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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. Identification and function of the second eIF4E-binding region in N-terminal domain of eIF4G: comparison with eIF4E-binding protein.

    PubMed

    Umenaga, Yu; Paku, Keum Soon; In, Yasuko; Ishida, Toshimasa; Tomoo, Koji

    2011-10-28

    The eukaryotic initiation factor 4E (eIF4E) serves as a master switch that controls mRNA translation through the promotive binding to eIF4G and the regulative binding with the endogenous inhibitor 4E-BP. Although the bindings of eIF4G and 4E-BP to eIF4E proceed through the common eIF4E recognition Y(X)(4)Lφ motif (X: variable, φ: hydrophobic) (first binding site), the relationship between their eIF4E binding mode and the functional difference is hardly known. Recently, we have clarified the existence and function of the second eIF4E binding site in 4E-BP. Surface plasmon resonance (SPR) analysis based on the sequential comparison between 4E-BP and eIF4GI clarified that eIF4G has the second binding site at the periphery of the (597)SDVVL(601) sequence and that it plays an auxiliary but indispensable function in stabilizing the binding of the first binding sequence (572)YDREFLL(578). The kinetic parameters of the interactions of the eIF4GI and 4E-BP2 fragment peptides with eIF4E showed that the association (ka) and dissociation (kd) rates of the former peptide are about three and two orders of magnitude lower than those of the latter peptide, respectively. This means that eIF4G has a potent resistive property for release from eIF4E, although its rate of binding to eIF4E is not as high as that of 4E-BP, that is, 4E-BP is apt to bind to and be released from eIF4E, as compared with eIF4G. Isothermal titration calorimetry (ITC) showed the opposite behavior between the second binding sites of eIF4GI and 4E-BP for the interaction with eIF4E. This clearly indicates the importance of the second binding region for the difference in function between eIF4G and 4E-BP for eIF4E translation. PMID:21964297

  3. Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins.

    PubMed

    Hagedorn, C H; Spivak-Kroizman, T; Friedland, D E; Goss, D J; Xie, Y

    1997-02-01

    Protein-mRNA cap interactions represent a critical point for regulating gene expression in vivo. For example, a rapid stimulation of gene expression at the mRNA level is mediated by insulin regulating the availability of functional cap-binding protein (eIF-4E). In addition, several viruses modify cap binding proteins to regulate host vs viral gene expression. However, little is known about the molecular details of eIF-4E interactions with m7GTP mRNA caps, with regulatory proteins (e.g., eIF-4E binding proteins), and with proteins within the eIF-4F complex. To study these protein-mRNA and protein-protein interactions in mammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E. Recombinant eIF-4Ehuman was purified in a functional state by m7GTP affinity chromatography and Mono Q FPLC. This recombinant protein has biological and physical characteristics that are similar or identical to native eIF-4E. Fluorescence titration studies determined the equilibrium constant for recombinant eIF-4E/m7GTP binding to be 10.1 +/- 0.3 x 10(5) M(-1). To isolate eIF-4E binding proteins, recombinant eIF-4E was linked to agarose beads and incubated with cell lysates. Several proteins were isolated, including a 220-kDa protein that was confirmed to be the p220 subunit of eIF-4F by its proteolysis during incubation with lysates of poliovirus-infected cells. We conclude that recombinant eIF-4E produced in Escherichia coli provides a useful tool for studying eIF-4E/protein and eIF-4E/mRNA cap interactions and their role in regulating mammalian gene expression.

  4. The translational repressor eIF4E-binding protein 2 (4E-BP2) correlates with selective delayed neuronal death after ischemia

    PubMed Central

    Ayuso, María Irene; Martínez-Alonso, Emma; Cid, Cristina; de Leciñana, Maria Alonso; Alcázar, Alberto

    2013-01-01

    Transient brain ischemia induces an inhibition of translational rates and causes delayed neuronal death in selective regions and cognitive deficits, whereas these effects do not occur in resistant areas. The translational repressor eukaryotic initiation factor (eIF) 4E-binding protein-2 (4E-BP2) specifically binds to eIF4E and is critical in the control of protein synthesis. To link neuronal death to translation inhibition, we study the eIF4E association with 4E-BP2 under ischemia reperfusion in a rat model of transient forebrain ischemia. Upon reperfusion, a selective neuronal apoptosis in the hippocampal cornu ammonis 1 (CA1) region was induced, while it did not occur in the cerebral cortex. Confocal microscopy analysis showed a decrease in 4E-BP2/eIF4E colocalization in resistant cortical neurons after reperfusion. In contrast, in vulnerable CA1 neurons, 4E-BP2 remains associated to eIF4E with a higher degree of 4E-BP2/eIF4E colocalization and translation inhibition. Furthermore, the binding of a 4E-BP2 peptide to eIF4E induced neuronal apoptosis in the CA1 region. Finally, pharmacological-induced protection of CA1 neurons inhibited neuronal apoptosis, decreased 4E-BP2/eIF4E association, and recovered translation. These findings documented specific changes in 4E-BP2/eIF4E association during ischemic reperfusion, linking the translation inhibition to selective neuronal death, and identifying 4E-BP2 as a novel target for protection of vulnerable neurons in ischemic injury. PMID:23591646

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

    PubMed

    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

    2010-12-01

    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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Inhibition of mRNA turnover in yeast by an xrn1 mutation enhances the requirement for eIF4E binding to eIF4G and for proper capping of transcripts by Ceg1p.

    PubMed Central

    Brown, J T; Yang, X; Johnson, A W

    2000-01-01

    Null mutants of XRN1, encoding the major cytoplasmic exoribonuclease in yeast, are viable but accumulate decapped, deadenylated transcripts. A screen for mutations synthetic lethal with xrn1Delta identified a mutation in CDC33, encoding eIF4E. This mutation (glutamate to glycine at position 72) affected a highly conserved residue involved in interaction with eIF4G. Synthetic lethality between xrn1 and cdc33 was not relieved by high-copy expression of eIF4G or by disruption of the yeast eIF4E binding protein Caf20p. High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrn1 mutant. Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not. Mutations in CDC33, CEG1, PRT1, PAB1, and TIF4631, encoding eIF4G1, have been shown to lead to destabilization of mRNAs. Although such destabilization in cdc33, ceg1, and pab1 mutants can be partially suppressed by an xrn1 mutation, we observed synthetic lethality between xrn1 and either cdc33 or ceg1 and no suppression of the inviability of a pab1 null mutation by xrn1Delta. Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for (7)mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex. PMID:10790382

  11. Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection.

    PubMed

    Sukarieh, R; Sonenberg, N; Pelletier, J

    2010-05-01

    Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

  12. Mextli is a novel eukaryotic translation initiation factor 4E-binding protein that promotes translation in Drosophila melanogaster.

    PubMed

    Hernández, Greco; Miron, Mathieu; Han, Hong; Liu, Niankun; Magescas, Jérémy; Tettweiler, Gritta; Frank, Filipp; Siddiqui, Nadeem; Sonenberg, Nahum; Lasko, Paul

    2013-08-01

    Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5'-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.

  13. Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing

    PubMed Central

    Kamenska, Anastasiia; Lu, Wei-Ting; Kubacka, Dorota; Broomhead, Helen; Minshall, Nicola; Bushell, Martin; Standart, Nancy

    2014-01-01

    A key player in translation initiation is eIF4E, the mRNA 5′ cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4Lϕ site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4Lϕ, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs. PMID:24335285

  14. 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

    2013-12-01

    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.

  15. The translational repressor 4E-BP called to order by eIF4E: new structural insights by SAXS.

    PubMed

    Gosselin, Pauline; Oulhen, Nathalie; Jam, Murielle; Ronzca, Justyna; Cormier, Patrick; Czjzek, Mirjam; Cosson, Bertrand

    2011-04-01

    eIF4E binding protein (4E-BP) inhibits translation of capped mRNA by binding to the initiation factor eIF4E and is known to be mostly or completely unstructured in both free and bound states. Using small angle X-ray scattering (SAXS), we report here the analysis of 4E-BP structure in solution, which reveals that while 4E-BP is intrinsically disordered in the free state, it undergoes a dramatic compaction in the bound state. Our results demonstrate that 4E-BP and eIF4E form a 'fuzzy complex', challenging current visions of eIF4E/4E-BP complex regulation.

  16. 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

  17. Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-rasLA1 mice.

    PubMed

    Chang, S-H; Kim, J-E; Lee, J-H; Minai-Tehrani, A; Han, K; Chae, C; Cho, Y-H; Yun, J-H; Park, K; Kim, Y-S; Cho, M-H

    2013-06-01

    Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-rasLA1 lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-rasLA1 mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-rasLA1 mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment. PMID:23640516

  18. A new mechanism of regulation of p21 by the mTORC1/4E-BP1 pathway predicts clinical outcome of head and neck cancer.

    PubMed

    Llanos, Susana; García-Pedrero, Juana M

    2016-07-01

    We have identified a novel mechanism of regulation of the protein CDKN1A (also known as p21) by the serine/threonine kinase complex mammalian target of rapamycin complex 1 (mTORC1). Our results demonstrate that the mTORC1 substrate EIF4E-binding protein 1 (4E-BP1) in its non-phosphorylated state interacts with p21 and promotes p21 degradation. In addition, we demonstrate the prevalence of this mechanism in head and neck squamous cell carcinomas and show that it strongly and significantly associates with improved disease-specific survival, providing evidence for its clinical relevance. PMID:27652314

  19. Phosphorylation of eukaryotic translation initiation factor 4E and eukaryotic translation initiation factor 4E-binding protein (4EBP) and their upstream signaling components undergo diurnal oscillation in the mouse hippocampus: implications for memory persistence.

    PubMed

    Saraf, Amit; Luo, Jie; Morris, David R; Storm, Daniel R

    2014-07-18

    Translation of mRNA plays a critical role in consolidation of long-term memory. Here, we report that markers of initiation of mRNA translation are activated during training for contextual memory and that they undergo diurnal oscillation in the mouse hippocampus with maximal activity observed during the daytime (zeitgeber time 4-8 h). Phosphorylation and activation of eukaryotic translation initiation factor 4E (eIF4E), eIF4E-binding protein 1 (4EBP1), ribosomal protein S6, and eIF4F cap-complex formation, all of which are markers for translation initiation, were higher in the hippocampus during the daytime compared with night. The circadian oscillation in markers of mRNA translation was lost in memory-deficient transgenic mice lacking calmodulin-stimulated adenylyl cyclases. Moreover, disruption of the circadian rhythm blocked diurnal oscillations in eIF4E, 4EBP1, rpS6, Akt, and ERK1/2 phosphorylation and impaired memory consolidation. Furthermore, repeated inhibition of translation in the hippocampus 48 h after contextual training with the protein synthesis inhibitor anisomycin impaired memory persistence. We conclude that repeated activation of markers of translation initiation in hippocampus during the circadian cycle might be critical for memory persistence.

  20. Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5' cap.

    PubMed

    Modrak-Wojcik, Anna; Gorka, Michal; Niedzwiecka, Katarzyna; Zdanowski, Konrad; Zuberek, Joanna; Niedzwiecka, Anna; Stolarski, Ryszard

    2013-12-11

    Initiation is the rate-limiting step during mRNA 5' cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.

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

    PubMed

    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. PMID:27525480

  2. A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.

    PubMed

    Panda, Swarupa; Vedagiri, Dhiviya; Viveka, Thangaraj Soundara; Harshan, Krishnan Harinivas

    2014-09-01

    We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.

  3. 4E-BP1 participates in maintaining spindle integrity and genomic stability via interacting with PLK1

    PubMed Central

    Shang, Zeng-Fu; Yu, Lan; Li, Bing; Tu, Wen-Zhi; Wang, Yu; Liu, Xiao-Dan; Guan, Hua; Huang, Bo; Rang, Wei-Qing; Zhou, Ping-Kun

    2012-01-01

    The essential function of eIF4E-binding protein 1 (4E-BP1) in translation initiation has been well established; however, the role of 4E-BP1 in normal cell cycle progression is coming to attention. Here, we revealed the role of 4E-BP1 on mitotic regulation and chromosomal DNA dynamics during mitosis. First, we have observed the co-localization of the phosphorylated 4E-BP1 at T37/46 with Polo-like kinase 1 (PLK1) at the centrosomes during. Depression of 4E-BP1 by small interfering RNA in HepG2 or HeLa cells resulted in an increased outcome of polyploidy and aberrant mitosis, including chromosomal DNA misaligned and multi-polar spindles or multiple centrosomes. We observed that 4E-BP1 interacted with PLK1 directly in vitro and in vivo in mitotic cells, and the C-terminal aa 77–118 of 4E-BP1 mediates its interaction with PLK1. PLK1 can phosphorylate 4E-BP1 in vitro. Furthermore, the depletion of 4E-BP1 sensitized HepG2 and HeLa cells to the microtubule disruption agent paclitaxel. These results demonstrate that 4E-BP1, beyond its role in translation regulation, can function as a regulator of mitosis via interacting with PLK1, and possibly plays a role in genomic stability maintaining. PMID:22918237

  4. 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

  5. Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export-competent RNP.

    PubMed

    Topisirovic, Ivan; Siddiqui, Nadeem; Lapointe, Vincent Leroux; Trost, Matthias; Thibault, Pierre; Bangeranye, Catherine; Piñol-Roma, Serafin; Borden, Katherine L B

    2009-04-22

    The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap-dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E-sensitivity element (4E-SE), and its overexpression alters the nuclear export of several eIF4E-sensitive mRNAs. LRPPRC-mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E-binding site and it coincides with the subcellular re-distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E- and eIF4E-sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.

  6. Approaches for analyzing the differential activities and functions of eIF4E family members.

    PubMed

    Rhoads, Robert E; Dinkova, Tzvetanka D; Jagus, Rosemary

    2007-01-01

    The translational initiation factor eIF4E binds to the m(7)G-containing cap of mRNA and participates in recruitment of mRNA to ribosomes for protein synthesis. eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol. Multiple eIF4E family members have been identified in a wide range of organisms that includes plants, flies, mammals, frogs, birds, nematodes, fish, and various protists. This chapter reviews methods that have been applied to learn the biochemical properties and physiological functions that differentiate eIF4E family members within a given organism. Much has been learned to date about approaches to discover new eIF4E family members, their in vitro properties (cap binding, stimulation of cell-free translation systems), tissue and developmental expression patterns, protein-binding partners, and their effects on the translation or repression of specific subsets of mRNA. Despite these advances, new eIF4E family members continue to be found and new physiological roles discovered.

  7. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

    PubMed Central

    Jones, R M; Branda, J; Johnston, K A; Polymenis, M; Gadd, M; Rustgi, A; Callanan, L; Schmidt, E V

    1996-01-01

    The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation. PMID:8756633

  8. A unique binding mode of the eukaryotic translation initiation factor 4E for guiding the design of novel peptide inhibitors.

    PubMed

    Di Marino, Daniele; D'Annessa, Ilda; Tancredi, Holly; Bagni, Claudia; Gallicchio, Emilio

    2015-09-01

    The interaction between the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E binding proteins (4E-BP) is a promising template for the inhibition of eIF4E and the treatment of diseases such as cancer and a spectrum of autism disorders, including the Fragile X syndrome (FXS). Here, we report an atomically detailed model of the complex between eIF4E and a peptide fragment of a 4E-BP, the cytoplasmic Fragile X interacting protein (CYFIP1). This model was generated using computer simulations with enhanced sampling from an alchemical replica exchange approach and validated using long molecular dynamics simulations. 4E-BP proteins act as post-transcriptional regulators by binding to eIF4E and preventing mRNA translation. Dysregulation of eIF4E activity has been linked to cancer, FXS, and autism spectrum disorders. Therefore, the study of the mechanism of inhibition of eIF4E by 4E-BPs is key to the development of drug therapies targeting this regulatory pathways. The results obtained in this work indicate that CYFIP1 interacts with eIF4E by an unique mode not shared by other 4E-BP proteins and elucidate the mechanism by which CYFIP1 interacts with eIF4E despite having a sequence binding motif significantly different from most 4E-BPs. Our study suggests an alternative strategy for the design of eIF4E inhibitor peptides with superior potency and specificity than currently available.

  9. 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

  10. Effects of 4E-BP1 expression on hypoxic cell cycle inhibition and tumor cell proliferation and survival

    PubMed Central

    Barnhart, Bryan C.; Lam, Jennifer C.; Young, Regina M.; Houghton, Peter J.; Keith, Brian; Simon, M. Celeste

    2008-01-01

    Elevated activity of the eIF4F complex, which controls initiation of cap-dependent mRNA translation, has been linked to cancer progression. eIF4E recruitment to eIF4F is the rate limiting step of complex assembly and is regulated by eIF4E-Binding Proteins (4E-BPs). When stimulated, the mammalian Target of Rapamycin complex 1 (mTORC1) phosphorylates 4E-BP1, which then releases eIF4E. Hypoxia inhibits mTORC1 activity and therefore cap-dependent protein synthesis. To establish a novel genetic test of the role of eIF4F activity in regulating cell division and viability within hypoxic tumor microenvironments, we generated shRNA mediated 4E-BP1 knock-down in Rh30 rhabdomyosarcoma cells. 4E-BP1 knock-down relieved hypoxia-mediated inhibition of cycle progression in vitro and was correlated with increased expression of cyclin D1 and c-Myc. Xenograft tumors derived from these cells also displayed enhanced expression of cyclin D1 and c-Myc along with antiapoptotic genes encoding Bcl-xL, and XIAP, and failed to develop the extensive necrotic zones and edema observed in control tumors. Surprisingly, 4E-BP1 knock-down also leads to a dramatic increase in aberrant mitoses in vivo and enhanced expression of Mad2 and securin. Thus, reduced expression of the negative regulator of eIF4E has significant effects on tumor development, and is associated with enhanced cell proliferation and survival. PMID:18708753

  11. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

    PubMed

    Klein, Cornelia; Terrao, Monica; Inchaustegui Gil, Diana; Clayton, Christine

    2015-01-01

    We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

  12. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

    PubMed Central

    Klein, Cornelia; Terrao, Monica; Inchaustegui Gil, Diana; Clayton, Christine

    2015-01-01

    We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites. PMID:26287607

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

    PubMed

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

    2013-08-01

    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.

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

    PubMed Central

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

    2015-01-01

    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 here 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. 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. PMID:26170285

  15. 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

  16. 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.

  17. 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.

  18. Leptin stimulates protein synthesis-activating translation machinery in human trophoblastic cells.

    PubMed

    Pérez-Pérez, Antonio; Maymó, Julieta; Gambino, Yésica; Dueñas, José L; Goberna, Raimundo; Varone, Cecilia; Sánchez-Margalet, Víctor

    2009-11-01

    Leptin was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it may work as an autocrine hormone, mediating angiogenesis, growth, and immunomodulation. Leptin receptor (LEPR, also known as Ob-R) shows sequence homology to members of the class I cytokine receptor (gp130) superfamily. In fact, leptin may function as a proinflammatory cytokine. We have previously found that leptin is a trophic and mitogenic factor for trophoblastic cells. In order to further investigate the mechanism by which leptin stimulates cell growth in JEG-3 cells and trophoblastic cells, we studied the phosphorylation state of different proteins of the initiation stage of translation and the total protein synthesis by [(3)H]leucine incorporation in JEG-3 cells. We have found that leptin dose-dependently stimulates the phosphorylation and activation of the translation initiation factor EIF4E as well as the phosphorylation of the EIF4E binding protein EIF4EBP1 (PHAS-I), which releases EIF4E to form active complexes. Moreover, leptin dose-dependently stimulates protein synthesis, and this effect can be partially prevented by blocking mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PIK3) pathways. In conclusion, leptin stimulates protein synthesis, at least in part activating the translation machinery, via the activation of MAPK and PIK3 pathways.

  19. Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

    PubMed Central

    Rousseau, D; Kaspar, R; Rosenwald, I; Gehrke, L; Sonenberg, N

    1996-01-01

    The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8577715

  20. Phylogenetic analysis of eIF4E-family members

    PubMed Central

    Joshi, Bhavesh; Lee, Kibwe; Maeder, Dennis L; Jagus, Rosemary

    2005-01-01

    Background Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m7Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the 7-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other. Results By combining sequence data deposited in the Genbank databases, we have identified sequences encoding 411 eIF4E-family members from 230 species. These sequences have been deposited into an internet-accessible database designed for sequence comparisons of eIF4E-family members. Most members can be grouped into one of three classes. Class I members carry Trp residues equivalent to Trp-43 and Trp-56 of H. sapiens eIF4E and appear to be present in all eukaryotes. Class II members, possess Trp→Tyr/Phe/Leu and Trp→Tyr/Phe substitutions relative to Trp-43 and Trp-56 of H. sapiens eIF4E, and can be identified in Metazoa, Viridiplantae, and Fungi. Class III members possess a Trp residue equivalent to Trp-43 of H. sapiens eIF4E but carry a Trp→Cys/Tyr substitution relative to Trp-56 of H. sapiens eIF4E, and can be identified in Coelomata and Cnidaria. Some eIF4E-family members from Protista show extension or compaction relative to prototypical eIF4E-family members. Conclusion The expansion of sequenced cDNAs and genomic DNAs from all eukaryotic kingdoms has revealed a variety of proteins related in structure to eIF4E. Evolutionarily it seems that a single early eIF4E gene has undergone multiple gene duplications generating multiple

  1. Viscera and Muscle Protein Synthesis in Neonatal Pigs Is Increased More by Intermittent Bolus than Continuous Feeding

    PubMed Central

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

    2014-01-01

    BACKGROUND Continuous and intermittent bolus orogastric feedings are strategies used in infants unable to tolerate normal feeds. METHODS To determine the effects of feeding modality on protein synthesis in different tissues, neonatal pigs received a balanced formula by orogastric tube either as an intermittent bolus feed every 4 h or as a continuous infusion, or were fasted overnight. RESULTS Compared to fasting, protein synthesis in gastrocnemius, masseter, and soleus muscles, left ventricle, liver, pancreas, jejunum, and kidney increased in bolus and continuously fed pigs, but the greatest increase occurred after a bolus meal. Tuberous sclerosis complex (TSC2), the proline-rich AKT substrate of 40 kDa (PRAS40), eukaryotic initiation factor (eIF) 4E binding protein (4EBP1) and rp S6 kinase 1 (S6K1) phosphorylation in all tissues and the proportion of ribosomal protein S4 in liver polysomes were enhanced 90 minutes following the bolus meal, but not immediately before the meal or during continuous feeding. Eukaryotic elongation factor 2 (eEF2) and eIF2α phosphorylation were unaffected by feeding. CONCLUSION These results suggest that intermittent bolus feeding increases protein synthesis in muscles of different fiber types and visceral tissues to a greater extent than continuous feeding by stimulating translation initiation. PMID:23736770

  2. Amino Acid Availability and Age Affect the Leucine Stimulation of Protein Synthesis and eIF4F Formation in Muscle

    PubMed Central

    Escobar, Jeffery; Frank, Jason W.; Suryawan, Agus; Nguyen, Hanh V.; Davis, Teresa A.

    2009-01-01

    We have previously shown that a physiological increase in plasma leucine for 60- and 120-min increases translation initiation factor activation in muscle of neonatal pigs. Although muscle protein synthesis is increased by leucine at 60 min, it is not maintained at 120 min, perhaps due to the decrease in plasma amino acids (AA). In the current study, 7- and 26-day-old pigs were fasted overnight and infused with leucine (0 or 400 µmol· kg−1· h−1) for 120 min to raise leucine within the postprandial range. The leucine was infused in the presence or absence of a replacement AA mixture (without leucine) to maintain baseline plasma AA levels. AA administration prevented the leucine-induced reduction in plasma AA in both age groups. At 7 days, leucine infusion alone increased eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) phosphorylation, decreased inactive 4E-BP1·eIF4E complex abundance, and increased active eIF4G·eIF4E complex formation in skeletal muscle; leucine infusion with replacement AA also stimulated these, as well as S6K1, rpS6, and eIF4G phosphorylation. At 26 days, leucine infusion alone increased 4E-BP1 phosphorylation and decreased the inactive 4E-BP1·eIF4E complex only; leucine with AA also stimulated these, as well as S6K1 and rpS6 phosphorylation. Muscle protein synthesis was increased in 7-day-old (+60%) and 26-day-old (+40%) pigs infused with leucine and replacement AA, but not with leucine alone. Thus, the ability of leucine to stimulate eIF4F formation and protein synthesis in skeletal muscle is dependent on AA availability and age. PMID:17878223

  3. 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.

  4. Lambda-cyhalothrin disrupts the up-regulation effect of 17β-estradiol on post-synaptic density 95 protein expression via estrogen receptor α-dependent Akt pathway.

    PubMed

    Wang, Qunan; Xia, Xin; Deng, Xiaomei; Li, Nian; Wu, Daji; Zhang, Long; Yang, Chengwei; Tao, Fangbiao; Zhou, Jiangning

    2016-03-01

    Lambda-cyhalothrin (LCT), one of the type II pyrethroids, has been widely used throughout the world. The estrogenic effect of LCT to increase cell proliferation has been well established. However, whether the estrogenic effect of LCT will influence neurodevelopment has not been investigated. In addition, 17β-Estradiol (E2) plays a crucial role in neurodevelopment and induces an increase in synaptic proteins. The post-synaptic density 95 (PSD95) protein, which is involved in the development of the structure and function of new spines and localized with estrogen receptor α (ERα) at the post-synaptic density (PSD), was detected in our study by using hippocampal neuron cell line HT22. We found that LCT up-regulated PSD95 and ERα expression, estrogen receptor (ER) antagonist ICI182,780 and phosphatidylinositol-4; 5-bisphosphate 3-kinase (PI3K) inhibitor LY294,002 blocked this effect. In addition, LCT disrupted the promotion effect of E2 on PSD95. To investigate whether the observed changes are caused by ERα-dependent signaling activation, we next detected the effects of LCT on the ERα-mediated PI3K-Protein kinase B (PKB/Akt)-eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) pathway. There existed an activation of Akt and the downstream factor 4E-BP1 after LCT treatment. In addition, LCT could disrupt the activation effect of E2 on the Akt pathway. However, no changes in cAMP response element-binding protein (CREB) activation and PSD95 messenger ribonucleic acid (mRNA) were observed. Our findings demonstrated that LCT could increase the PSD95 protein level via the ERα-dependent Akt pathway, and LCT might disrupt the up-regulation effect of E2 on PSD95 protein expression via this signaling pathway. PMID:26969072

  5. In vitro synthesis of human protein synthesis initiation factor 4 gamma and its localization on 43 and 48 S initiation complexes.

    PubMed

    Joshi, B; Yan, R; Rhoads, R E

    1994-01-21

    The rate of protein synthesis is controlled in a large number of physiological situations at the stage of 48 S initiation complex formation, a phase that involves the recruitment of mRNA to the 40 S ribosomal subunit. This process is mediated by the eukaryotic initiation factor-4 (eIF-4) group of translation initiation factors consisting of eIF-4E, eIF-4A, eIF-4B, and eIF-4 gamma. In order to develop a new tool to study this process, we have produced radiolabeled eIF-4 gamma by in vitro transcription and translation. Despite the fact that eIF-4 gamma is predicted from the cDNA sequence to be 154 kDa, the major synthetic product migrated on SDS-polyacrylamide gel electrophoresis at 205 kDa. Although this is similar to the migration of the fastest polypeptide of authentic eIF-4 gamma (approximately 206 kDa), no products were found to co-migrate with the slowest forms of authentic eIF-4 gamma (210-220 kDa), suggesting that these forms derive from extensive modification of the initial polypeptide. The in vitro product also formed a complex with eIF-4E, as judged by its ability to bind to m7GTP-Sepharose. Sucrose gradient sedimentation studies demonstrated that eIF-4 gamma was present on both 43 and 48 S initiation complexes but not 80 S complexes. This supports a model in which free eIF-4E binds to mRNA followed by binding of the eIF-4E.mRNA complex to a 43 S initiation complex already containing eIF-4 gamma.

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

    PubMed

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

    2016-03-11

    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.

  7. Leucine or carbohydrate supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle protein synthesis in rats.

    PubMed

    Wilson, Gabriel J; Layman, Donald K; Moulton, Christopher J; Norton, Layne E; Anthony, Tracy G; Proud, Christopher G; Rupassara, S Indu; Garlick, Peter J

    2011-12-01

    Muscle protein synthesis (MPS) increases after consumption of a protein-containing meal but returns to baseline values within 3 h despite continued elevations of plasma amino acids and mammalian target of rapamycin (mTORC1) signaling. This study evaluated the potential for supplemental leucine (Leu), carbohydrates (CHO), or both to prolong elevated MPS after a meal. Male Sprague-Dawley rats (∼270 g) trained to consume three meals daily were food deprived for 12 h, and then blood and gastrocnemius muscle were collected 0, 90, or 180 min after a standard 4-g test meal (20% whey protein). At 135 min postmeal, rats were orally administered 2.63 g of CHO, 270 mg of Leu, both, or water (sham control). Following test meal consumption, MPS peaked at 90 min and then returned to basal (time 0) rates at 180 min, although ribosomal protein S6 kinase and eIF4E-binding protein-1 phosphorylation remained elevated. In contrast, rats administered Leu and/or CHO supplements at 135 min postmeal maintained peak MPS through 180 min. MPS was inversely associated with the phosphorylation states of translation elongation factor 2, the "cellular energy sensor" adenosine monophosphate-activated protein kinase-α (AMPKα) and its substrate acetyl-CoA carboxylase, and increases in the ratio of AMP/ATP. We conclude that the incongruity between MPS and mTORC1 at 180 min reflects a block in translation elongation due to reduced cellular energy. Administering Leu or CHO supplements ∼2 h after a meal maintains cellular energy status and extends the postprandial duration of MPS.

  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. 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

  10. The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs

    PubMed Central

    Signer, Robert A.J.; Qi, Le; Zhao, Zhiyu; Thompson, David; Sigova, Alla A.; Fan, Zi Peng; DeMartino, George N.; Young, Richard A.; Sonenberg, Nahum; Morrison, Sean J.

    2016-01-01

    Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis. PMID:27492367

  11. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  12. Hyperactivation of 4E-binding protein 1 as a mediator of biguanide-induced cytotoxicity during glucose deprivation.

    PubMed

    Matsuo, Junichi; Tsukumo, Yoshinori; Saito, Sakae; Tsukahara, Satomi; Sakurai, Junko; Sato, Shigeo; Kondo, Hiromichi; Ushijima, Masaru; Matsuura, Masaaki; Watanabe, Toshiki; Tomida, Akihiro

    2012-05-01

    Biguanides, including metformin, buformin, and phenformin, are potential antitumorigenic agents and induce cell death during glucose deprivation, a cell condition that occurs in the tumor microenvironment. Here, we show that this selective killing of glucose-deprived cells is coupled with hyperactivation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation initiation. We found, in fact, that the 4E-BP1 hyperactivation led to failure of the unfolded protein response (UPR), an endoplasmic reticulum-originated stress signaling pathway for cell survival. We also found that the 4E-BP1-mediated UPR inhibition occurred through a strong inhibition of the mTOR signaling pathway, a proven antitumor target. Importantly, the 4E-BP1 hyperactivation can be also seen in xenografted cancer cells through an in vivo biguanide treatment. Our findings indicate that antitumor action of biguanides can be mediated by 4E-BP1 hyperactivation, which results in UPR inhibition and selective cell killing when glucose is withdrawn.

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

    PubMed Central

    Gardner, Thomas W.; Abcouwer, Steven F.; Losiewicz, Mandy K.

    2015-01-01

    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. PMID:26199279

  14. 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.

  15. Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure.

    PubMed

    Carberry, S E; Friedland, D E; Rhoads, R E; Goss, D J

    1992-02-11

    The binding of rabbit globin mRNA to the 25-kDa cap binding protein eIF-4E from human erythrocytes was found to be 5.3-fold stronger than the binding of the cap analogue m7GpppG to eIF-4E [Gross et al. (1990) Biochemistry 29, 5008-5012]. In order to investigate whether this effect is due to the longer sequence of nucleotides in globin mRNA or to other features such as cap accessibility or secondary structure, oligoribonucleotide analogues of rabbit alpha-globin mRNA were synthesized by T7 RNA polymerase from a synthetic oligodeoxynucleotide template in the presence of m7GpppG; these oligoribonucleotide analogues possess varying degrees of cap accessibility and secondary structure. Equilibrium association constants for the interaction of these oligoribonucleotides and purified human erythrocyte eIF-4E were obtained from direct fluorescence titration experiments. The data indicate that while the presence of the m7G cap is required for efficient recognition by eIF-4E, the cap need not be completely sterically accessible, since other structural features within the mRNA also influence binding.

  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. 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...

  18. Structure-activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein-protein interaction inhibitors

    PubMed Central

    Takrouri, Khuloud; Chen, Ting; Papadopoulos, Evangelos; Sahoo, Rupam; Kabha, Eihab; Chen, Han; Cantel, Sonia; Wagner, Gerhard; Halperin, Jose A; Aktas, Bertal H; Chorev, Michael

    2014-01-01

    Protein-protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein-protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities. PMID:24675136

  19. 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.

  20. Turnip Mosaic Virus Genome-Linked Protein VPg Binds C-Terminal Region of Cap-Bound Initiation Factor 4E Orthologue Without Exhibiting Host Cellular Specificity

    PubMed Central

    Okade, Hayato; Fujita, Yuki; Miyamoto, Saori; Tomoo, Koji; Muto, Shinji; Miyoshi, Hiroshi; Natsuaki, Tomohide; Rhoads, Robert E.; Ishida, Toshimasa

    2014-01-01

    To investigate the binding specificity of turnip mosaic virus (TuMV) viral protein-genome linked (VPg) with translation initiation factor 4E, we evaluated here the kinetic parameters for the interactions of human eIF4E, Caenorhabditis elegans IFE-3 and IFE-5 and Arabidopsis eIFiso4E, by surface plasmon resonance (SPR). The results indicated that TuMV VPg does not show a binding preference for Arabidopsis eIFiso4E, even though it is from a host species whereas the other eIF4E orthologues are not. Surprisingly, the effect of m7GTP on both the rate constants and equilibrium binding constants for the interactions of VPg differed for the four eIF4E orthologues. In the case of eIFiso4E and IFE-3, m7GTP increased kon, but for eIF4E and IFE-5, it decreased kon. To provide insight into the structural basis for these differences in VPg binding, tertiary structures of the eIF4E orthologues were predicted on the basis of the previously determined crystal structure of m7GpppA-bound human eIF4E. The results suggested that in cap-bound eIF4E orthologues, the VPg binds to the C-terminal region, which constitutes one side of the entrance to the cap-binding pocket, whereas in the cap-free state, VPg binds to the widely opened cap-binding pocket and its surrounding region. The binding of VPg to the C-terminal region was confirmed by the SPR analyses of N- or C-terminal residues-deleted eIF4E orthologues. PMID:19122207

  1. Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells.

    PubMed Central

    Senthil, D; Faulkner, J L; Choudhury, G G; Abboud, H E; Kasinath, B S

    2001-01-01

    Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1 nM) or insulin (1 nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/-2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor beta-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/-2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II. PMID:11695995

  2. 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

  3. 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.

  4. Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol.

    PubMed

    Hackl, Ellen V

    2015-02-01

    Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein--hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour.

  5. THE CANNABINOID WIN 55,212-2 DECREASES SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS AND THE ONCOGENIC CAP PROTEIN eIF4E IN COLON CANCER CELLS

    PubMed Central

    Sreevalsan, Sandeep; Safe, Stephen

    2013-01-01

    2,3-Dihydro-5-methyl-3-([morpholinyl]methyl)pyrollo(1,2,3-de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone [WIN 55,212-2 (WIN)] is a synthetic cannabinoid that inhibits RKO, HT-29 and SW480 cell growth, induced apoptosis, and downregulated expression of survivin, cyclin D1, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and its receptor (VEGFR1). WIN also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and this is consistent with the observed downregulation of the aforementioned Sp-regulated genes. In addition, we also observed by RNA interference (RNAi) that the oncogenic cap protein eIF4E was an Sp-regulated gene also downregulated by WIN in colon cancer cells. WIN-mediated repression of Sp proteins was not affected by CB receptor antagonists or by knockdown of the receptor but was attenuated by the phosphatase inhibitor sodium orthovanadate or by knockdown of protein phosphatase 2A (PP2A). WIN-mediated repression of Sp1, Sp3 and Sp4 was due to PP2A-dependent downregulation of microRNA-27a (miR-27a) and induction of miR-27a-regulated ZBTB10 which has previously been characterized as an “Sp repressor”. The results demonstrate that the anticancer activity of WIN is due, in part, to PP2A-dependent disruption of miR-27a:ZBTB10 and ZBTB10-mediated repression of Sp transcription factors and Sp-regulated genes including eIF4E. PMID:24030632

  6. IGF-I/IGFBP-3 ameliorates alterations in protein synthesis, eIF4E availability, and myostatin in alcohol-fed rats.

    PubMed

    Lang, Charles H; Frost, Robert A; Svanberg, Elisabeth; Vary, Thomas C

    2004-06-01

    Chronic alcohol consumption decreases the concentration of the anabolic hormone IGF-I, and this change is associated with impaired muscle protein synthesis. The present study evaluated the ability of IGF-I complexed with IGF-binding protein (IGFBP)-3 to modulate the alcohol-induced inhibition of muscle protein synthesis in gastrocnemius. After 16 wk on an alcohol-containing diet, either the IGF-I/IGFBP-3 binary complex (BC) or saline was injected two times daily for three consecutive days. After the final injection of BC (3 h), plasma IGF-I concentrations were elevated in alcohol-fed rats to values not different from those of similarly treated control animals. Alcohol feeding decreased the basal rate of muscle protein synthesis by limiting translational efficiency. BC treatment of alcohol-fed rats increased protein synthesis back to basal control values, but the rate remained lower than that of BC-injected control rats. The BC partially reversed the alcohol-induced decrease in the binding of eukaryotic initiation factor (eIF)4E with eIF4G. This change was associated with reversal of the alcohol-induced dephosphorylation of eIF4G but was independent of changes in the phosphorylation of either 4E-BP1 or eIF4E. However, BC reversed the alcohol-induced increase in IGFBP-1 and muscle myostatin, known negative regulators of IGF-I action and muscle mass. Hence, exogenous IGF-I, administered as part of a BC to increase its circulating half-life, can in part reverse the decreased protein synthesis observed in muscle from chronic alcohol-fed rats by stimulating selected components of translation initiation. The data support the role of IGF-I as a mediator of chronic alcohol myopathy in rats.

  7. The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

    PubMed Central

    Halter, David; Collart, Martine A.; Panasenko, Olesya O.

    2014-01-01

    Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

  8. 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

  9. Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance

    PubMed Central

    Duluc, Camille; Moatassim-Billah, Siham; Chalabi-Dchar, Mounira; Perraud, Aurélie; Samain, Rémi; Breibach, Florence; Gayral, Marion; Cordelier, Pierre; Delisle, Marie-Bernadette; Bousquet-Dubouch, Marie-Pierre; Tomasini, Richard; Schmid, Herbert; Mathonnet, Muriel; Pyronnet, Stéphane; Martineau, Yvan; Bousquet, Corinne

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of α-SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1-directed pharmacological compounds represents an anti-stromal-targeted therapy with promising chemosensitization potential. PMID:25834145

  10. Up-regulation of Translation Eukaryotic Initiation Factor 4E in Nucleophosmin 1 Haploinsufficient Cells Results in Changes in CCAAT Enhancer-binding Protein α Activity

    PubMed Central

    Khanna-Gupta, Arati; Abayasekara, Nirmalee; Levine, Michelle; Sun, Hong; Virgilio, Maria; Nia, Navid; Halene, Stephanie; Sportoletti, Paolo; Jeong, Jee-Yeong; Pandolfi, Pier Paolo; Berliner, Nancy

    2012-01-01

    NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1+/− mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1+/− cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1+/− cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1+/− cells contributes to the MDS phenotype associated with NPM1 deficiency. PMID:22851180

  11. Enterovirus-induced miR-141 contributes to shutoff of host protein translation by targeting the translation initiation factor eIF4E.

    PubMed

    Ho, Bing-Ching; Yu, Sung-Liang; Chen, Jeremy J W; Chang, Sui-Yuan; Yan, Bo-Shiun; Hong, Qi-Sheng; Singh, Sher; Kao, Chuan-Liang; Chen, Hsuan-Yu; Su, Kang-Yi; Li, Ker-Chau; Cheng, Chiou-Ling; Cheng, Hao-Wei; Lee, Jen-Yi; Lee, Chun-Nan; Yang, Pan-Chyr

    2011-01-20

    Viruses rely on the host translation machinery to complete their life cycles. Picornaviruses use an internal ribosome entry site to initiate cap-independent protein translation and in parallel host cap-dependent translation is shut off. This process is thought to occur primarily via cleavage of host translation initiation factors eIF4GI and eIF4GII by viral proteases. Here we describe another mechanism whereby miR-141 induced upon enterovirus infection targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis. Knockdown of miR-141 reduces viral propagation, and silencing of eIF4E can completely reverse the inhibitory effect of the miR-141 antagomiR on viral propagation. Ectopic expression of miR-141 promotes the switch from cap-dependent to cap-independent translation. Moreover, we identified a transcription factor, EGR1, which is partly responsible for miR-141 induction in response to enterovirus infection. Our results suggest that upregulation of miR-141 upon enterovirus infection can facilitate viral propagation by expediting the translational switch.

  12. 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

  13. Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5 prime -triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

    SciTech Connect

    Chavan, A.J.; Rychlik, W.; Watt, D.S.; Rhoads, R.E. ); Blaas, D.; Kuechler, E. )

    1990-06-12

    Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, ({gamma}-{sup 32}P)-{gamma}-(((4-benzoylphenyl)methyl)amido)-7-methyl-GTP. A second probe was synthesized that was an azidophenyltyrosine derivative of m{sup 7}GTP (({sup 125}I)APTM), the monoanhydride of m{sup 7}GDP with ({sup 125}I)-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodophenyl)propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 {mu}M was determined for ({sup 125}I)APTM, which is comparable to the published values for m{sup 7}GTP. A third probe, an azidophenylglycine derivative of m{sup 7}GTP (({sup 32}P)APGM), the monoanhydride of m{sup 7}GDP with ({sup 32}P)-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike ({sup 32}P)BPM and ({sup 125}I)APTM, however, ({sup 32}P)APGM labeled eIF-4E{sup *} approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E{sup *} consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

  14. Regulation of mTORC1 by growth factors, energy status, amino acids and mechanical stimuli at a glance.

    PubMed

    Bond, Peter

    2016-01-01

    The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) plays a pivotal role in the regulation of skeletal muscle protein synthesis. Activation of the complex leads to phosphorylation of two important sets of substrates, namely eIF4E binding proteins and ribosomal S6 kinases. Phosphorylation of these substrates then leads to an increase in protein synthesis, mainly by enhancing translation initiation. mTORC1 activity is regulated by several inputs, such as growth factors, energy status, amino acids and mechanical stimuli. Research in this field is rapidly evolving and unraveling how these inputs regulate the complex. Therefore this review attempts to provide a brief and up-to-date narrative on the regulation of this marvelous protein complex. Additionally, some sports supplements which have been shown to regulate mTORC1 activity are discussed. PMID:26937223

  15. Stat1 stimulates cap-independent mRNA translation to inhibit cell proliferation and promote survival in response to antitumor drugs

    PubMed Central

    Wang, Shuo; Patsis, Christos; Koromilas, Antonis E.

    2015-01-01

    The signal transducer and activator of transcription 1 (Stat1) functions as a tumor suppressor via immune regulatory and cell-autonomous pathways. Herein, we report a previously unidentified cell-autonomous Stat1 function, which is its ability to exhibit both antiproliferative and prosurvival properties by facilitating translation of mRNAs encoding for the cyclin-dependent kinase inhibitor p27Kip1 and antiapoptotic proteins X-linked inhibitor of apoptosis and B-cell lymphoma xl. Translation of the select mRNAs requires the transcriptional function of Stat1, resulting in the up-regulation of the p110γ subunit of phosphoinositide 3-kinase (PI3K) class IB and increased expression of the translational repressor translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). Increased PI3Kγ signaling promotes the degradation of the eIF4A inhibitor programmed cell death protein 4, which favors the cap-independent translation of the select mRNAs under conditions of general inhibition of protein synthesis by up-regulated eIF4E-binding protein 1. As such, Stat1 inhibits cell proliferation but also renders cells increasingly resistant to antiproliferative effects of pharmacological inhibitors of PI3K and/or mammalian target of rapamycin. Stat1 also protects Ras-transformed cells from the genotoxic effects of doxorubicin in culture and immune-deficient mice. Our findings demonstrate an important role of mRNA translation in the cell-autonomous Stat1 functions, with implications in tumor growth and treatment with chemotherapeutic drugs. PMID:25870277

  16. 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.

  17. Knock-Down of Both eIF4E1 and eIF4E2 Genes Confers Broad-Spectrum Resistance against Potyviruses in Tomato

    PubMed Central

    Mazier, Marianne; Flamain, Fabrice; Nicolaï, Maryse; Sarnette, Verane; Caranta, Carole

    2011-01-01

    Background The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance. Methodology/Principal Findings To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2. Conclusion/Significance These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato

  18. The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

    PubMed Central

    Ohlmann, T; Rau, M; Pain, V M; Morley, S J

    1996-01-01

    The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule. Images PMID:8635470

  19. An eIF4E1/4E-T complex determines the genesis of neurons from precursors by translationally repressing a proneurogenic transcription program.

    PubMed

    Yang, Guang; Smibert, Craig A; Kaplan, David R; Miller, Freda D

    2014-11-19

    Here, we have addressed the mechanisms that determine genesis of the correct numbers of neurons during development, focusing on the embryonic cortex. We identify in neural precursors a repressive complex involving eIF4E1 and its binding partner 4E-T that coordinately represses translation of proteins that determine neurogenesis. This eIF4E1/4E-T complex is present in granules with the processing body proteins Lsm1 and Rck, and disruption of this complex causes premature and enhanced neurogenesis and neural precursor depletion. Analysis of the 4E-T complex shows that it is highly enriched in mRNAs encoding transcription factors and differentiation-related proteins. These include the proneurogenic bHLH mRNAs, which colocalize with 4E-T in granules and whose protein products are aberrantly upregulated following knockdown of eIF4E, 4E-T, or processing body proteins. Thus, neural precursors are transcriptionally primed to generate neurons, but an eIF4E/4E-T complex sequesters and represses translation of proneurogenic proteins to determine appropriate neurogenesis.

  20. Pore-forming activity of Coxiella burnetii outer membrane protein oligomer comprised of 29.5- and 31-kDa polypeptides. Inhibition of porin activity by monoclonal antibodies 4E8 and 4D6.

    PubMed

    Banerjee-Bhatnagar, N; Bolt, C R; Williams, J C

    1996-07-23

    Envelopes of large-cell variant Coxiella burnetii, the agent of Q fever, were the starting material for purification of an outer membrane protein (OMP) oligomer with aggregate molecular mass of approximately 2 x 10(4) kDa. The oligomer was resistant to trypsin and dissociation by SDS at 100 degrees C. Reducing agents dissociated the oligomer into monomers of 29.5 and 31 kDa, which migrated as a doublet during SDS-polyacrylamide gel electrophoresis. Both monomers were reactive in an immunoblot assay with monoclonal antibodies (mAbs) 4E8 and 4D6, which were previously selected for their reactivity with purified and SDS-denatured 29.5 kDa protein. Proteoliposomes were functional in an equilibrium assay at pH 7 and a swelling assay at pH 7 and 4.5. The pores in proteoliposomes allowed the passage of arabinose, glucose, and sucrose, but restricted stachyose. Polyclonal antibodies to C. burnetii cells and the mAbs were able to bind C. burnetii at pH 7 and 4.5. The uptake of 14C-glucose at pH 4.5 was inhibited by polyclonal antibodies and mAbs after binding to cells at pH 7. The mAbs did not inhibit 14C-glucose uptake at pH 4.5 after binding to cells at pH 4.5. Although the mAbs bind C. burnetii porin epitopes before and after acid activation, the mAbs bound under acidic conditions were unable to inhibit porin function. The inhibition of porin channel function by mAbs confirms the role of porin as a permeability barrier for the subsequent active transport of glucose by C. burnetii. In another study, we showed that the 29.5 kDa OMP antigen induced active immunity against virulent challenge. This information, combined with the recent confirmation that porins are important antigens in the induction of specific protective immune responses against infection by gram-negative bacteria, suggests that humoral immunity directed against C. burnetii porins might play an important role in immunity against Q fever (human infection) and coxiellosis (animal infection), global enzootic

  1. Human Immunodeficiency Virus Type 1 Vpr-Binding Protein VprBP, a WD40 Protein Associated with the DDB1-CUL4 E3 Ubiquitin Ligase, Is Essential for DNA Replication and Embryonic Development▿

    PubMed Central

    McCall, Chad M.; Miliani de Marval, Paula L.; Chastain, Paul D.; Jackson, Sarah C.; He, Yizhou J.; Kotake, Yojiro; Cook, Jeanette Gowen; Xiong, Yue

    2008-01-01

    Damaged DNA binding protein 1, DDB1, bridges an estimated 90 or more WD40 repeats (DDB1-binding WD40, or DWD proteins) to the CUL4-ROC1 catalytic core to constitute a potentially large number of E3 ligase complexes. Among these DWD proteins is the human immunodeficiency virus type 1 (HIV-1) Vpr-binding protein VprBP, whose cellular function has yet to be characterized but has recently been found to mediate Vpr-induced G2 cell cycle arrest. We demonstrate here that VprBP binds stoichiometrically with DDB1 through its WD40 domain and through DDB1 to CUL4A, subunits of the COP9/signalsome, and DDA1. The steady-state level of VprBP remains constant during interphase and decreases during mitosis. VprBP binds to chromatin in a DDB1-independent and cell cycle-dependent manner, increasing from early S through G2 before decreasing to undetectable levels in mitotic and G1 cells. Silencing VprBP reduced the rate of DNA replication, blocked cells from progressing through the S phase, and inhibited proliferation. VprBP ablation in mice results in early embryonic lethality. Conditional deletion of the VprBP gene in mouse embryonic fibroblasts results in severely defective progression through S phase and subsequent apoptosis. Our studies identify a previously unknown function of VprBP in S-phase progression and suggest the possibility that HIV-1 Vpr may divert an ongoing chromosomal replication activity to facilitate viral replication. PMID:18606781

  2. Two Arabidopsis loci encode novel eukaryotic initiation factor 4E isoforms that are functionally distinct from the conserved plant eukaryotic initiation factor 4E.

    PubMed

    Patrick, Ryan M; Mayberry, Laura K; Choy, Grace; Woodard, Lauren E; Liu, Joceline S; White, Allyson; Mullen, Rebecca A; Tanavin, Toug M; Latz, Christopher A; Browning, Karen S

    2014-04-01

    Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation.

  3. Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E1[W][OPEN

    PubMed Central

    Patrick, Ryan M.; Mayberry, Laura K.; Choy, Grace; Woodard, Lauren E.; Liu, Joceline S.; White, Allyson; Mullen, Rebecca A.; Tanavin, Toug M.; Latz, Christopher A.; Browning, Karen S.

    2014-01-01

    Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation. PMID:24501003

  4. Double mutations in eIF4E and eIFiso4E confer recessive resistance to Chilli veinal mottle virus in pepper.

    PubMed

    Hwang, Jeena; Li, Jinjie; Liu, Wing-Yee; An, Song-Ji; Cho, Hwajin; Her, Nam Han; Yeam, Inhwa; Kim, Dosun; Kang, Byoung-Cheorl

    2009-03-31

    To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinai mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum 'Dempsey' containing an eIF4E mutation (pvr1(2)) and C. annuum 'Perennial' containing an eIFiso4E mutation (pvr6). C. annuum 'Dempsey' was susceptible and C. annuum 'Perennial' was resistant to ChiVMV. All F(1) plants showed resistance, and F(2) individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five F(2) and 329 F(3) plants of 17 families were genotyped with pvr1(2) and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both pvr1(2) and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in eIF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of F2 plants revealed that all plants containing homozygous genotypes of both pvr1(2) and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of eIF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper.

  5. Synthesis of Rigidified eIF4E/eIF4G Inhibitor-1 (4EGI-1) Mimetic and Their in Vitro Characterization as Inhibitors of Protein–Protein Interaction

    PubMed Central

    2015-01-01

    The 4EGI-1 is the prototypic inhibitor of eIF4E/eIF4G interaction, a potent inhibitor of translation initiation in vitro and in vivo and an efficacious anticancer agent in animal models of human cancers. We report on the design, synthesis, and in vitro characterization of a series of rigidified mimetic of this prototypic inhibitor in which the phenyl in the 2-(4-(3,4-dichlorophenyl)thiazol-2-yl) moiety was bridged into a tricyclic system. The bridge consisted one of the following: ethylene, methylene oxide, methylenesulfide, methylenesulfoxide, and methylenesulfone. Numerous analogues in this series were found to be markedly more potent than the parent prototypic inhibitor in the inhibition of eIF4E/eIF4G interaction, thus preventing the eIF4F complex formation, a rate limiting step in the translation initiation cascade in eukaryotes, and in inhibition of human cancer cell proliferation. PMID:24827861

  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. 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

  8. Nutritional and regulatory roles of leucine in muscle growth and fat reduction.

    PubMed

    Duan, Yehui; Li, Fengna; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Zhang, Yuzhe; Deng, Dun; Tang, Yulong; Feng, Zemeng; Wu, Guoyao; Yin, Yulong

    2015-01-01

    The metabolic roles for L-leucine, an essential branched-chain amino acid (BCAA), go far beyond serving exclusively as a building block for de novo protein synthesis. Growing evidence shows that leucine regulates protein and lipid metabolism in animals. Specifically, leucine activates the mammalian target of rapamycin (mTOR) signaling pathway, including the 70 kDa ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1) to stimulate protein synthesis in skeletal muscle and adipose tissue and to promote mitochondrial biogenesis, resulting in enhanced cellular respiration and energy partitioning. Activation of cellular energy metabolism favors fatty acid oxidation to CO2 and water in adipocytes, lean tissue gain in young animals, and alleviation of muscle protein loss in aging adults, lactating mammals, and food-deprived subjects. As a functional amino acid, leucine holds great promise to enhance the growth, efficiency of food utilization, and health of animals and humans.  PMID:25553480

  9. A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E).

    PubMed

    Ruffel, Sandrine; Dussault, Marie-Hélène; Palloix, Alain; Moury, Benoît; Bendahmane, Abdelhafid; Robaglia, Christophe; Caranta, Carole

    2002-12-01

    We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype. PMID:12492847

  10. 4E-BP1 regulates the differentiation of white adipose tissue.

    PubMed

    Tsukiyama-Kohara, Kyoko; Katsume, Asao; Kimura, Kazuhiro; Saito, Masayuki; Kohara, Michinori

    2013-07-01

    4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1(-/-) mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1(-/-) MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues.

  11. 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.

  12. eIF4E as a Control Target for Viruses

    PubMed Central

    Montero, Hilda; García-Román, Rebeca; Mora, Silvia I.

    2015-01-01

    Translation is a complex process involving diverse cellular proteins, including the translation initiation factor eIF4E, which has been shown to be a protein that is a point for translational regulation. Viruses require components from the host cell to complete their replication cycles. Various studies show how eIF4E and its regulatory cellular proteins are manipulated during viral infections. Interestingly, viral action mechanisms in eIF4E are diverse and have an impact not only on viral protein synthesis, but also on other aspects that are important for the replication cycle, such as the proliferation of infected cells and stimulation of viral reactivation. This review shows how some viruses use eIF4E and its regulatory proteins for their own benefit in order to spread themselves. PMID:25690796

  13. Requirement of the eukaryotic translation initiation factor 4F complex in hepatitis E virus replication.

    PubMed

    Zhou, Xinying; Xu, Lei; Wang, Yijin; Wang, Wenshi; Sprengers, Dave; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-12-01

    Hepatitis E virus (HEV) infection, one of the foremost causes of acute hepatitis, is becoming a health problem of increasing magnitude. As other viruses, HEV exploits elements from host cell biochemistry, but we understand little as to which components of the human hepatocellular machinery are perverted for HEV multiplication. It is, however, known that the eukaryotic translation initiation factors 4F (eIF4F) complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, serves as an important component for the translation and replication of many viruses. Here we aim to investigate the role of three subunits of the eIF4F complex: eukaryotic translation initiation factor 4A (eIF4A), eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E) in HEV replication. We found that efficient replication of HEV requires eIF4A, eIF4G and eIF4E. Consistently, the negative regulatory factors of this complex: programmed cell death 4 (PDCD4) and eIF4E-binding protein 1 (4E-BP1) exert anti-HEV activities, which further illustrates the requirement for eIF4A and eIF4E in supporting HEV replication. Notably, phosphorylation of eIF4E induced by MNK1/2 activation is not involved in HEV replication. Although ribavirin and interferon-α (IFN-α), the most often-used off-label drugs for treating hepatitis E, interact with this complex, their antiviral activities are independent of eIF4E. In contrast, eIF4E silencing provokes enhanced anti-HEV activity of these compounds. Thus, HEV replication requires eIF4F complex and targeting essential elements of this complex provides important clues for the development of novel antiviral therapy against HEV.

  14. 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

  15. Gcn4 Is Required for the Response to Peroxide Stress in the Yeast Saccharomyces cerevisiae

    PubMed Central

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

    2008-01-01

    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 α 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 H2O2, 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 H2O2-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. PMID:18417611

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

    PubMed

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

    2012-03-15

    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.

  17. 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.

  18. 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.

  19. 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

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

    PubMed

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

    2016-07-27

    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

  1. 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

  2. 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

  3. eIF4E is a central node of an RNA regulon that governs cellular proliferation.

    PubMed

    Culjkovic, Biljana; Topisirovic, Ivan; Skrabanek, Lucy; Ruiz-Gutierrez, Melisa; Borden, Katherine L B

    2006-11-01

    This study demonstrates that the eukaryotic translation initiation factor eIF4E is a critical node in an RNA regulon that impacts nearly every stage of cell cycle progression. Specifically, eIF4E coordinately promotes the messenger RNA (mRNA) export of several genes involved in the cell cycle. A common feature of these mRNAs is a structurally conserved, approximately 50-nucleotide element in the 3' untranslated region denoted as an eIF4E sensitivity element. This element is sufficient for localization of capped mRNAs to eIF4E nuclear bodies, formation of eIF4E-specific ribonucleoproteins in the nucleus, and eIF4E-dependent mRNA export. The roles of eIF4E in translation and mRNA export are distinct, as they rely on different mRNA elements. Furthermore, eIF4E-dependent mRNA export is independent of ongoing RNA or protein synthesis. Unlike the NXF1-mediated export of bulk mRNAs, eIF4E-dependent mRNA export is CRM1 dependent. Finally, the growth-suppressive promyelocytic leukemia protein (PML) inhibits this RNA regulon. These data provide novel perspectives into the proliferative and oncogenic properties of eIF4E.

  4. 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. PMID:27053525

  5. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

    PubMed Central

    Brunn, G J; Williams, J; Sabers, C; Wiederrecht, G; Lawrence, J C; Abraham, R T

    1996-01-01

    The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002. Images PMID:8895571

  6. 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

  7. 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.

  8. Regulation of cap-dependent translation initiation in the early stage porcine parthenotes.

    PubMed

    Susor, Andrej; Jelínková, Lucie; Karabínová, Pavla; Torner, Helmut; Tomek, Wolfgang; Kovárová, Hana; Kubelka, Michal

    2008-12-01

    The binding of mRNAs to ribosomes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5'-cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and association with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regulator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphorylation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9-15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes decreases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development.

  9. Specific requirement for translation initiation factor 4E or its isoform drives plant host susceptibility to Tobacco etch virus

    PubMed Central

    2014-01-01

    Background In plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species. Results A complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific. Conclusion We report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for

  10. ELAVL1 regulates alternative splicing of eIF4E transporter to promote postnatal angiogenesis

    PubMed Central

    Chang, Sung-Hee; Elemento, Olivier; Zhang, Jiasheng; Zhuang, Zhen W.; Simons, Michael; Hla, Timothy

    2014-01-01

    Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), which encodes an eukaryotic translation initiation factor 4E transporter (4E-T) protein and suppresses the expression of capped mRNAs. In the absence of ELAVL1, skipping of exon 11 of Eif4enif1 forms the stable, short isoform, 4E-Ts. This alternative splicing event results in the formation of RNA processing bodies (PBs), enhanced turnover of angiogenic mRNAs, and suppressed sprouting behavior of vascular endothelial cells. Further, endothelial-specific Elavl1 knockout mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in oncogene-induced mammary cancer, resulting in attenuated blood flow and tumor growth, respectively. ELAVL1-regulated alternative splicing of Eif4enif1 leading to enhanced formation of PB and mRNA turnover constitutes a novel posttranscriptional mechanism critical for pathological angiogenesis. PMID:25422430

  11. Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg.

    PubMed

    Charron, Carine; Nicolaï, Maryse; Gallois, Jean-Luc; Robaglia, Christophe; Moury, Benoît; Palloix, Alain; Caranta, Carole

    2008-04-01

    Amino acid substitutions in the eukaryotic translation initiation factor 4E (eIF4E) result in recessive resistance to potyviruses in a range of plant species, including Capsicum spp. Correspondingly, amino acid changes in the central part of the viral genome-linked protein (VPg) are responsible for the potyvirus's ability to overcome eIF4E-mediated resistance. A key observation was that physical interaction between eIF4E and the VPg is required for viral infection, and eIF4E mutations that cause resistance prevent VPg binding and inhibit the viral cycle. In this study, polymorphism analysis of the pvr2-eIF4E coding sequence in a worldwide sample of 25 C. annuum accessions identified 10 allelic variants with exclusively non-synonymous variations clustered in two surface loops of eIF4E. Resistance and genetic complementation assays demonstrated that pvr2 variants, each with signature amino acid changes, corresponded to potyvirus resistance alleles. Systematic analysis of the interactions between eIF4E proteins encoded by the 10 pvr2 alleles and VPgs of virulent and avirulent potato virus Y (PVY) and tobacco etch virus (TEV) strains demonstrated that resistance phenotypes arose from disruption of the interaction between eIF4E and VPg, and that viral adaptation to eIF4E-mediated resistance resulted from restored interaction with the resistance protein. Complementation of an eIF4E knockout yeast strain by C. annuum eIF4E proteins further shows that amino acid changes did not impede essential eIF4E functions. Altogether, these results argue in favour of a co-evolutionary 'arms race' between Capsicum eIF4E and potyviral VPg.

  12. 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

  13. An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses

    PubMed Central

    Minoïa, Silvia; Piednoir, Elodie; Moretti, André; Salgues, Aurélie; Zamir, Dani; Caranta, Carole; Bendahmane, Abdelhafid

    2010-01-01

    Background The characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function. Methodology/Principal Findings To investigate further the role of translation initiation factors in virus resistance we set up a TILLING platform in tomato, cloned genes encoding for translation initiation factors eIF4E and eIF4G and screened for induced mutations that lead to virus resistance. A splicing mutant of the eukaryotic translation initiation factor, S.l_eIF4E1 G1485A, was identified and characterized with respect to cap binding activity and resistance spectrum. Molecular analysis of the transcript of the mutant form showed that both the second and the third exons were miss-spliced, leading to a truncated mRNA. The resulting truncated eIF4E1 protein is also impaired in cap-binding activity. The mutant line had no growth defect, likely because of functional redundancy with others eIF4E isoforms. When infected with different potyviruses, the mutant line was immune to two strains of Potato virus Y and Pepper mottle virus and susceptible to Tobacco each virus. Conclusions/Significance Mutation analysis of translation initiation factors shows that translation initiation factors of the eIF4E family are determinants of plant susceptibility to RNA viruses and viruses have adopted strategies to use different isoforms. This work also demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. We have also developed a complete tool that can be used for both forward and reverse genetics in tomato, for both basic science and crop improvement. By opening it to the community, we hope to fulfill the expectations of both crop breeders and scientists who are using tomato as their model of study. PMID:20593023

  14. 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

  15. Characterization of the Raptor/4E-BP1 Interaction by Chemical Cross-linking Coupled with Mass Spectrometry Analysis*

    PubMed Central

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

    2014-01-01

    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. PMID:24403073

  16. Vitamin E succinate induces apoptosis via the PI3K/AKT signaling pathways in EC109 esophageal cancer cells

    PubMed Central

    Yang, Peng; Zhao, Jiaying; Hou, Liying; Yang, Lei; Wu, Kun; Zhang, Linyou

    2016-01-01

    Esophageal cancer is the fourth most common gastrointestinal cancer, it generally has a poor prognosis and novel strategies are required for prevention and treatment. Vitamin E succinate (VES) is a potential chemical agent for cancer prevention and therapy as it exerts anti-tumor effects in a variety of cancers. However, the role of VES in tumorigenesis and progression of cancer remains to be elucidated. The present study aimed to determine the effects of VES in regulating the survival and apoptosis of human esophageal cancer cells. EC109 human esophageal cancer cells were used to investigate the anti-proliferative effects of VES. The MTT and Annexin V-fluorescein isothiocyanate/propidium iodide assays demonstrated that VES inhibited cell proliferation and induced apoptosis in esophageal cancer cells. Furthermore, VES downregulated constitutively active basal levels of phosphorylated (p)-serine-threonine kinase AKT (AKT) and p-mammalian target of rapamycin (mTOR), and decreased the phosphorylation of AKT substrates Bcl-2-associated death receptor and caspase-9, in addition to mTOR effectors, ribosomal protein S6 kinase β1 and eIF4E-binding protein 1. Phosphoinositide-3-kinase (PI3K) inhibitor, LY294002 suppressed p-AKT and p-mTOR, indicating PI3K is a common upstream mediator. The apoptosis induced by VES was increased by inhibition of AKT or mTOR with their respective inhibitor in esophageal cancer cells. The results of the present study suggested that VES targeted the PI3K/AKT signaling pathways and induced apoptosis in esophageal cancer cells. Furthermore, the current study suggests that VES may be useful in a combinational therapeutic strategy employing an mTOR inhibitor. PMID:27357907

  17. Targeting Eukaryotic Translation in Mesothelioma Cells with an eIF4E-Specific Antisense Oligonucleotide

    PubMed Central

    Jacobson, Blake A.; Thumma, Saritha C.; Jay-Dixon, Joseph; Patel, Manish R.; Dubear Kroening, K.; Kratzke, Marian G.; Etchison, Ryan G.; Konicek, Bruce W.; Graff, Jeremy R.; Kratzke, Robert A.

    2013-01-01

    Background Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma. Methods Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. Results eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. Conclusion 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity. PMID:24260583

  18. 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.

  19. The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond1[OA

    PubMed Central

    Monzingo, Arthur F.; Dhaliwal, Simrit; Dutt-Chaudhuri, Anirvan; Lyon, Angeline; Sadow, Jennifer H.; Hoffman, David W.; Robertus, Jon D.; Browning, Karen S.

    2007-01-01

    Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m7 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 β-strands, three α-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m7GTP 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 m7GDP 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 m7GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s−1. PMID:17322339

  20. The eukaryotic translation initiation factor eIF4E in the nucleus: taking the road less traveled

    PubMed Central

    Osborne, Michael J.; Borden, Katherine L.B.

    2014-01-01

    Summary The eukaryotic translation initiation factor eIF4E is a potent oncogene. Although eIF4E has traditional roles in translation initiation in the cytoplasm, it is also found in the nucleus, suggesting that it has activities beyond its role in protein synthesis. The road less traveled has been taken to study these nuclear activities and to understand their contribution to the oncogenic potential of eIF4E. The molecular features and biological pathways underpinning eIF4E’s nuclear mRNA export are described. New classes of eIF4E regulators have been identified and their relevance to cancer shown. The studies presented here reveal the molecular, biophysical, and structural bases for eIF4E regulation. Finally, recent clinical work targeting eIF4E in acute myeloid leukemia patients with ribavirin is discussed. In summary, these findings provide a novel paradigm for eIF4E function and the molecular basis for targeting it in leukemia patients. PMID:25510279

  1. 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

  2. Methionine improves the performance and breast muscle growth of broilers with lower hatching weight by altering the expression of genes associated with the insulin-like growth factor-I signalling pathway.

    PubMed

    Wen, Chao; Wu, Ping; Chen, Yueping; Wang, Tian; Zhou, Yanmin

    2014-01-28

    The present study aimed to investigate the responses of broilers with different hatching weights (HW) to dietary methionine (Met). A total of 192 1-d-old Arbor Acres broiler chicks with different HW (heavy: 48·3 (sem 0·1) g and light: 41·7 (sem 0·1) g) were allocated to a 2 (HW) × 2 (Met) factorial arrangement with six replicates of eight chicks. Control starter (1-21 d) and finisher (22-42 d) diets contained 0·50 and 0·43 % Met, respectively. Corresponding values for a high-Met treatment were 0·60 and 0·53 %. Light chicks had poorer (P< 0·05) growth performance and breast muscle weight and lower (P< 0·05) insulin-like growth factor-I (IGF-I) concentration and mRNA level in breast muscle than heavy chicks when both were fed the control diets. High-Met diets improved performance and promoted breast muscle growth and IGF-I concentration in light chicks (P< 0·05). Increased IGF-I and target of rapamycin (TOR) mRNA levels as well as decreased eIF4E-binding protein 1 (4EBP1), atrogin-1 and forkhead box O 4 (FOXO4) mRNA levels were induced by high-Met diets in light chicks (P< 0·05). In conclusion, the Met requirement of broilers might depend on their HW and Met levels used in the control diets in the present study were adequate for heavy chicks but inadequate for light chicks, resulting in poorer performance and breast muscle growth, which were improved by increasing dietary Met supply presumably through alterations in IGF-I synthesis and gene expression of the TOR/4EBP1 and FOXO4/atrogin-1 pathway.

  3. Functional dissection of naturally occurring amino acid substitutions in eIF4E that confers recessive potyvirus resistance in plants.

    PubMed

    Yeam, Inhwa; Cavatorta, Jason R; Ripoll, Daniel R; Kang, Byoung-Cheorl; Jahn, Molly M

    2007-09-01

    Naturally existing variation in the eukaryotic translation initiation factor 4E (eIF4E) homolog encoded at the pvr1 locus in Capsicum results in recessively inherited resistance against several potyviruses. Previously reported data indicate that the physical interaction between Capsicum-eIF4E and the viral genome-linked protein (VPg) is required for the viral infection in the Capsicum-Tobacco etch virus (TEV) pathosystem. In this study, the potential structural role(s) of natural variation in the eIF4E protein encoded by recessive resistance alleles and their biological consequences have been assessed. Using high-resolution three-dimensional structural models based on the available crystallographic structures of eIF4E, we show that the amino acid substitution G107R, found in many recessive plant virus resistance genes encoding eIF4E, is predicted to result in a substantial modification in the protein binding pocket. The G107R change was shown to not only be responsible for the interruption of VPg binding in planta but also for the loss of cap binding ability in vitro, the principal function of eIF4E in the host. Overexpression of the Capsicum-eIF4E protein containing the G107R amino acid substitution in Solanum lycopersicum indicated that this polymorphism alone is sufficient for the acquisition of resistance against several TEV strains.

  4. Integrin β6 can be translationally regulated by eukaryotic initiation factor 4E: Contributing to colonic tumor malignancy.

    PubMed

    Enyu, Liu; Zhengchuan, Niu; Jiayong, Wang; Benjia, Liang; Qi, Sun; Ruixi, Qin; Cheng, Peng; Khan, Abdul Qadir; Wei, Song; Jun, Niu

    2015-08-01

    It is well known that both eukaryotic initiation factor 4E (eIF4E) and integrin αvβ6 can contribute to malignant behavior of colon cancer. We have found that integrin αvβ6 and eIF4E were co-expressed and positively correlated in colon cancer tissues. Recently, deregulation of the protein synthesis apparatus has begun to gain attention as a major participant in cancer development and progression. However, the regulation of integrin β6 expression at translational level has never been investigated before. In present study, gene-silencing technique for eIF4E by small interfering RNA (siRNA) was used in all the subsequent experiments, in order to investigate whether eIF4E could translationally regulate expression of integrin β6 in colon cancer SW480 and HT-29 cell lines. Additionally, the subsequent effects of eIF4E knockdown on cellular malignant behavior were observed. siRNA in SW480 and HT-29 transfectants. Subsequently, protein expression of β6 was markedly suppressed, while mRNA expression of β6 showed no significant variation before and after eIF4E RNA interfering. Therefore, it could be seen that eIF4E could upregulate the expression of β6, without effect on β6 mRNA expression. More importantly, after treated with eIF4E siRNA, cellular migratory capacity on fibronectin of HT-29 and β6-transfected SW480 as well as their survival to 5-FU was decreased distinctly. Expression of integrin β6 could be translationally regulated by eIF4E, which subsequently contributed to tumor malignancy through enhancing cellular migration, survival, anti-apoptosis, and chemoresistance of colon cancer in vitro. Thus, targeting eIF4E in integrin αvβ6 expressing tumors can be a potential therapeutic strategy for patients with colon cancer.

  5. Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development.

    PubMed

    Siemer, Corinna; Smiljakovic, Tatjana; Bhojwani, Monika; Leiding, Claus; Kanitz, Wilhelm; Kubelka, Michal; Tomek, Wolfgang

    2009-12-01

    Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed-loop model of translational initiation, we have analyzed components of the 5 -mRNA cap-binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E-BP1, and 3 -mRNA poly-(A) tail-associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16-cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA-binding factors occur at the transition from M II stage oocytes to 2-cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5 -end and 3 -end mRNA associated factors, mainly the poly-(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin-like protein with distinct eIF4E-binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16-cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by (7)m-GTP-Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development.

  6. 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.

  7. eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition.

    PubMed

    Osborne, Michael J; Volpon, Laurent; Kornblatt, Jack A; Culjkovic-Kraljacic, Biljana; Baguet, Aurélie; Borden, Katherine L B

    2013-03-01

    Recognition of the methyl-7-guanosine (m(7)G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m(7)G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m(7)G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growth-promoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.

  8. Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells.

    PubMed

    Thumma, S C; Jacobson, B A; Patel, M R; Konicek, B W; Franklin, M J; Jay-Dixon, J; Sadiq, A; De, A; Graff, J R; Kratzke, R A

    2015-08-01

    Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of β-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

  9. Muscle-specific 4E-BP1 signaling activation improves metabolic parameters during aging and obesity

    PubMed Central

    Tsai, Shihyin; Sitzmann, Joanna M.; Dastidar, Somasish G.; Rodriguez, Ariana A.; Vu, Stephanie L.; McDonald, Circe E.; Academia, Emmeline C.; O’Leary, Monique N.; Ashe, Travis D.; La Spada, Albert R.; Kennedy, Brian K.

    2015-01-01

    Eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) is a key downstream effector of mTOR complex 1 (mTORC1) that represses cap-dependent mRNA translation initiation by sequestering the translation initiation factor eIF4E. Reduced mTORC1 signaling is associated with life span extension and improved metabolic homeostasis, yet the downstream targets that mediate these benefits are unclear. Here, we demonstrated that enhanced 4E-BP1 activity in mouse skeletal muscle protects against age- and diet-induced insulin resistance and metabolic rate decline. Transgenic animals displayed increased energy expenditure; altered adipose tissue distribution, including reduced white adipose accumulation and preserved brown adipose mass; and were protected from hepatic steatosis. Skeletal muscle–specific 4E-BP1 mediated metabolic protection directly through increased translation of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) and enhanced respiratory function. Non–cell autonomous protection was through preservation of brown adipose tissue metabolism, which was increased in 4E-BP1 transgenic animals during normal aging and in a response to diet-induced type 2 diabetes. Adipose phenotypes may derive from enhanced skeletal muscle expression and secretion of the known myokine FGF21. Unlike skeletal muscle, enhanced adipose-specific 4E-BP1 activity was not protective but instead was deleterious in response to the same challenges. These findings indicate that regulation of 4E-BP1 in skeletal muscle may serve as an important conduit through which mTORC1 controls metabolism. PMID:26121750

  10. 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

    2014-09-01

    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.

  11. Immunohistochemical analysis of the Akt/mTOR/4E-BP1 signalling pathway in canine haemangiomas and haemangiosarcomas.

    PubMed

    Murai, A; Abou Asa, S; Kodama, A; Sakai, H; Hirata, A; Yanai, T

    2012-11-01

    The specific signalling pathways that are deregulated in canine endothelial tumours have not yet fully elucidated. Therefore, the aim of the present study was to examine activation of the Akt/mammalian target of rapamycin (mTOR)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) signalling pathway in spontaneously arising canine haemangiomas (HAs) and haemangiosarcomas (HSAs) in order to identify novel molecular targets for treatment. Surgically-resected samples of HA (n = 27), HSA (n = 37), granulation tissue (n = 4) and normal skin (n = 4) were investigated by immunohistochemistry. Approximately 80% of the HSA samples had moderate to intense expression of phosphorylated Akt at Ser473 (p-Akt Ser473), p-Akt Thr308, p-4E-BP1 Thr37/46 and eukaryotic initiation factor 4E, which was significantly higher than in the HAs and was similar to the expression in activated endothelial cells (ECs). Although p-mTOR complex1 (p-mTORC1) Ser2448 was expressed by most of the activated ECs, only 35% of the HSA samples had weak to moderate expression. Because mTORC2 and phosphorylates Akt Ser473 was activated in HSA samples, the present findings suggest that the mTORC2/Akt/4E-BP1 pathway, regulated independently of mTORC1, may be important for targeting therapy in canine HSAs. PMID:22789858

  12. Epigenetic Activation of a Subset of mRNAs by eIF4E Explains Its Effects on Cell Proliferation

    PubMed Central

    Mamane, Yaël; Petroulakis, Emmanuel; Martineau, Yvan; Sato, Taka-Aki; Larsson, Ola; Rajasekhar, Vinagolu K.; Sonenberg, Nahum

    2007-01-01

    Background Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5′ cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. Methodology/Principal Findings Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. Conclusions/Significance Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs. PMID:17311107

  13. Mnk1 and 2 are dispensable for T-cell development and activation but important for the pathogenesis of experimental autoimmune encephalitis

    PubMed Central

    Gorentla, Balachandra K; Krishna, Sruti; Shin, Jinwook; Inoue, Makoto; Shinohara, Mari L.; Grayson, Jason M.; Fukunaga, Rikiro; Zhong, Xiao-Ping

    2012-01-01

    T-cell development and activation are usually accompanied by expansion and production of numerous proteins that require active translation. The eukaryotic translation initiation factor 4E (eIF4E) binds to the 5' cap structure of mRNA and is critical for cap-dependent translational initiation. It has been hypothesized that MAPK-interacting kinase 1 and 2 (Mnk1/2) promote cap-dependent translation by phosphorylating eIF4E at serine 209 (S209). Pharmacological studies utilizing inhibitors have suggested that Mnk1/2 play important roles in T-cells. However, genetic evidence supporting such conclusions is lacking. Moreover, the signaling pathways that regulate Mnk1/2 in T-cells remain unclear. We demonstrated here that T-cell receptor (TCR) engagement activates Mnk1/2 in primary T-cells. Such activation is dependent on Ras-Erk1/2 signaling and is inhibited by diacylglycerol kinases α and ζ. Mnk1/2 double deficiency in mice abolishes TCR-induced eIF4E S209 phosphorylation, indicating their absolute requirement for eIF4E S209 phosphorylation. However, Mnk1/2 double deficiency does not affect the development of conventional αβ T-cells, regulatory T-cells, or NKT-cells. Furthermore, T-cell activation, in vivo primary and memory CD8 T-cell responses to microbial infection, and NKT-cell cytokine production were not obviously altered by Mnk1/2 deficiency. Although Mnk1/2 deficiency causes decreased IL-17 and IFNγ production by CD4 T-cells following immunization of mice with myelin oligodendrocyte glycoprotein peptide in complete Freud's adjuvant, correlating with milder experimental autoimmune encephalitis scores, it does not affect T helper cell differentiation in vitro. Together, these data suggest that Mnk1/2 play a minimal role in T-cell development and activation but may regulate non-T-cell lineages to control Th1/Th17 differentiation in vivo. PMID:23269249

  14. 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. PMID:24417952

  15. 49 CFR 178.68 - Specification 4E welded aluminum cylinders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Specification 4E welded aluminum cylinders. 178.68... FOR 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. Human inducible nitric oxide synthase (iNOS) expression depends on chromosome region maintenance 1 (CRM1)- and eukaryotic translation initiation factor 4E (elF4E)-mediated nucleocytoplasmic mRNA transport.

    PubMed

    Bollmann, Franziska; Fechir, Katrin; Nowag, Sebastian; Koch, Kathrin; Art, Julia; Kleinert, Hartmut; Pautz, Andrea

    2013-04-01

    Human inducible nitric oxide synthase (iNOS) is regulated on the expressional level mostly by post-transcriptional mechanisms modulating the mRNA stability. Another important step in the control of eukaryotic gene expression is the nucleocytoplasmic mRNA transport. Most cellular mRNAs are exported via the TAP/Nxt complex of proteins. However, some mRNAs are transported by a different mechanism involving the nuclear export receptor CRM1. Treatment of DLD-1 cells with the CRM1 inhibitor leptomycin B (LMB) or anti-CRM1 siRNAs reduced cytokine-induced iNOS expression. We could demonstrate that the iNOS mRNA is exported from the nucleus in a CRM1-dependent manner. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed to mediate CRM1-dependent mRNA export. Western blot experiments showed that the eukaryotic translation initiation factor eIF4E is retained in the nucleus after LMB treatment. Blockade of eIF4E by ribavirin or overexpression of the promyelocytic leukemia protein (PML) decreased iNOS expression due to reduced iNOS mRNA export from the nucleus. Transfection experiments provide evidence that the 3'-untranslated region of the iNOS mRNA is involved in eIF4E-mediated iNOS mRNA transport. In summary, CRM1 and eIF4E seem to play an important role in the nucleocytoplasmic export of human iNOS mRNA.

  1. 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

  2. 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

  3. The Translation Initiation Factor eIF4E Regulates the Sex-Specific Expression of the Master Switch Gene Sxl in Drosophila melanogaster

    PubMed Central

    Graham, Patricia L.; Yanowitz, Judith L.; Penn, Jill K. M.; Deshpande, Girish; Schedl, Paul

    2011-01-01

    In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors—the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d—that have been directly implicated in Sxl splicing regulation. PMID:21829374

  4. RhoE inhibits 4E-BP1 phosphorylation and eIF4E function impairing cap-dependent translation.

    PubMed

    Villalonga, Priam; Fernández de Mattos, Silvia; Ridley, Anne J

    2009-12-18

    The Rho GTPase family member RhoE inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have previously reported that RhoE also inhibits cell cycle progression and Ras-induced transformation, specifically preventing cyclin D1 translation. Here we investigate the molecular mechanisms underlying those observations. RhoE inhibits the phosphorylation of the translational repressor 4E-BP1 in response to extracellular stimuli. However, RhoE does not affect the activation of mTOR, the major kinase regulating 4E-BP1 phosphorylation, as indicated by the phosphorylation levels of the mTOR substrate S6K, the dynamics of mTOR/Raptor association, and the observation that RhoE, as opposed to rapamycin, does not impair cellular growth. Interestingly, RhoE prevents the release of the eukaryotic initiation factor eIF4E from 4E-BP1, inhibiting cap-dependent translation. Accordingly, RhoE also inhibits the expression and the transcriptional activity of the eIF4E target c-Myc. Consistent with its crucial role in cell proliferation, we show that eIF4E can rescue both cell cycle progression and Ras-induced transformation in RhoE-expressing cells, indicating that the inhibition of eIF4E function is critical to mediate the anti-proliferative effects of RhoE.

  5. Trypanosoma brucei translation initiation factor homolog EIF4E6 forms a tripartite cytosolic complex with EIF4G5 and a capping enzyme homolog.

    PubMed

    Freire, Eden R; Malvezzi, Amaranta M; Vashisht, Ajay A; Zuberek, Joanna; Saada, Edwin A; Langousis, Gerasimos; Nascimento, Janaína D F; Moura, Danielle; Darzynkiewicz, Edward; Hill, Kent; de Melo Neto, Osvaldo P; Wohlschlegel, James A; Sturm, Nancy R; Campbell, David A

    2014-07-01

    Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene-one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5' cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules. PMID:24839125

  6. Trypanosoma brucei translation initiation factor homolog EIF4E6 forms a tripartite cytosolic complex with EIF4G5 and a capping enzyme homolog.

    PubMed

    Freire, Eden R; Malvezzi, Amaranta M; Vashisht, Ajay A; Zuberek, Joanna; Saada, Edwin A; Langousis, Gerasimos; Nascimento, Janaína D F; Moura, Danielle; Darzynkiewicz, Edward; Hill, Kent; de Melo Neto, Osvaldo P; Wohlschlegel, James A; Sturm, Nancy R; Campbell, David A

    2014-07-01

    Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene-one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5' cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.

  7. Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog

    PubMed Central

    Freire, Eden R.; Malvezzi, Amaranta M.; Vashisht, Ajay A.; Zuberek, Joanna; Saada, Edwin A.; Langousis, Gerasimos; Nascimento, Janaína D. F.; Moura, Danielle; Darzynkiewicz, Edward; Hill, Kent; de Melo Neto, Osvaldo P.; Wohlschlegel, James A.; Sturm, Nancy R.

    2014-01-01

    Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules. PMID:24839125

  8. Eukaryotic Initiation Factor 4E Binding Protien Family of Protiens: Sentinels at a Translational Control Checkpoint in Lung Tumor Defense

    PubMed Central

    Kim, Yong Y; Von Weymarn, Linda; Larsson, Ola; Fan, Danhua; Underwood, Jon M; Hecht, Stephen S; Polunovsky, Vitaly A; Bitterman, Peter B

    2009-01-01

    The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here we show that the primary negative regulators of these oncogenic initiation factors - the 4E-BP protein family - operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen NNK, 4ebp1−/−/4ebp2−/− mice showed increased sensitivity to tumorigenesis compared to their wild type counterparts. The 4E-BP deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape - with translational activation of genes governing angiogenesis, growth and proliferation; and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense. PMID:19843855

  9. Resistance to discodermolide, a microtubule-stabilizing agent and senescence inducer, is 4E-BP1-dependent.

    PubMed

    Chao, Suzan K; Lin, Juan; Brouwer-Visser, Jurriaan; Smith, Amos B; Horwitz, Susan Band; McDaid, Hayley M

    2011-01-01

    Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild-type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, reintroduction of a nonphosphorylatable mutant (Thr-37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32-resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation, and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence.

  10. 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. PMID:26339794

  11. Mechanistic target of rapamycin (mTOR) signaling genes in decapod crustaceans: cloning and tissue expression of mTOR, Akt, Rheb, and p70 S6 kinase in the green crab, Carcinus maenas, and blackback land crab, Gecarcinus lateralis.

    PubMed

    Abuhagr, Ali M; Maclea, Kyle S; Chang, Ernest S; Mykles, Donald L

    2014-02-01

    Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031bp), Akt (855bp), and S6K (918bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas. PMID:24269559

  12. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    PubMed

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

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

    PubMed

    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. 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 (mammalian 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

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

    PubMed

    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

    2015-04-14

    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. 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 (mammalian 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.

  15. 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

  16. 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

    2015-11-01

    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

  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. Pathophysiology of the pancreas after oral infection of genetically diverse mice with coxsackievirus B4-E2.

    PubMed

    Precechtelova, Jana; Borsanyiova, Maria; Stipalova, Darina; Sarmirova, Sona; Gomolcak, Pavol; Berakova, Katarina; Bopegamage, Shubhada

    2015-01-01

    Coxsackievirus B4 strain E2 (CVB4-E2) and its association with type 1 diabetes (T1D) have been studied in experimental in vitro and in vivo murine models. CVB4-E2, known to be pancreotropic and diabetogenic in nature, is associated with acute pancreatitis in mice but shows differences in the induction of glycemia after intraperitoneal (i.p.) infection. Therefore, the aim of this work was to study the outcome of oral infection with CVB4-E2 in five mouse strains with different genetic backgrounds: two outbred (Swiss albino, CD1), two inbred (SJL, NOD) and one transgenic (NOD.SCID). Survival rates, fasting blood glucose, histopathology, viral titres and persistence were studied in selected organs and stool samples. Viral protein (VP1), proinflammatory cytokines, and interferon alpha (IFN-α) were analyzed by immunohistochemistry. We observed mortality only in infected NOD and NOD.SCID mice, with differing survival rates implying initial innate protection in the NOD.SCID mice and low virus clearance with replicating virus titres in the studied organs and stool up to day 40 post infection (p.i.). Independent of the mouse strain hyperglycemia, proinflammatory cytokines and histopathological changes were absent in the endocrine pancreas of infected mice. Only the pancreata of the dead NOD.SCID mice showed inflammation even in presence of IFN-α. Host-dependent viral RNA persistence was observed in all outbred mice. In conclusion, oral infection with CVB4-E2, despite the known affinity of this strain towards the pancreatic tissue and the presence of replicating virus, conferred total protection to the endocrine pancreas in all mice and failed to induce the proinflammatory cytokines studied by us.

  19. 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

    2015-11-01

    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.

  20. L-serine deficiency caused by genetic Phgdh deletion leads to robust induction of 4E-BP1 and subsequent repression of translation initiation in the developing central nervous system.

    PubMed

    Sayano, Tomoko; Kawakami, Yuriko; Kusada, Wataru; Suzuki, Takeshi; Kawano, Yuki; Watanabe, Akihiro; Takashima, Kana; Arimoto, Yashiho; Esaki, Kayoko; Wada, Akira; Yoshizawa, Fumiaki; Watanabe, Masahiko; Okamoto, Masahiro; Hirabayashi, Yoshio; Furuya, Shigeki

    2013-03-01

    Targeted disruption in mice of the gene encoding D-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free L-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that L-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of L-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when L-serine was supplemented in the culture medium, indicating that reduced L-serine availability regulates the induction of Eif4ebp1/4E-BP1. These data suggest that elevated levels of 4E-BP1 may be involved in a mechanism to arrest brain development in Phgdh KO embryos. PMID:23350942

  1. Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum

    PubMed Central

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

    2009-01-01

    Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (ΔN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 Å from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection. PMID:19652353

  2. 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

  3. Overexpression of Phosphorylated 4E-BP1 Predicts for Tumor Recurrence and Reduced Survival in Cervical Carcinoma Treated With Postoperative Radiotherapy

    SciTech Connect

    Benavente, Sergio; Verges, Ramona; Hermosilla, Eduardo; Fumanal, Victor; Casanova, Nathalie; Garcia, Angel; Ramon y Cajal, Santiago; Giralt, Jordi

    2009-12-01

    Purpose: To examine the prognostic value of the 4E-BP1 activation state and related upstream/downstream signaling proteins on the clinical outcome of patients with intermediate- or high-risk early-stage cervical carcinoma treated with postoperative radiotherapy and to determine the optimal treatment of early-stage cervical carcinoma. Methods and Materials: Immunohistochemical staining was performed on 64 formalin-fixed, paraffin-embedded cervical carcinoma surgical specimens for each protein of the panel (p4E-BP1, phosphorylated mitogen-activated protein kinase, pAkt, vascular endothelial growth factor, KDR, Bcl-2, TP53, receptor for activated C-kinase 1). The expression patterns were related to the clinical data. All patients received postoperative radiotherapy. Concurrent chemotherapy was added if high-risk features were present. The median follow-up was 40 months. Results: Of the 64 patients, 13 received concomitant chemotherapy. p4E-BP1 overexpression in moderate/high-risk early-stage cervical carcinoma correlated significantly with disease-free survival (hazard ratio, 4.39; p = .009) and overall survival (hazard ratio, 4.88; p = .005). Vascular endothelial growth factor, and its receptor KDR, had positive immunoreactivity in all tumor samples. No correlation with clinical outcome was found for the remaining proteins evaluated. Conclusion: In this study, moderate/high-risk early-stage cervical carcinoma with low p4E-BP1 expression was highly curable with the current postoperative treatments. For tumors with p4E-BP1 overexpression, new investigational strategies are needed.

  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/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

    PubMed

    Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

    2015-11-01

    Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD.

  6. The MNK-1/eIF4E pathway as a new therapeutic pathway to target inflammation and remodelling in asthma.

    PubMed

    Seidel, Petra; Sun, Qingzhu; Costa, Luigi; Lardinois, Didier; Tamm, Michael; Roth, Michael

    2016-10-01

    Therapeutic targets in asthma are reduction of airway inflammation and remodelling, the latter is not affected by available drugs. Here we present data that inhibition of MAPK-activated protein kinase (MNK)-1 reduces inflammation and remodelling. MNK-1 regulates protein expression by controlling mRNA stability, nuclear export and translation through the eukaryotic initiation factor 4E (eIF4E). Airway smooth muscle cells were derived from asthmatic and non-asthmatic donors. Cells were pre-treated with CGP57380 (MNK-1 inhibitor) or MNK-1 siRNA, before TNF-α stimulation. Cytokine and protein expression was analysed by ELISA, real time PCR and immunoblotting. Proliferation was monitored by cell counts. TNF-α activated MNK-1 phosphorylation between 15 and 30min. and subsequently eIF4E between 15 and 60min. EIF4E activity was inhibited by CGP57380 dose-dependently. Inhibition of MNK-1 by CGP57380 or MNK-1 siRNA significantly reduced TNF-α induced CXCL10 and eotaxin mRNA expression and secretion, but had no effect on IL-8. However, CXCL10 mRNA stability or NF-κB activity were not affected by MNK-1 inhibition. Furthermore, eIF4E was detected in the cytosol and the nucleus, but TNF-α did not affected its export from the nucleus. Cytokine array assessment showed that in addition to eotaxin and CXCL10, asthma relevant GRO α and RANTES were down-regulated by MNK-1 inhibition. In addition, MNK-1 inhibition significantly reduced FCS and PDGF-BB induced cell proliferation. We are the first to report that MNK-1 controls chemokine secretion and proliferation in human airway smooth muscle cells. Therefore we suggest that MNK-1 inhibition may present a new target to limit inflammation and remodelling in asthmatic airways. PMID:27418099

  7. The MNK-1/eIF4E pathway as a new therapeutic pathway to target inflammation and remodelling in asthma.

    PubMed

    Seidel, Petra; Sun, Qingzhu; Costa, Luigi; Lardinois, Didier; Tamm, Michael; Roth, Michael

    2016-10-01

    Therapeutic targets in asthma are reduction of airway inflammation and remodelling, the latter is not affected by available drugs. Here we present data that inhibition of MAPK-activated protein kinase (MNK)-1 reduces inflammation and remodelling. MNK-1 regulates protein expression by controlling mRNA stability, nuclear export and translation through the eukaryotic initiation factor 4E (eIF4E). Airway smooth muscle cells were derived from asthmatic and non-asthmatic donors. Cells were pre-treated with CGP57380 (MNK-1 inhibitor) or MNK-1 siRNA, before TNF-α stimulation. Cytokine and protein expression was analysed by ELISA, real time PCR and immunoblotting. Proliferation was monitored by cell counts. TNF-α activated MNK-1 phosphorylation between 15 and 30min. and subsequently eIF4E between 15 and 60min. EIF4E activity was inhibited by CGP57380 dose-dependently. Inhibition of MNK-1 by CGP57380 or MNK-1 siRNA significantly reduced TNF-α induced CXCL10 and eotaxin mRNA expression and secretion, but had no effect on IL-8. However, CXCL10 mRNA stability or NF-κB activity were not affected by MNK-1 inhibition. Furthermore, eIF4E was detected in the cytosol and the nucleus, but TNF-α did not affected its export from the nucleus. Cytokine array assessment showed that in addition to eotaxin and CXCL10, asthma relevant GRO α and RANTES were down-regulated by MNK-1 inhibition. In addition, MNK-1 inhibition significantly reduced FCS and PDGF-BB induced cell proliferation. We are the first to report that MNK-1 controls chemokine secretion and proliferation in human airway smooth muscle cells. Therefore we suggest that MNK-1 inhibition may present a new target to limit inflammation and remodelling in asthmatic airways.

  8. Migration and epithelial-to-mesenchymal transition of lung cancer can be targeted via translation initiation factors eIF4E and eIF4GI.

    PubMed

    Attar-Schneider, Oshrat; Drucker, Liat; Gottfried, Maya

    2016-09-01

    Metastasis underlies cancer morbidity and accounts for disease progression and significant death rates generally and in non-small cell lung cancer (NSCLC) particularly. Therefore, it is critically important to understand the molecular events that regulate metastasis. Accumulating data portray a central role for protein synthesis, particularly translation initiation (TI) factors eIF4E and eIF4G in tumorigenesis and patients' survival. We have published that eIF4E/eIF4GI activities and consequently NSCLC cell migration are modulated by bone-marrow mesenchymal stem cell secretomes, suggesting a role for TI in metastasis. Here, we aimed to expand our understanding of the TI factors significance to NSCLC characteristics, particularly epithelial-to-mesenchymal transition (EMT) and migration, supportive of metastasis. In a model of NSCLC cell lines (H1299, H460), we inhibited eIF4E/eIF4GI's expressions (siRNA, ribavirin) and assessed NSCLC cell lines' migration (scratch), differentiation (EMT, immunoblotting), and expression of select microRNAs (qPCR). Initially, we determined an overexpression of several TI factors (eIF4E, eIF4GI, eIF4B, and DHX29) and their respective targets in NSCLC compared with normal lung samples (70-350%↑, P<0.05). Knockdown (KD) of eIF4E/eIF4GI in NSCLC cell lines (70%↓, P<0.05) also manifested in decreased target levels (ERα, SMAD5, NFkB, CyclinD1, c-MYC, and HIF1α) (20-50%↓, P<0.05). eIF4E/eIF4GI KD also attenuated cell migration (60-75%↓, P<0.05), EMT promoters (15-90%↓, P<0.05), and enhanced EMT suppressors (30-380%↑, P<0.05). The importance of eIF4E KD to NSCLC phenotype was further corroborated with its inhibitor, ribavirin. Changes in expression of essential microRNAs implicated in NSCLC cell migration concluded the study (20-100%, P<0.05). In summary, targeting eIF4E/eIF4GI reduces migration and EMT, both essential for metastasis, thereby underscoring the potential of TI targeting in NSCLC therapy, especially the already

  9. 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

  10. The pvr1 locus in Capsicum encodes a translation initiation factor eIF4E that interacts with Tobacco etch virus VPg.

    PubMed

    Kang, Byoung-Cheorl; Yeam, Inhwa; Frantz, James D; Murphy, John F; Jahn, Molly M

    2005-05-01

    Mutations in the eIF4E homolog encoded at the pvr1 locus in Capsicum result in broad-spectrum potyvirus resistance attributed to the pvr1 resistance allele, a gene widely deployed in agriculture for more than 50 years. We show that two other resistance genes, previously known to be eIF4E with narrower resistance spectra, pvr2(1) and pvr2(2), are alleles at the pvr1 locus. Based on these data and current nomenclature guidelines, we have re-designated these alleles, pvr1(1) and pvr1(2), respectively. Point mutations in pvr1, pvr1(1), and pvr1(2) grouped to similar regions of eIF4E and were predicted by protein homology models to cause conformational shifts in the encoded proteins. The avirulence determinant in this potyvirus system has previously been identified as VPg, therefore yeast two-hybrid and GST pull-down assays were carried out with proteins encoded by the pvr1 alleles and VPg from two different strains of Tobacco etch virus (TEV) that differentially infected Capsicum lines carrying these genes. While the protein encoded by the susceptible allele pvr1+ interacted strongly, proteins translated from all three resistance alleles (pvr1, pvr1(1), and pvr1(2)) failed to bind VPg from either strain of TEV. This failure to bind correlated with resistance or reduced susceptibility, suggesting that interruption of the interaction between VPg and this eIF4E paralog may be necessary, but is not sufficient for potyvirus resistance in vivo. Among the three resistance alleles, only the pvr1 gene product failed to bind m7-GTP cap-analog columns, suggesting that disrupted cap binding is not required for potyvirus resistance.

  11. Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase.

    PubMed

    Panasenko, Olesya O; Collart, Martine A

    2012-02-01

    In this study, we determine that Saccharomyces cerevisiae Not4 E3 ligase ubiquitinates Rps7A in vivo and in vitro, but not its paralogue, Rps7B. Ubiquitinated Rps7A is detectable only in 80S and polysomes, but not in free 40S fractions. A different role of the Rps7 paralogues in vivo is supported by the observation that the deletion of Rps7A but not Rps7B is sensitive to translational inhibitors and leads to an accumulation of aggregated proteins. An important accumulation of aggregated proteins that include ribosomal proteins and ribosome-associated chaperones is also observed in cells lacking Not4. A contribution of Not4 to ribosomal function extending beyond Rps7A ubiquitination is supported by the observation that the deletion of Not4 displays a synthetic slow growth phenotype when combined with the deletion of either one of the two Rps7 paralogues. Not4 is detectable in polysome fractions, as are other subunits of the Ccr4-Not complex such as Not5. The optimal presence of Not5 in polysomes is dependent upon Not4 and the deletion of Not5 leads to a dramatic reduction of polysomes. These results lead us to suggest that Not4 contributes to normal polysome levels and is important for cellular protein solubility maybe in part by ubiquitination of Rps7A.

  12. Turnip mosaic virus (TuMV) is able to use alleles of both eIF4E and eIF(iso)4E from multiple loci of the diploid Brassica rapa.

    PubMed

    Jenner, Carol E; Nellist, Charlotte F; Barker, Guy C; Walsh, John A

    2010-11-01

    Three copies of eIF4E and three copies of eIF(iso)4E have been identified and sequenced from a Turnip mosaic virus (TuMV)-susceptible, inbred, diploid Brassica rapa line, R-o-18. One of the copies of eIF4E lacked exons 2 and 3 and appeared to be a pseudogene. The two other copies of eIF4E and two of the three copies of eIF(iso)4E were isolated from a bacterial artificial chromosome library of R-o-18. Using an Arabidopsis line (Col-0::dSpm) with a transposon knock-out of the eIF(iso)4E gene which resulted in a change from complete susceptibility to complete resistance to TuMV, complementation experiments were carried out with the two versions of eIF4E and the two versions of eIF(iso)4E. When transformed into Col-0::dSpm, all four Brassica transgenes complemented the Arabidopsis eIF(iso)4E knock-out, conferring susceptibility to both mechanical and aphid challenge with TuMV. One of the copies of eIF4E did not appear to support viral replication as successfully as the other copy of eIF4E or the two copies of eIF(iso)4E. The results show that TuMV can use both eIF4E and eIF(iso)4E from B. rapa for replication and, for the first time, that a virus can use eIF4E and eIF(iso)4E from multiple loci of a single host plant.

  13. Turnip mosaic virus (TuMV) is able to use alleles of both eIF4E and eIF(iso)4E from multiple loci of the diploid Brassica rapa.

    PubMed

    Jenner, Carol E; Nellist, Charlotte F; Barker, Guy C; Walsh, John A

    2010-11-01

    Three copies of eIF4E and three copies of eIF(iso)4E have been identified and sequenced from a Turnip mosaic virus (TuMV)-susceptible, inbred, diploid Brassica rapa line, R-o-18. One of the copies of eIF4E lacked exons 2 and 3 and appeared to be a pseudogene. The two other copies of eIF4E and two of the three copies of eIF(iso)4E were isolated from a bacterial artificial chromosome library of R-o-18. Using an Arabidopsis line (Col-0::dSpm) with a transposon knock-out of the eIF(iso)4E gene which resulted in a change from complete susceptibility to complete resistance to TuMV, complementation experiments were carried out with the two versions of eIF4E and the two versions of eIF(iso)4E. When transformed into Col-0::dSpm, all four Brassica transgenes complemented the Arabidopsis eIF(iso)4E knock-out, conferring susceptibility to both mechanical and aphid challenge with TuMV. One of the copies of eIF4E did not appear to support viral replication as successfully as the other copy of eIF4E or the two copies of eIF(iso)4E. The results show that TuMV can use both eIF4E and eIF(iso)4E from B. rapa for replication and, for the first time, that a virus can use eIF4E and eIF(iso)4E from multiple loci of a single host plant. PMID:20672877

  14. 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. PMID:26292231

  15. Initial testing of two DEMI (Driesbach Electromotive Inc. ) Model 4E zinc-air rechargeable cells

    SciTech Connect

    Hardin, J.E.; Martin, M.E.

    1989-10-23

    The purpose of this document is to report the results of INEL laboratory testing of two DEMI 4E Aerobic Power Battery Cells (collectively designated Pack 46 in INEL records). The 4E Aerobic Power Battery is a secondary battery developed privately by Driesbach Electromotive Inc. (DEMI). The battery employs zinc as the anode and a bifunctional air cathode. This testing was performed as the first phase of a cooperative agreement between INEL and DEMI leading to the construction and testing of electric vehicle-size cells, to be followed eventually by a battery pack. 3 refs., 3 figs., 5 tabs.

  16. 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

  17. Activation of mTOR in a subgroup of ovarian carcinomas: correlation with p-eIF-4E and prognosis.

    PubMed

    Noske, Aurelia; Lindenberg, Juliane Lena; Darb-Esfahani, Silvia; Weichert, Wilko; Buckendahl, Ann-Christin; Röske, Annika; Sehouli, Jalid; Dietel, Manfred; Denkert, Carsten

    2008-12-01

    Ovarian carcinoma patients have an extremely poor prognosis; therefore, new molecular therapeutic approaches are urgently needed. The mTOR pathway, which may be targeted by substances such as Rapamycin or RAD001, is emerging as a promising target for anticancer therapy. So far, the expression and prognostic impact of mTOR signalling elements have not been completely studied in ovarian tumors. We analyzed p-mTOR, p-4E-BP1 and p-eIF-4E in 107 human ovarian lesions and observed an overexpression of p-mTOR (47%) and p-eIF-4E (56%) protein in primary ovarian carcinomas as compared to borderline tumors. Phospho-mTOR expression was significantly related to p-eIF-4E (p< or =0.001) and serous histological type (p=0.03). Increased p-4E-BP1 (31%) was associated with poor differentiation (p=0.04) and higher mitotic rate (p=0.004). In univariate analysis, increased expression of p-mTOR and p-eIF-4E was significantly associated with better overall survival (p=0.003, p=0.029). To connect the expression data with mechanistic studies, a set of 10 ovarian cancer cell lines was used. Expression of p-mTOR was increased in all cancer cell lines as compared to ovarian surface epithelial (HOSE) cells. Rapamycin treatment revealed a reduction of p-mTOR and p-4E-BP1 but increased p-AKT levels. We show for the first time an association of p-mTOR and p-eIF-4E with better overall survival for ovarian cancer patients. The combined results of our in vivo and cell culture studies suggest that a subpopulation of these patients may benefit from mTOR inhibition. The design of future clinical trials should incorporate biomarker testing to determine predictive markers for response to mTOR inhibitors.

  18. 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. PMID:27194579

  19. 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.

  20. Knockdown of eukaryotic translation initiation factor 4E suppresses cell growth and invasion, and induces apoptosis and cell cycle arrest in a human lung adenocarcinoma cell line.

    PubMed

    Chen, Baofu; Zhang, Bo; Xia, Lilong; Zhang, Jian; Chen, Yu; Hu, Quanteng; Zhu, Chengchu

    2015-12-01

    Eukaryotic translation initiation factor 4E (eIF4E) was shown to be upregulated in malignant human tumors. To assess the effect of downregulation of eIF4E on the proliferation and invasiveness of a human lung adenocarcinoma cell line, a short hairpin (sh)RNA targeting eIF4E was constructed and transfected into A549 human lung adenocarcinoma cells. The expression of eIF4E was determined by reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell viability was assessed using a Cell Counting kit‑8, and apoptosis levels and cell cycle distribution were assessed by flow cytometry. Invasiveness was assessed using Transwell chambers. Transfection of the A549 cells with eIF4E targeting shRNA reduced the mRNA and protein expression levels of eIF4E by >70% 48 and 72 h following transfection, and eIF4E targeting shRNA‑transfected cells were significantly less viable compared with the cells transfected with scrambled shRNA. The rate of apoptosis was also significantly increased, significantly more cells were in the G0/G1 phase and fewer were in the S phase, indicating cell cycle arrest. The fraction of transfected cells migrating across Transwell inserts were also reduced. In conclusion, inhibition of eIF4E suppressed cell growth and invasion, induced apoptosis and cell cycle arrest, suggesting that eIF4E may be a potential therapeutic target in lung adenocarcinoma.

  1. A Phase 1 Dose-Escalation, Pharmacokinetic, and Pharmacodynamic Evaluation of eIF-4E Antisense Oligonucleotide LY2275796 in Patients with Advanced Cancer

    PubMed Central

    Hong, David S.; Kurzrock, Razelle; Oh, Yun; Wheler, Jennifer; Naing, Aung; Brail, Les; Callies, Sophie; André, Valérie; Kadam, Sunil K; Nasir, Aejaz; Holzer, Timothy R.; Meric-Bernstam, Funda; Fishman, Mayer; Simon, George

    2016-01-01

    Purpose The antisense oligonucleotide, LY2275796, blocks expression of eIF-4E, an mRNA translation regulator upregulated in tumors. This Phase I study sought an appropriate LY2275796 dose in patients with advanced tumors. Experimental Design A 3-day loading dose, then weekly maintenance doses, were given to 1–3 patient cohorts, beginning with 100 mg and escalating. Plasma samples were collected to determine LY2275796 concentrations; tumor biopsies, to quantify eIF-4E mRNA/protein. Results Thirty patients with Stage 4 disease received ≥1 LY2275796 dose. A dose-limiting toxicity was observed at 1200 mg, with 1000 mg the maximum-tolerated dose. Across all dose levels, most patients (87%) had only grade 1–2 toxicities. LY2275796 pharmacokinetics supported the dosing regimen. Comparison of pre- and post-dose biopsies showed eIF-4E decreased in most patients. Fifteen patients had progressive disease, and seven patients achieved stable disease (minimum of 6 weeks) as best response, with two patients on therapy >3 months (one with melanoma, one with cystadenocarcinoma of the head/neck). Conclusions LY2275796 was well tolerated up to 1000 mg. Since tumor eIF-4E expression was decreased, but no tumor response observed, LY2275796 should be studied combined with other treatment modalities. PMID:21831956

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

    PubMed

    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

    2016-05-10

    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.

  3. 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

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

    PubMed

    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

    2016-05-10

    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

  5. 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

  6. 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.

  7. 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)

  8. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  9. Skyrmions with quadratic band touching fermions: A way to achieve charge 4e superconductivity

    NASA Astrophysics Data System (ADS)

    Moon, Eun-Gook

    2012-06-01

    We study Skyrmion quantum numbers, charge, and statistics, in (2+1) dimension induced by quadratic band touching (QBT) fermions. It is shown that induced charge of Skyrmions is twice bigger than corresponding Dirac particles’ and their statistics are always bosonic. Applying to the Bernal stacking bilayer graphene, we show that Skyrmions of quantum spin Hall are charge 4e bosons, so their condensation realizes charge 4e superconductivity. The phase transition could be of second order, and one candidate theory of the transition is an O(5) nonlinear sigma model with a nonzero Wess-Zumino-Witten term. We calculate the renormalization group beta function of the model perturbatively and propose a possible phase diagram. We also discuss how QBT fermions are different from two copies of Dirac particles.

  10. 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.

  11. Electron-impact excitation of the 31. 4-eV band in N sub 2

    SciTech Connect

    de Souza, G.G.B.; Bielschowsky, C.E.; Lucas, C.A.; Souza, A.C.A. )

    1990-08-01

    Generalized oscillator strengths (GOS) for the dipole-forbidden 31.4-eV band in N{sub 2} have been determined both experimentally and theoretically. The experimental values for the GOS were obtained using a crossed-beam electron spectrometer at 1-keV impact energy. The theoretical results were determined using the first Born approximation with {ital ab} {ital initio} configuration-interaction target wave functions.

  12. 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.

  13. 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

  14. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  15. 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

  16. 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

  17. Methionine hydroxy analogue enhanced fish immunity via modulation of NF-κB, TOR, MLCK, MAPKs and Nrf2 signaling in young grass carp (Ctenopharyngodon idella).

    PubMed

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

    2016-09-01

    Our study investigated the effect of dietary methionine hydroxy analogue (MHA) on growth and immunity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (259.70 ± 0.47 g) were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one dl-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. The results indicated that optimal MHA increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and up-regulated mRNA levels of liver expressed antimicrobial peptide 2, hepcidin (head kidney), β-defensin-1 in the immune organs (P < 0.05), suggesting that MHA could enhance antimicrobial ability of fish. Meanwhile, optimal MHA enhanced the immune function of immune organs via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase β, p38 mitogen activated protein kinase, eIF4E-binding protein1 (4E-BP1) and 4E-BP2 mRNA levels and up-regulation of inhibitor of κBα, ribosomal protein S6 kinase 1 and target of rapamycin mRNA levels (P < 0.05). In addition, optimal MHA improved cellular structure integrity of immune organs via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < 0.05). Simultaneously, optimal MHA improved cellular structure integrity of immune organs via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < 0.05). Besides, optimal MHA improved

  18. Methionine hydroxy analogue enhanced fish immunity via modulation of NF-κB, TOR, MLCK, MAPKs and Nrf2 signaling in young grass carp (Ctenopharyngodon idella).

    PubMed

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

    2016-09-01

    Our study investigated the effect of dietary methionine hydroxy analogue (MHA) on growth and immunity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (259.70 ± 0.47 g) were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one dl-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. The results indicated that optimal MHA increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and up-regulated mRNA levels of liver expressed antimicrobial peptide 2, hepcidin (head kidney), β-defensin-1 in the immune organs (P < 0.05), suggesting that MHA could enhance antimicrobial ability of fish. Meanwhile, optimal MHA enhanced the immune function of immune organs via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase β, p38 mitogen activated protein kinase, eIF4E-binding protein1 (4E-BP1) and 4E-BP2 mRNA levels and up-regulation of inhibitor of κBα, ribosomal protein S6 kinase 1 and target of rapamycin mRNA levels (P < 0.05). In addition, optimal MHA improved cellular structure integrity of immune organs via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < 0.05). Simultaneously, optimal MHA improved cellular structure integrity of immune organs via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < 0.05). Besides, optimal MHA improved

  19. 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...

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

    PubMed

    Doyle-Cooper, Colleen; Hudson, Krystalyn E; Cooper, Anthony B; Ota, Takayuki; Skog, Patrick; Dawson, Phillip E; Zwick, Michael B; Schief, William R; Burton, Dennis R; Nemazee, David

    2013-09-15

    A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. 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 companion article (Ota et al. 2013. J. Immunol. 191: 3179-3185), 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 Ig 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 a membrane proximal external region-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

  1. 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.

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

    PubMed

    Doyle-Cooper, Colleen; Hudson, Krystalyn E; Cooper, Anthony B; Ota, Takayuki; Skog, Patrick; Dawson, Phillip E; Zwick, Michael B; Schief, William R; Burton, Dennis R; Nemazee, David

    2013-09-15

    A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. 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 companion article (Ota et al. 2013. J. Immunol. 191: 3179-3185), 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 Ig 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 a membrane proximal external region-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.

  3. Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing

    PubMed Central

    Guo, Wei Mei; Kong, Kiat Whye; Brown, Christopher John; Quah, Soo Tng; Yeo, Hui Ling; Hoon, Shawn; Seow, Yiqi

    2014-01-01

    Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High eIF4e levels contribute to malignancies, thus eIF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. Here, we demonstrate a method for the rapid selection of looped DNA aptamers against eIF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to eIF4e and inhibit translation initiation in biochemical assays. When transfected into cells, eIF4e aptamers cause a dramatic loss of cell proliferation in tumor cells as seen with eIF4e knockdown with antisense oligonucleotides, shRNAs, and siRNAs, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, we demonstrate that selection happens in waves and that sequencing data can be used to infer aptamer structure. Lastly, we show that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest. PMID:25514650

  4. 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.

  5. 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

  6. Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

    PubMed

    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.

  7. 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.

  8. A novel homozygous p.R1105X mutation of the AP4E1 gene in twins with hereditary spastic paraplegia and mycobacterial disease.

    PubMed

    Kong, Xiao-Fei; Bousfiha, Aziz; Rouissi, Abdelfettah; Itan, Yuval; Abhyankar, Avinash; Bryant, Vanessa; Okada, Satoshi; Ailal, Fatima; Bustamante, Jacinta; Casanova, Jean-Laurent; Hirst, Jennifer; Boisson-Dupuis, Stéphanie

    2013-01-01

    We report identical twins with intellectual disability, progressive spastic paraplegia and short stature, born to a consanguineous family. Intriguingly, both children presented with lymphadenitis caused by the live Bacillus Calmette-Guérin (BCG) vaccine. Two syndromes - hereditary spastic paraplegia (HSP) and mycobacterial disease - thus occurred simultaneously. Whole-exome sequencing (WES) revealed a homozygous nonsense mutation (p.R1105X) of the AP4E1 gene, which was confirmed by Sanger sequencing. The p.R1105X mutation has no effect on AP4E1 mRNA levels, but results in lower levels of AP-4ε protein and of the other components of the AP-4 complex, as shown by western blotting, immunoprecipitation and immunofluorescence. Thus, the C-terminal part of the AP-4ε subunit plays an important role in maintaining the integrity of the AP-4 complex. No abnormalities of the IL-12/IFN-γ axis or oxidative burst pathways were identified. In conclusion, we identified twins with autosomal recessive AP-4 deficiency associated with HSP and mycobacterial disease, suggesting that AP-4 may play important role in the neurological and immunological systems. PMID:23472171

  9. 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

  10. 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.

  11. Vaginal Lactobacillus gasseri CMUL57 can inhibit herpes simplex type 2 but not Coxsackievirus B4E2.

    PubMed

    Kassaa, Imad Al; Hober, Didier; Hamze, Monzer; Caloone, Delphine; Dewilde, Anny; Chihib, Nour-Eddine; Drider, Djamel

    2015-06-01

    This study aimed at demonstrating the antiviral activity of Lactobacillus gasseri CMUL57 (L. gasseri CMUL57), L. acidophilus CMUL67 and L. plantarum CMUL140 against herpes simplex type 2 (HSV-2) and Coxsackievirus B4E2 (CVB4E2), which are enveloped and naked viruses, respectively. These lactobacilli were non-cytotoxic and were able to reduce the cytopathic effect induced by HSV-2 in Vero cell monolayers. However, lactobacilli were not active against CVB4E2. Tested lactobacilli displayed anti-HSV-2 activity when they were co-incubated with the virus prior to inoculating the mixture to Vero cell monolayers. The detection of HSV-2 DNA by PCR in pellets of bacteria/virus mixtures let us to hypothesize that anti-HSV-2 activity of lactobacilli resulted from the viruses' entrapment. This study showed the capabilities of vaginal lactobacilli to inhibit enveloped viruses such as HSV-2.

  12. 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.

  13. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

    PubMed Central

    Graves, L M; Bornfeldt, K E; Argast, G M; Krebs, E G; Kong, X; Lin, T A; Lawrence, J C

    1995-01-01

    Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7638171

  14. Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

    PubMed

    Park, Jungyun; Ahn, Seyoung; Jayabalan, Aravinth K; Ohn, Takbum; Koh, Hyun Chul; Hwang, Jungwook

    2016-07-01

    Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets. PMID:26708722

  15. Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis.

    PubMed

    Zhou, Qian; Liu, Chunxiao; Liu, Wen; Zhang, Hai; Zhang, Ruijie; Liu, Jia; Zhang, Jinfei; Xu, Chong; Liu, Lei; Huang, Shile; Chen, Long

    2015-01-01

    Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson's disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H₂O₂) involved, which was further verified by imaging intracellular H₂O₂ using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H₂O₂-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H₂O₂ and cell apoptosis. Furthermore, we noticed that rotenone-induced H₂O₂ was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H₂O₂ inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants.

  16. 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. PMID:17358756

  17. Kalman filter design for the long range intercept function of the F-4E/G fire control system

    NASA Astrophysics Data System (ADS)

    Halbert, R. C.

    1985-12-01

    This study examines reduced-order Kalman filters designed to improve performance of the F-4E/G long range air-to-air missile capability (LRI function). Operational requirements dictate a high degree of accuracy and constraints imposed by existing hardware mandate minimal complexity. Two linear dynamics models are proposed, one based on constant target velocity, and the other based on time-correlated target acceleration. Both are defined in inertial Cartesian coordinates aligned with north, east, and down. A nonlinear model is developed for measurements available in the existing F-4E/G hardware, including range, range rate, radar antenna gimbal angles, and radar antenna rates. The models are implemented in extended Kalman filter formulations employing linear propagation equations to avoid on-line numerical integration. Performance evaluations are performed on three test trajectories using Monte Carlo analysis. Filter tuning, error budgets, adaptive techniques, and observability issues are addressed during filter evaluation. Results of the evaluation indicate the filter designs can meet the requirements of the F-4E/G fire control system. Recommendations are made for continued testing and for operational implementation.

  18. Mobile chemical detector (AP2C+SP4E) as an aid for medical decision making in the battlefield.

    PubMed

    Eisenkraft, Arik; Markel, Gal; Simovich, Shirley; Layish, Ido; Hoffman, Azik; Finkelstein, Arseny; Rotman, Eran; Dushnitsky, Tsvika; Krivoy, Amir

    2007-09-01

    The combination of the AP2C unit with the SP4E kit composes a lightweight mobile detector of chemical warfare agents (CWA), such as nerve and mustard agents, with both vapor- and liquid-sampling capabilities. This apparatus was recently introduced into our military medical units as an aid for detection of CWA on casualties. Importantly, critical information regarding the applicability in the battlefield was absent. In view of the serious consequences that might follow a proclamation of CWA recognition in battlefield, a high false-positive rate positions the utilization of this apparatus as a medical decision tool in question. We have therefore conducted a field experiment to test the false-positive rate as well as analyze possible factors leading to false-positive readings with this device. The experiment was carried out before and after a 4-day army field exercise, using a standard AP2C device, a SP4E surface sampling kit, and a specially designed medical sampling kit for casualties, intended for medical teams. Soldiers were examined at rest, after mild exercise, and after 4 days in the field. The readings with AP2C alone were compared to the combination of AP2C and SP4E and to the medical sampling kit. Various body fluids served as negative controls. Remarkably, we found a false-positive rate of 57% at rest and after mild exercise, and an even higher rate of 64% after the 4-day field exercise with the AP2C detector alone, as compared to almost no false-positive readings with the combination of AP2C and SP4E. Strikingly, the medical sampling kit has yielded numerous false-positive readings, even in normal body fluids such as blood, urine, and saliva. We therefore see no place for using the medical sampling kit due to an unaccepted high rate of false-positive readings. Finally, we have designed an algorithm that uses the entire apparatus of AP2C and SP4E as a reliable validation tool for medical triage in the setting of exposure to nerve agents in the battlefield.

  19. Atrogin-1, MuRF1, and FoXO, as well as phosphorylated GSK-3beta and 4E-BP1 are reduced in skeletal muscle of chronic spinal cord-injured patients.

    PubMed

    Léger, Bertrand; Senese, Rosalba; Al-Khodairy, Abdul W; Dériaz, Olivier; Gobelet, Charles; Giacobino, Jean-Paul; Russell, Aaron P

    2009-07-01

    Chronic complete spinal cord injury (SCI) is associated with severe skeletal muscle atrophy as well several atrophy and physical-inactivity-related comorbidity factors such as diabetes, obesity, lipid disorders, and cardiovascular diseases. Intracellular mechanisms associated with chronic complete SCI-related muscle atrophy are not well understood, and thus their characterization may assist with developing strategies to reduce the risk of comorbidity factors. Therefore, the aim of this study was to determine whether there was an increase in catabolic signaling targets, such as atrogin-1, muscle ring finger-1 (MuRF1), forkhead transcription factor (FoXO), and myostatin, and decreases in anabolic signaling targets, such as insulin-like growth factor (IGF), v-akt murine thymoma viral oncogene (Akt), glycogen synthase kinase-beta (GSK-3beta), mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and p70(s6kinase) in chronic complete SCI patients. In SCI patients, when compared with controls, there was a significant reduction in mRNA levels of atrogin-1 (59%; P < 0.05), MuRF1 (55%; P < 0.05), and myostatin (46%; P < 0.01), and in protein levels of FoXO1 (72%; P < 0.05), FoXO3a (60%; P < 0.05), and atrogin-1 (36%; P < 0.05). Decreases in the protein levels of IGF-1 (48%; P < 0.001) and phosphorylated GSK-3beta (54%; P < 0.05), 4E-BP1 (48%; P < 0.05), and p70(s6kinase) (60%; P = 0.1) were also observed, the latter three in an Akt- and mTOR-independent manner. Reductions in atrogin-1, MuRF1, FoXO, and myostatin suggest the existence of an internal mechanism aimed at reducing further loss of muscle proteins during chronic SCI. The downregulation of signaling proteins that regulate anabolism, such as IGF, GSK-3beta, and 4E-BP1, would reduce the ability to increase protein synthesis rates.

  20. The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro.

    PubMed

    Westman, Belinda; Beeren, Lisa; Grudzien, Ewa; Stepinski, Janusz; Worch, Remigiusz; Zuberek, Joanna; Jemielity, Jacek; Stolarski, Ryszard; Darzynkiewicz, Edward; Rhoads, Robert E; Preiss, Thomas

    2005-10-01

    The eukaryotic initiation factor eIF4E binds the mRNA 5' cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m(7)GTP and m(7)GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.

  1. The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

    PubMed Central

    WESTMAN, BELINDA; BEEREN, LISA; GRUDZIEN, EWA; STEPINSKI, JANUSZ; WORCH, REMIGIUSZ; ZUBEREK, JOANNA; JEMIELITY, JACEK; STOLARSKI, RYSZARD; DARZYNKIEWICZ, EDWARD; RHOADS, ROBERT E.; PREISS, THOMAS

    2005-01-01

    The eukaryotic initiation factor eIF4E binds the mRNA 5′ cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m7GTP and m7GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap. PMID:16131589

  2. 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.

  3. 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. PMID:26063273

  4. 17 CFR 249.820 - Form 19b-4(e) for the listing and trading of new derivative securities products by self...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Forms for Self-Regulatory Organization Rule Changes and Forms for Registration of and Reporting by... deemed a proposed rule change, pursuant to Rule 19b-4(e) under the Act (17 CFR 240.19b-4(e)). Editorial... and trading of new derivative securities products by self-regulatory organizations that are not...

  5. 17 CFR 249.820 - Form 19b-4(e) for the listing and trading of new derivative securities products by self...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Forms for Self-Regulatory Organization Rule Changes and Forms for Registration of and Reporting by... deemed a proposed rule change, pursuant to Rule 19b-4(e) under the Act (17 CFR 240.19b-4(e)). Editorial... and trading of new derivative securities products by self-regulatory organizations that are not...

  6. 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...

  7. Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

    PubMed

    Igoucheva, Olga; Alexeev, Vitali; Halabi, Carmen M; Adams, Sheila M; Stoilov, Ivan; Sasaki, Takako; Arita, Machiko; Donahue, Adele; Mecham, Robert P; Birk, David E; Chu, Mon-Li

    2015-08-28

    Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

  8. Isolation of Flavonoids from Deguelia duckeana and Their Effect on Cellular Viability, AMPK, eEF2, eIF2 and eIF4E.

    PubMed

    Cursino, Lorena M C; Lima, Nerilson M; Murillo, Renato; Nunez, Cecilia V; Merfort, Irmgard; Humar, Matjaz

    2016-01-01

    Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4'-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpine (3), 4-hydroxylonchocarpine (4), 4-methoxylonchocarpine (5), 5-hydroxy-4',7-dimethoxy-6-prenylflavanone (6), 4'-hydroxyisolonchocarpine (7), 4'-methoxyisolonchocarpine (8), 3',4',7-trimethoxyflavone (9), 3',4'-methylenedioxy-7-methoxyflavone (10), and 2,2-dimethyl-chromone-5,4'-hydroxy-5'-methoxyflavone (11). Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH) release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK) and the eukaryotic elongation factor 2 (eEF2). The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives. PMID:26861281

  9. Interaction Patterns between Potato Virus Y and eIF4E-Mediated Recessive Resistance in the Solanaceae

    PubMed Central

    Janzac, Bérenger; Ruellan, Youna; Simon, Vincent; Ben Khalifa, Mekki; Fakhfakh, Hatem; Fabre, Frédéric; Palloix, Alain

    2014-01-01

    ABSTRACT The structural pattern of infectivity matrices, which contains infection data resulting from inoculations of a set of hosts by a set of parasites, is a key parameter for our understanding of biological interactions and their evolution. This pattern determines the evolution of parasite pathogenicity and host resistance, the spatiotemporal distribution of host and parasite genotypes, and the efficiency of disease control strategies. Two major patterns have been proposed for plant-virus genotype infectivity matrices. In the gene-for-gene model, infectivity matrices show a nested pattern, where the host ranges of specialist virus genotypes are subsets of the host ranges of less specialized viruses. In contrast, in the matching-allele (MA) model, each virus genotype is specialized to infect one (or a small set of) host genotype(s). The corresponding infectivity matrix shows a modular pattern where infection is frequent for plants and viruses belonging to the same module but rare for those belonging to different modules. We analyzed the structure of infectivity matrices between Potato virus Y (PVY) and plant genotypes in the family Solanaceae carrying different eukaryotic initiation factor 4E (eIF4E)-coding alleles conferring recessive resistance. Whereas this system corresponds mechanistically to an MA model, the expected modular pattern was rejected based on our experimental data. This was mostly because PVY mutations involved in adaptation to a particular plant genotype displayed frequent pleiotropic effects, conferring simultaneously an adaptation to additional plant genotypes with different eIF4E alleles. Such effects should be taken into account for the design of strategies of sustainable control of PVY through plant varietal mixtures or rotations. IMPORTANCE The interaction pattern between host and virus genotypes has important consequences on their respective evolution and on issues regarding the application of disease control strategies. We found that

  10. 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

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

    PubMed

    Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen; Schneider, Robert J

    2015-08-01

    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.

  12. Ozonolysis of alkanes and reactions of polyfunctional compounds. XXXVI. Synthesis of ethyl 3,7,10-trimethyl-2E,4E-dodecadienoate

    SciTech Connect

    Odinokov, V.N.; Kukovinets, O.S.; Zainullin, R.A.; Sultanmuratova, V.R.; Tolstikov, G.A.

    1988-12-10

    Hydroprene was obtained with an overall yield of 16% in the form of 77:23 mixture of the 2E,4E and 2Z,4E isomers by ozonolysis of 1,5-dimethyl-1-cyclooctene and selective transformations of the 4-methyl-1,1-dimethoxy-8-oxononane, including ethoxycarbonylmethylenation of the keto group, alkylation of the formyl group by isopropylmagnesium bromide followed by deoxygenation, and allylic bromination-dehydrobromination. The individual 2E,4E isomer was isolated by HPLC.

  13. The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in Acute Myeloid Leukemia

    PubMed Central

    Hariri, Fadi; Arguello, Meztli; Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Nielsen, Torsten Holm; Hiscott, John; Mann, Koren K.; Borden, Katherine L.B.

    2015-01-01

    The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers including the M4 and M5 subtypes of acute myeloid leukemia (AML). While eIF4E RNA levels are elevated 3–10 fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of NF-κB that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the transcriptional start site. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens. PMID:23467026

  14. Evolution of the excited electron bubble in liquid H4e and the appearance of fission-like processes

    NASA Astrophysics Data System (ADS)

    Mateo, David; Pi, Martí; Barranco, Manuel

    2010-05-01

    We have studied the evolution of an excited electron bubble in superfluid H4e for several tens of picosecond combining the dynamics of the liquid with an adiabatic evolution for the electron. The path followed by the excited bubble in its decay to the ground state is shown to strongly depend on pressure. While for pressures below 1 bar the 1P excited electron bubble has allowance for radiatively decay to the deformed ground state, evolving then nonradiatively toward the ground state of the spherical electron bubble, we have found that above 1 bar two distinct baby bubbles appear in the course of the dynamical evolution, pointing to a different relaxation path in which the electron may be localized in one of the baby bubbles while the other collapses, allowing for a pure radiationless de-excitation. Our calculations are in agreement with experiments indicating that relaxed 1P bubbles are only observed for pressures smaller than a critical one, on the order of 1 bar, and that above this value the decay of the excited bubble has to proceed differently. A similar analysis carried out for the 2P bubble shows that the adiabatic approximation fails at an early stage of its dynamical evolution due to the crossing of the 2P and 1F states.

  15. 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).

  16. 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.

  17. 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. PMID:27428868

  18. 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.

  19. Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10

    PubMed Central

    Rujas, Edurne; Gulzar, Naveed; Morante, Koldo; Tsumoto, Kouhei; Scott, Jamie K.

    2015-01-01

    ABSTRACT The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed ΔLoop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Collectively, the data ruled out major conformational changes of CDR-H3 at any stage during the binding process (equilibrium or transition state). Although these mutations did not impact the affinity of wild-type Fab for the 4E10ep in solution, the two nonneutralizing versions of 4E10 were deficient in binding to MPER inserted in the plasma membrane (mimicking the environment faced by the antibody in vivo). The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids. IMPORTANCE The broadly neutralizing anti-HIV-1 4E10 antibody blocks infection caused by nearly all viral strains and isolates examined thus far. However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination

  20. 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.

  1. 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.

  2. The Role of Dynamics and Allostery in the Inhibition of the eIF4E/eIF4G Translation Initiation Factor Complex

    PubMed Central

    Papadopoulos, Evangelos; Blackledge, Martin

    2016-01-01

    Lack of regulation of the eIF4E/eIF4G interaction is a hallmark of cancer. 4EGI-1 binds to eIF4E preventing association to eIF4G via an allosteric mechanism. We use NMR spectroscopy and MD simulations to obtain a mechanistic description of the role of correlated dynamics in this allosteric regulation. We show that binding of 4EGI-1 perturbs native correlated motions and increases correlated fluctuations in part of the eIF4G binding site. PMID:27162083

  3. 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

  4. Expression of a synthetic antimicrobial peptide, D4E1, in Gladiolus plants for resistance to Fusarium oxysporum f. sp. gladioli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The main pathogen of Gladiolus plants is Fusarium oxysporum, a soilborne fungus that infects roots and corms and kills the plant. Purified D4E1, a synthetic antimicrobial peptide, was found to effectively inhibit 100% of F. oxysporum f. sp. gladioli germinated spores from forming a mycelial mass in ...

  5. 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

  6. 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...

  7. 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

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

    PubMed

    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

  9. 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.

  10. Bean common mosaic virus Isolate Exhibits a Novel Pathogenicity Profile in Common Bean, Overcoming the bc-3 Resistance Allele Coding for the Mutated eIF4E Translation Initiation Factor.

    PubMed

    Feng, Xue; Myers, James R; Karasev, Alexander V

    2015-11-01

    Resistance against Bean common mosaic virus (BCMV) in Phaseolus vulgaris is governed by six recessive resistance alleles at four loci. One of these alleles, bc-3, is able to protect P. vulgaris against all BCMV strains and against other potyviruses; bc-3 was identified as the eIF4E allele carrying mutated eukaryotic translation initiation factor gene. Here, we characterized a novel BCMV isolate 1755a that was able to overcome bc-2 and bc-3 alleles in common bean. Thus, it displayed a novel pattern of interactions with resistance genes in P. vulgaris, and was assigned to a new pathogroup, PG-VIII. The IVT7214 cultivar supporting the replication of BCMV-1755a was found to have the intact homozygous bc-3 cleaved amplified polymorphic sequences marker and corresponding mutations in the eIF4E allele that confer resistance to BCMV isolates from all other pathogroups as well as to other potyviruses. The VPg protein of 1755a had seven amino acid substitutions relative to VPgs of other BCMV isolates unable to overcome bc-3. The 1755a genome was found to be a recombinant between NL1, US1 (both PG-I), and a yet unknown BCMV strain. Analysis of the recombination patterns in the genomes of NL1 and US1 (PG-I), NY15P (PG-V), US10 and RU1-OR (PG-VII), and 1755a (PG-VIII), indicated that P1/HC-Pro cistrons of BCMV strains may interact with most resistance genes. This is the first report of a BCMV isolate able to overcome the bc-3 resistance allele, suggesting that the virus has evolved mechanisms to overcome multiple resistance genes available in common bean. PMID:26196181

  11. 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).

  12. Molecular variants of human papilloma virus 16 E2, E4, E5, E6 and E7 genes associated with cervical neoplasia in Romanian patients.

    PubMed

    Plesa, Adriana; Anton, Gabriela; Iancu, Iulia V; Diaconu, Carmen C; Huica, Irina; Stanescu, Anca D; Socolov, Demetra; Nistor, Elena; Popa, Elena; Stoian, Mihai; Botezatu, Anca

    2014-12-01

    The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p < 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis. PMID:25143263

  13. 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

  14. Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6

    PubMed Central

    Schmitt, J; Noble, A; Otsuka, M; Berry, P; Maitland, N J; Rumsby, M G

    2014-01-01

    Background: Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium. Methods: Prostate epithelial cell lines P4E6, LNCaP and PC3 were models of prostate cancer (PCa). PNT2C2 and PNT1A were models of benign prostate epithelia. Cellular EtnPGs were labelled with [1-3H]-Etn hydrochloride. PKC was activated with phorbol ester (TPA) and inhibited with Ro31-8220 and GF109203X. D609 was used to inhibit PLD (phospholipase D). [3H]-labelled Etn metabolites were resolved by ion-exchange chromatography. Sodium oleate and mastoparan were tested as activators of PLD2. Phospholipase D activity was measured by a transphosphatidylation reaction. Cells were treated with ionomycin to raise intracellular Ca2+ levels. Results: Unstimulated cell lines release mainly Etn and glycerylphosphorylEtn (GPEtn) to the medium. Phorbol ester treatment over 3h increased Etn metabolite release from the metastatic PC3 cell line and the benign cell lines PNT2C2 and PNT1A but not from the tumour-derived cell lines P4E6 and LNCaP; this effect was blocked by Ro31-8220 and GF109203X as well as by D609, which inhibited PLD in a transphosphatidylation reaction. Only metastatic PC3 cells specifically upregulated Etn release in response to TPA treatment. Oleate and mastoparan increased GPEtn release from all cell lines at the expense of Etn. Ionomycin stimulated GPEtn release from benign PNT2C2 cells but not from cancer-derived cell lines P4E6 or PC3. Ethanolamine did not stimulate the proliferation of LNCaP or PC3 cell lines but decreased the uptake of choline (Cho). Conclusions: Only the metastatic basal PC3 cell line specifically increased the release of Etn on TPA treatment most probably by PKC activation of

  15. Very slow decay of a defect related emission band at 2.4 eV in AlN: Signatures of the Si related shallow DX state

    NASA Astrophysics Data System (ADS)

    Lamprecht, M.; Grund, C.; Neuschl, B.; Thonke, K.; Bryan, Z.; Collazo, R.; Sitar, Z.

    2016-04-01

    We report on a defect related luminescence band at 2.4 eV in aluminum nitride bulk crystals, for which we find strong indications to be related to silicon DX centers. Time resolved photoluminescence spectroscopy using a sub-bandgap excitation reveals two different recombination processes with very long decay times of 13 ms and 153 ms at low temperature. Based on the results of temperature and excitation dependent photoluminescence experiments, the process with the shorter lifetime is assigned to a donor-acceptor pair transition involving the shallow silicon donor state, which can be emptied with a thermal dissociation energy of 65 meV. The slower process with a thermal quenching energy of 15 meV is assigned to the slightly deeper Si DX state known from electron paramagnetic resonance experiments, which is transferred back to the shallow donor state.

  16. Azaphilones and p-terphenyls from the mangrove endophytic fungus Penicillium chermesinum (ZH4-E2) isolated from the South China Sea.

    PubMed

    Huang, Hongbo; Feng, Xiaojun; Xiao, Ze'en; Liu, Lan; Li, Hanxiang; Ma, Lin; Lu, Yongjun; Ju, Jianhua; She, Zhigang; Lin, Yongcheng

    2011-05-27

    Eight secondary metabolites, including three new azaphilones (chermesinones A-C, 1-3), three new p-terphenyls (6'-O-desmethylterphenyllin, 4; 3-hydroxy-6'-O-desmethylterphenyllin, 5; 3''-deoxy-6'-O-desmethylcandidusin B, 7), and two known p-terphenyls (6, 8), were isolated from the culture of the mangrove endophytic fungus Penicillium chermesinum (ZH4-E2). Their structures were established by spectroscopic analysis. The absolute configuration of 1 was determined by X-ray crystallography. Terphenyls 4, 5, and 6 exhibited strong inhibitory effects against α-glucosidase with IC50 values of 0.9, 4.9, and 2.5 μM, respectively. Terphenyls 7 and 8 showed inhibitory activity toward acetylcholinesterase with IC50 values of 7.8 and 5.2 μM.

  17. 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.

  18. 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

    2015-01-01

    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. PMID:25849803

  19. Plant Cap-binding Complexes Eukaryotic Initiation Factors eIF4F and eIFISO4F

    PubMed Central

    Mayberry, Laura K.; Allen, M. Leah; Nitka, Kelley R.; Campbell, Lara; Murphy, Patricia A.; Browning, Karen S.

    2011-01-01

    The initiation of translation in eukaryotes requires a suite of eIFs that include the cap-binding complex, eIF4F. eIF4F is comprised of the subunits eIF4G and eIF4E and often the helicase, eIF4A. The eIF4G subunit serves as an assembly point for other initiation factors, whereas eIF4E binds to the 7-methyl guanosine cap of mRNA. Plants have an isozyme form of eIF4F (eIFiso4F) with comparable subunits, eIFiso4E and eIFiso4G. Plant eIF4A is very loosely associated with the plant cap-binding complexes. The specificity of interaction of the individual subunits of the two complexes was previously unknown. To address this issue, mixed complexes (eIF4E-eIFiso4G or eIFiso4E-eIF4G) were expressed and purified from Escherichia coli for biochemical analysis. The activity of the mixed complexes in in vitro translation assays correlated with the large subunit of the respective correct complex. These results suggest that the eIF4G or eIFiso4G subunits influence translational efficiency more than the cap-binding subunits. The translation assays also showed varying responses of the mRNA templates to eIF4F or eIFiso4F, suggesting that some level of mRNA discrimination is possible. The dissociation constants for the correct complexes have KD values in the subnanomolar range, whereas the mixed complexes were found to have KD values in the ∼10 nm range. Displacement assays showed that the correct binding partner readily displaces the incorrect binding partner in a manner consistent with the difference in KD values. These results show molecular specificity for the formation of plant eIF4F and eIFiso4F complexes and suggest a role in mRNA discrimination during initiation of translation. PMID:21965660

  20. Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).

    PubMed

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

    2016-05-01

    This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7

  1. Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).

    PubMed

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

    2016-05-01

    This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7

  2. 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

    2014-08-01

    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.

  3. 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; 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 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

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

    PubMed

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

    2010-10-21

    Electron attachment to clusters of N(2)O in the energy range of 0-4 eV yields the ionic complexes [(N(2)O)(n)O](-), [(N(2)O)(n)NO](-), and (N(2)O)(n) (-) . The shape of the ion yields of the three homologous series differs substantially reflecting the different formation mechanisms. While the generation of [(N(2)O)(n)O](-) can be assigned to dissociative electron attachment (DEA) of an individual N(2)O molecule in the target cluster, the formation of [(N(2)O)(n)NO](-) is interpreted via a sequence of ion molecule reactions involving the formation of O(-) via DEA in the first step. The nondecomposed complexes (N(2)O)(n) (-) 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(2)O)(n)O](-) and (N(2)O)(n) (-) 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(2)O clusters [E. Leber, S. Barsotti, J. Bömmels, J. M. Weber, I. I. Fabrikant, M.-W. Ruf, and H. Hotop, Chem. Phys. Lett. 325, 345 (2000)]. PMID:20969408

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

    PubMed

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

    2015-06-18

    Chemical bonding interactions are the main driving force for the formation of molecules and materials from atoms. The two-electron/multicenter pancake π-π bonding found in phenalenyl (PLY, 1) radical π-dimers is intriguing due to its unconventional nature of covalent bonding for molecular aggregations and its propensity to induce unique optical, electronic, and magnetic properties. By using high-level quantum chemistry calculations, we show that the B- or N-doped PLYs (2 and 4), usually considered closed-shell and therefore trifling, can be rendered open-shell singlet by proper edge substitutions (3 and 5). The resulting two unpaired valence electrons on each molecular unit contribute to the formation of a genuine pancake-shaped 4e/all-sites double π-π bonding upon intermolecular π-dimerization, in contrast to the 2e/half-sites single π-π bonding in the parent PLY π-dimers. The unusual double π-π bonding motif discovered in these PLY analogues may broaden the landscape of, and find new applications for, intermolecular covalent bonding interactions.

  6. Reactive scattering of O and H2 and quenching of OH at collision energies up to 4.4 eV

    NASA Astrophysics Data System (ADS)

    Gacesa, Marko; Kharchenko, Vasili

    2016-05-01

    We report new cross sections for the O(3 P) + H2 reactive scattering as well as quenching rates for rotationally and vibrationally excited OH by H atoms for a range of collision energies from 0.4 and 4.4 eV. These processes are important for understanding non-local thermal equilibrium (non-LTE) regime in astrophysical environment such as photon-dominated regions (PDRs) and evolution of planetary atmospheres in time, including the atmospheres of Earth and Mars. The cross sections were calculated quantum mechanically using coupled-channel formalism implemented in MOLSCAT and ABC computer codes on refitted recent potential energy surfaces for 3A' and 3A'' , while the surface-hopping effects were estimated from models and similar atom-molecule reactions. A large basis set was used to ensure the convergence at higher energies. Our results agree well with the published data at lower energies and indicate that reduced-dimensionality approach at collision energies higher than about 1.5 eV may not be adequate. Differential cross sections and diffusion cross sections, of interest in transport calculations, are also reported. This work was been supported by NASA grant NNX10AB88G.

  7. miR-124 downregulation leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal activation

    PubMed Central

    Feng, Tongbao; Shao, Fang; Wu, Qiyong; Zhang, Xiaohang; Xu, Dongqin; Qian, Keqing; Xie, Yewen; Wang, Shizhong; Xu, Ning; Wang, Yong; Qi, Chunjian

    2016-01-01

    The long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recently shown to be dysregulated in several cancers. However, the mechanisms underlying the role of MALAT1 in breast cancer remain unclear. Herein, we showed that MALAT1 was aberrantly increased in breast cancer tissues and cells. MALAT1-siRNA inhibited breast cancer cell proliferation and cell cycle progression in vitro and in vivo. Furthermore, MALAT1 acted as an endogenous potent regulator by directly binding to miR-124 and down-regulating miR-124 expression. In addition, MALAT1 reversed the inhibitory effect of miR-124 on breast cancer proliferation and was involved in the cyclin-dependent kinase 4 (CDK4) expression. Taken together, our data highlight the pivotal role of MALAT1 in breast cancer tumorigenesis. Moreover, the present study elucidated the MALAT1-miR-124-CDK4/E2F1 signaling pathway in breast cancer, which might provide a new approach for tackling breast cancer. PMID:26918449

  8. miR-124 downregulation leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal activation.

    PubMed

    Feng, Tongbao; Shao, Fang; Wu, Qiyong; Zhang, Xiaohang; Xu, Dongqin; Qian, Keqing; Xie, Yewen; Wang, Shizhong; Xu, Ning; Wang, Yong; Qi, Chunjian

    2016-03-29

    The long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recently shown to be dysregulated in several cancers. However, the mechanisms underlying the role of MALAT1 in breast cancer remain unclear. Herein, we showed that MALAT1 was aberrantly increased in breast cancer tissues and cells. MALAT1-siRNA inhibited breast cancer cell proliferation and cell cycle progression in vitro and in vivo. Furthermore, MALAT1 acted as an endogenous potent regulator by directly binding to miR-124 and down-regulating miR-124 expression. In addition, MALAT1 reversed the inhibitory effect of miR-124 on breast cancer proliferation and was involved in the cyclin-dependent kinase 4 (CDK4) expression. Taken together, our data highlight the pivotal role of MALAT1 in breast cancer tumorigenesis. Moreover, the present study elucidated the MALAT1-miR-124-CDK4/E2F1 signaling pathway in breast cancer, which might provide a new approach for tackling breast cancer.

  9. 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.

  10. Molecular Characterization of Plant Ubiquitin-Conjugating Enzymes Belonging to the UbcP4/E2-C/UBCx/UbcH10 Gene Family1

    PubMed Central

    Criqui, Marie Claire; de Almeida Engler, Janice; Camasses, Alain; Capron, Arnaud; Parmentier, Yves; Inzé, Dirk; Genschik, Pascal

    2002-01-01

    The anaphase promoting complex or cyclosome is the ubiquitin-ligase that targets destruction box-containing proteins for proteolysis during the cell cycle. Anaphase promoting complex or cyclosome and its activator (the fizzy and fizzy-related) proteins work together with ubiquitin-conjugating enzymes (UBCs) (E2s). One class of E2s (called E2-C) seems specifically involved in cyclin B1 degradation. Although it has recently been shown that mammalian E2-C is regulated at the protein level during the cell cycle, not much is known concerning the expression of these genes. Arabidopsis encodes two genes belonging to the E2-C gene family (called UBC19 and UBC20). We found that UBC19 is able to complement fission yeast (Schizosaccharomyces pombe) UbcP4-140 mutant, indicating that the plant protein can functionally replace its yeast ortholog for protein degradation during mitosis. In situ hybridization experiments were performed to study the expression of the E2-C genes in various tissues of plants. Their transcripts were always, but not exclusively, found in tissues active for cell division. Thus, the UBC19/20 E2s may have a key function during cell cycle, but may also be involved in ubiquitylation reactions occurring during differentiation and/or in differentiated cells. Finally, we showed that a translational fusion protein between UBC19 and green fluorescent protein localized both in the cytosol and the nucleus in stable transformed tobacco (Nicotiana tabacum cv Bright Yellow 2) cells. PMID:12427990

  11. CGP57380 enhances efficacy of RAD001 in non-small cell lung cancer through abrogating mTOR inhibition-induced phosphorylation of eIF4E and activating mitochondrial apoptotic pathway

    PubMed Central

    Wen, Qiuyuan; Wang, Weiyuan; Luo, Jiadi; Chu, Shuzhou; Chen, Lingjiao; Xu, Lina; Zang, Hongjing; Alnemah, Mohannad Ma; Ma, Jian; Fan, Songqing

    2016-01-01

    The mammalian target of rapamycin (mTOR) is a potentially important therapeutic target in a broad range of cancer types. mTOR inhibitors such as rapamycin and its analogs (rapalogs) have been proven effective as anticancer agents in non-small cell lung cancer (NSCLC), whereas they strongly enhance phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and activation of Akt, which cause resistance to mTOR-targeted therapy after an initial response. Rapamycin induces eIF4E phosphorylation by activating MAPK-interacting kinases (Mnks), and therefore targeting Mnk/eIF4E pathway represents a potential therapeutic strategy for the treatment of NSCLC. Here, our results showed that over-expression of p-Mnk1 and p-eIF4E was significantly associated with poor overall survival of NSCLC patients and high expression of p-Mnk1 might act as an independent prognostic biomarker for these patients. Meanwhile, inhibiting Mnk1 expression by Mnk inhibitor (CGP57380) could abrogate rapalogs (RAD001)-induced eIF4E phosphorylation and Akt activation. Furthermore, combination of CGP57380 and RAD001 could induce NSCLC cells apoptosis via activating intrinsic mitochondrial pathway, and exert synergistic antitumor efficacy both in vitro and in vivo. In conclusion, combination of targeting both mTOR and Mnk/eIF4E signaling pathways to enhance effectiveness of mTOR-targeted cancer therapy might be significant innovation for the personalized treatment of NSCLC. PMID:27050281

  12. Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum.

    PubMed

    Yeam, Inhwa; Kang, Byoung-Cheorl; Lindeman, Wouter; Frantz, James D; Faber, Nanne; Jahn, Molly M

    2005-12-01

    Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 (1), and pvr1 (2). These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.

  13. 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

  14. 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.

  15. 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)].

  16. 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.

  17. 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.

  18. 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).

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

    PubMed Central

    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

    2014-01-01

    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. PMID:25049413

  20. Alcohol myopathy: impairment of protein synthesis and translation initiation.

    PubMed

    Lang, C H; Kimball, S R; Frost, R A; Vary, T C

    2001-05-01

    Alcohol consumption leads to numerous morphological, biochemical and functional changes in skeletal and cardiac muscle. One such change observed in both tissues after either acute alcohol intoxication or chronic alcohol consumption is a characteristic decrease in the rate of protein synthesis. A decrease in translation efficiency appears to be responsible for at least part of the reduction. This review highlights advances in determining the molecular mechanisms by which alcohol impairs protein synthesis and places these observations in context of earlier studies on alcoholic myopathy. Both acute and chronic alcohol administration impairs translational control by modulating various aspects of peptide-chain initiation. Moreover, this alcohol-induced impairment in initiation is associated with a decreased availability of eukaryotic initiation factor (eIF) 4E in striated muscle, as evidenced by an increase in the amount of the inactive eIF4E.4E-BP1 complex and decrease in the active eIF4E.eIF4G complex. In contrast, alcohol does not produce consistent alterations in the control of translation initiation by the eIF2 system. The etiology of these changes remain unresolved. However, defects in the availability or effectiveness of various anabolic hormones, particularly insulin-like growth factor-I, are consistent with the alcohol-induced decrease in protein synthesis and translation initiation.

  1. 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

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

    PubMed

    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 eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before 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

  3. Complete Genome Sequences of vB_LmoS_188 and vB_LmoS_293, Two Bacteriophages with Specificity for Listeria monocytogenes Strains of Serotypes 4b and 4e

    PubMed Central

    Casey, Aidan; Jordan, Kieran; Coffey, Aidan

    2015-01-01

    Listeria monocytogenes is responsible for the rare disease listeriosis, which is associated with the consumption of contaminated food products. We report here the complete genome sequences of vB_LmoS_188 and vB_LmoS_293, phages isolated from environmental sources and that have host specificity for L. monocytogenes strains of the 4b and 4e serotypes. PMID:25858822

  4. 40 CFR 180.1281 - S-Abscisic Acid, (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-1-cyclohex-2-enyl)-3-methyl-penta-(2Z,4E...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false S-Abscisic Acid, (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-1-cyclohex-2-enyl)-3-methyl-penta-(2Z,4E)-dienoic Acid; exemption from the... Exemptions From Tolerances § 180.1281 S-Abscisic Acid,...

  5. 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

  6. Total protein

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  7. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  8. Dietary Proteins

    MedlinePlus

    ... meat, dairy products, nuts, and certain grains and beans. Proteins from meat and other animal products are complete proteins. This means they supply all of the amino acids the body can't make on its own. Most plant proteins are incomplete. You should eat different types ...

  9. 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…

  10. 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

  11. An Australian retrospective study to evaluate the prognostic role of p53 and eIF4E cancer markers in patients with head and neck squamous cell carcinoma (HNSCC): study protocol.

    PubMed

    Singh, Jagtar; Jayaraj, Rama; Baxi, Siddhartha; Mileva, Mariana; Curtin, Justin; Thomas, Mahiban

    2013-01-01

    Complete surgical resection of the primary tumour is a crucial predictive step for head and neck squamous cell carcinoma (HNSCC), because incomplete resection may lead to increase in the recurrence rate. Molecular cancer markers have been investigated as potential predictors of prognosis marker, to identify patients who are at high risk of local recurrence. This retrospective study aimed to determine the prognostic correlation between p53 and eIF4E expression and clinical characteristics, recurrence and overall survival. Forty eight HNSCC patients were selected between 2006 and 2009 diagnosed at the Royal Darwin Hospital, Darwin, Northern Territory, Australia. Out of 48, only those 24 with negative surgical margins with hematoxylin and eosin (HandE) were chosen for further analysis. A total of 77 surgical margins were obtained and subsequently analysed by immunohistochemical (IHC) staining with monoclonal p53 and polyclonal eIF4E antibodies. Contingency table and χ2-test were used to investigate the correlation between p53 and eIF4E expression and clinical characteristics, recurrence and overall survival of the HNSCC patients. The follow up period was 74 months (range 1-74 months). The Kaplan-Meier method was used to generate recurrence and survival curves. This is a first retrospective study of Northern Territory patients, including Indigenous and non-Indigenous Australians. Molecular study of surgical margins could help to identify patients with and without clear margins after surgery and help in choice of the most appropriate adjuvant treatment for HNSCC patients.

  12. The Kissing-Loop T-Shaped Structure Translational Enhancer of Pea Enation Mosaic Virus Can Bind Simultaneously to Ribosomes and a 5′ Proximal Hairpin

    PubMed Central

    Gao, Feng; Gulay, Suna P.; Kasprzak, Wojciech; Dinman, Jonathan D.

    2013-01-01

    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. PMID:23986599

  13. Skeletal muscle protein synthesis and degradation exhibit sexual dimorphism after chronic alcohol consumption but not acute intoxication.

    PubMed

    Lang, Charles H; Frost, Robert A; Vary, Thomas C

    2007-06-01

    Epidemiological evidence suggests alcoholic myopathy is more severe in females than males, but comparable animal studies are lacking that make elucidating the biochemical locus for this defect problematic. The present study determined whether skeletal muscle protein synthesis and markers of degradation exhibit a sexual dimorphic response to either chronic alcohol consumption or acute intoxication. Male and female rats were fed an alcohol-containing diet, pair-fed for 26 wk (chronic), or received an intraperitoneal injection of alcohol (acute). In males, chronic alcohol decreased gastrocnemius protein synthesis by 20%. This reduction was associated with a twofold increase in the inactive eukaryotic initiation factor (eIF) 4E.4E-binding protein 1 (4E-BP1) complex and a 60% reduction in the active eIF4E.eIF4G complex. This redistribution of eIF4E was associated with decreased phosphorylation of both 4E-BP1 and eIF4G (50-55%). The phosphorylation of ribosomal protein S6 was also reduced 60% in alcohol-consuming male rats. In contrast, neither rates of protein synthesis nor indexes of translation initiation in muscle were altered in alcohol-fed female rats despite blood alcohol levels comparable to males. Chronic alcohol ingestion did not alter atrogin-1 or muscle RING finger-1 mRNA content (biomarkers of muscle proteolysis) in males but increased their expression in females 50-100%. Acute alcohol intoxication produced a comparable decrease in muscle protein synthesis and translation initiation in both male and female rats. Our data demonstrate a sexual dimorphism for muscle protein synthesis, translation initiation, and proteolysis in response to chronic, but not acute, alcohol intoxication; however, they do not support evidence indicating females are more sensitive toward the development of alcoholic skeletal muscle myopathy.

  14. Differential effects of insulin and dietary amino acids on muscle protein synthesis in adult and old rats

    PubMed Central

    Prod'homme, Magali; Balage, Michèle; Debras, Elisabeth; Farges, Marie-Chantal; Kimball, Scott; Jefferson, Leonard; Grizard, Jean

    2005-01-01

    The potential roles of insulin and dietary amino acids in the regulation of skeletal muscle protein synthesis were examined in adult and old rats. Animals were fed over 1 h with either a 25% or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained or blocked with diazoxide injections. Protein synthesis in gastrocnemius and soleus muscles was assessed in vivo using the flooding dose method. Insulin suppression decreased protein synthesis in both muscles irrespective of the nutritional condition and age of the rats. Moreover, reduced insulinaemia was associated with 4E-BP1 dephosphorylation, enhanced assembly of the 4E-BP1−eIF4E inactive complex and hypophosphorylation of eIF4E, p70S6k and protein kinase B, key intermediates in the regulation of translation initiation and protein synthesis. Old rats did not differ from adult rats. The lack of amino acids in the meal of insulin-suppressed rats did not result in any additional decrease in protein synthesis. In the presence of insulin secretion, dietary amino acid suppression significantly decreased gastrocnemius protein synthesis in adult but not in old rats. Amino acid suppression was associated with reduced phosphorylation of 4E-BP1 and p70S6k in adults. Along with protein synthesis, only the inhibition of p70S6k phosphorylation was abolished in old rats. We concluded that insulin is required for the regulation of muscle protein synthesis irrespective of age and that the effect of dietary amino acids is blunted in old rats. PMID:15513948

  15. The soy isoflavone equol may increase cancer malignancy via up-regulation of eukaryotic protein synthesis initiation factor eIF4G.

    PubMed

    de la Parra, Columba; Otero-Franqui, Elisa; Martinez-Montemayor, Michelle; Dharmawardhane, Suranganie

    2012-12-01

    Dietary soy is thought to be cancer-preventive; however, the beneficial effects of soy on established breast cancer is controversial. We recently demonstrated that dietary daidzein or combined soy isoflavones (genistein, daidzein, and glycitein) increased primary mammary tumor growth and metastasis. Cancer-promoting molecules, including eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E, were up-regulated in mammary tumors from mice that received dietary daidzein. Herein, we show that increased eIF expression in tumor extracts of mice after daidzein diets is associated with protein expression of mRNAs with internal ribosome entry sites (IRES) that are sensitive to eIF4E and eIF4G levels. Results with metastatic cancer cell lines show that some of the effects of daidzein in vivo can be recapitulated by the daidzein metabolite equol. In vitro, equol, but not daidzein, up-regulated eIF4G without affecting eIF4E or its regulator, 4E-binding protein (4E-BP), levels. Equol also increased metastatic cancer cell viability. Equol specifically increased the protein expression of IRES containing cell survival and proliferation-promoting molecules and up-regulated gene and protein expression of the transcription factor c-Myc. Moreover, equol increased the polysomal association of mRNAs for p 120 catenin and eIF4G. The elevated eIF4G in response to equol was not associated with eIF4E or 4E-binding protein in 5' cap co-capture assays or co-immunoprecipitations. In dual luciferase assays, IRES-dependent protein synthesis was increased by equol. Therefore, up-regulation of eIF4G by equol may result in increased translation of pro-cancer mRNAs with IRESs and, thus, promote cancer malignancy.

  16. Transport proteins.

    PubMed

    Thatcher, Jack D

    2013-04-16

    This Teaching Resource provides and describes two animated lessons that illustrate general properties of transport proteins. The lesson called "transport protein classes" depicts major classes and subclasses of transport proteins. The "transporters, mechanism of action" lesson explains how transporters and P class ATPase (adenosine triphosphatase) pumps function. These animations serve as valuable resources for any collegiate-level course that describes these important factors. Courses that might use them include introductory biology, biochemistry, cell biology, physiology, and biophysics.

  17. Proteins wriggle.

    PubMed Central

    Cahill, Michael; Cahill, Sean; Cahill, Kevin

    2002-01-01

    We propose an algorithmic strategy for improving the efficiency of Monte Carlo searches for the low-energy states of proteins. Our strategy is motivated by a model of how proteins alter their shapes. In our model, when proteins fold under physiological conditions, their backbone dihedral angles change synchronously in groups of four or more to avoid steric clashes and respect the kinematic conservation laws. They wriggle; they do not thrash. We describe a simple algorithm that can be used to incorporate wriggling in Monte Carlo simulations of protein folding. We have tested this wriggling algorithm against a code in which the dihedral angles are varied independently (thrashing). Our standard of success is the average root-mean-square distance (rmsd) between the alpha-carbons of the folding protein and those of its native structure. After 100,000 Monte Carlo sweeps, the relative decrease in the mean rmsd, as one switches from thrashing to wriggling, rises from 11% for the protein 3LZM with 164 amino acids (aa) to 40% for the protein 1A1S with 313 aa and 47% for the protein 16PK with 415 aa. These results suggest that wriggling is useful and that its utility increases with the size of the protein. One may implement wriggling on a parallel computer or a computer farm. PMID:11964253

  18. Mouse monoclonal antibodies against Phytolacca americana antiviral protein PAP I.

    PubMed

    Kaloyanova, D; Kyurkchiev, S; Xu, J; Abouhaidar, M; Ivanov, I

    1999-08-01

    Four hybridoma lines are constructed producing monoclonal antibodies against the pokeweed (Phytolacca americana) antiviral protein PAP I. Two of the antibodies, 4E8 and 5D3, are characterized in more detail. They recognize amino acid sequences rather than conformational changes and their epitopes are 65% distinct. One of these antibodies (5D3) is used to study localization of recombinant PAP I in Escherichia coli cells by immuno-gold electron microscopy.

  19. Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

    PubMed

    Rabe, Patrick; Barra, Lena; Rinkel, Jan; Riclea, Ramona; Citron, Christian A; Klapschinski, Tim A; Janusko, Aron; Dickschat, Jeroen S

    2015-11-01

    An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product. PMID:26361082

  20. Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by (13)C-Labeling Experiments.

    PubMed

    Rabe, Patrick; Barra, Lena; Rinkel, Jan; Riclea, Ramona; Citron, Christian A; Klapschinski, Tim A; Janusko, Aron; Dickschat, Jeroen S

    2015-11-01

    An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)C COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.

  1. 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.

  2. Selective 4e-/4H+ O2 reduction by an iron(tetraferrocenyl)porphyrin complex: from proton transfer followed by electron transfer in organic solvent to proton coupled electron transfer in aqueous medium.

    PubMed

    Mittra, Kaustuv; Chatterjee, Sudipta; Samanta, Subhra; Dey, Abhishek

    2013-12-16

    An iron porphyrin catalyst bearing four ferrocenes and a hydrogen bonding distal pocket is found to catalyze 4e(-)/4H(+) oxygen reduction reaction (ORR) in organic solvent under homogeneous conditions in the presence of 2-3 equiv of Trifluoromethanesulphonic acid. Absorption spectroscopy, electron paramagnetic resonance (EPR), and resonance Raman data along with H2O2 assay indicate that one out of the four electrons necessary to reduce O2 to H2O is donated by the ferrous porphyrin while three are donated by the distal ferrocene residues. The same catalyst shows 4e(-)/4H(+) reduction of O2 in an aqueous medium, under heterogeneous conditions, over a wide range of pH. Both the selectivity and the rate of ORR are found to be pH independent in an aqueous medium. The ORR proceeds via a proton transfer followed by electron transfer (PET) step in an organic medium and while a 2e(-)/1H(+) proton coupled electron transfer (PCET) step determines the electrochemical potential of ORR in an aqueous medium.

  3. 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. PMID:23668610

  4. 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.

  5. 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.

  6. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  7. 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...

  8. The structure at 2.5 Å resolution of human basophilic leukemia-expressed protein BLES03

    PubMed Central

    Bitto, Eduard; Bingman, Craig A.; Robinson, Howard; Allard, Simon T. M.; Wesenberg, Gary E.; Phillips, George N.

    2005-01-01

    The crystal structure of the human basophilic leukemia-expressed protein (BLES03, p5326, Hs.433573) was determined by single-wavelength anomalous diffraction and refined to an R factor of 18.8% (R free = 24.5%) at 2.5 Å resolution. BLES03 shows no detectable sequence similarity to any functionally characterized proteins using state-of-the-art sequence-comparison tools. The structure of BLES03 adopts a fold similar to that of eukaryotic transcription initiation factor 4E (eIF4E), a protein involved in the recognition of the cap structure of eukaryotic mRNA. In addition to fold similarity, the electrostatic surface potentials of BLES03 and eIF4E show a clear conservation of basic and acidic patches. In the crystal lattice, the acidic amino-terminal helices of BLES03 monomers are bound within the basic cavity of symmetry-related monomers in a manner analogous to the binding of mRNA by eIF4E. Interestingly, the gene locus encoding BLES03 is located between genes encoding the proteins DRAP1 and FOSL1, both of which are involved in transcription initiation. It is hypothesized that BLES03 itself may be involved in a biochemical process that requires recognition of nucleic acids. PMID:16511166

  9. (2E)-1-(5-Chlorothiophen-2-yl)-3-{4-[(E)-2-phenylethenyl]phenyl}prop-2-en-1-one: Synthesis, XRD, FT-IR, Raman and DFT studies.

    PubMed

    Parlak, Cemal; Ramasami, Ponnadurai; Kumar, Chandraju Sadolalu Chidan; Tursun, Mahir; Quah, Ching Kheng; Rhyman, Lydia; Bilge, Metin; Fun, Hoong-Kun; Chandraju, Siddegowda

    2015-01-01

    A novel (2E)-1-(5-chlorothiophen-2-yl)-3-{4-[(E)-2-phenylethenyl]phenyl}prop-2-en-1-one [C21H15ClOS] compound has been synthesized and its structure has been characterized by FT-IR, Raman and single-crystal X-ray diffraction techniques. The conformational isomers, optimized geometric parameters, normal mode frequencies and corresponding vibrational assignments of the compound have been examined by means of HF, MP2, BP86, BLYP, BMK, B3LYP, B3PW91, B3P86 and M06-2X functionals. Reliable vibrational assignments and molecular orbitals have been investigated by the potential energy distribution and natural bonding orbital analyses, respectively. The compound crystallizes in the triclinic space group P-1 with the cis-trans-trans form. There is a good agreement between the experimentally determined structural parameters and vibrational frequencies of the compound and those predicted theoretically using the density functional theory with the BLYP and BP86 functionals.

  10. Design, Synthesis and Evaluation of New 2, 6-Dihydroimidazo[1, 2-c]Pyrimido[5, 4-e]-Pyrimidine-5(3H)-thiones as Possible Antihistaminic/Antiasthmatic Agents.

    PubMed

    Sirisha, K; Achaiah, G; Ram Rao, A Raghu

    2014-01-01

    A series of new 10-(alkylamino)-8-methyl-2, 6-dihydroimidazo[1, 2-c]pyrimido[5, 4-e]pyrimidine-5(3H)-thiones (4a-g) were subjected to molecular property prediction (drug-likeness, lipophilicity and solubility parameters) using Osiris Property Explorer, ALOGPS 2.1, Molinspiration and ACD/Chemsketch 12.0 software programmes. The calculated drug-related properties of the designed molecules were similar to those found in most marketed drugs. Amongst the proposed analogues, four promising candidates were chosen (4a-d) for synthesis on the basis of Lipinski's 'Rule of Five' and drug-likeness scores. The significant biological activity of the test compounds in two in vitro modes (isolated guinea pig tracheal chain preparation, isolated guinea pig ileum) supports the promise and accuracy of the prediction. Among them, 4a was the most potent antihistaminic (IC50 value of 30.2 μM; standard, chlorpheniramine maleate showed an IC50 of 14.1 μM).

  11. 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

  12. 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.

  13. 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

  14. Protein electrophoresis - serum

    MedlinePlus

    ... of protein and fat, called lipoproteins (such as LDL cholesterol). ... globulin proteins may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone ...

  15. Maintaining adequate nutrition, not probiotic administration, prevents growth stunting and maintains skeletal muscle protein synthesis rates in a piglet model of colitis.

    PubMed

    Harding, Scott V; Adegoke, Olasunkanmi A J; Fraser, Keely G; Marliss, Errol B; Chevalier, Stéphanie; Kimball, Scot R; Jefferson, Leonard S; Wykes, Linda J

    2010-03-01

    Malnutrition and cytokine-induced catabolism are pervasive in children with inflammatory bowel diseases (IBD), however, the benefits of aggressive nutrition support or of probiotics on nutrient and functional deficiencies and growth remain unclear. Piglets with dextran sulfate (DS)-induced colitis consuming a 50% macronutrient restricted diet (C-MR) were compared with those receiving probiotics (C-MRP) or adequate nutrition (C-WN) and with healthy well-nourished controls (REF). C-WN versus REF had reduced growth (-34% chest circumference and -22% snout-to-rump length gain) and a tendency toward lesser weight gain, but no differences in skeletal muscle protein fractional synthesis rates (FSR) or initiation of translation via the mTOR pathway were observed. Compared with C-WN, the C-MR and C-MRP piglets had lower weight gain, growth, and skeletal muscle FSR, and lower phosphorylated p70S6K1 with higher eIF4E*4E-BP1, indicative of reduced initiation of protein translation. Finally, plasma leucine concentrations were positively correlated with weight and phosphorylated p70S6K1, whereas negatively correlated with eIF4E*4E-BP1. In conclusion, reductions in weight gain, growth, protein turnover, skeletal muscle FSR, and initiation of protein translation with moderate macronutrient restriction in colitis are not ameliorated by probiotic supplementation. However, maintaining adequate nutrient intake during colitis preserves whole body protein metabolism, but growth remains compromised.

  16. Carbon monoxide releasing molecule-2 CORM-2 represses global protein synthesis by inhibition of eukaryotic elongation factor eEF2.

    PubMed

    Schwer, Christian Ingo; Stoll, Patrick; Rospert, Sabine; Fitzke, Edith; Schallner, Nils; Bürkle, Hartmut; Schmidt, Rene; Humar, Matjaz

    2013-02-01

    Carbon monoxide (CO) is an endogenous gaseous transmitter that exerts antiproliferative effects in many cell types, but effects of CO on the translational machinery are not described. We examined the effects of the carbon monoxide releasing molecule-2 (CORM-2) on critical steps in translational signaling and global protein synthesis in pancreatic stellate cells (PSCs), the most prominent collagen-producing cells in the pancreas, whose activation is associated with pancreatic fibrosis. PSCs were isolated from rat pancreatic tissue and incubated with CORM-2. CORM-2 prevented the decrease in the phosphorylation of eukaryotic elongation factor 2 (eEF2) caused by serum. By contrast, the activation dependent phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1) was inhibited by CORM-2 treatment. The phosphorylation of eukaryotic initiation factor 2α (eIF2α) and eukaryotic initiation factor 4E (eIF4E) were not affected by CORM-2 treatment. In consequence, CORM-2 mediated eEF2 phosphorylation and inactivation of 4E-BP1 suppressed global protein synthesis. These observations were associated with inhibition of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling and increased intracellular calcium and cAMP levels. The CORM-2 mediated inhibition of protein synthesis resulted in downregulation of cyclin D1 and cyclin E expression, a subsequent decline in the phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and cell growth arrest at the G(0)/G(1) phase checkpoint of the cell cycle. Our results suggest the therapeutic application of CO releasing molecules such as CORM-2 for the treatment of fibrosis, inflammation, cancer, or other pathologic states associated with excessive protein synthesis or hyperproliferation. However, prolonged exogenous application of CO might also have negative effects on cellular protein homeostasis.

  17. Protein sulfhydration.

    PubMed

    Paul, Bindu D; Snyder, Solomon H

    2015-01-01

    Hydrogen sulfide (H2S) is one of the gasotransmitters that modulates various biological processes and participates in multiple signaling pathways. H2S signals by a process termed sulfhydration. Sulfhydration has recently been recognized as a posttranslational modification similar to nitrosylation. Sulfhydration occurs at reactive cysteine residues in proteins and results in the conversion of an -SH group of cysteine to an -SSH or a persulfide group. Sulfhydration is highly prevalent in vivo, and aberrant sulfhydration patterns have been observed under several pathological conditions ranging from heart disease to neurodegenerative diseases such as Parkinson's disease. The biotin switch assay, originally developed to detect nitrosylation, has been modified to detect sulfhydration. In this chapter, we discuss the physiological roles of sulfhydration and the methodologies used to detect this modification.

  18. Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells.

    PubMed

    Lee, Myung-Ja; Feliers, Denis; Sataranatarajan, Kavithalakshmi; Mariappan, Meenalakshmi M; Li, Manli; Barnes, Jeffrey L; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

    2010-01-01

    High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1-AMPK axis.

  19. Glucocorticoids and 11β-HSD1 are major regulators of intramyocellular protein metabolism

    PubMed Central

    Hassan-Smith, Zaki K; Doig, Craig L; Sherlock, Mark; Stewart, Paul M; Lavery, Gareth G

    2016-01-01

    The adverse metabolic effects of prescribed and endogenous glucocorticoid excess, ‘Cushing’s syndrome’, create a significant health burden. While skeletal muscle atrophy and resultant myopathy is a clinical feature, the molecular mechanisms underpinning these changes are not fully defined. We have characterized the impact of glucocorticoids upon key metabolic pathways and processes regulating muscle size and mass including: protein synthesis, protein degradation, and myoblast proliferation in both murine C2C12 and human primary myotube cultures. Furthermore, we have investigated the role of pre-receptor modulation of glucocorticoid availability by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in these processes. Corticosterone (CORT) decreased myotube area, decreased protein synthesis, and increased protein degradation in murine myotubes. This was supported by decreased mRNA expression of insulin-like growth factor (IGF1), decreased activating phosphorylation of mammalian target of rapamycin (mTOR), decreased phosphorylation of 4E binding protein 1 (4E-BP1), and increased mRNA expression of key atrophy markers including: atrogin-1, forkhead box O3a (FOXO3a), myostatin (MSTN), and muscle-ring finger protein-1 (MuRF1). These findings were endorsed in human primary myotubes, where cortisol also decreased protein synthesis and increased protein degradation. The effects of 11-dehydrocorticosterone (11DHC) (in murine myotubes) and cortisone (in human myotubes) on protein metabolism were indistinguishable from that of CORT/cortisol treatments. Selective 11β-HSD1 inhibition blocked the decrease in protein synthesis, increase in protein degradation, and reduction in myotube area induced by 11DHC/cortisone. Furthermore, CORT/cortisol, but not 11DHC/cortisone, decreased murine and human myoblast proliferative capacity. Glucocorticoids are potent regulators of skeletal muscle protein homeostasis and myoblast proliferation. Our data underscores the potential use

  20. Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue.

    PubMed

    Hudelist, Gernot; Pacher-Zavisin, Margit; Singer, Christian F; Holper, Tina; Kubista, Ernst; Schreiber, Martin; Manavi, Mahmood; Bilban, Martin; Czerwenka, Klaus

    2004-08-01

    cDNA arrays provide a powerful tool to identify gene expression pattern that are potentially associated with tumor invasion and metastasis. However, genes work at the protein level and, since the transcriptional activity of a gene does not necessarily reflect cellular protein expression, the identification and quantification of proteins is essential for the understanding of molecular events leading to malignant transformation. We have therefore employed a high-throughput protein microarray system which contains 378 well-characterized monoclonal antibodies in order to compare the gene expression pattern of malignant and adjacent normal breast tissue in a patient with primary breast cancer. Using this technique, we have identified a number of proteins that show increased expression levels in malignant breast tissues such as casein kinase Ie, p53, annexin XI, CDC25C, eIF-4E and MAP kinase 7. The expression of other proteins, such as the multifunctional regulator 14-3-3e was found to be decreased in malignant breast tissue, whereas the majority of proteins remained unchanged when compared to the corresponding non-malignant samples. The protein expression pattern was confirmed by immunohistochemistry, in which antibodies against 8 representative proteins known to be involved in carcinogenesis were employed in paraffin-embedded normal and malignant tissue sections deriving from the same patient. In each case, the results obtained by IHC matched the data obtained by antibody microarray system. Taken together, we have described for the first time a tumor cell specificity protein expression pattern by use of a novel commercially available antibody microarray system. We have thus demonstrated the feasibility of high-throughput protein arrays in the proteomic analysis of human breast tissue. We hypothesize that the use of protein arrays will not only increase our understanding of the molecular events, but could prove useful in evaluating prognosis and in determining optimal

  1. Fusion-protein-assisted protein crystallization.

    PubMed

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  2. General RNA binding proteins render translation cap dependent.

    PubMed Central

    Svitkin, Y V; Ovchinnikov, L P; Dreyfuss, G; Sonenberg, N

    1996-01-01

    Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF-4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap-dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5' end, cap-mediated mechanism, and prevent spurious initiations at aberrant translation start sites. Images PMID:9003790

  3. A mechanistic model of nutritional control of protein synthesis in animal tissues.

    PubMed

    El-Haroun, Ehab R; Bureau, Dominique P; Cant, John P

    2010-01-21

    Regulation of mRNA translation has been held responsible for effects of diet, age, alcohol, hormones, hibernation, disease and hypoxia on protein synthesis in animal tissues. Dietary effects are due to concentrations of amino acids and insulin in circulation that affect activities of two key translational regulators, eukaryotic initiation factor 2 (F2) and eukaryotic initiation factor 4E binding protein 1 (Bp). To construct a platform for prediction of global protein synthesis to nutritional stimuli, a dynamic, mechanistic model of translational control in whole tissues was developed. The model was composed of a set of differential equations which describe the dynamics of 11 state variables: tRNA and acyl-tRNA for leucine (Leu), limiting (Laa) and other amino acids (Oaa), inactivated F2 with GDP (F2d), activated F2 with GTP (F2t), F4e, Bp and its complex with F4e (4eBp), available mRNA start codons (AUG), and active ribosomes (Arib). Material was assumed to flow from one variable to another according to mass-action kinetics or Michaelis-Menten form. Uncharged tRNA inhibit GTP exchange on eIF2, and free amino acids and insulin inhibit reversible sequestration of F4e by Bp. Initial conditions and parameters were set for a skeletal muscle fractional synthesis rate of 10%/d and ribosome transit time of 80s. Between amino acid concentrations of 500 and 4000x10(3)nM, protein synthesis increased from 0.9 to 11.7%/d at 0microU/mL insulin, and from 5.0 to 12.8%/d at 30microU/mL insulin. Predicted responses to graded levels of a deficient amino acid were asymptotic. A single parameter accomodated differences between tissues in insulin sensitivity. Seven parameters must be changed to simulate initiation and elongation rates in more active tissues such as liver, or in tissues of older mature animals. An increase in uncharged tRNA during insulin stimulation highlighted the physiological importance of coordinated regulation of amino acid supply by insulin. In conclusion, the

  4. 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

  5. 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

  6. BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis

    PubMed Central

    Grespi, Francesca; Soratroi, Claudia; Krumschnabel, Gerhard; Sohm, Benedicte; Ploner, Christian; Geley, Stephan; Hengst, Ludger; Häcker, Georg; Villunger, Andreas

    2010-01-01

    Tight transcriptional regulation, post-translational modifications and/or alternative splicing of BH3-only proteins fine-tune their pro-apoptotic function. Here, we characterize the gene locus of the BH3-only protein Bmf (Bcl-2 modifying factor) and describe the generation of two major isoforms from a common transcript where initiation of protein synthesis involves leucine-coding CUG. BmfCUG and the originally described isoform, Bmf short (BmfS), display comparable binding affinities to pro-survival Bcl-2 family members, localize preferentially to the outer mitochondrial membrane and induce rapid Bcl-2-blockable apoptosis. Notably, endogenous Bmf expression is induced upon forms of cell stress known to cause the repression of the CAP-dependent translation machinery such as serum-deprivation, hypoxia, inhibition of the PI3K/AKT pathway or mTOR, as well as direct pharmacological inhibition of eukaryotic translation initiation factor eIF-4E. Knock-down or deletion of Bmf reduces apoptosis under some of these conditions demonstrating that Bmf can act as a sentinel for the stress-impaired CAP-dependent protein translation machinery (150). PMID:20706276

  7. 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.

  8. 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.

  9. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control.

    PubMed

    Piñeiro, David; Fernandez-Chamorro, Javier; Francisco-Velilla, Rosario; Martinez-Salas, Encarna

    2015-01-01

    Gemin5 is a RNA-binding protein (RBP) that was first identified as a peripheral component of the survival of motor neurons (SMN) complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs) through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs). Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E). Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES) elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

  10. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

    PubMed Central

    Piñeiro, David; Fernandez-Chamorro, Javier; Francisco-Velilla, Rosario; Martinez-Salas, Encarna

    2015-01-01

    Gemin5 is a RNA-binding protein (RBP) that was first identified as a peripheral component of the survival of motor neurons (SMN) complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs) through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs). Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E). Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES) elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis. PMID:25898402

  11. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control.

    PubMed

    Piñeiro, David; Fernandez-Chamorro, Javier; Francisco-Velilla, Rosario; Martinez-Salas, Encarna

    2015-01-01

    Gemin5 is a RNA-binding protein (RBP) that was first identified as a peripheral component of the survival of motor neurons (SMN) complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs) through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs). Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E). Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES) elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis. PMID:25898402

  12. Protein immobilization strategies for protein biochips.

    PubMed

    Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-06-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein immobilization, in order to fully realize the potential of protein biochips. In fact, protein immobilization is the key to the success of microarray technology. Proteins need to be immobilized onto surfaces with high density in order to allow the usage of small amount of sample solution. Nonspecific protein adsorption needs to be avoided or at least minimized in order to improve detection performances. Moreover, full retention of protein conformation and activity is a challenging task to be accomplished. Although a large number of review papers on protein biochips have been published in recent years, few have focused on protein immobilization technology. In this review, current protein immobilization strategies, including physical, covalent, and bioaffinity immobilization for the fabrication of protein biochips, are described. Particular consideration has been given to oriented immobilization, also referred to as site-specific immobilization, which is believed will improve homogeneous surface covering and accessibility of the active site.

  13. 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 ...

  14. 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 ...

  15. 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

  16. Acetylation of Human TCF4 (TCF7L2) Proteins Attenuates Inhibition by the HBP1 Repressor and Induces a Conformational Change in the TCF4::DNA Complex

    PubMed Central

    Elfert, Susanne; Weise, Andreas; Bruser, Katja; Biniossek, Martin L.; Jägle, Sabine; Senghaas, Niklas; Hecht, Andreas

    2013-01-01

    The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/β-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us to investigate whether vertebrate TCF/LEF proteins are subject to acetylation. Through co-expression with p300 and CBP and subsequent analyses using mass spectrometry and immunodetection with anti-acetyl-lysine antibodies we show that TCF4 can be acetylated at lysine K150 by CBP. K150 acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of β-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K150 acetylation we substituted K150 with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/β-catenin-responsive promoter regions did not indicate a general role of K150 acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K150R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K150 using a bacterial expression system or amino acid substitutions at K150 alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K150 acetylation leads to a conformational change that may also represent the mechanism whereby acetylated TCF4E acquires resistance against HBP1. In summary, TCF4 not only recruits acetyltransferases but is also a substrate for these enzymes. The fact that acetylation affects only a subset of TCF4 splice variants and is mediated preferentially by CBP suggests that the conditional acetylation of TCF4E is a novel

  17. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  18. Protein domain architectures.

    PubMed

    Mulder, Nicola J

    2010-01-01

    Proteins are composed of functional units, or domains, that can be found alone or in combination with other domains. Analysis of protein domain architectures and the movement of protein domains within and across different genomes provide clues about the evolution of protein function. The classification of proteins into families and domains is provided through publicly available tools and databases that use known protein domains to predict other members in new proteins sequences. Currently at least 80% of the main protein sequence databases can be classified using these tools, thus providing a large data set to work from for analyzing protein domain architectures. Each of the protein domain databases provide intuitive web interfaces for viewing and analyzing their domain classifications and provide their data freely for downloading. Some of the main protein family and domain databases are described here, along with their Web-based tools for analyzing domain architectures.

  19. mTORC1-Independent Reduction of Retinal Protein Synthesis in Type 1 Diabetes

    PubMed Central

    Losiewicz, Mandy K.; Pennathur, Subramaniam; Jefferson, Leonard S.; Kimball, Scot R.; Abcouwer, Steven F.; Gardner, Thomas W.

    2014-01-01

    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. Ins2Akita 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. PMID:24740573

  20. 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

  1. 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.

  2. Inferring Protein Associations Using Protein Pulldown Assays

    SciTech Connect

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  3. 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

  4. Translation factors and ribosomal proteins control tumor onset and progression: how?

    PubMed

    Loreni, F; Mancino, M; Biffo, S

    2014-04-24

    Gene expression is shaped by translational control. The modalities and the extent by which translation factors modify gene expression have revealed therapeutic scenarios. For instance, eukaryotic initiation factor (eIF)4E activity is controlled by the signaling cascade of growth factors, and drives tumorigenesis by favoring the translation of specific mRNAs. Highly specific drugs target the activity of eIF4E. Indeed, the antitumor action of mTOR complex 1 (mTORc1) blockers like rapamycin relies on their capability to inhibit eIF4E assembly into functional eIF4F complexes. eIF4E biology, from its inception to recent pharmacological targeting, is proof-of-principle that translational control is druggable. The case for eIF4E is not isolated. The translational machinery is involved in the biology of cancer through many other mechanisms. First, untranslated sequences on mRNAs as well as noncoding RNAs regulate the translational efficiency of mRNAs that are central for tumor progression. Second, other initiation factors like eIF6 show a tumorigenic potential by acting downstream of oncogenic pathways. Third, genetic alterations in components of the translational apparatus underlie an entire class of inherited syndromes known as 'ribosomopathies' that are associated with increased cancer risk. Taken together, data suggest that in spite of their evolutionary conservation and ubiquitous nature, variations in the activity and levels of ribosomal proteins and translation factors generate highly specific effects. Beside, as the structures and biochemical activities of several noncoding RNAs and initiation factors are known, these factors may be amenable to rational pharmacological targeting. The future is to design highly specific drugs targeting the translational apparatus. PMID:23644661

  5. Mirror image proteins.

    PubMed

    Zhao, Le; Lu, Wuyuan

    2014-10-01

    Proteins composed entirely of unnatural d-amino acids and the achiral amino acid glycine are mirror image forms of their native l-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image d-proteins, enabling protein research to be conducted through 'the looking glass' and in a way previously unattainable. d-Proteins can facilitate structure determination of their native l-forms that are difficult to crystallize (racemic X-ray crystallography); d-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior d-peptide/d-protein therapeutics (mirror-image phage display); d-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology.

  6. High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

    PubMed

    Sakanyan, Vehary

    2005-02-01

    Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

  7. The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

    PubMed Central

    Wang, Jingang; Zhou, Dan; Prabhu, Anjali; Schlegel, Richard; Yuan, Hang

    2010-01-01

    The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb. PMID:20824099

  8. Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

    PubMed Central

    Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael

    2011-01-01

    Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful

  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. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  11. Urine Protein and Urine Protein to Creatinine Ratio

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  12. Designing Fluorinated Proteins.

    PubMed

    Marsh, E N G

    2016-01-01

    As methods to incorporate noncanonical amino acid residues into proteins have become more powerful, interest in their use to modify the physical and biological properties of proteins and enzymes has increased. This chapter discusses the use of highly fluorinated analogs of hydrophobic amino acids, for example, hexafluoroleucine, in protein design. In particular, fluorinated residues have proven to be generally effective in increasing the thermodynamic stability of proteins. The chapter provides an overview of the different fluorinated amino acids that have been used in protein design and the various methods available for producing fluorinated proteins. It discusses model proteins systems into which highly fluorinated amino acids have been introduced and the reasons why fluorinated residues are generally stabilizing, with particular reference to thermodynamic and structural studies from our laboratory. Lastly, details of the methodology we have developed to measure the thermodynamic stability of oligomeric fluorinated proteins are presented, as this may be generally applicable to many proteins. PMID:27586337

  13. Activity-dependent neuroprotective protein (ADNP) exhibits striking sexual dichotomy impacting on autistic and Alzheimer's pathologies.

    PubMed

    Malishkevich, A; Amram, N; Hacohen-Kleiman, G; Magen, I; Giladi, E; Gozes, I

    2015-02-03

    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.

  14. 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

  15. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  16. 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.

  17. 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

  18. Protein and protein hydrolysates in sports nutrition.

    PubMed

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  19. Engineering fluorescent proteins.

    PubMed

    Miyawaki, Atsushi; Nagai, Takeharu; Mizuno, Hideaki

    2005-01-01

    Green fluorescent protein from the jellyfish Aequorea victora (GFP) and GFP-like proteins from Anthozoa species encode light-absorbing chromophores intrinsically within their respective protein sequences. Recent studies have made progress in obtaining bright variants of these proteins which develop chromophores quickly and efficiently, as well as novel fluorescent proteins that photoactivate or photoconvert, i.e., become fluorescent or change colors upon illumination at specific wavelengths. Also, monomeric versions of these proteins have been engineered for fusion protein applications. Simple GFP variants and circularly permuted GFP variants have been used to develop fluorescent probes that sense physiological signals such as membrane potential and concentrations of free calcium. Further molecular characterization of the structure and maturation of these proteins is in progress, aimed at providing information for rational design of variants with desired fluorescence properties.

  20. 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 ...

  1. 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 ...

  2. 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 ...

  3. [Protein-losing enteropathy].

    PubMed

    Amiot, A

    2015-07-01

    Protein-losing enteropathy is a rare syndrome of gastrointestinal protein loss. The primary causes can be classified into lymphatic leakage due to increased interstitial pressure and increased leakage of protein-rich fluids due to erosive or non-erosive gastrointestinal disorders. The diagnosis of protein-losing enteropathy should be considered in patients with chronic diarrhea and peripheral oedema. The diagnosis of protein-losing enteropathy is most commonly based on the determination of fecal alpha-1 antitrypsin clearance. Most protein-losing enteropathy cases are the result of either lymphatic obstruction or a variety of gastrointestinal disorders and cardiac diseases, while primary intestinal lymphangiectasia (Waldmann's disease) is less common. Treatment of protein-losing enteropathy targets the underlying disease but also includes dietary modification, such as high-protein and low-fat diet along with medium-chain triglyceride supplementation. PMID:25618488

  4. Viral Genome-Linked Protein (VPg) Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV)

    PubMed Central

    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. PMID:26599265

  5. 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. PMID:26599265

  6. Protein and older adults.

    PubMed

    Chernoff, Ronni

    2004-12-01

    Body composition changes as people get older. One of the noteworthy alterations is the reduction in total body protein. A decrease in skeletal muscle is the most noticeable manifestation of this change but there is also a reduction in other physiologic proteins such as organ tissue, blood components, and immune bodies as well as declines in total body potassium and water. This contributes to impaired wound healing, loss of skin elasticity, and an inability to fight infection. The recommended dietary allowance (RDA) for adults for protein is 0.8 grams of protein per kilogram of body weight. Protein tissue accounts for 30% of whole-body protein turnover but that rate declines to 20% or less by age 70. The result of this phenomenon is that older adults require more protein/kilogram body weight than do younger adults. Recently, it has become clear that the requirement for exogenous protein is at least 1.0 gram/kilogram body weight. Adequate dietary intake of protein may be more difficult for older adults to obtain. Dietary animal protein is the primary source of high biological value protein, iron, vitamin B(12), folic acid, biotin and other essential nutrients. In fact, egg protein is the standard against which all other proteins are compared. Compared to other high-quality protein sources like meat, poultry and seafood, eggs are the least expensive. The importance of dietary protein cannot be underestimated in the diets of older adults; inadequate protein intake contributes to a decrease in reserve capacity, increased skin fragility, decreased immune function, poorer healing, and longer recuperation from illness.

  7. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-09-04

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of {sup 35}S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

  8. 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...

  9. 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…

  10. 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.

  11. 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…

  12. 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 mg/dL. Note: mg/dL = ...

  13. 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.

  14. 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

  15. Viral complement regulatory proteins.

    PubMed

    Rosengard, A M; Ahearn, J M

    1999-05-01

    The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats. PMID:10408371

  16. 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.

  17. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    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.

  18. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    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.

  19. Selective Precipitation of Proteins.

    PubMed

    Matulis, Daumantas

    2016-01-01

    Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.

  20. 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.

  1. Forces stabilizing proteins.

    PubMed

    Nick Pace, C; Scholtz, J Martin; Grimsley, Gerald R

    2014-06-27

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a -CH2- group on folding contributes 1.1±0.5 kcal/mol to protein stability. (2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. (3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1±0.8 kcal/mol to protein stability. (4) The contribution of hydrogen bonds to protein stability is strongly context dependent. (5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (6) Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. (7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability.

  2. Clinical protein mass spectrometry.

    PubMed

    Scherl, Alexander

    2015-06-15

    Quantitative protein analysis is routinely performed in clinical chemistry laboratories for diagnosis, therapeutic monitoring, and prognosis. Today, protein assays are mostly performed either with non-specific detection methods or immunoassays. Mass spectrometry (MS) is a very specific analytical method potentially very well suited for clinical laboratories. Its unique advantage relies in the high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein. On the other hand, protein MS is a relatively young technique, demanding specialized personnel and expensive instrumentation. Many scientists and opinion leaders predict MS to replace immunoassays for routine protein analysis, but there are only few protein MS applications routinely used in clinical chemistry laboratories today. The present review consists of a didactical introduction summarizing the pros and cons of MS assays compared to immunoassays, the different instrumentations, and various MS protein assays that have been proposed and/or are used in clinical laboratories. An important distinction is made between full length protein analysis (top-down method) and peptide analysis after enzymatic digestion of the proteins (bottom-up method) and its implication for the protein assay. The document ends with an outlook on what type of analyses could be used in the future, and for what type of applications MS has a clear advantage compared to immunoassays.

  3. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  4. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  5. 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.

  6. 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.

  7. Racemic protein crystallography.

    PubMed

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  8. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  9. Resveratrol up-regulates AMPA receptor expression via AMP-activated protein kinase-mediated protein translation.

    PubMed

    Wang, Guan; Amato, Stephen; Gilbert, James; Man, Heng-Ye

    2015-08-01

    Resveratrol is a phytoalexin that confers overall health benefits including positive regulation in brain function such as learning and cognition. However, whether and how resveratrol affects synaptic activity remains largely unknown. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are glutamatergic receptors that mediate the majority of fast excitatory transmission and synaptic plasticity, and thus play a critical role in higher brain functions, including learning and memory. We find that in rat primary neurons, resveratrol can rapidly increase AMPAR protein level, AMPAR synaptic accumulation and the strength of excitatory synaptic transmission. The resveratrol effect on AMPAR protein expression is independent of sirtuin 1 (SIRT1), the conventional downstream target of resveratrol, but rather is mediated by AMP-activated protein kinase (AMPK) and subsequent downstream phosphoinositide 3-kinase (PI3K)/Akt signaling. Application of the AMPK specific activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis via regulation of the eukaryotic initiation factor (eIF) 4E/4G complex. Disruption of the translation initiation complex completely blocks resveratrol-dependent AMPAR up-regulation. These findings indicate that resveratrol may regulate brain function through facilitation of AMPAR biogenesis and synaptic transmission.

  10. 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.

  11. Toxic proteins in plants.

    PubMed

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed.

  12. Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2013-01-01

    Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame. PMID:24418875

  13. Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2013-01-01

    Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p=0.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame.

  14. 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

  15. Energy design for protein-protein interactions

    PubMed Central

    Ravikant, D. V. S.; Elber, Ron

    2011-01-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

  16. Avian reovirus nonstructural protein p17-induced G(2)/M cell cycle arrest and host cellular protein translation shutoff involve activation of p53-dependent pathways.

    PubMed

    Chulu, Julius L C; Huang, Wei R; Wang, L; Shih, Wen L; Liu, Hung J

    2010-08-01

    The effects of avian reovirus (ARV) p17 protein on cell cycle progression and host cellular protein translation were studied. ARV infection and ARV p17 transfection resulted in the accumulation of infected and/or transfected cells in the G(2)/M phase of the cell cycle. The accumulation of cells in the G(2)/M phase was accompanied by upregulation and phosphorylation of the G(2)/M-phase proteins ATM, p53, p21(cip1/waf1), Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C, suggesting that p17 induces a G(2)/M cell cycle arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/cyclin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G(2)/M cell cycle arrest resulted in increased virus replication. In the present study, we also provide evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2alpha and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G, as well as 4E-BP1 and Mnk-1 in p17-transfected cells, demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together, our data indicate that ARV p17-induced G(2)/M arrest and host cellular translation shutoff resulted in increased ARV replication.

  17. Principles of Flexible Protein-Protein Docking

    PubMed Central

    Andrusier, Nelly; Mashiach, Efrat; Nussinov, Ruth; Wolfson, Haim J.

    2008-01-01

    Treating flexibility in molecular docking is a major challenge in cell biology research. Here we describe the background and the principles of existing flexible protein-protein docking methods, focusing on the algorithms and their rational. We describe how protein flexibility is treated in different stages of the docking process: in the preprocessing stage, rigid and flexible parts are identified and their possible conformations are modeled. This preprocessing provides information for the subsequent docking and refinement stages. In the docking stage, an ensemble of pre-generated conformations or the identified rigid domains may be docked separately. In the refinement stage, small-scale movements of the backbone and side-chains are modeled and the binding orientation is improved by rigid-body adjustments. For clarity of presentation, we divide the different methods into categories. This should allow the reader to focus on the most suitable method for a particular docking problem. PMID:18655061

  18. 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. PMID:26320628

  19. Electrophoretic separation of proteins.

    PubMed

    Chakavarti, Bulbul; Chakavarti, Deb

    2008-01-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). PMID:19066548

  20. Functional Protein Microarray Technology

    PubMed Central

    Hu, Shaohui; Xie, Zhi; Qian, Jiang; Blackshaw, Seth; Zhu, Heng

    2010-01-01

    Functional protein microarrays are emerging as a promising new tool for large-scale and high-throughput studies. In this article, we will review their applications in basic proteomics research, where various types of assays have been developed to probe binding activities to other biomolecules, such as proteins, DNA, RNA, small molecules, and glycans. We will also report recent progress of using functional protein microarrays in profiling protein posttranslational modifications, including phosphorylation, ubiquitylation, acetylation, and nitrosylation. Finally, we will discuss potential of functional protein microarrays in biomarker identification and clinical diagnostics. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology. PMID:20872749

  1. 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

  2. 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)

  3. 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.

  4. Protein oxidation and peroxidation.

    PubMed

    Davies, Michael J

    2016-04-01

    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.

  5. 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

  6. 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.

  7. Isolation and characterization of anti-inflammatory peptides derived from whey protein.

    PubMed

    Ma, Ye; Liu, Jie; Shi, Haiming; Yu, Liangli Lucy

    2016-09-01

    The present study was conducted to isolate and characterize anti-inflammatory peptides from whey protein hydrolysates using alcalase. Nine subfractions were obtained after sequential purification by ultrafiltration, Sephadex G-25 gel (GE Healthcare, Uppsala, Sweden) filtration chromatography, and preparative HPLC. Among them, subfraction F4e showed the strongest inhibitory activity on interleukin-1β (IL-1β), cyclooxygenase-2, and tumor necrosis factor-α (TNF-α) mRNA expression in lipopolysaccharide-induced RAW 264.7 mouse macrophages. Eight peptides, including 2 new peptides-Asp-Tyr-Lys-Lys-Tyr (DYKKY) and Asp-Gln-Trp-Leu (DQWL)-were identified from subfractions F4c and F4e, respectively, using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Peptide DQWL showed the strongest inhibitory ability on IL-1β, cyclooxygenase-2, and TNF-α mRNA expression and production of IL-1β and TNF-α proteins at concentrations of 10 and 100μg/mL, respectively. Additionally, DQWL treatment significantly inhibited nuclear factor-κB activation by suppressing nuclear translocation of nuclear factor-κB p65 and blocking inhibitor κB kinase phosphorylation and inhibitor κB degradation together with p38 mitogen-activated protein kinase activation. Our study suggests that peptide DQWL has anti-inflammatory potential; further confirmation using an in vivo model is needed. PMID:27394940

  8. Protein-Losing Gastroenteropathy

    PubMed Central

    Pops, Martin A.

    1966-01-01

    In the past 10 years with the development of improved methods, particularly radioisotope techniques, it has been demonstrated that a number of patients with gastrointestinal disease and depletion of plasma proteins become hypoproteinemic because of actual leakage of albumin and other plasma proteins into the lumen of the gastrointestinal tract. The site of protein leakage is variable depending on the underlying pathological state but the loss of protein-containing lymph through the gastrointestinal lymphatic channels seems to be the major mechanism for hypoproteinemia. It has become apparent that there exists a normal mechanism for secretion of plasma proteins into the gastrointestinal tract as part of the overall metabolism of the plasma proteins. When the process is exaggerated so that resynthesis of plasma protein cannot keep pace with its degradation, sometimes severe hypoproteinemia is the result. Such a pathological process has now been described in approximately 40 disease states. A review of all the techniques which can demonstrate gastroenteric protein loss reveals that there are no widely available quantitative tests but that accurate quantitation is not necessary for the diagnosis of protein losing gastroenteropathy. PMID:18730025

  9. 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.

  10. Acanthamoeba castellanii STAT protein.

    PubMed

    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

  11. The Halophile protein database.

    PubMed

    Sharma, Naveen; Farooqi, Mohammad Samir; Chaturvedi, Krishna Kumar; Lal, Shashi Bhushan; Grover, Monendra; Rai, Anil; Pandey, Pankaj

    2014-01-01

    Halophilic archaea/bacteria adapt to different salt concentration, namely extreme, moderate and low. These type of adaptations may occur as a result of modification of protein structure and other changes in different cell organelles. Thus proteins may play an important role in the adaptation of halophilic archaea/bacteria to saline conditions. The Halophile protein database (HProtDB) is a systematic attempt to document the biochemical and biophysical properties of proteins from halophilic archaea/bacteria which may be involved in adaptation of these organisms to saline conditions. In this database, various physicochemical properties such as molecular weight, theoretical pI, amino acid composition, atomic composition, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (Gravy) have been listed. These physicochemical properties play an important role in identifying the protein structure, bonding pattern and function of the specific proteins. This database is comprehensive, manually curated, non-redundant catalogue of proteins. The database currently contains 59 897 proteins properties extracted from 21 different strains of halophilic archaea/bacteria. The database can be accessed through link. Database URL: http://webapp.cabgrid.res.in/protein/

  12. Moonlighting proteins in cancer.

    PubMed

    Min, Kyung-Won; Lee, Seong-Ho; Baek, Seung Joon

    2016-01-01

    Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.

  13. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  14. Glycolipid transfer proteins

    PubMed Central

    Brown, Rhoderick E.; Mattjus, Peter

    2007-01-01

    Glycolipid transfer proteins (GLTPs) are small (24 kD), soluble, ubiquitous proteins characterized by their ability to accelerate the intermembrane transfer of glycolipids in vitro. GLTP specificity encompasses both sphingoid- and glycerol-based glycolipids, but with a strict requirement that the initial sugar residue be beta-linked to the hydrophobic lipid backbone. The 3D protein structures of GLTP reveal liganded structures with unique lipid binding modes. The biochemical properties of GLTP action at the membrane surface have been studied rather comprehensively, but the biological role of GLTP remains enigmatic. What is clear is that GLTP differs distinctly from other known glycolipid-binding proteins, such as nonspecific lipid transfer proteins, lysosomal sphingolipid activator proteins, lectins, lung surfactant proteins as well as other lipid binding/transfer proteins. Based on the unique conformational architecture that targets GLTP to membranes and enables glycolipid binding, GLTP is now considered the prototypical and founding member of a new protein superfamily in eukaryotes. PMID:17320476

  15. 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

  16. 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

  17. 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.

  18. Protein metabolism and requirements.

    PubMed

    Biolo, Gianni

    2013-01-01

    Skeletal muscle adaptation to critical illness includes insulin resistance, accelerated proteolysis, and increased release of glutamine and the other amino acids. Such amino acid efflux from skeletal muscle provides precursors for protein synthesis and energy fuel to the liver and to the rapidly dividing cells of the intestinal mucosa and the immune system. From these adaptation mechanisms, severe muscle wasting, glutamine depletion, and hyperglycemia, with increased patient morbidity and mortality, may ensue. Protein/amino acid nutrition, through either enteral or parenteral routes, plays a pivotal role in treatment of metabolic abnormalities in critical illness. In contrast to energy requirement, which can be accurately assessed by indirect calorimetry, methods to determine individual protein/amino acid needs are not currently available. In critical illness, a decreased ability of protein/amino acid intake to promote body protein synthesis is defined as anabolic resistance. This abnormality leads to increased protein/amino acid requirement and relative inefficiency of nutritional interventions. In addition to stress mediators, immobility and physical inactivity are key determinants of anabolic resistance. The development of mobility protocols in the intensive care unit should be encouraged to enhance the efficacy of nutrition. In critical illness, protein/amino acid requirement has been defined as the intake level associated with the lowest rate of catabolism. The optimal protein-sparing effects in patients receiving adequate energy are achieved when protein/amino acids are administered at rates between 1.3 and 1.5 g/kg/day. Extra glutamine supplementation is required in conditions of severe systemic inflammatory response. Protein requirement increases during hypocaloric feeding and in patients with acute renal failure on continuous renal replacement therapy. Evidence suggests that receiving adequate protein/amino acid intake may be more important than achieving

  19. 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

  20. Interolog interfaces in protein-protein docking.

    PubMed

    Alsop, James D; Mitchell, Julie C

    2015-11-01

    Proteins are essential elements of biological systems, and their function typically relies on their ability to successfully bind to specific partners. Recently, an emphasis of study into protein interactions has been on hot spots, or residues in the binding interface that make a significant contribution to the binding energetics. In this study, we investigate how conservation of hot spots can be used to guide docking prediction. We show that the use of evolutionary data combined with hot spot prediction highlights near-native structures across a range of benchmark examples. Our approach explores various strategies for using hot spots and evolutionary data to score protein complexes, using both absolute and chemical definitions of conservation along with refinements to these strategies that look at windowed conservation and filtering to ensure a minimum number of hot spots in each binding partner. Finally, structure-based models of orthologs were generated for comparison with sequence-based scoring. Using two data sets of 22 and 85 examples, a high rate of top 10 and top 1 predictions are observed, with up to 82% of examples returning a top 10 hit and 35% returning top 1 hit depending on the data set and strategy applied; upon inclusion of the native structure among the decoys, up to 55% of examples yielded a top 1 hit. The 20 common examples between data sets show that more carefully curated interolog data yields better predictions, particularly in achieving top 1 hits. Proteins 2015; 83:1940-1946. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  1. The folate binding proteins.

    PubMed

    Corrocher, R; Olivieri, O; Pacor, M L

    1991-01-01

    Folates are essential molecules for cell life and, not surprisingly, their transport in biological fluids and their transfer to cells are finely regulated. Folate binding proteins play a major role in this regulation. This paper will review our knowledge on these proteins and examine the most recent advances in this field. PMID:1820987

  2. 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

  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. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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…

  15. 1-Fluoro-4-[(E)-2-nitro-vin-yl]benzene.

    PubMed

    Sreenivasa, S; Nanjundaswamy, M S; Manojkumar, K E; Madankumar, S; Lokanath, N K; Suchetan, P A

    2014-02-01

    The title compound, C8H6FNO2, is almost planar (r.m.s. deviation for the non-H atoms = 0.019 Å) and the conformation across the C=C bond is trans. The C and H atoms of the side chain are disordered over two sets of sites in a 0.56 (3):0.44 (3) ratio. In the crystal, mol-ecules are linked by C-H⋯O inter-actions, thus forming C(5) chains propagating in [001].

  16. Yeast mRNA cap-binding protein Cbc1/Sto1 is necessary for the rapid reprogramming of translation after hyperosmotic shock

    PubMed Central

    Garre, Elena; Romero-Santacreu, Lorena; De Clercq, Nikki; Blasco-Angulo, Nati; Sunnerhagen, Per; Alepuz, Paula

    2012-01-01

    In response to osmotic stress, global translation is inhibited, but the mRNAs encoding stress-protective proteins are selectively translated to allow cell survival. To date, the mechanisms and factors involved in the specific translation of osmostress-responsive genes in Saccharomyces cerevisiae are unknown. We find that the mRNA cap-binding protein Cbc1 is important for yeast survival under osmotic stress. Our results provide new evidence supporting a role of Cbc1 in translation initiation. Cbc1 associates with polysomes, while the deletion of the CBC1 gene causes hypersensitivity to the translation inhibitor cycloheximide and yields synthetic “sickness” in cells with limiting amounts of translation initiator factor eIF4E. In cbc1Δ mutants, translation drops sharply under osmotic stress, the subsequent reinitiation of translation is retarded, and “processing bodies” containing untranslating mRNAs remain for long periods. Furthermore, osmostress-responsive mRNAs are transcriptionally induced after osmotic stress in cbc1Δ cells, but their rapid association with polysomes is delayed. However, in cells containing a thermosensitive eIF4E allele, their inability to grow at 37ºC is suppressed by hyperosmosis, and Cbc1 relocalizes from nucleus to cytoplasm. These data support a model in which eIF4E-translation could be stress-sensitive, while Cbc1-mediated translation is necessary for the rapid translation of osmostress-protective proteins under osmotic stress. PMID:22072789

  17. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  18. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  19. Fullerene sorting proteins.

    PubMed

    Calvaresi, Matteo; Zerbetto, Francesco

    2011-07-01

    Proteins bind fullerenes. Hydrophobic pockets can accommodate a carbon cage either in full or in part. However, the identification of proteins able to discriminate between different cages is an open issue. Prediction of candidates able to perform this function is desirable and is achieved with an inverse docking procedure that accurately accounts for (i) van der Waals interactions between the cage and the protein surface, (ii) desolvation free energy, (iii) shape complementarity, and (iv) minimization of the number of steric clashes through conformational variations. A set of more than 1000 protein structures is divided into four categories that either select C(60) or C(70) (p-C(60) or p-C(70)) and either accommodate the cages in the same pocket (homosaccic proteins, from σακκoζ meaning pocket) or in different pockets (heterosaccic proteins). In agreement with the experiments, the KcsA Potassium Channel is predicted to have one of the best performances for both cages. Possible ways to exploit the results and efficiently separate the two cages with proteins are also discussed.

  20. 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

  1. PSC: protein surface classification.

    PubMed

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  2. 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

  3. 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. PMID:24579057

  4. Protein microarrays: prospects and problems.

    PubMed

    Kodadek, T

    2001-02-01

    Protein microarrays are potentially powerful tools in biochemistry and molecular biology. Two types of protein microarrays are defined. One, termed a protein function array, will consist of thousands of native proteins immobilized in a defined pattern. Such arrays can be utilized for massively parallel testing of protein function, hence the name. The other type is termed a protein-detecting array. This will consist of large numbers of arrayed protein-binding agents. These arrays will allow for expression profiling to be done at the protein level. In this article, some of the major technological challenges to the development of protein arrays are discussed, along with potential solutions.

  5. Phospholipid transfer proteins revisited.

    PubMed Central

    Wirtz, K W

    1997-01-01

    Phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) (identical with sterol carrier protein 2) belong to the large and diverse family of intracellular lipid-binding proteins. Although these two proteins may express a comparable phospholipid transfer activity in vitro, recent studies in yeast and mammalian cells have indicated that they serve completely different functions. PI-TP (identical with yeast SEC14p) plays an important role in vesicle flow both in the budding reaction from the trans-Golgi network and in the fusion reaction with the plasma membrane. In yeast, vesicle budding is linked to PI-TP regulating Golgi phosphatidylcholine (PC) biosynthesis with the apparent purpose of maintaining an optimal PI/PC ratio of the Golgi complex. In mammalian cells, vesicle flow appears to be dependent on PI-TP stimulating phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis. This latter process may also be linked to the ability of PI-TP to reconstitute the receptor-controlled PIP2-specific phospholipase C activity. The nsL-TP is a peroxisomal protein which, by its ability to bind fatty acyl-CoAs, is most likely involved in the beta-oxidation of fatty acids in this organelle. This protein constitutes the N-terminus of the 58 kDa protein which is one of the peroxisomal 3-oxo-acyl-CoA thiolases. Further studies on these and other known phospholipid transfer proteins are bound to reveal new insights in their important role as mediators between lipid metabolism and cell functions. PMID:9182690

  6. (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.

  7. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  8. Piezoelectric allostery of protein.

    PubMed

    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. PMID:27575163

  9. 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.

  10. 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.

  11. 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.

  12. 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)

  13. 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

  14. Amino acids and proteins.

    PubMed

    van Goudoever, Johannes B; Vlaardingerbroek, Hester; van den Akker, Chris H; de Groof, Femke; van der Schoor, Sophie R D

    2014-01-01

    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional requirements are not met, resulting in a postnatal growth restriction. However, current knowledge on adequate levels of both amino acid as well as protein intake can avoid under nutrition in the direct postnatal phase, avoid the need for subsequent catch-up growth and improve later outcome.

  15. 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.

  16. The protein-protein interaction map of Helicobacter pylori.

    PubMed

    Rain, J C; Selig, L; De Reuse, H; Battaglia, V; Reverdy, C; Simon, S; Lenzen, G; Petel, F; Wojcik, J; Schächter, V; Chemama, Y; Labigne, A; Legrain, P

    2001-01-11

    With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score fo