuivre l'etude. Les donnees recueillies par ces instruments font ensuite l'objet d'une analyse de contenu. En premier lieu, les verbatims de l'epreuve et de l'entretien ont ete transcrits, puis codifies. La correction des reponses fournies pour les problemes resolus s'est faite a partir d'une grille d

  17. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  18. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  19. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  20. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    PubMed Central

    Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David

    2016-01-01

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553

  1. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis.

  2. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  3. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation.

    PubMed

    Timpano, Sara; Uniacke, James

    2016-05-13

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins.

  4. Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion.

    PubMed

    Carroll, Chad C; Fluckey, James D; Williams, Rick H; Sullivan, Dennis H; Trappe, Todd A

    2005-03-01

    The calf muscles, compared with the thigh, are less responsive to resistance exercise in ambulatory and bed-rested individuals, apparently due to muscle-specific differences in protein metabolism. We chose to evaluate the efficacy of using amino acids to elevate protein synthesis in the soleus, because amino acids have been shown to have a potent anabolic effect in the vastus lateralis. Mixed muscle protein synthesis in the soleus and vastus lateralis was measured before and after infusion of mixed amino acids in 10 individuals (28 +/- 1 yr). Phosphorylation of ribosomal protein p70 S6 kinase (p70S6K; Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1; Thr37/46) was also evaluated at rest and after 3 h of amino acid infusion. Basal protein synthesis was similar (P = 0.126), and amino acids stimulated protein synthesis to a similar extent (P = 0.004) in the vastus lateralis (0.043 +/- 0.011%/h) and soleus (0.032 +/- 0.017%/h). Phosphorylation of p70S6K (P = 0.443) and 4E-BP1 (P = 0.192) was not increased in either muscle; however, the soleus contained more total (P = 0.002) and phosphorylated (P = 0.013) 4E-BP1 than the vastus lateralis. These data support the need for further study of amino acid supplementation as a means to compensate for the reduced effectiveness of calf resistance exercise in ambulatory individuals and those exposed to extended periods of unloading. The greater 4E-BP1 in the soleus suggests that there is a muscle-specific distribution of general translational initiation machinery in human skeletal muscle.

  5. Low-energy laser irradiation enhances de novo protein synthesis via its effects on translation-regulatory proteins in skeletal muscle myoblasts.

    PubMed

    Shefer, Gavriela; Barash, Itamar; Oron, Uri; Halevy, Orna

    2003-02-17

    Low-energy laser irradiation (LELI) drives quiescent skeletal muscle satellite cells into the cell cycle and enhances their proliferation, thereby promoting skeletal muscle regeneration. Ongoing protein synthesis is a prerequisite for these processes. Here, we studied the signaling pathways involved in the LELI regulation of protein synthesis. High levels of labeled [35S]methionine incorporation were detected in LELI cells as early as 20 min after irradiation, suggesting translation of pre-existing mRNAs. Induced levels of protein synthesis were detected up until 8 h after LELI implying a role for LELI in de novo protein synthesis. Elevated levels of cyclin D1, associated with augmented phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding protein PHAS-I, suggested the involvement of LELI in the initiation steps of protein translation. In the presence of the MEK inhibitor, PD98059, eIF4E phosphorylation was abolished and levels of cyclin D1 were dramatically reduced. The LELI-induced PHAS-I phosphorylation was abolished after preincubation with the PI3K inhibitor, Wortmannin. Concomitantly, LELI enhanced Akt phosphorylation, which was attenuated in the presence of Wortmannin. Taken together, these results suggest that LELI induces protein translation via the PI3K/Akt and Ras/Raf/ERK pathways.

  6. Whey Protein

    MedlinePlus

    ... inflammation (polymyalgia rheumatica). Taking whey protein in a dairy product twice daily for 8 weeks does not improve muscle function, walking speed, or other movement tests in people with polymyalgia rheumatica. Other conditions. More evidence is needed to rate whey protein for these uses.

  7. Prolonged submaximal exercise induces isoform-specific Na+-K+-ATPase mRNA and protein responses in human skeletal muscle.

    PubMed

    Murphy, K T; Petersen, A C; Goodman, C; Gong, X; Leppik, J A; Garnham, A P; Cameron-Smith, D; Snow, R J; McKenna, M J

    2006-02-01

    This study investigated effects of prolonged submaximal exercise on Na+-K+-ATPase mRNA and protein expression, maximal activity, and content in human skeletal muscle. We also investigated the effects on mRNA expression of the transcription initiator gene, RNA polymerase II (RNAP II), and key genes involved in protein translation, eukaryotic initiation factor-4E (eIF-4E) and 4E-binding protein 1 (4E-BP1). Eleven subjects (6 men, 5 women) cycled at 75.5% (SD 4.8%) peak O2 uptake and continued until fatigue. A vastus lateralis muscle biopsy was taken at rest, fatigue, and 3 and 24 h postexercise. We analyzed muscle for Na+-K+-ATPase alpha1, alpha2, alpha3, beta1, beta2, and beta3, as well for RNAP II, eIF-4E, and 4E-BP1 mRNA expression by real-time RT-PCR and Na+-K+-ATPase isoform protein abundance using immunoblotting. Muscle homogenate maximal Na+-K+-ATPase activity was determined by 3-O-methylfluorescein phosphatase activity and Na+-K+-ATPase content by [3H]ouabain binding. Cycling to fatigue [54.5 (SD 20.6) min] immediately increased alpha3 (P = 0.044) and beta2 mRNA (P = 0.042) by 2.2- and 1.9-fold, respectively, whereas alpha1 mRNA was elevated by 2.0-fold at 24 h postexercise (P = 0.036). A significant time main effect was found for alpha3 protein abundance (P = 0.046). Exercise transiently depressed maximal Na+-K+-ATPase activity (P = 0.004), but Na+-K+-ATPase content was unaltered throughout recovery. Exercise immediately increased RNAP II mRNA by 2.6-fold (P = 0.011) but had no effect on eIF-4E and 4E-BP1 mRNA. Thus a single bout of prolonged submaximal exercise induced isoform-specific Na+-K+-ATPase responses, increasing alpha1, alpha3, and beta2 mRNA but only alpha3 protein expression. Exercise also increased mRNA expression of RNAP II, a gene initiating transcription, but not of eIF-4E and 4E-BP1, key genes initiating protein translation.

  8. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

    PubMed

    Manjarín, Rodrigo; Columbus, Daniel A; Suryawan, Agus; Nguyen, Hanh V; Hernandez-García, Adriana D; Hoang, Nguyet-Minh; Fiorotto, Marta L; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.

  9. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  10. Retrieval of transmembrane proteins to the endoplasmic reticulum

    PubMed Central

    1993-01-01

    A COOH-terminal double lysine motif maintains type I transmembrane proteins in the ER. Proteins tagged with this motif, eg., CD8/E19 and CD4/E19, rapidly receive post-translational modifications characteristic of the intermediate compartment and partially colocalized to this organelle. These proteins also received modifications characteristic of the Golgi but much more slowly. Lectin staining localized these Golgi modified proteins to ER indicating that this motif is a retrieval signal. Differences in the subcellular distribution and rate of post-translational modification of CD8 maintained in the ER by sequences derived from a variety of ER resident proteins suggested that the efficiency of retrieval was dependent on the sequence context of the double lysine motif and that retrieval may be initiated from multiple positions along the exocytotic pathway. PMID:8468349

  11. Larvicidal activity of Blumea eriantha essential oil and its components against six mosquito species, including Zika virus vectors: the promising potential of (4E,6Z)-allo-ocimene, carvotanacetone and dodecyl acetate.

    PubMed

    Benelli, Giovanni; Govindarajan, Marimuthu; Rajeswary, Mohan; Senthilmurugan, Sengamalai; Vijayan, Periasamy; Alharbi, Naiyf S; Kadaikunnan, Shine; Khaled, Jamal M

    2017-04-01

    The effective and environmentally sustainable control of mosquitoes is a challenge of essential importance. This is due to the fact that some invasive mosquitoes, with special reference to the Aedes genus, are particularly difficult to control, due to their high ecological plasticity. Moreover, the indiscriminate overuse of synthetic insecticides resulted in undesirable effects on human health and non-target organisms, as well as resistance development in targeted vectors. Here, the leaf essential oil (EO) extracted from a scarcely studied plant of ethno-medicinal interest, Blumea eriantha (Asteraceae), was tested on the larvae of six mosquitoes, including Zika virus vectors. The B. eriantha EO was analyzed by GC and GC-MS. The B. eriantha EO showed high toxicity against 3rd instar larvae of six important mosquito species: Anopheles stephensi (LC50=41.61 μg/ml), Aedes aegypti (LC50=44.82 μg/ml), Culex quinquefasciatus (LC50 =48.92 μg/ml), Anopheles subpictus (LC50=51.21 μg/ml), Ae. albopictus (LC50=56.33 μg/ml) and Culex tritaeniorhynchus (LC50=61.33 μg/ml). The major components found in B. eriantha EO were (4E,6Z)-allo-ocimene (12.8%), carvotanacetone (10.6%), and dodecyl acetate (8.9%). Interestingly, two of the main EO components, (4E,6Z)-allo-ocimene and carvotanacetone, achieved LC50 lower than 10 μg/ml on all tested mosquito species. The acute toxicity of B. eriantha EO and its major constituents on four aquatic predators of mosquito larval instars was limited, with LC50 ranging from 519 to 11.431 μg/ml. Overall, the larvicidal activity of (4E,6Z)-allo-ocimene and carvotanacetone far exceed most of the LC50 calculated in current literature on mosquito botanical larvicides, allowing us to propose both of them as potentially alternatives for developing eco-friendly mosquito control tools.

  12. Expression and immunological characterization of cardamom mosaic virus coat protein displaying HIV gp41 epitopes.

    PubMed

    Damodharan, Subha; Gujar, Ravindra; Pattabiraman, Sathyamurthy; Nesakumar, Manohar; Hanna, Luke Elizabeth; Vadakkuppattu, Ramanathan D; Usha, Ramakrishnan

    2013-05-01

    The coat protein of cardamom mosaic virus (CdMV), a member of the genus Macluravirus, assembles into virus-like particles when expressed in an Escherichia coli expression system. The N and C-termini of the coat protein were engineered with the Kennedy peptide and the 2F5 and 4E10 epitopes of gp41 of HIV. The chimeric proteins reacted with sera from HIV positive persons and also stimulated secretion of cytokines by peripheral blood mononuclear cells from these persons. Thus, a system based on the coat protein of CdMV can be used to display HIV-1 antigens.

  13. Role of PY Motif Containing Protein, WBP-2 in ER, PR Signaling and Breast Tumorigenesis

    DTIC Science & Technology

    2009-03-01

    WWP1 D1 D2 D3 D4 ++ ++ ++ ++ WW-domain contain- ing protein 1 Nedd-4-like ubiquitin protein ligase NEDD4 D1 ++ Neuronal precursor...Ubiquitin protein 10 D2 D3 D4 ++ ++ ++ cell-expressed devel- opmentally down- regulated 4 ligase Caveolin-3 + Membrane pro- tein PABPN1 + Poly...Membrane associ- ated guanylaste ki- nase-1 ITCH D1 D2 D3 D4 + + + + Atropin-1 interacting protein 4 E3 ubiquitin li- gase TAZ

  14. Role of PY Motif Containing Protein, WBP-2 in ER, PR Signaling and Breast Tumorigenesis

    DTIC Science & Technology

    2009-09-01

    D2 D3 D4 ++ ++ ++ ++ WW-domain contain- ing protein 1 Nedd-4-like ubi- quitin protein li- gase NEDD4 D1 D2 D3 D4... D2 - + Membrane asso- ciated guanylaste ki- nase-1 ITCH D1 D2 D3 D4 + + + + Atropin-1 interacting protein 4 E3 ubiquitin li- gase...Gene Symbol WW- domain(s) Interaction Protein name Function SMURF1 D1 D2 - + E3 ubiquitin li- gase SMURF2 D1 D2 - +

  15. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  16. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  17. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  18. Translational repression by RNA-binding protein TIAR.

    PubMed

    Mazan-Mamczarz, Krystyna; Lal, Ashish; Martindale, Jennifer L; Kawai, Tomoko; Gorospe, Myriam

    2006-04-01

    The RNA-binding protein TIAR has been proposed to inhibit protein synthesis transiently by promoting the formation of translationally silent stress granules. Here, we report the selective binding of TIAR to several mRNAs encoding translation factors such as eukaryotic initiation factor 4A (eIF4A) and eIF4E (translation initiation factors), eEF1B (a translation elongation factor), and c-Myc (which transcriptionally controls the expression of numerous translation regulatory proteins). TIAR bound the 3'-untranslated regions of these mRNAs and potently suppressed their translation, particularly in response to low levels of short-wavelength UV (UVC) irradiation. The UVC-imposed global inhibition of the cellular translation machinery was significantly relieved after silencing of TIAR expression. We propose that the TIAR-mediated inhibition of translation factor expression elicits a sustained repression of protein biosynthesis in cells responding to stress.

  19. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  20. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/.

  1. Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

    PubMed

    Toselli, F; Matthias, A; Bone, K M; Gillam, E M J; Lehmann, R P

    2010-08-01

    Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.

  2. Evidence of Polymorphism on the Antitrypanosomal Naphthoquinone (4E)-2-(1H-Pyrazol-3-ylamino)-4-(1H-pyrazol-3-ylimino)naphthalen-1(4H)-one

    PubMed Central

    Sperandeo, Norma R.; Faudone, Sonia N.

    2013-01-01

    The aim of this study was to characterize the solid state properties of (4E)-2-(1H-pyrazol-3-ylamino)-4-(1H-pyrazol-3-ylimino)naphthalen-1(4H)-one (BiPNQ), a compound with a significant inhibitory activity against Trypanosoma cruzi, the etiological agent of Chagas disease (American trypanosomiasis). Methods used included Differential Scanning Calorimetry (DSC), Thermogravimetry (TG), Fourier Transform Infrared Spectroscopy (FTIR), Powder X-Ray Diffraction (PXRD), Hot Stage, and Confocal Microscopy. Two BiPNQ samples were obtained by crystallization from absolute methanol and 2-propanol-water that exhibited different thermal behaviours, PXRD patterns, and FTIR spectra, indicating the existence of an anhydrous form (BiPNQ-I) and a solvate (BIPNQ-s), which on heating desolvated leading to the anhydrous modification BiPNQ-I. It was determined that FTIR, DSC, and PXRD are useful techniques for the characterization and identification of the crystalline modifications of BiPNQ. PMID:24106678

  3. In vitro drug release studies from the polymeric hydrogels based on HEA and HPMA using 4-[(E)-[(3Z)-3-(4-(acryloyloxy)benzylidene)-2-hexylidene]methyl]phenyl acrylate as a crosslinker.

    PubMed

    Arun, A; Reddy, B S R

    2005-04-01

    Novel crosslinker, 4-[(E)-[(3Z)-3-(4-(acryloyloxy)benzylidene)-2-hexylidene]methyl]phenyl acrylate (AMA) was synthesized using (2Z, 6E)-2,6-bis(4-hydroxybenzylidene)cyclohexanone (HBC) and acryloyl chloride. Two types of crosslinked polymeric hydrogels were prepared from 2-hydroxyethyl acrylate (HEA) and 2-hydroxypropyl methacrylate (HPMA) monomers using AMA as a crosslinking agent. 2',4-dichloro-5'-fluoro-1-ene-2-(4-hydroxyphenyl)phenone (EHP) (J. Bio Active Compat. Polym. 18 (2003) 219) was used as a drug molecule for monitoring the releasing behaviour of the hydrogels. Morphology of the hydrogels was characterized using optical microscopy (OM) and Scanning Electron Microscopy (SEM) techniques. Several modifications were made in the experimental sections to study the effect of crosslinking percentage (CLP), drug loading percentage (DLP), monomer type (HEA and HPMA) and the pH. Totally 18 experiments were carried out to study the desired parameters in the hydrogels. The drug-releasing rate was monitored by the absorption appeared at 330.5 nm using UV spectrometer. It was found that the releasing rate of the drug from the polymeric hydrogels was dependent on the crosslinking density, drug loading percentage, monomer type and pH of the medium.

  4. FT-IR, HOMO-LUMO, NBO, MEP analysis and molecular docking study of 3-Methyl-4-{(E)-[4-(methylsulfanyl)-benzylidene]amino}1H-1,2,4-triazole-5(4H)-thione.

    PubMed

    Panicker, C Yohannan; Varghese, Hema Tresa; Manjula, P S; Sarojini, B K; Narayana, B; War, Javeed Ahamad; Srivastava, S K; Van Alsenoy, C; Al-Saadi, Abdulaziz A

    2015-01-01

    FT-IR spectrum of 3-Methyl-4-{(E)-[4-(methylsulfanyl)-benzylidene]amino}1H-1,2,4-triazole-5(4H)-thione was recorded and analysed. The vibrational wavenumbers were computed and at HF and DFT levels of theory. The data obtained from wavenumber calculations are used to assign the vibrational bands obtained in the IR spectrum. The NH stretching wavenumber is red shifted in the IR spectrum from the computed value, which indicates the weakening of the NH bond. The geometrical parameters of the title compound are in agreement with the XRD results. NBO analysis, HOMO-LUMO, first and second order hyperpolarizability and molecular electrostatic potential results are also reported. From the MEP map it is evident that the negative regions are localized over the sulphur atoms and N3 atom of triazole ring and the maximum positive region is localized on NH group, indicating a possible site for nucleophilic attack. Prediction of Activity Spectra analysis of the title compound predicts anti-tuberculostic activity with probability to be active value of 0.543. Molecular docking studies reveal that the triazole nitrogen atoms and the thione sulphur atom play vital role in bonding and results draw us to the conclusion that the compound might exhibit anti-tuberculostic activity.

  5. Synthesis, Characterization, Antioxidant, and Antibacterial Studies of Some Metal(II) Complexes of Tetradentate Schiff Base Ligand: (4E)-4-[(2-{(E)-[1-(2,4-Dihydroxyphenyl)ethylidene]amino}ethyl)imino]pentan-2-one

    PubMed Central

    Ejidike, Ikechukwu P.; Ajibade, Peter A.

    2015-01-01

    Co(II), Ni(II), Cu(II), and Zn(II) complexes of (4E)-4-[(2-{(E)-[1-(2,4-dihydroxyphenyl)ethylidene]amino}ethyl)imino]pentan-2-one have been synthesized and characterized by elemental analyses, molar conductance, electronic and IR spectral studies, and XRD. FTIR confirmed the ligand coordinates the metal ion to form mononuclear complex via the oxygen and nitrogen atoms of the phenolic group and azomethine group, respectively. Tetrahedral geometry is proposed for Co(II) complex and square-planar geometry for Ni(II) and Cu(II) complexes. The antibacterial studies of the compounds were determined and they show that the metal complexes are more active than the free ligands. The antioxidant activity by DPPH and ABTS method was examined and it shows Cu(II); IC50 = 2.31 ± 1.54 µM for DPPH and Co(II); IC50 = 1.83 ± 1.08 µM for ABTS were the most active. PMID:26074738

  6. Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein

    PubMed Central

    Casperson, Shanon L.; Sheffield-Moore, Melinda; Hewlings, Susan J.; Paddon-Jones, Douglas

    2013-01-01

    SUMMARY Background & aim Protein-energy supplementation is routinely employed to combat muscle loss. However, success is often compromised by increased satiety, poor palatability, high costs and low compliance. Methods For 2-weeks we supplemented meals of older individuals with leucine (4 g/meal; 3 meals/day; days 2–14). Metabolic studies were performed prior to (Day 1) and following (Day 15) supplementation. Leucine was not provided on metabolic study days. Venous blood and vastus lateralis muscle biopsies were obtained during a primed constant infusion of L-[ring-13C6] phenylalanine. Mixed muscle fractional synthesis rate (FSR), body composition and markers of nutrient signaling (mTOR, 4E-BP1 and p70S6K1 phosphorylation) were measured before and after a low protein/carbohydrate simulated meal. Results The meal modestly increased FSR on Day 1 (postabsorptive: 0.063 ± 0.004 vs. postprandial: 0.075 ± 0.006%/h; p = 0.03), however, two weeks of leucine supplementation increased postabsorptive FSR (p = 0.004) and the response to the meal (p = 0.01) (postabsorptive: 0.074 ± 0.007 vs. postprandial: 0.10 ± 0.007%/h). Changes in FSR were mirrored by increased phosphorylation of mTOR, 4E-BP1 and p70S6K1 (p ≤ 0.1). No change in fat free mass was observed (p > 0.05). Conclusions In older adults, leucine supplementation may improve muscle protein synthesis in response to lower protein meals. PMID:22357161

  7. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  8. Protein synthesis during sleep consolidates cortical plasticity in vivo

    PubMed Central

    Seibt, Julie; Dumoulin, Michelle C.; Aton, Sara J.; Coleman, Tammi; Watson, Adam; Naidoo, Nirinjini; Frank, Marcos G.

    2012-01-01

    SUMMARY Sleep consolidates experience-dependent brain plasticity, but the precise cellular mechanisms mediating this process are unknown [1]. De novo cortical protein synthesis is one possible mechanism. In support of this hypothesis, sleep is associated with increased brain protein synthesis [2, 3] and transcription of mRNAs involved in protein synthesis regulation [4, 5]. Protein synthesis in turn is critical for memory consolidation and persistent forms of plasticity in vitro and in vivo [6, 7]. However, it is unknown if cortical protein synthesis in sleep serves similar functions. We investigated the role of protein synthesis in the sleep-dependent consolidation of a classic form of cortical plasticity in vivo (ocular dominance plasticity: ODP [8, 9]) in the cat visual cortex. We show that intracortical inhibition of mammalian target of rapamycin (mTOR)-dependent protein synthesis during sleep abolishes consolidation, but has no effect on plasticity induced during wakefulness. Sleep also promotes phosphorylation of protein synthesis regulators (i.e. 4E-BP1 and eEF2) and the translation (but not transcription) of key plasticity-related mRNAs (ARC and BDNF). These findings show that sleep promotes cortical mRNA translation. Interruption of this process has functional consequences, as it abolishes the consolidation of experience in the cortex. PMID:22386312

  9. [The synthesis of proteins in unnucleated blood platelets].

    PubMed

    Bijak, Michał; Saluk, Joanna; Ponczek, Michał Błażej Ponczek; Nowak, Paweł; Wachowicz, Barbara

    2013-07-23

    Platelets are the smallest, unnucleated blood cells that play a key role in maintaining normal hemostasis. In the human body about 1x1011 platelets are formed every day, as a the result of complex processes of differentiation, maturation and fragmentation of megakaryocytes. Studies done over 4 decades ago demonstrated that circulating in blood mature platelets can synthesize proteins. Recent discoveries confirm protein synthesis by unnucleated platelets in response to activation. Moreover, protein synthesis alters the phenotype and function of platelets. Platelets synthesize several proteins involved in hemostasis (COX, αIIbβ3, TF PAI-1, Factor XI, protein C inhibitor) and in inflammatory process (IL-1β, CCL5/RANTES). In spite of lack of transcription platelets have a stable mRNA transcripts with a long life correlated with platelet life span. Platelets also show expression of two important key regulators of translation eIF4E and EIF-2α and have a variety of miRNA molecules responsible for translational regulation. This article describes the historical overview of research on protein synthesis by platelets and presents the molecular mechanisms of protein synthesis in activated platelets (and synthesis of the most important platelet proteins).

  10. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  11. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs

    PubMed Central

    Suryawan, Agus; Nguyen, Hanh V.; Hernandez-García, Adriana D.; Hoang, Nguyet-Minh; Fiorotto, Marta L.; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight−1·day−1) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E·eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs. PMID:26334346

  12. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    PubMed

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

  13. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  14. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  15. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  16. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  17. Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

    PubMed Central

    Maroto, F G; Sierra, J M

    1989-01-01

    A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos. Images PMID:2501660

  18. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  19. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  20. Restorative Mechanisms Regulating Protein Balance in Skeletal Muscle During Recovery From Sepsis.

    PubMed

    Crowell, Kristen T; Soybel, David I; Lang, Charles H

    2017-04-01

    Muscle deconditioning is commonly observed in patients surviving sepsis. Little is known regarding the molecular mechanisms regulating muscle protein homeostasis during the recovery or convalescence phase. We adapted a sepsis-recovery mouse model that uses cecal ligation and puncture (CLP), followed 24 h later by cecal resection and antibiotic treatment, to identify putative cellular pathways regulating protein synthesis and breakdown in skeletal muscle. Ten days after CLP, body weight and food consumption did not differ between control and sepsis-recovery mice, but gastrocnemius weight was reduced. During sepsis-recovery, muscle protein synthesis was increased 2-fold and associated with enhanced mTOR kinase activity (4E-BP1 and S6K1 phosphorylation). The sepsis-induced increase in 4E-BP1 was associated with enhanced formation of the eIF4E-eIF4G active cap-dependent complex, while the increased S6K1 was associated with increased phosphorylation of downstream targets S6 and eIF4B. Proximal to mTOR, sepsis-recovery increased Akt and TSC2 phosphorylation, did not alter AMPK phosphorylation, and decreased REDD1 protein content. Despite the decreased mRNA content for the E3 ubiquitin ligases atrogin-1 and muscle RING-finger 1, proteasomal activity was increased 50%. In contrast, sepsis-recovery was associated with an apparent decrease in autophagy (e.g., increased ULK-1 phosphorylation, decreased LCB3-II, and increased p62). The mRNA content for IL-1β, IL-18, TNFα, and IL-6 in muscle was elevated in sepsis-recovery. During recovery after sepsis skeletal muscle responds with an increase in Akt-TSC2-mTOR-dependent protein synthesis and decreased autophagy, but full restoration of muscle protein content may be slowed by the continued stimulation of ubiquitin-proteasome activity.

  1. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  2. JIP60-mediated, jasmonate- and senescence-induced molecular switch in translation toward stress and defense protein synthesis

    PubMed Central

    Rustgi, Sachin; Pollmann, Stephan; Buhr, Frank; Springer, Armin; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

    2014-01-01

    Two closely related genes encoding the jasmonate-induced protein 60 (JIP60) were identified in the barley genome. The gene on chromosome arm 4HL encodes the previously identified protein encoded by the cDNA X66376.1. This JIP60 protein is characterized here and shown to consist of two domains: an NH2-terminal domain related to ribosome-inactivating proteins and a COOH-terminal domain, which displays similarity to eukaryotic translation initiation factor 4E (eIF4E). JIP60 undergoes processing in vivo, as a result of which JIP60’s COOH-terminal eIF4E domain is released and functions in recruiting a subset of cellular messengers for translation. This effect was observed for both MeJA-treated and naturally senescing plants. Because the JIP60 gene is in close proximity to several quantitative trait loci for both biotic and abiotic stress resistance, our results identify a unique target for future breeding programs. PMID:25225401

  3. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  4. mTORC1-independent reduction of retinal protein synthesis in type 1 diabetes.

    PubMed

    Fort, Patrice E; Losiewicz, Mandy K; Pennathur, Subramaniam; Jefferson, Leonard S; Kimball, Scot R; Abcouwer, Steven F; Gardner, Thomas W

    2014-09-01

    Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy.

  5. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  6. Whey protein fractionation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  7. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    PubMed Central

    Kim, Cy Hyun; Shin, Jin-Hong; Hwang, Sung Jun; Choi, Yung Hyun; Kim, Dae-Seong; Kim, Cheol Min

    2016-01-01

    Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy. PMID:27330287

  8. Castration alters protein balance after high-frequency muscle contraction.

    PubMed

    Steiner, Jennifer L; Fukuda, David H; Rossetti, Michael L; Hoffman, Jay R; Gordon, Bradley S

    2017-02-01

    Resistance exercise increases muscle mass by shifting protein balance in favor of protein accretion. Androgens independently alter protein balance, but it is unknown whether androgens alter this measure after resistance exercise. To answer this, male mice were subjected to sham or castration surgery 7-8 wk before undergoing a bout of unilateral, high-frequency, electrically induced muscle contractions in the fasted or refed state. Puromycin was injected 30 min before euthanasia to measure protein synthesis. The tibialis anterior was analyzed 4 h postcontraction. In fasted mice, neither basal nor stimulated rates of protein synthesis were affected by castration despite lower phosphorylation of mechanistic target of rapamycin in complex 1 (mTORC1) substrates [p70S6K1 (Thr389) and 4E-BP1 (Ser65)]. Markers of autophagy (LC3 II/I ratio and p62 protein content) were elevated by castration, and these measures remained elevated above sham values after contractions. Furthermore, in fasted mice, the protein content of Regulated in Development and DNA Damage 1 (REDD1) was correlated with LC3 II/I in noncontracted muscle, whereas phosphorylation of uncoordinated like kinase 1 (ULK1) (Ser757) was correlated with LC3 II/I in the contracted muscle. When mice were refed before contractions, protein synthesis and mTORC1 signaling were not affected by castration in either the noncontracted or contracted muscle. Conversely, markers of autophagy remained elevated in the muscles of refed, castrated mice even after contractions. These data suggest the castration-mediated elevation in baseline autophagy reduces the absolute positive shift in protein balance after muscle contractions in the refed or fasted states.

  9. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  10. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

  11. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  12. Activity-dependent neuroprotective protein (ADNP) exhibits striking sexual dichotomy impacting on autistic and Alzheimer's pathologies

    PubMed Central

    Malishkevich, A; Amram, N; Hacohen-Kleiman, G; Magen, I; Giladi, E; Gozes, I

    2015-01-01

    Activity-dependent neuroprotective protein (ADNP) is a most frequent autism spectrum disorder (ASD)-associated gene and the only protein significantly decreasing in the serum of Alzheimer's disease (AD) patients. Is ADNP associated with ASD being more prevalent in boys and AD more prevalent in women? Our results revealed sex-related learning/memory differences in mice, reflecting hippocampal expression changes in ADNP and ADNP-controlled AD/ASD risk genes. Hippocampal ADNP transcript content was doubled in male vs female mice, with females showing equal expression to ADNP haploinsufficient (ADNP+/−) males and no significant genotype-associated reduction. Increased male ADNP expression was replicated in human postmortem hippocampal samples. The hippocampal transcript for apolipoprotein E (the major risk gene for AD) was doubled in female mice compared with males, and further doubled in the ADNP+/− females, contrasting a decrease in ADNP+/− males. Previously, overexpression of the eukaryotic translation initiation factor 4E (eIF4E) led to ASD-like phenotype in mice. Here, we identified binding sites on ADNP for eIF4E and co-immunoprecipitation. Furthermore, hippocampal eIF4E expression was specifically increased in young ADNP+/− male mice. Behaviorally, ADNP+/− male mice exhibited deficiencies in object recognition and social memory compared with ADNP+/+ mice, while ADNP+/− females were partially spared. Contrasting males, which preferred novel over familiar mice, ADNP+/+ females showed no preference to novel mice and ADNP+/− females did not prefer mice over object. ADNP expression, positioned as a master regulator of key ASD and AD risk genes, introduces a novel concept of hippocampal gene-regulated sexual dimorphism and an ADNP+/− animal model for translational psychiatry. PMID:25646590

  13. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    PubMed

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  14. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  15. Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content.

    PubMed

    Kanda, Atsushi; Nakayama, Kyosuke; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2013-09-28

    It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague–Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.

  16. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  17. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  18. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  19. Viral Genome-Linked Protein (VPg) Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV).

    PubMed

    Zhu, Jie; Wang, Binbin; Miao, Qiuhong; Tan, Yonggui; Li, Chuanfeng; Chen, Zongyan; Guo, Huimin; Liu, Guangqing

    2015-01-01

    Rabbit hemorrhagic disease virus (RHDV), the causative agent of rabbit hemorrhagic disease, is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, limiting the study of the pathogenesis of RHDV. In addition, the mechanisms underlying RHDV translation and replication are largely unknown compared with other caliciviridae viruses. The RHDV replicon recently constructed in our laboratory provides an appropriate model to study the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon, we demonstrated that the viral genome-linked protein (VPg) is essential for RHDV translation in RK-13 cells for the first time. In addition, we showed that VPg interacts with eukaryotic initiation factor 4E (eIF4E) in vivo and in vitro and that eIF4E silencing inhibits RHDV translation, suggesting the interaction between VPg and eIF4E is involved in RHDV translation. Our results support the hypothesis that VPg serves as a novel cap substitute during the initiation of RHDV translation.

  20. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  1. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  2. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  3. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  4. Texturized dairy proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  5. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  6. The E5 Proteins

    PubMed Central

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 is the PDGF β receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis. PMID:23731971

  7. Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines.

    PubMed

    Ye, Ling; Wen, Zhiyuan; Dong, Ke; Wang, Xi; Bu, Zhigao; Zhang, Huizhong; Compans, Richard W; Yang, Chinglai

    2011-01-01

    Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.

  8. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  9. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  10. Protein-protein interactions in multienzyme megasynthetases.

    PubMed

    Weissman, Kira J; Müller, Rolf

    2008-04-14

    The multienzyme polyketide synthases (PKSs), nonribosomal polypeptide synthetases (NRPSs), and their hybrids are responsible for the construction in bacteria of numerous natural products of clinical value. These systems generate high structural complexity by using a simple biosynthetic logic--that of the assembly line. Each of the individual steps in building the metabolites is designated to an independently folded domain within gigantic polypeptides. The domains are clustered into functional modules, and the modules are strung out along the proteins in the order in which they act. Every metabolite results, therefore, from the successive action of up to 100 individual catalysts. Despite the conceptual simplicity of this division-of-labor organization, we are only beginning to decipher the molecular details of the numerous protein-protein interactions that support assembly-line biosynthesis, and which are critical to attempts to re-engineer these systems as a tool in drug discovery. This review aims to summarize the state of knowledge about several aspects of protein-protein interactions, including current architectural models for PKS and NRPS systems, the central role of carrier proteins, and the structural basis for intersubunit recognition.

  11. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  12. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  13. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  14. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  15. [Atypical ubiquitination of proteins].

    PubMed

    Buneeva, O A; Medvedev, A E

    2016-07-01

    Ubiquitination is a type of posttranslational modification of intracellular proteins characterized by covalent attachment of one (monoubiquitination) or several (polyubiquitination) of ubiquitin molecules to target proteins. In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins not necessarily targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes and altered polyubiquitination of certain proteins is crucial for development of serious diseases.

  16. Protein and vegetarian diets.

    PubMed

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  17. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  18. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  19. The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius.

    PubMed

    Yang, D; Kusser, I; Köpke, A K; Koop, B F; Matheson, A T

    1999-07-01

    The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

  20. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  1. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  2. Protein flexibility as a biosignal.

    PubMed

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  3. Antimicrobial proteins: From old proteins, new tricks.

    PubMed

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis.

  4. Protein-protein Interactions using Radiolytic Footprinting

    SciTech Connect

    Takamoto,K.; Chance, M.

    2006-01-01

    Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.

  5. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation.

  6. Mayaro virus proteins.

    PubMed

    Mezencio, J M; Rebello, M A

    1993-01-01

    Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

  7. TRIM proteins and diseases.

    PubMed

    Watanabe, Masashi; Hatakeyama, Shigetsugu

    2017-01-07

    Ubiquitination is one of the posttranslational modifications that regulates a number of intracellular events including signal transduction, protein quality control, transcription, cell cycle, apoptosis and development. The ubiquitin system functions as a garbage machine to degrade target proteins and as a regulator for several signalling pathways. Biochemical reaction of ubiquitination requires several enzymes including E1, E2 and E3, and E3 ubiquitin ligases play roles as receptors for recognizing target proteins. Most of the tripartite motif (TRIM) proteins are E3 ubiquitin ligases. Recent studies have shown that some TRIM proteins function as important regulators for a variety of diseases including cancer, inflammatory diseases, infectious diseases, neuropsychiatric disorders, chromosomal abnormalities and developmental diseases. In this review, we summarize the involvement of TRIM proteins in the aetiology of various diseases.

  8. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  9. eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    PubMed Central

    Lee, Amy S.Y.; Kranzusch, Philip J.; Doudna, Jennifer A.; Cate, Jamie H.D.

    2016-01-01

    Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation. PMID:27462815

  10. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  11. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  12. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  13. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  14. Chemical Synthesis of Proteins

    PubMed Central

    Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

    2010-01-01

    Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

  15. PIC: Protein Interactions Calculator

    PubMed Central

    Tina, K. G.; Bhadra, R.; Srinivasan, N.

    2007-01-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic–aromatic interactions, aromatic–sulphur interactions and cation–π interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar–apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  16. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  17. Dietary proteins and angiogenesis.

    PubMed

    Medina, Miguel Ángel; Quesada, Ana R

    2014-01-17

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

  18. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  19. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  20. TRIM proteins in development.

    PubMed

    Petrera, Francesca; Meroni, Germana

    2012-01-01

    TRIM proteins play important roles in several patho-physiological processes. Their common activity within the ubiquitylation pathway makes them amenable to a number of diverse biological roles. Many of the TRIM genes are highly and sometimes specifically expressed during embryogenesis, it is therefore not surprising that several of them might be involved in developmental processes. Here, we primarily discuss the developmental implications of two subgroups of TRIM proteins that conserved domain composition and functions from their invertebrate ancestors. The two groups are: the TRIM-NHL proteins implicated in miRNA processing regulation and the TRIM-FN3 proteins involved in ventral midline development.

  1. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  2. Acanthamoeba castellanii STAT Protein

    PubMed Central

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  3. Protein intakes in India.

    PubMed

    Swaminathan, Sumathi; Vaz, Mario; Kurpad, Anura V

    2012-08-01

    Indian diets derive almost 60 % of their protein from cereals with relatively low digestibility and quality. There have been several surveys of diets and protein intakes in India by the National Nutrition Monitoring Board (NNMB) over the last 25 years, in urban and rural, as well as in slum dwellers and tribal populations. Data of disadvantaged populations from slums, tribals and sedentary rural Indian populations show that the protein intake (mainly from cereals) is about 1 gm/kg/day. However, the protein intake looks less promising in terms of the protein digestibility corrected amino acid score (PDCAAS), using lysine as the first limiting amino acid, where all populations, particularly rural and tribal, appear to have an inadequate quality to their protein intake. The protein: energy (PE) ratio is a measure of dietary quality, and has been used in the 2007 WHO/FAO/UNU report to define reference requirement values with which the adequacy of diets can be evaluated in terms of a protein quality corrected PE ratio. It is likely that about one third of this sedentary rural population is at risk of not meeting their requirements. These levels of risk of deficiency are in a population with relatively low BMI populations, whose diets are also inadequate in fruits and vegetables. Therefore, while the burden of enhancing the quality of protein intake in rural India exists, the quality of the diet, in general, represents a challenge that must be met.

  4. Self assembling proteins

    DOEpatents

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  5. Ultrafiltration of pegylated proteins

    NASA Astrophysics Data System (ADS)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  6. Regulation of protein secretion by ... protein secretion?

    PubMed

    Atmakuri, Krishnamohan; Fortune, Sarah M

    2008-09-11

    Mycobacterium tuberculosis (Mtb) requires an alternative protein secretion system, ESX1, for virulence. Recently, Raghavan et al. (2008) reported a new regulatory circuit that may explain how ESX1 activity is controlled during infection. Mtb appears to regulate ESX1 by modulating transcription of associated genes rather than structural components of the secretion system itself.

  7. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  8. Literacy's Beginnings: Supporting Young Readers and Writers, 4/E

    ERIC Educational Resources Information Center

    McGee, Lea M.; Richgels, Donald J.

    2004-01-01

    McGee and Richgels have set the standard in this new edition by clearly and simply explaining the issues addressed in Reading First and Early Reading First legislation that affect the reading instruction of young children. Aligned with the findings of the National Reading Panel, this edition focuses on child-centered instruction in phonemic…

  9. US EPA, Pesticide Product Label, PLAINSMAN BRAND 4-E ...

    EPA Pesticide Factsheets

    2011-04-14

    ... r • '\\ ii' j i, 1d III 1. 11 ., j' < II' _'., I' 11'PII,tli',II'" 1\\ rl, "" : 01" ,.,' II "j" tIt' '/"'fOll, ,., " It I" .i ..•. I I" , "I , . J" u" l. ill', ,I· 1,· ... 1. h"p ,,, ' . _. '1.... ,II JI "I ,: I .", I .. ...

  10. US EPA, Pesticide Product Label, PRATT DIAZINON AG 4E ...

    EPA Pesticide Factsheets

    2011-04-14

    ... A'-hi;s~ e~~'~(n: c.~r·~~ Bud !\\jik~. carnat~~n--I-- ~ r ..... tGs. tiev.;; ;!iif~~,cyc!arrq!n fAne!' • Oip~'·(y;;!'> i ea' : .·Ikl,. r:;ropea'1 Pine S1vot I MO~hS. ...

  11. Engineered Protein Polymers

    DTIC Science & Technology

    2010-05-31

    of each pure polymer, we plan to combine the various polymer solutions in different ratios to tune the composition and physico-chemical properties...protein materials as vehicles for storage and delivery of small molecules. Each protein polymer under concentrations for particle formation ( vida

  12. Multidomain proteins under force.

    PubMed

    Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel

    2017-04-28

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  13. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  14. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  15. Poxviral Ankyrin Proteins

    PubMed Central

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  16. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  17. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  18. Proteins in unexpected locations.

    PubMed Central

    Smalheiser, N R

    1996-01-01

    Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer. PMID:8862516

  19. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  20. Transdermal delivery of proteins.

    PubMed

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  1. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  2. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  3. Protein Unfolding and Alzheimer's

    NASA Astrophysics Data System (ADS)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  4. Junin virus structural proteins.

    PubMed Central

    De Martínez Segovia, Z M; De Mitri, M I

    1977-01-01

    Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined. PMID:189088

  5. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so

  6. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  7. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  8. [Controversies around diet proteins].

    PubMed

    Cichosz, Grazyna; Czeczot, Hanna

    2013-12-01

    Critical theories regarding proteins of anima origin are still and still popularized, though they are ungrounded from scientific point of view. Predominance of soya proteins over the animal ones in relation to their influence on calcium metabolism, bone break risk or risk of osteoporosis morbidity has not been confirmed in any honest, reliable research experiment. Statement, that sulphur amino acids influence disadvantageously on calcium metabolism of human organism and bone status, is completely groundless, the more so as presence of sulphur amino acids in diet (animal proteins are their best source) is the condition of endogenic synthesis of glutathione, the key antioxidant of the organism, and taurine stimulating brain functioning. Deficiency of proteins in the diet produce weakness of intellectual effectiveness and immune response. There is no doubt that limitation of consumption of animal proteins of standard value is not good for health.

  9. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  10. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  11. Regulation of protein turnover by heat shock proteins.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.

  12. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  13. Prediction of protein-protein interactions: unifying evolution and structure at protein interfaces.

    PubMed

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-06-01

    The vast majority of the chores in the living cell involve protein-protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein-protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations.

  14. NMCP/LINC proteins

    PubMed Central

    Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

    2013-01-01

    Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins. PMID:24128696

  15. TRIM proteins in cancer.

    PubMed

    Cambiaghi, Valeria; Giuliani, Virginia; Lombardi, Sara; Marinelli, Cristiano; Toffalorio, Francesca; Pelicci, Pier Giuseppe

    2012-01-01

    Some members of the tripartite motif (TRIM/RBCC) protein family are thought to be important regulators of carcinogenesis. This is not surprising as the TRIM proteins are involved in several biological processes, such as cell growth, development and cellular differentiation and alteration of these proteins can affect transcriptional regulation, cell proliferation and apoptosis. In particular, four TRIM family genes are frequently translocated to other genes, generating fusion proteins implicated in cancer initiation and progression. Among these the most famous is the promyelocytic leukaemia gene PML, which encodes the protein TRIM19. PML is involved in the t(15;17) translocation that specifically occurs in Acute Promyelocytic Leukaemia (APL), resulting in a PML-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. Other members of the TRIM family are linked to cancer development without being involved in chromosomal re-arrangements, possibly through ubiquitination or loss of tumour suppression functions. This chapter discusses the biological functions of TRIM proteins in cancer.

  16. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  17. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  18. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  19. Piezoelectric allostery of protein

    NASA Astrophysics Data System (ADS)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

  20. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  1. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  2. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  3. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  4. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  5. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression.

  6. Evolution of proteins.

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1971-01-01

    The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

  7. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  8. Teaching resources. Protein phosphatases.

    PubMed

    Salton, Stephen R

    2005-03-01

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

  9. Electrochromatographic separation of proteins

    NASA Technical Reports Server (NTRS)

    Basak, S. K.; Velayudhan, A.; Kohlmann, K.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

    1995-01-01

    We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

  10. Molecular cloning, gene expression analysis, and recombinant protein expression of novel silk proteins from larvae of a retreat-maker caddisfly, Stenopsyche marmorata.

    PubMed

    Bai, Xue; Sakaguchi, Mayo; Yamaguchi, Yuko; Ishihara, Shiori; Tsukada, Masuhiro; Hirabayashi, Kimio; Ohkawa, Kousaku; Nomura, Takaomi; Arai, Ryoichi

    2015-08-28

    Retreat-maker larvae of Stenopsyche marmorata, one of the major caddisfly species in Japan, produce silk threads and adhesives to build food capture nets and protective nests in water. Research on these underwater adhesive silk proteins potentially leads to the development of new functional biofiber materials. Recently, we identified four major S. marmorata silk proteins (Smsps), Smsp-1, Smsp-2, Smsp-3, and Smsp-4 from silk glands of S. marmorata larvae. In this study, we cloned full-length cDNAs of Smsp-2, Smsp-3, and Smsp-4 from the cDNA library of the S. marmorata silk glands to reveal the primary sequences of Smsps. Homology search results of the deduced amino acid sequences indicate that Smsp-2 and Smsp-4 are novel proteins. The Smsp-2 sequence [167 amino acids (aa)] has an array of GYD-rich repeat motifs and two (SX)4E motifs. The Smsp-4 sequence (132 aa) contains a number of GW-rich repeat motifs and three (SX)4E motifs. The Smsp-3 sequence (248 aa) exhibits high homology with fibroin light chain of other caddisflies. Gene expression analysis of Smsps by real-time PCR suggested that the gene expression of Smsp-1 and Smsp-3 was relatively stable throughout the year, whereas that of Smsp-2 and Smsp-4 varied seasonally. Furthermore, Smsps recombinant protein expression was successfully performed in Escherichia coli. The study provides new molecular insights into caddisfly aquatic silk and its potential for future applications.

  11. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  12. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

    SciTech Connect

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-03-30

    Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.

  13. Plant protein glycosylation

    PubMed Central

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  14. Protein Model Database

    SciTech Connect

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  15. Protein Colloidal Aggregation Project

    NASA Technical Reports Server (NTRS)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  16. Fully automated protein purification

    PubMed Central

    Camper, DeMarco V.; Viola, Ronald E.

    2009-01-01

    Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol. PMID:19595984

  17. Protein fabrication automation

    PubMed Central

    Cox, J. Colin; Lape, Janel; Sayed, Mahmood A.; Hellinga, Homme W.

    2007-01-01

    Facile “writing” of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable. PMID:17242375

  18. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  19. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  20. Occupational protein contact dermatitis.

    PubMed

    Barbaud, Annick; Poreaux, Claire; Penven, Emmanuelle; Waton, Julie

    2015-01-01

    Occupational contact dermatitis is generally caused by haptens but can also be induced by proteins causing mainly immunological contact urticaria (ICU); chronic hand eczema in the context of protein contact dermatitis (PCD). In a monocentric retrospective study, from our database, only 31 (0.41%) of patients with contact dermatitis had positive skin tests with proteins: 22 had occupational PCD, 3 had non-occupational PCD, 5 occupational ICU and 1 cook had a neutrophilic fixed food eruption (NFFE) due to fish. From these results and analysis of literature, the characteristics of PCD can be summarized as follows. It is a chronic eczematous dermatitis, possibly exacerbated by work, suggestive if associated with inflammatory perionyxix and immediate erythema with pruritis, to be investigated when the patient resumes work after a period of interruption. Prick tests with the suspected protein-containing material are essential, as patch tests have negative results. In case of multisensitisation revealed by prick tests, it is advisable to analyse IgE against recombinant allergens. A history of atopy, found in 56 to 68% of the patients, has to be checked for. Most of the cases are observed among food-handlers but PCD can also be due to non-edible plants, latex, hydrolysed proteins or animal proteins. Occupational exposure to proteins can thus lead to the development of ICU. Reflecting hypersensitivity to very low concentrations of allergens, investigating ICU therefore requires caution and prick tests should be performed with a diluted form of the causative protein-containing product. Causes are food, especially fruit peel, non-edible plants, cosmetic products, latex, animals.

  1. Chirality and protein biosynthesis.

    PubMed

    Banik, Sindrila Dutta; Nandi, Nilashis

    2013-01-01

    Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

  2. Protein Nitrogen Determination

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  3. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  4. Protein conducting nanopores

    NASA Astrophysics Data System (ADS)

    Harsman, Anke; Krüger, Vivien; Bartsch, Philipp; Honigmann, Alf; Schmidt, Oliver; Rao, Sanjana; Meisinger, Christof; Wagner, Richard

    2010-11-01

    About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40SC as well as a mutant Tom40SC (\\mathrm {S}_{54} \\to E ) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40SC corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40SC S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with \\bar {t}_{\\mathrm {off}} \\cong 1.1 ms for the wildtype, whereas the mutant Tom40SC S54E displayed a biphasic dwelltime distribution (\\bar {t}_{\\mathrm {off}}^1 \\cong 0.4 ms \\bar {t}_{\\mathrm {off}}^2 \\cong 4.6 ms).

  5. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  6. Motor proteins 1: kinesins.

    PubMed

    Bloom, G S; Endow, S A

    1995-01-01

    Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles [17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292] or books [49, 227, 293] that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.

  7. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, pr