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Sample records for c-terminal acidic tail

  1. Cystoviral polymerase complex protein P7 uses its acidic C-terminal tail to regulate the RNA-directed RNA polymerase P2.

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    Alphonse, Sébastien; Arnold, Jamie J; Bhattacharya, Shibani; Wang, Hsin; Kloss, Brian; Cameron, Craig E; Ghose, Ranajeet

    2014-07-15

    In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former toward template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in cystoviruses.

  2. C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors.

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    Wu, Xinle; Lemon, Bryan; Li, XiaoFan; Gupte, Jamila; Weiszmann, Jennifer; Stevens, Jennitte; Hawkins, Nessa; Shen, Wenyan; Lindberg, Richard; Chen, Jin-Long; Tian, Hui; Li, Yang

    2008-11-28

    FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of alpha or betaKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both alpha and betaKlotho, whereas FGF21 and FGF23 can bind only to either betaKlotho or alphaKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with betaKlotho and a chimera with the FGF23 C-terminal tail interacts only with alphaKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between alpha and betaKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact alpha or betaKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of alphaKlotho and the C terminus of betaKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.

  3. Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing.

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    Bhattacharyya, M K; Matthews, K M; Lustig, A J

    2008-08-01

    Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (DeltaC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. DeltaC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.

  4. TubZ filament assembly dynamics requires the flexible C-terminal tail

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    Fuentes-Pérez, Maria E.; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A.

    2017-01-01

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY. PMID:28230082

  5. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

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    Fernández-Fueyo, Elena [CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Acebes, Sandra [Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona (Spain); Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier, E-mail: fjmedrano@cib.csic.es [CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Guallar, Victor, E-mail: fjmedrano@cib.csic.es [Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona (Spain); ICREA, Passeig Lluís Companys 23, 08010 Barcelona (Spain); Martínez, Angel T., E-mail: fjmedrano@cib.csic.es [CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-12-01

    The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized. The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn{sup 2+}-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn{sup 2+} oxidation by the internal propionate, but prevents the oxidation of 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd{sup 2+} binds at the Mn{sup 2+}-oxidation site and competitively inhibits oxidation of both Mn{sup 2+} and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their

  6. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

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    Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

    2013-06-01

    The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

  7. The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

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    Coceres, V M; Alonso, A M; Nievas, Y R; Midlej, V; Frontera, L; Benchimol, M; Johnson, P J; de Miguel, N

    2015-08-01

    The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction.

  8. Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

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    Brophy, Megan Brunjes; Nakashige, Toshiki G; Gaillard, Aleth; Nolan, Elizabeth M

    2013-11-27

    Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103-105 to 104-106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

  9. The role of the S4-S5 linker and C-terminal tail in inositol 1,4,5-trisphosphate receptor function.

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    Schug, Zachary T; Joseph, Suresh K

    2006-08-25

    In previous studies we have suggested that spatial proximity of the C- and N-terminal domains of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) may be critical for the channel gating mechanism. In the present study we have examined the sites of C-N interaction in more detail. We report that deletion mutations within the S4-S5 linker (amino acids 2418-2437) prevent co-immunoprecipitation of the C- and N-terminal domains, inhibit channel activity and enhance IP(3) binding. We also show that a region of the C-terminal tail (amino acids 2694-2721), predicted to be a coiled-coil, is also required for channel activity. Circular dichroism spectroscopy and gel filtration studies confirm that this region has a helical structure with the ability to form tetramers. We propose a model in which IP(3)-induced conformational changes in the N-terminal domain are mechanically transmitted to the opening of the pore through an attachment to the S4-S5 linker. The coiled-coil domain in the C-terminal tail may play a critical role in maintaining the structural integrity of the channel.

  10. Ligand-induced Ordering of the C-terminal Tail Primes STING for Phosphorylation by TBK1

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    Yuko Tsuchiya

    2016-07-01

    Full Text Available The innate immune protein Stimulator of interferon genes (STING promotes the induction of interferon beta (IFN-β production via the phosphorylation of its C-terminal tail (CTT by TANK-binding kinase 1 (TBK1. Potent ligands of STING are, therefore, promising candidates for novel anti-cancer drugs or vaccine adjuvants. However, the intrinsically flexible CTT poses serious problems in in silico drug discovery. Here, we performed molecular dynamics simulations of the STING fragment containing the CTT in ligand-bound and unbound forms and observed that the binding of a potent ligand cyclic GMP-AMP (cGAMP induced a local structure in the CTT, reminiscent of the known structure of a TBK1 substrate. The subsequent molecular biological experiments confirmed the observed dynamics of the CTT and identified essential residues for the activation of the IFN-β promoter, leading us to propose a new mechanism of STING activation.

  11. Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

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    Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

    2003-08-29

    One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

  12. Two Distinct Binding Modes Define the Interaction of Brox with the C-Terminal Tails of CHMP5 and CHMP4B

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    Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F.; Bouamr, Fadila; Xiao, Tsan Sam (NIH)

    2012-05-21

    Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic {alpha} helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an {alpha} helix, the CHMP5 C-terminal tail adopts a tandem {beta}-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a {beta}-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.

  13. Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.

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    Panchaud, Alexandre; Guillaume, Elisabeth; Affolter, Michael; Robert, Fabien; Moreillon, Philippe; Kussmann, Martin

    2006-01-01

    Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

  14. The Epstein-Barr virus (EBV) glycoprotein B cytoplasmic C-terminal tail domain regulates the energy requirement for EBV-induced membrane fusion.

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    Chen, Jia; Zhang, Xianming; Jardetzky, Theodore S; Longnecker, Richard

    2014-10-01

    The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoprotein B (gB) from a prefusion to a postfusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) mutant truncated at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue the fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion, and can function in fusion at a lower temperature than wild-type gB, indicating a lower energy requirement for fusion activation. Since a key step in fusion is the conversion of gB from a prefusion to an active postfusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in the fusion function by maintaining gB in an inactive prefusion form prior to activation by receptor binding. Importance: Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign disease infectious mononucleosis to life-threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection by using glycoproteins gH/gL, gB, and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutant and studied its fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion

  15. Emi2 inhibition of the anaphase-promoting complex/cyclosome absolutely requires Emi2 binding via the C-terminal RL tail.

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    Ohe, Munemichi; Kawamura, Yoshiko; Ueno, Hiroyuki; Inoue, Daigo; Kanemori, Yoshinori; Senoo, Chiharu; Isoda, Michitaka; Nakajo, Nobushige; Sagata, Noriyuki

    2010-03-15

    Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.

  16. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

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    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  17. The C-Terminal Region of G72 Increases D-Amino Acid Oxidase Activity

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    Sunny Li-Yun Chang

    2013-12-01

    Full Text Available The schizophrenia-related protein G72 plays a unique role in the regulation of D-amino acid oxidase (DAO in great apes. Several psychiatric diseases, including schizophrenia and bipolar disorder, are linked to overexpression of DAO and G72. Whether G72 plays a positive or negative regulatory role in DAO activity, however, has been controversial. Exploring the molecular basis of the relationship between G72 and DAO is thus important to understand how G72 regulates DAO activity. We performed yeast two-hybrid experiments and determined enzymatic activity to identify potential sites in G72 involved in binding DAO. Our results demonstrate that residues 123–153 and 138–153 in the long isoform of G72 bind to DAO and enhance its activity by 22% and 32%, respectively. A docking exercise indicated that these G72 peptides can interact with loops in DAO that abut the entrance of the tunnel that substrate and cofactor must traverse to reach the active site. We propose that a unique gating mechanism underlies the ability of G72 to increase the activity of DAO. Because upregulation of DAO activity decreases d-serine levels, which may lead to psychiatric abnormalities, our results suggest a molecular mechanism involving interaction between DAO and the C-terminal region of G72 that can regulate N-methyl-d-aspartate receptor-mediated neurotransmission.

  18. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

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    Steckbeck, Jonathan D; Sun, Chengqun; Sturgeon, Timothy J; Montelaro, Ronald C

    2010-01-01

    The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  19. Detailed topology mapping reveals substantial exposure of the "cytoplasmic" C-terminal tail (CTT sequences in HIV-1 Env proteins at the cell surface.

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    Jonathan D Steckbeck

    Full Text Available Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT. While few studies have been designed to directly address the topology of the CTT, results from envelope (Env protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

  20. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

    Directory of Open Access Journals (Sweden)

    Jonathan D Steckbeck

    Full Text Available The C-terminal tail (CTT of the HIV-1 gp41 envelope (Env protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs. Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  1. A C-terminal acidic domain regulates degradation of the transcriptional coactivator Bob1.

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    Lindner, John M; Wong, Christina S F; Möller, Andreas; Nielsen, Peter J

    2013-12-01

    Bob1 (Obf-1 or OCA-B) is a 34-kDa transcriptional coactivator encoded by the Pou2af1 gene that is essential for normal B-cell development and immune responses in mice. During lymphocyte activation, Bob1 protein levels dramatically increase independently of mRNA levels, suggesting that the stability of Bob1 is regulated. We used a fluorescent protein-based reporter system to analyze protein stability in response to genetic and physiological perturbations and show that, while Bob1 degradation is proteasome mediated, it does not require ubiquitination of Bob1. Furthermore, degradation of Bob1 in B cells appears to be largely independent of the E3 ubiquitin ligase Siah. We propose a novel mechanism of Bob1 turnover in B cells, whereby an acidic region in the C terminus of Bob1 regulates the activity of degron signals elsewhere in the protein. Changes that make the C terminus more acidic, including tyrosine phosphorylation-mimetic mutations, stabilize the instable murine Bob1 protein, indicating that B cells may regulate Bob1 stability and activity via signaling pathways. Finally, we show that expressing a stable Bob1 mutant in B cells suppresses cell proliferation and induces changes in surface marker expression commonly seen during B-cell differentiation.

  2. Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.

    Directory of Open Access Journals (Sweden)

    Sarah E Boyce

    Full Text Available Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B--the C-terminal tail and β-loop--in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor-NS5B complex are absent in the inhibitor-bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.

  3. Active and accurate trans-translation requires distinct determinants in the C-terminal tail of SmpB protein and the mRNA-like domain of transfer messenger RNA (tmRNA).

    Science.gov (United States)

    Camenares, Devin; Dulebohn, Daniel P; Svetlanov, Anton; Karzai, A Wali

    2013-10-18

    Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.

  4. Identification of two Amino Acids in the C-terminal Domain of Mouse CRY2 Essential for PER2 Interaction

    Directory of Open Access Journals (Sweden)

    Ozber Natali

    2010-09-01

    Full Text Available Abstract Background Cryptochromes (CRYs are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the Per and Cry genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of Per and Cry core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins. Results We identified a region on mCRY2 (between residues 493 and 512 responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2. Conclusions Our results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches.

  5. Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition.

    Science.gov (United States)

    Christian, Claiborne M; Sokolowski, Mark; deHaro, Dawn; Kines, Kristine J; Belancio, Victoria P

    2017-03-01

    Long interspersed element 1 (L1) is the only currently active autonomous retroelement in the human genome. Along with the parasitic SVA and short interspersed element Alu, L1 is the source of DNA damage induced by retrotransposition: a copy-and-paste process that has the potential to disrupt gene function and cause human disease. The retrotransposition process is dependent upon the ORF2 protein (ORF2p). However, it is unknown whether most of the protein is important for retrotransposition. In particular, other than the Cys motif, the C terminus of the protein has not been intensely examined in the context of retrotransposition. Using evolutionary analysis and the Alu retrotransposition assay, we sought to identify additional amino acids in the C terminus important for retrotransposition. Here, we demonstrate that Gal4-tagged and untagged C-terminally truncated ORF2p fragments possess residual potential to drive Alu retrotransposition. Using sight-directed mutagenesis we identify that while the Y1180 amino acid is important for ORF2p- and L1-driven Alu retrotransposition, a mutation at this position improves L1 retrotransposition. Even though the mechanism of the contribution of Y1180 to Alu and L1 mobilization remains unknown, experimental evidence rules out its direct involvement in the ability of the ORF2p reverse transcriptase to generate complementary DNA. Additionally, our data support that ORF2p amino acids 1180 and 1250-1262 may be involved in the reported ORF1p-mediated increase in ORF2p-driven Alu retrotransposition.

  6. Identification of potent 11mer glucagon-like peptide-1 receptor agonist peptides with novel C-terminal amino acids: Homohomophenylalanine analogs.

    Science.gov (United States)

    Haque, Tasir S; Lee, Ving G; Riexinger, Douglas; Lei, Ming; Malmstrom, Sarah; Xin, Li; Han, Songping; Mapelli, Claudio; Cooper, Christopher B; Zhang, Ge; Ewing, William R; Krupinski, John

    2010-05-01

    We report the identification of potent agonists of the Glucagon-Like Peptide-1 Receptor (GLP-1R). These compounds are short, 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of homohomophenylalanine (hhPhe) at the C-terminal position. Typically the functional activity of the more potent peptides in this class is in the low picomolar range in an in vitro cAMP assay, with one example demonstrating excellent in vivo activity in an ob/ob mouse model of diabetes.

  7. Direct influence of C-terminally substituted amino acids in the Dmt-Tic pharmacophore on delta-opioid receptor selectivity and antagonism.

    Science.gov (United States)

    Balboni, Gianfranco; Salvadori, Severo; Guerrini, Remo; Negri, Lucia; Giannini, Elisa; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H

    2004-07-29

    A series of 17 analogues were developed on the basis of the general formula H-Dmt-Tic-NH-CH(R)-R' (denotes chirality; R = charged, neutral, or aromatic functional group; R' = -OH or -NH(2)). These compounds were designed to test the following hypothesis: the physicochemical properties of third-residue substitutions C-terminal to Tic in the Dmt-Tic pharmacophore modify delta-opioid receptor selectivity and delta-opioid receptor antagonism through enhanced interactions with the mu-opioid receptor. The data substantiate the following conclusions: (i) all compounds had high receptor affinity [K(i)(delta) = 0.034-1.1 nM], while that for the mu-opioid receptor fluctuated by orders of magnitude [K(i)(mu) = 15.1-3966 nM]; (ii) delta-opioid receptor selectivity [K(i)(mu)/K(i)(delta)] declined 1000-fold from 22,600 to 21; (iii) a C-terminal carboxyl group enhanced selectivity but only as a consequence of the specific residue; (iv) amidated, positive charged residues [Lys-NH(2) (6), Arg-NH(2) (7)], and a negatively charged aromatic residue [Trp-OH (11)] enhanced mu-opioid affinity [K(i)(mu) = 17.0, 15.1, and 15.7 nM, respectively], while Gly-NH(2) (8), Ser-NH(2) (10), and His-OH (12) were nearly one-tenth as active; and (v) D-isomers exhibited mixed effects on mu-opioid receptor affinity (2' 1 microM) except H-Dmt-Tic-Glu-NH(2) (3), which was a partial delta-opioid receptor agonist (IC(50) = 2.5 nM). Thus, these C-terminally extended analogues indicated that an amino acid residue containing a single charge, amino or guanidino functionality, or aromatic group substantially altered the delta-opioid receptor activity profile (selectivity and antagonism) of the Dmt-Tic pharmacophore, which suggests that the C-terminal constituent plays a major role in determining opioid receptor activity as an "address domain".

  8. Truncation of the MSH2 C-terminal 60 amino acids disrupts effective DNA mismatch repair and is causative for Lynch syndrome.

    Science.gov (United States)

    Wielders, Eva; Delzenne-Goette, Elly; Dekker, Rob; van der Valk, Martin; Te Riele, Hein

    2017-04-01

    Missense variants of DNA mismatch repair (MMR) genes pose a problem in clinical genetics as long as they cannot unambiguously be assigned as the cause of Lynch syndrome (LS). To study such variants of uncertain clinical significance, we have developed a functional assay based on direct measurement of MMR activity in mouse embryonic stem cells expressing mutant protein from the endogenous alleles. We have applied this protocol to a specific truncation mutant of MSH2 that removes 60 C-terminal amino acids and has been found in suspected LS families. We show that the stability of the MSH2/MSH6 heterodimer is severely perturbed, causing attenuated MMR in in vitro assays and cancer predisposition in mice. This mutation can therefore unambiguously be considered as deleterious and causative for LS.

  9. Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA.

    Science.gov (United States)

    Gite, S; Li, Y; Ramesh, V; RajBhandary, U L

    2000-03-01

    The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.

  10. Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment.

    Science.gov (United States)

    Souza, Andre L A; Díaz-Dellavalle, Paola; Cabrera, Andrea; Larrañaga, Patricia; Dalla-Rizza, Marco; De-Simone, Salvatore G

    2013-07-01

    An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.

  11. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Birkelund, S; Holm, A;

    1996-01-01

    , in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N...

  12. Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C-Terminal Signaling in the Blue Light Sensor Plant Cryptochrome.

    Science.gov (United States)

    Thöing, Christian; Oldemeyer, Sabine; Kottke, Tilman

    2015-05-13

    Plant cryptochromes are photosensory receptors that regulate various central aspects of plant growth and development. These receptors consist of a photolyase homology region (PHR) carrying the oxidized flavin adenine dinucleotide (FAD) cofactor, and a cryptochrome C-terminal extension (CCT), which is essential for signaling. Absorption of blue/UVA light leads to formation of the FAD neutral radical as the likely signaling state, and ultimately activates the CCT. Little is known about the signal transfer from the flavin to the CCT. Here, we investigated the photoreaction of the PHR by time-resolved step-scan FT-IR spectroscopy complemented by UV-vis spectroscopy. The first spectrum at 500 ns shows major contributions from the FAD anion radical, which is demonstrated to then be protonated by aspartic acid 396 to the neutral radical within 3.5 μs. The analysis revealed the existence of three intermediates characterized by changes in secondary structure. A marked loss of β-sheet structure is observed in the second intermediate evolving with a time constant of 500 μs. This change is accompanied by a conversion of a tyrosine residue, which is identified as the formation of a tyrosine radical in the UV-vis. The only β-sheet in the PHR is located within the α/β subdomain, ∼25 Å away from the flavin. This subdomain has been previously attributed a role as a putative antenna binding site, but is now suggested to have evolved to a component in the signaling of plant cryptochromes by mediating the interaction with the CCT.

  13. A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

    2004-01-01

    -coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed......Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...

  14. Amyloidogenic Properties of a D/N Mutated 12 Amino Acid Fragment of the C-Terminal Domain of the Cholesteryl-Ester Transfer Protein (CETP

    Directory of Open Access Journals (Sweden)

    Victor García-González

    2011-03-01

    Full Text Available The cholesteryl-ester transfer protein (CETP facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D470N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when β-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

  15. A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

    2004-01-01

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequ...... that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor....... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...

  16. Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env

    Directory of Open Access Journals (Sweden)

    Bosch Valerie

    2006-05-01

    Full Text Available Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT. Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD. We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

  17. Influences of wetland plants on weathered acidic mine tailings

    Energy Technology Data Exchange (ETDEWEB)

    Eva Stoltz; Maria Greger [Stockholm University, Stockholm (Sweden). Department of Botany

    2006-11-15

    Establishment of Carex rostrata, Eriophorum angustifolium and Phragmites australis on weathered, acidic mine tailings (pH {approximately} 3) and their effect on pH in tailings were investigated in a field experiment. The amendments, sewage sludge and an ashes-sewage sludge mixture, were used as plant nutrition and their influence on the metal and As concentrations of plant shoots was analysed. An additional experiment was performed in greenhouse with E. angustifolium and sewage sludge as amendments in both weathered and unweathered tailings. After one year, plants grew better in amendments containing ashes in the field, also in those plants the metal and As shoot concentrations were generally lower than in other treatments. After two years, the only surviving plants were found in sewage sludge mixed with ashes. No effect on pH by plants was found in weathered acidic mine tailings in either field- or greenhouse experiment.

  18. Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase.

    Science.gov (United States)

    Mason, A Brett; Allen, Kenneth E; Slayman, Carolyn W

    2006-08-18

    Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.

  19. Evolution of Acid Mine Drainage Formation in Sulphidic Mine Tailings

    Directory of Open Access Journals (Sweden)

    Bernhard Dold

    2014-07-01

    Full Text Available Sulphidic mine tailings are among the largest mining wastes on Earth and are prone to produce acid mine drainage (AMD. The formation of AMD is a sequence of complex biogeochemical and mineral dissolution processes. It can be classified in three main steps occurring from the operational phase of a tailings impoundment until the final appearance of AMD after operations ceased: (1 During the operational phase of a tailings impoundment the pH-Eh regime is normally alkaline to neutral and reducing (water-saturated. Associated environmental problems include the presence of high sulphate concentrations due to dissolution of gypsum-anhydrite, and/or effluents enriched in elements such as Mo and As, which desorbed from primary ferric hydroxides during the alkaline flotation process. (2 Once mining-related operations of the tailings impoundment has ceased, sulphide oxidation starts, resulting in the formation of an acidic oxidation zone and a ferrous iron-rich plume below the oxidation front, that re-oxidises once it surfaces, producing the first visible sign of AMD, i.e., the precipitation of ferrihydrite and concomitant acidification. (3 Consumption of the (reactive neutralization potential of the gangue minerals and subsequent outflow of acidic, heavy metal-rich leachates from the tailings is the final step in the evolution of an AMD system. The formation of multi-colour efflorescent salts can be a visible sign of this stage.

  20. Influences of wetland plants on weathered acidic mine tailings

    Energy Technology Data Exchange (ETDEWEB)

    Stoltz, Eva [Department of Botany, Stockholm University, Lilla Frescativaegen 5, S-106 91 Stockholm (Sweden)]. E-mail: eva.stoltz@botan.su.se; Greger, Maria [Department of Botany, Stockholm University, Lilla Frescativaegen 5, S-106 91 Stockholm (Sweden)]. E-mail: maria.greger@botan.su.se

    2006-11-15

    Establishment of Carex rostrata, Eriophorum angustifolium and Phragmites australis on weathered, acidic mine tailings (pH {approx}3) and their effect on pH in tailings were investigated in a field experiment. The amendments, sewage sludge and an ashes-sewage sludge mixture, were used as plant nutrition and their influence on the metal and As concentrations of plant shoots was analysed. An additional experiment was performed in greenhouse with E. angustifolium and sewage sludge as amendments in both weathered and unweathered tailings. After one year, plants grew better in amendments containing ashes in the field, also in those plants the metal and As shoot concentrations were generally lower than in other treatments. After two years, the only surviving plants were found in sewage sludge mixed with ashes. No effect on pH by plants was found in weathered acidic mine tailings in either field- or greenhouse experiment. - Wetland plant establishment on acidic mine tailings may contribute to a reduced metal release and a stabilisation of pH.

  1. Assessment of Phytostabilization Success in Metalliferous Acid Mine Tailings

    Science.gov (United States)

    Wang, Y.; Root, R. A.; Hammond, C.; Amistadi, M. K.; Maier, R. M.; Chorover, J.

    2014-12-01

    Legacy mine tailings are a significant source of metal(loid)s due to wind and water erosion, especially in the arid southwest, and exposure to fugative dusts presents a health risk to surrounding populations. Compost assisted phytostabilization has been implemented to reduce off site emissions at the Iron King Mine U.S. Superfund Site in central Arizona, concurrent with a greenhouse mesocosm study for detailed study of subsurface mechanisms. Quantification of plant available toxic metal(loid)s in the amended tailings was accessed with a targeted single extraction of diethylenetriaminepentaactic acid (DTPA). Greenhouse mesocosms (1m dia, 0.4 m deep), run in triplicate, mimicked field treatments with: i) tailings only control (TO), ii) tailings plus 15 wt% compost (TC), iii) TC + quailbush seeds (TCA), and iv) TC + buffalo grass seeds (TCB). Core samples collected at 3-month intervals for 1 year were dissected by depth (10 cm each) for analysis. DTPA results indicated that compost treated samples decreased plant availability of Al, As, Cd, Cu, Fe, and Pb but increased Mn and Zn compared with TO. TCB decreased plant available metal(loid)s at all depths, whereas TCA plant available Al, As, Cd, Cu, Fe, Mn and Zn increased in the deeper 20-30cm and 30-40 cm relative to TCB. Samples from the greenhouse were compared to tailings from both the field site and tailings impacted soils used to grow vegetables. Mineral transformations and metal complexation, in the pre- and post-extracted tailings were analyzed by synchrotron transmission XRD and FTIR spectroscopy. The temporal change in plant available metal(loid)s in response to phytostabilization indicates mineralogical alteration that improves soil quality by reducing plant available metal(loid)s. These results will aid in the understanding and efficacy of phytostabilization as a means of remediating and reducing toxicity on mine tailings as well as providing information on health risk management in the region.

  2. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain

    DEFF Research Database (Denmark)

    Pedersen, LB; Birkelund, Svend; Holm, A

    1996-01-01

    , in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N...... retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect...

  3. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

    Science.gov (United States)

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  4. PICK1蛋白C端酸性区域对其功能的调节%C-terminal Acidic Region Regulates PICK1 Function

    Institute of Scientific and Technical Information of China (English)

    贾原; 黄玉民; 刘俊林; 石亚伟

    2010-01-01

    蛋白激酶C相互作用蛋白1(protein interacting with C kinase l,PICKl)是调节AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)受体在细胞膜上的数量与分布,引起 LTP与LTD现象的重要蛋白.本文利用基因克隆、荧光光谱以及免疫分析等方法,分析了PICK1蛋白C末端酸性区对BAR结构域与膜脂结合能力以及PICK1分子内BAR(Bin/amphiphysin/RVS)结构域与PDZ结构域相互作用的影响,研究了钙离子结合C末端酸性区后对上述相互作用的调节.结果显示,C末端酸性区的存在使BAR结构域与膜脂的结合能力减弱大约10倍,但PICK1分子内的BAR与PDZ结构域的相互作用与不含C末端的酸性区相比增强了大约4倍.另一方面,C末端酸性区的存在,伴随钙离子浓度的提高,有助于增强BAR与膜脂的结合,却削弱了PDZ和BAR结构域的作用.当钙离子浓度增加到500 μmoL/L时,BARC的脂质结合能力以及和PDZ的亲和力与不合酸性区相当.

  5. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds

    Science.gov (United States)

    Feng, Zi-Ming; Zhan, Zhi-Lai; Yang, Ya-Nan; Jiang, Jian-Shuang; Zhang, Pei-Cheng

    2016-05-01

    The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of γ-amino acids and sugars from the roots of Ranunculus ternatus, which possess potential tail to tail ether-connected (6,6-ether-connected) modes in the sugar moiety. The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method.

  6. C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells.

    Science.gov (United States)

    Zhang, Xinquan; Bilic, Ivana; Marek, Ana; Glösmann, Martin; Hess, Michael

    2016-01-01

    The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 - ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.

  7. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    Science.gov (United States)

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  8. The impact of the human DNA topoisomerase II C-terminal domain on activity.

    Directory of Open Access Journals (Sweden)

    Emma L Meczes

    Full Text Available BACKGROUND: Type II DNA topoisomerases (topos are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity. METHODOLOGY/PRINCIPLE FINDINGS: We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity. CONCLUSIONS/SIGNIFICANCE: In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro

  9. Geophysical delineation of acidity and salinity in the Central Manitoba gold mine tailings pile, Manitoba, Canada

    Science.gov (United States)

    Tycholiz, C.; Ferguson, I. J.; Sherriff, B. L.; Cordeiro, M.; Sri Ranjan, R.; Pérez-Flores, M. A.

    2016-08-01

    Surface electrical and electromagnetic geophysical methods can map enhanced electrical conductivity caused by acid mine drainage in mine tailings piles. In this case study, we investigate quantitative relationships between geophysical responses and the electrical conductivity, acidity and salinity of tailing samples at the Central Manitoba Mine tailings in Manitoba, Canada. Previous electromagnetic surveys at the site identified zones of enhanced conductivity that were hypothesized to be caused by acid mine drainage. In the present study, high-resolution EM31 and DC-resistivity measurements were made on a profile through a zone of enhanced conductivity and laboratory measurements of salinity and pH were made on saturation paste extracts from an array of tailing samples collected from the upper 2 m of tailings along the profile. Observed spatial correlation of pH and pore-fluid salinity in the tailings samples confirms that the enhanced conductivity in the Central Manitoba Mine tailings is due to acid mine drainage. Contoured cross-sections of the data indicate that the acid mine drainage is concentrated near the base of the oxidized zone in the thicker parts of the tailings pile. The zone of increased acidity extends to the surface on sloping margins causing an increase in apparent conductivity in shallow penetrating geophysical responses. The quantitative relationship between measured pH and salinity shows that the conductivity increase associated with the acid mine drainage is due only in part to conduction by ions produced from dissociation of sulfuric acid. Comparison of the observations with fluid conductivity estimates based on statistical relationships of pH and ion concentrations in water samples from across the tailings pile shows that Ca2 + and Mg2 + ions also make significant contributions to the conductivity at all values of pH and Cu2 +, Al3 + and Fe3 + ions make additional contributions at low pH. Variability in the measured conductivity at constant

  10. Recombinant C-terminal 311 amino acids of HapS adhesin as a vaccine candidate for nontypeable Haemophilus influenzae: A study on immunoreactivity in Balb/C mouse.

    Science.gov (United States)

    Tabatabaee Bafroee, Akram Sadat; Siadat, Seyed Davar; Mousavi, Seyed Fazlollah; Aghasadeghi, Mohammad Reza; Khorsand, Hashem; Nejati, Mehdi; Sadat, Seyed Mehdi; Mahdavi, Mehdi

    2016-09-01

    Hap, an auto-transporter protein, is an antigenically conserved adhesion protein which is present on both typeable and nontypeable Haemophilus influenzae. This protein has central role in bacterial attachment to respiratory tract epithelial cells. A 1000bp C-terminal fragment of Hap passenger domain (HapS) from nontypeable Haemophilus influenzae was cloned into a prokaryotic expression vector, pET-24a. BALB/c mice were immunized subcutaneously with purified rC-HapS. Serum IgG responses to purified rC-HapS, serum IgG subclasses were determined by ELISA and functional activity of antibodies was examined by Serum Bactericidal Assay. The output of rC-HapS was approximately 62% of the total bacterial proteins. Serum IgG responses were significantly increased in immunized group with rC-HapS mixed with Freund's adjuvant in comparison with control groups. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after subcutaneous immunization in BALB/c mice (IgG2a/IgG1 < 1). The sera from rC-HapS immunized animals were strongly bactericidal against nontypeable Haemophilus influenzae. These results suggest that rC-HapS may be a potential vaccine candidate for nontypeable Haemophilus influenzae.

  11. Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    1996-01-01

    when the free amino acid does not, and that hydroperoxides can be formed on both the backbone (at alpha-carbon positions) and the side chain. Decomposition of alpha-carbon hydroperoxides by Fe(II)-EDTA gives initially an alkoxyl radical via a pseudo-Fenton reaction; these radicals fragment rapidly...... with k estimated as > or = 10(7) s(-1). With N-acetyl amino acids and dipeptides beta-scission of an alkoxyl radical at the C-terminal alpha-carbon results in C-terminal decarboxylation, with release of CO2.-; the corresponding amides undergo deamidation with release of .C(O)NH2. Cyclic dipeptides...... undergo analogous reactions with cleavage of the alpha-carbon to carbonyl-carbon bond and formation of .C(O)NHR radicals. With substrates with large aliphatic side chains, radicals from side-chain hydroperoxides are also observed. C-terminal decarboxylation and backbone fragmentation are also observed...

  12. Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Knudsen, Lotte Bjerre; Garibay, Patrick W.;

    2013-01-01

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally trun......The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C......-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys174, Cys226, Cys296 and Cys403 are important for the GLP-1-mediated response, whereas Cys236, Cys329, Cys341, Cys347, Cys438...... that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function....

  13. C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

    Science.gov (United States)

    Zhang, Jihua; Markiewicz, Matthew J; Mowery, Brendan P; Weisblum, Bernard; Stahl, Shannon S; Gellman, Samuel H

    2012-02-13

    Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.

  14. Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain.

    Science.gov (United States)

    Tišáková, Lenka; Vidová, Barbora; Farkašovská, Jarmila; Godány, Andrej

    2014-01-01

    The gene product of orf50 from actinophage μ1/6 of Streptomyces aureofaciens is a putative endolysin, Lyt μ1/6. It has a two-domain modular structure, consisting of an N-terminal catalytic and a C-terminal cell wall binding domain (CBD). Comparative analysis of Streptomyces phage endolysins revealed that they all have a modular structure and contain functional C-terminal domains with conserved amino acids, probably associated with their binding function. A blast analysis of Lyt μ1/6 in conjunction with secondary and tertiary structure prediction disclosed the presence of a PG_binding_1 domain within the CBD. The sequence of the C-terminal domain of lyt μ1/6 and truncated forms of it were cloned and expressed in Escherichia coli. The ability of these CBD variants fused to GFP to bind to the surface of S. aureofaciens NMU was shown by specific binding assays.

  15. Synthesis of Metal Porphyrins Tailed with Salicylic Acid and their Interaction with Bovine Serum Albumin

    Institute of Scientific and Technical Information of China (English)

    Tao JIA; Kai WANG; Yi Mei ZHAO; Zao Ying LI

    2004-01-01

    A synthetic method of porphyrins tailed with salicylic substituents is described. Reaction of bromoalkoxyphenyl porphyrin 1 with salicylic acid gave porphyrins 2~5. These new compounds were confirmed by 1H NMR, IR, UV-vis, MS and elemental analysis, and observed their interaction with bovine serum albumin (BSA) in fluorescence spectrum.

  16. Design and synthesis of peptide YY analogues with c-terminal backbone amide-to-ester modifications

    DEFF Research Database (Denmark)

    Albertsen, Louise; Andersen, J.J.; Paulsson, J.F.;

    2013-01-01

    Peptide YY (PYY) is a gut hormone that activates the G protein-coupled neuropeptide Y (NPY) receptors, and because of its appetite reducing actions, it is evaluated as an antiobesity drug candidate. The C-terminal tail of PYY is crucial for activation of the NPY receptors. Here, we describe...... the design and preparation of a series of PYY(3-36) depsipeptide analogues, in which backbone amide-to-ester modifications were systematically introduced in the C-terminal. Functional NPY receptor assays and circular dichroism revealed that the ψ(CONH) bonds at positions 30-31 and 33-34 are particularly...

  17. Docking Studies of Binding of Ethambutol to the C-Terminal Domain of the Arabinosyltransferase from Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Guillermo Salgado-Moran

    2013-01-01

    Full Text Available The binding of ethambutol to the C-terminal domain of the arabinosyltransferase from Mycobacterium tuberculosis was studied. The analysis was performed using an in silico approach in order to find out, by docking calculations and energy descriptors, the conformer of Ethambutol that forms the most stable complex with the C-terminal domain of arabinosyltransferase. The complex shows that location of the Ethambutol coincides with the cocrystallization ligand position and that amino acid residues ASH1051, ASN740, ASP1052, and ARG1055 should be critical in the binding of Ethambutol to C-terminal domain EmbC.

  18. Functional role of C-terminal domain of Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Tukalo M. A.

    2010-11-01

    Full Text Available Aim. To study a role of C-terminal domain of T. thermophilus leucyl-tRNA synthetase (LeuRSTT in the reactions of aminoacylation and editing. Methods. A mutant of LeuRSTT without C- terminal domain (ΔС was obtained by the method of mutagenesis. The kinetic constants in aminoacylation reaction catalyzed by LeuRS and its mutant (ΔС were determined by the methods of equilibrium enzyme kinetics. To evaluate the contribution of C-terminal domain to interaction of the enzyme with tRNALeu, Kd of a complex between tRNA and LeuRSTT and its mutant ΔС was determined by fluorescence titration. Results. The C-terminal domain is shown to play a significant role in the aminoacylation and editing reactions of LeuRSTT and not essential for the activity in the reaction of amino acid activation. The kinetic parameters of aminoacylation of tRNALeu and tRNATyr by LeuRS and ΔС mutant were also determined, their analysis suggests that the C-domain is not critical for the manifestation of specificity of the enzyme in the recognition of homologous RNAs. At the same time a significant influence of the C-terminal domain on the value of catalytic constant was shown. At the domain deletion the kcat value is lower by 152-fold. Conclusion. The C-terminal domain of LeuRSTT is evolutionarily acquired to enhance the rate of catalysis in the aminoacylation and editing reactions, and makes no significant contribution to the specificity of the enzyme in the recognition of tRNA.

  19. Docking Studies of Binding of Ethambutol to the C-Terminal Domain of the Arabinosyltransferase from Mycobacterium tuberculosis

    OpenAIRE

    Guillermo Salgado-Moran; Rodrigo Ramirez-Tagle; Daniel Glossman-Mitnik; Samuel Ruiz-Nieto; Pran Kishore-Deb; Marta Bunster; Francisco Lobos-Gonzalez

    2013-01-01

    The binding of ethambutol to the C-terminal domain of the arabinosyltransferase from Mycobacterium tuberculosis was studied. The analysis was performed using an in silico approach in order to find out, by docking calculations and energy descriptors, the conformer of Ethambutol that forms the most stable complex with the C-terminal domain of arabinosyltransferase. The complex shows that location of the Ethambutol coincides with the cocrystallization ligand position and that amino acid residu...

  20. A plasma membrane association module in yeast amino acid transporters

    NARCIS (Netherlands)

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert

    2016-01-01

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in sili

  1. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.

    Science.gov (United States)

    Altai, Mohamed; Wållberg, Helena; Orlova, Anna; Rosestedt, Maria; Hosseinimehr, Seyed Jalal; Tolmachev, Vladimir; Ståhl, Stefan

    2012-05-01

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  2. Utilization of Atikokan coal fly ash in acid rock drainage control from Musselwhite Mine tailings

    Energy Technology Data Exchange (ETDEWEB)

    Wang, H.L.; Shang, J.Q. [Western Ontario Univ., London, ON (Canada). Dept. of Civil and Environmental Engineering; Kovac, V. [Ontario Power Generation Inc., Toronto, ON (Canada). By-products, Fuel Division; Ho, K.S. [Trow Consulting Engineers, Brampton, ON (Canada)

    2006-03-15

    Acid rock drainage (ARD) is the greatest environmental liability facing the mining industry. Mines produce acidic effluents that are generated from the chemical reaction of sulphide containing minerals and atmospheric oxygen. The effluents have a pH value as low as 2 to 4 and their movement is accompanied by heavy metals which damage the ecosystem. This paper described some of the ARD-preventing technologies that are under investigation. In particular, it examined the feasibility of using Atikokan coal fly ash (AFA) as a buffering material to control and mitigate the generation of ARD from reactive Musselwhite Mine gold mine tailings. Coal fly ash is the residue resulting from the combustion of coal at electric generating plants. It consists of organic and inorganic matter, including silica, alumina, iron and calcium oxide with various amounts of carbon. More than 40,000 tons of fly ash is generated each year from the Atikokan Generating Station located 190 km west of the mine, of which 80 per cent is used for concrete manufacturing. In this study, experiments were conducted to determine the physical, chemical and mineralogical properties of both the fly ash residue and mine tailings. Six kinetic column permeation tests were then performed to monitor the leaching properties of the fly ash and the coal fly ash-mine tailings mixtures to determine the hydraulic conductivities resulting from pozzolanic reactions. The potential impacts of the disposal of AFA and mine tailings were also assessed. The study showed that the hydraulic conductivities of high-calcium AFA and the ash-tailings mixtures were greatly reduced upon contact with ARD. The pH of the pore fluid increased from acidic to alkaline. The concentration of regulated elements in the leachate from the ash-tailings mixtures were also below the limits set by the Ontario Ministry of Environment. The results indicate that AFA could mitigate the generation of ARD from reactive Musselwhite Mine gold mine tailings. 1

  3. Oil sands thickened froth treatment tailings exhibit acid rock drainage potential during evaporative drying.

    Science.gov (United States)

    Kuznetsov, Petr; Kuznetsova, Alsu; Foght, Julia M; Siddique, Tariq

    2015-02-01

    Bitumen extraction from oil sands ores after surface mining produces different tailings waste streams: 'froth treatment tailings' are enriched in pyrite relative to other streams. Tailings treatment can include addition of organic polymers to produce thickened tailings (TT). TT may be further de-watered by deposition into geotechnical cells for evaporative drying to increase shear strength prior to reclamation. To examine the acid rock drainage (ARD) potential of TT, we performed predictive analyses and laboratory experiments on material from field trials of two types of thickened froth treatment tailings (TT1 and TT2). Acid-base accounting (ABA) of initial samples showed that both TT1 and TT2 initially had net acid-producing potential, with ABA values of -141 and -230 t CaCO₃ equiv. 1000 t(-1) of TT, respectively. In long-term kinetic experiments, duplicate ~2-kg samples of TT were incubated in shallow trays and intermittently irrigated under air flow for 459 days to simulate evaporative field drying. Leachates collected from both TT samples initially had pH~6.8 that began decreasing after ~50 days (TT2) or ~250 days (TT1), stabilizing at pH~2. Correspondingly, the redox potential of leachates increased from 100-200 mV to 500-580 mV and electrical conductivity increased from 2-5 dS m(-1) to 26 dS m(-1), indicating dissolution of minerals during ARD. The rapid onset and prolonged ARD observed with TT2 is attributed to its greater pyrite (13.4%) and lower carbonate (1.4%) contents versus the slower onset of ARD in TT1 (initially 6.0% pyrite and 2.5% carbonates). 16S rRNA gene pyrosequencing analysis revealed rapid shift in microbial community when conditions became strongly acidic (pH~2) favoring the enrichment of Acidithiobacillus and Sulfobacillus bacteria in TT. This is the first report showing ARD potential of TT and the results have significant implications for effective management of pyrite-enriched oil sands tailings streams/deposits.

  4. NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

    Science.gov (United States)

    Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

    2013-10-01

    The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

  5. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    Directory of Open Access Journals (Sweden)

    Raats Jos MH

    2009-07-01

    Full Text Available Abstract Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL. However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

  6. Structure discrimination for the C-terminal domain of Escherichia coli trigger factor in solution

    Energy Technology Data Exchange (ETDEWEB)

    Yao Yong; Bhabha, Gira; Kroon, Gerard; Landes, Mindy; Dyson, H. Jane [Scripps Research Institute, Department of Molecular Biology (United States)], E-mail: dyson@scripps.edu

    2008-01-15

    NMR measurements can give important information on solution structure, without the necessity for a full-scale solution structure determination. The C-terminal protein binding domain of the ribosome-associated chaperone protein trigger factor is composed of non-contiguous parts of the polypeptide chain, with an interpolated prolyl isomerase domain. A construct of the C-terminal domain of Escherichia coli trigger factor containing residues 113-149 and 247-432, joined by a Gly-Ser-Gly-Ser linker, is well folded and gives excellent NMR spectra in solution. We have used NMR measurements on this construct, and on a longer construct that includes the prolyl isomerase domain, to distinguish between two possible structures for the C-terminal domain of trigger factor, and to assess the behavior of the trigger factor C-terminal domain in solution. Two X-ray crystal structures, of intact trigger factor from E. coli (Ferbitz et al., Nature 431:590-596, 2004), and of a truncated trigger factor from Vibrio cholerae (Ludlam et al., Proc Natl Acad Sci USA 101:13436-13441, 2004) showed significant differences in the structure of the C-terminal domain, such that the two structures could not be superimposed. We show using NMR chemical shifts and long range nuclear Overhauser effects that the secondary and tertiary structure of the E. coli C-terminal domain in solution is consistent with the crystal structure of the E. coli trigger factor and not with the V. cholerae protein. Given the similarity of the amino acid sequences of the E. coli and V. cholerae proteins, it appears likely that the structure of the V. cholerae protein has been distorted as a result of truncation of a 44-amino acid segment at the C-terminus. Analysis of residual dipolar coupling measurements shows that the overall topology of the solution structure is completely inconsistent with both structures. Dynamics analysis of the C-terminal domain using T{sub 1}, T{sub 2} and heteronuclear NOE parameters show that the

  7. Comparison of the Amino-Acid Content in Pharmacopuncture Extracts Taken from a Scorpion's Body and from Its Tail

    Directory of Open Access Journals (Sweden)

    Lee Jin-Ho

    2013-06-01

    Full Text Available Objective: This study was conducted to investigate the amino-acid compositions of pharmacopuncture extracts taken from the body and from the tail of Buthus martensii Karsch, which are frequently prescribed in Oriental medicine. Methods: Amino acids in hot water and 70% ethanol extracts taken from the scorpion’s whole body and from its tail were screened by using high performance liquid chromatography (HPLC. The experiments were performed with linearity, precision and accuracy. Results: The results of the amino-acid-composition analysis showed that the Buthus martensii Karsch extracts contained various amino acids such as aspartic acid, histidine, alanine, tyrosine, and cystine. The amino-acid analysis showed that the hot water extract was more beneficial than the ethanol extract, except for histidine. The amino acids from the tail and the body of the scorpion were compared, and the concentration of aspartic acid in the extract from the scorpion’s tail was two times that found in the extract from its body. The results of validation experiments were all satisfactory. Conclusion: Studies on the ingredients in extracts from a scorpion other than buthotoxin may demonstrate that the antiepileptic efficacy, anticancer activity, antithrombotic action and analgesic effect are enhanced. Using only the tail of the scorpion when pharmacopuncture is dispensed may be beneficial because the extracts from the tail of the scorpion have higher potency than those from the whole body.

  8. Secretin and its C-terminal hexapeptide potentiates insulin release in mouse islets

    DEFF Research Database (Denmark)

    Kofod, Hans; Hansen, B; Lernmark, A;

    1986-01-01

    /ml; the maximal effect was obtained with 1 microgram/ml secretin. This effect was mimicked by 50-500 micrograms/ml NH2-Leu-Leu-Gln-Gly-Leu-Val-NH2, [S-(22-27)], which represents an amidated C-terminal sequence of the secretin molecule. The consecutive smaller secretin C-terminal peptides had either no effects...... no stimulatory effect on islet glutamate dehydrogenase activity. In fact, S-(23-27), S-(24-27), and S-(25-27) inhibited the islet glutamate dehydrogenase activity, the activation by which amino acids and amino acid derivatives are known to elicit a potentiation of insulin release. Our results suggest that the C...

  9. Tailings management

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-05-01

    Careful planning and water management is essential to the proper management of conventional tailings impoundments. Alternatives, like dry stacking and tailings backfill are becoming more widely used. Effective closure is important, especially if acid rock drainage (ARD) is to be avoided. 11 refs., 3 photos.

  10. Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

    DEFF Research Database (Denmark)

    Clausen, Johannes D; Vandecaetsbeek, Ilse; Wuytack, Frank

    2012-01-01

    The molecular mechanism underlying the characteristic high apparent Ca2+ affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11 amino-acid luminal extension (LE). The effects...... from Ca2E1. Addition of the SERCA2b tail to SERCA1a slowed Ca2+ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca2E1. The interaction of LE with L7/8 is also important for the low rate...

  11. The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

    Science.gov (United States)

    Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

    2013-07-10

    NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions.

  12. Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23.

    Science.gov (United States)

    Lo, Huei-Fen; Lin, Long-Liu; Chiang, Wen-Ying; Chie, Meng-Chun; Hsu, Wen-Hwei; Chang, Chen-Tien

    2002-08-01

    The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.

  13. Evolution of plant colonization in acid and alkaline mine tailing ponds after amendments and microorganisms application

    Science.gov (United States)

    Acosta, Jose Alberto; Faz, Ángel; Kabas, Sebla; Zornoza, Raúl; Martínez-Martínez, Silvia

    2014-05-01

    Intense mining activities in the past were carried out in Cartagena-La Unión mining district, SE Spain, and caused excessive accumulation of toxic metals in tailing ponds which poses a high environmental and ecological risk. One of the remediation options gaining considerable interest in recent years is the in situ immobilization of metals. A corresponding reduction in the plant-available metal fraction allows re-vegetation and ecosystem restoration of the heavily contaminated sites. In addition, the use of microorganisms to improve the soil condition is a new tool used to increase spontaneous plant colonization. The aim of this research was to assess the effect of amendments (pig manure, sewage sludge, and lime) and microorganisms on plant cover establishment, as a consequence of metal immobilization and the improvement of soil properties. The study was carried out in two mine ponds (acid and alkaline). Twenty seven square field plots, each one consisting of 4 m2, were located in each pond. Four different doses of microorganism (0 ml, 20 ml, 100 ml and 200 ml of microorganism solution in each plot) and one dose of pig manure (5 kg per plot), sewage sludge (4 kg per plot) and lime (22 kg per plot) were used. Organic amendment doses were calculated according to European nitrogen legislations, and lime dose was calculated according with the potential acid production through total sulphur oxidation. Three replicates of each treatment (organic amendment + lime + microorganism dose 0, 1, 2, or 3) and control soil (with no amendments) were carried out. Plots were left to the semi-arid climate conditions after the addition of amendments to simulate real potential applications of the results. Identification of plant species and biodiversity was determined on each plot, after 2, 4, 6 and 8 months of amendment addition. The results showed that, in those plots without application of microorganism, 8 months after applications the number of species and individuals of each

  14. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2003-05-01

    Full Text Available Abstract Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102, only the bidirectional (Synechocystis PCC 6803 or both NiFe-hydrogenases (Anabaena PCC 7120 prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41% to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known

  15. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  16. Modules for C-terminal epitope tagging of Tetrahymena genes

    Science.gov (United States)

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  17. Structure-activity studies on the C-terminal amide of substance P.

    Science.gov (United States)

    Escher, E; Couture, R; Poulos, C; Pinas, N; Mizrahi, J; Theodoropoulos, D; Regoli, D

    1982-11-01

    Twelve C-terminal heptapeptide analogues of substance P have been synthesized by solid phase and by the classical solution method. The modifications concerned all the C-terminal primary amide of SP and should therefore help to understand the biological significance of this carboxamide, as evaluated by in vivo and in vitro bioassays. From the results it can be seen that not the slightest change of the two amide protons is tolerated without an important loss of activity: replacement of one or two amide protons with alkyl groups, extension of the amide to the hydrazide and its alkyl analogues, and exchange of the amide with an ester or a carboxylic acid all reduce the relative activity/affinity at least by 2-fold. It is not clear for what reason all these modifications produce such a drastic activity reduction.

  18. Efficient degradation of Acid Orange 7 in aqueous solution by iron ore tailing Fenton-like process.

    Science.gov (United States)

    Zheng, Jianming; Gao, Zhanqi; He, Huan; Yang, Shaogui; Sun, Cheng

    2016-05-01

    An effective method based on iron ore tailing Fenton-like process was studied for removing an azo dye, Acid Orange 7 (AO7) in aqueous solution. Five tailings were characterized by X-ray fluorescence spectroscope (XFS), Brunner-Emmet-Teller (BET) measurement, and Scanning Electron Microscope (SEM). The result of XFS showed that Fe, Si and Ca were the most abundant elements and some toxic heavy metals were also present in the studied tailings. The result of BET analysis indicated that the studied tailings had very low surface areas (0.64-5.68 m(2) g(-1)). The degradation efficiencies of AO7 were positively correlated with the content of iron oxide and cupric oxide, and not related with the BET surface area of the tailings. The co-existing metal elements, particularly Cu, might accelerate the heterogeneous Fenton-like reaction. The effects of other parameters on heterogeneous Fenton-like degradation of AO7 by a converter slag iron tailing (tailing E) which contains highest iron oxide were also investigated. The tailing could be reused 10 times without significant decrease of the catalytic capacity. Very low amount of iron species and almost undetectable toxic elements were leached in the catalytic degradation of AO7 by the tailing E. The reaction products were identified by gas chromatography-mass spectrometry and a possible pathway of AO7 degradation was proposed. This study not only provides an effective method for removing azo dyes in polluted water by employing waste tailings as Fenton-like catalysts, but also uses waste tailings as the secondary resource.

  19. Response of Key Soil Parameters During Compost-Assisted Phytostabilization in Extremely Acidic Tailings: Effect of Plant Species

    OpenAIRE

    Solís-Dominguez, Fernando A.; White, Scott A.; Hutter, Travis Borrillo; Amistadi, Mary Kay; Root, Robert A.; Chorover, Jon; Maier, Raina M.

    2012-01-01

    Phytostabilization of mine tailings acts to mitigate both eolian dispersion and water erosion events which can disseminate barren tailings over large distances. This technology uses plants to establish a vegetative cover to permanently immobilize contaminants in the rooting zone, often requiring addition of an amendment to assist plant growth. Here we report the results of a greenhouse study that evaluated the ability of six native plant species to grow in extremely acidic (pH ~ 2.5) metallif...

  20. Metabolism of branched-chain amino acids in leg muscles from tail-cast suspended intact and adrenalectomized rats

    Science.gov (United States)

    Jaspers, Stephen R.; Henriksen, Erik; Jacob, Stephan; Tischler, Marc E.

    1989-01-01

    The effects of muscle unloading, adrenalectomy, and cortisol treatment on the metabolism of branched-chain amino acids in the soleus and extensor digitorum longus of tail-cast suspended rats were investigated using C-14-labeled lucine, isoleucine, and valine in incubation studies. It was found that, compared to not suspended controls, the degradation of branched-chain amino acids in hind limb muscles was accelerated in tail-cast suspended rats. Adrenalectomy was found to abolish the aminotransferase flux and to diminish the dehydrogenase flux in the soleus. The data also suggest that cortisol treatment increases the rate of metabolism of branched-chain amino acids at the dehydrogenase step.

  1. Resonance assignments and secondary structure of apolipoprotein E C-terminal domain in DHPC micelles.

    Science.gov (United States)

    Lo, Chi-Jen; Chyan, Chia-Lin; Chen, Yi-Chen; Chang, Chi-Fon; Huang, Hsien-Bin; Lin, Ta-Hsien

    2015-04-01

    Human apolipoprotein E (apoE) has been known to play a key role in the transport of plasma cholesterol and lipoprotein metabolism. It is an apolipoprotein of 299 amino acids with a molecular mass, ~34 kDa. ApoE has three major isoforms, apoE2, apoE3, and apoE4 which differ only at residue 112 or 158. ApoE consists of two independently folded domains (N-terminal and C-terminal domain) separated by a hinge region. The N-terminal domain and C-terminal domain of apoE are responsible for the binding to receptor and to lipid, respectively. Since the high resolution structures of apoE in lipids are still unavailable to date, we therefore aim to resolve the structures in lipids by NMR. Here, we reported the resonance assignments and secondary structure distribution of the C-terminal domain of wild-type human apoE (residue 195-299) in the micelles formed by dihexanoylphosphatidylcholine. Our results may provide a novel structural model of apoE in micelles and may shed new light on the molecular mechanisms underlying the apoE related biological processes.

  2. 傅里叶显微红外研究C-末端酸性蛋白对α-硫素原核表达过程的影响%Study on the Effect of C-Terminal Acidic Protein on the Prokaryotic Expression of α-Thionin by FTIR Microspectroscopy

    Institute of Scientific and Technical Information of China (English)

    刘艳; 冯娟; 陶栋梁; 翁诗甫; 任正隆

    2009-01-01

    Fourier transform infrared (FTIR) microspectroscopy was used to investigate the effects of C-terminal acidic protein on the secondary structure of wheat α-thionin in the absence of signal peptide during the prokaryotic expression process. SDS-PAGE analysis revealed that the presence of acidic protein gave rise to the formation of inclusion body, however, the absence of acidic protein greatly enhanced the solubility of the heterogenous protein expressed in E. coli BL21(DE3) with the induction of 1 containing S and Sc, which corresponds to the absence and presence of C-terminal acidic proteins, respectively. The second de-rivative of the difference spectra measured 2 h after induction showed one principal component at ~1 630 cm~(-1) , while no signifi-cant peak appeared at the same peak position when the spectra before induction were compared. Combined with SD~PAGE of recombinant protein, the authors presumed that the peak absorption at ~1 630 cm~(-1) is most likey to be assigned to protein ag-gregate within inclusion body. Gaussian curve-fitting was done on the Fourier self-deconvolution spectra within amide I region of intact cells containing S and Sc. The experimental data revealed that the relative content of aggregate absorption at (1 629 ± 1) cm~(-1) gradually increased with induction time, which is consistent with the results of SDS-PAGE. Simutaneously, the formation of aggregate gave rise to the increase of a-helix, as well as the decrease of fl-turn and random coil in the ease of Sc. It was not the case for S, however, where random coil experienced the increase in the relative average fractions, while β-turn and β-sheet at (1 629±1) cm~(-1) behaved in different ways. The above mentioned phenomenon indicated that fl-sheet and random coil are most likely to transform into aggregate and α-helix with the introduction of C-terminal acidic protein.%采用傅里叶变换显微红外手段研究了在信号肽缺失的情况下,C-末端酸性蛋白的

  3. Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte; Munch-Petersen, Sune; Berenstein, Dvora;

    2006-01-01

    and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication. The cell cycle specific activity of TK1 is regulated at the transcriptional level......, but posttranslational mechanisms seem to play an important role for the level of functional TK1 protein as well. Thus, the C-terminal of TK1 is known to be essential for the specific degradation of the enzyme at the G2/M phase. In this work, we have studied the effect of deletion of the C-terminal 20, 40, and 44 amino...... acids of TK1 on in vitro stability, oligomerization, and enzyme kinetics. We found that deletion of the C-terminal fold markedly increased the stability as well as the catalytic activity....

  4. Cytochrome oxidase and ascorbic acid in the normal and regenerating tail of the scincid lizard, Mabuya carinata. A histophysiological study.

    Science.gov (United States)

    Ramachandran, A V; Radhakrishnan, N; Shah, R V

    1975-01-01

    The concentration of ascorbic acid (AA) and the histochemical distribution of this vitamin together with cytochrome oxidase have been investigated in the normal and regenerating tail of the Scincid lizard, Mabuya carinata. An interesting aspect of this investigation is the observation of a total lack of cytochrome oxidase in both the normal and regenerating tail of the lizard, except for the differentiating phase. On the other hand, AA has been found to be present in the normal and regenerating tail with above normal levels during wound healing (twofold) and differentiation (fivefold). In the light of the poor cytochrome oxidase activity, the higher content of AA noted during regeneration has been construed to play a possible role in the respiratory mechanics of the regenerating lizard tail. Further, the importance of AA in cellular metabolism and the wound healing and differentiative processes have also been discussed.

  5. Trace metal mobilization from oil sands froth treatment thickened tailings exhibiting acid rock drainage.

    Science.gov (United States)

    Kuznetsova, Alsu; Kuznetsov, Petr; Foght, Julia M; Siddique, Tariq

    2016-11-15

    Froth treatment thickened tailings (TT) are a waste product of bitumen extraction from surface-mined oil sands ores. When incubated in a laboratory under simulated moist oxic environmental conditions for ~450d, two different types of TT (TT1 and TT2) exhibited the potential to generate acid rock drainage (ARD) by producing acid leachate after 250 and 50d, respectively. We report here the release of toxic metals from TT via ARD, which could pose an environmental threat if oil sands TT deposits are not properly managed. Trace metal concentrations in leachate samples collected periodically revealed that Mn and Sr were released immediately even before the onset of ARD. Spikes in Co and Ni concentrations were observed both pre-ARD and during active ARD, particularly in TT1. For most elements measured (Fe, Cr, V, As, Cu, Pb, Zn, Cd, and Se), leaching was associated with ARD production. Though equivalent acidification (pH2) was achieved in leachate from both TT types, greater metal release was observed from TT2 where concentrations reached 10,000ppb for Ni, 5000ppb for Co, 3000ppb for As, 2000ppb for V, and 1000ppb for Cr. Generally, metal concentrations decreased in leachate with time during ARD and became negligible by the end of incubation (~450d) despite appreciable metals remaining in the leached TT. These results suggest that using TT for land reclamation purposes or surface deposition for volume reduction may unfavorably impact the environment, and warrants application of appropriate strategies for management of pyrite-enriched oil sands tailings streams.

  6. Efficient inhibition of heavy metal release from mine tailings against acid rain exposure by triethylenetetramine intercalated montmorillonite (TETA-Mt).

    Science.gov (United States)

    Gong, Beini; Wu, Pingxiao; Huang, Zhujian; Li, Yuanyuan; Yang, Shanshan; Dang, Zhi; Ruan, Bo; Kang, Chunxi

    2016-11-15

    The potential application of triethylenetetramine intercalated montmorillonite (TETA-Mt) in mine tailings treatment and AMD (acid mine drainage) remediation was investigated with batch experiments. The structural and morphological characteristics of TETA-Mt were analyzed with XRD, FTIR, DTG-TG and SEM. The inhibition efficiencies of TETA-Mt against heavy metal release from mine tailings when exposed to acid rain leaching was examined and compared with that of triethylenetetramine (TETA) and Mt. Results showed that the overall inhibition by TETA-Mt surpassed that by TETA or Mt for various heavy metal ions over an acid rain pH range of 3-5.6 and a temperature range of 25-40°C. When mine tailings were exposed to acid rain of pH 4.8 (the average rain pH of the mining site where the mine tailings were from), TETA-Mt achieved an inhibition efficiency of over 90% for Cu(2+), Zn(2+), Cd(2+) and Mn(2+) release, and 70% for Pb(2+) at 25°C. It was shown that TETA-Mt has a strong buffering capacity. Moreover, TETA-Mt was able to adsorb heavy metal ions and the adsorption process was fast, suggesting that coordination was mainly responsible. These results showed the potential of TETA-Mt in AMD mitigation, especially in acid rain affected mining area.

  7. Manganese ore tailing: optimization of acid leaching conditions and recovery of soluble manganese.

    Science.gov (United States)

    Santos, Olívia de Souza Heleno; Carvalho, Cornélio de Freitas; Silva, Gilmare Antônia da; Santos, Cláudio Gouvêa Dos

    2015-01-01

    Manganese recovery from industrial ore processing waste by means of leaching with sulfuric acid was the objective of this study. Experimental conditions were optimized by multivariate experimental design approaches. In order to study the factors affecting leaching, a screening step was used involving a full factorial design with central point for three variables in two levels (2(3)). The three variables studied were leaching time, concentration of sulfuric acid and sample amount. The three factors screened were shown to be relevant and therefore a Doehlert design was applied to determine the best working conditions for leaching and to build the response surface. By applying the best leaching conditions, the concentrations of 12.80 and 13.64 %w/w of manganese for the global sample and for the fraction -44 + 37 μm, respectively, were found. Microbeads of chitosan were tested for removal of leachate acidity and recovering of soluble manganese. Manganese recovery from the leachate was 95.4%. Upon drying the leachate, a solid containing mostly manganese sulfate was obtained, showing that the proposed optimized method is efficient for manganese recovery from ore tailings.

  8. Development of Noviomimetics as C-Terminal Hsp90 Inhibitors.

    Science.gov (United States)

    Anyika, Mercy; McMullen, Mason; Forsberg, Leah K; Dobrowsky, Rick T; Blagg, Brian S J

    2016-01-14

    KU-32 and KU-596 are novobiocin-derived, C-terminal heat shock protein 90 (Hsp90) modulators that induce Hsp70 levels and manifest neuroprotective activity. However, the synthetically complex noviose sugar requires 10 steps to prepare, which makes translational development difficult. In this study, we developed a series of "noviomimetic" analogues of KU-596, which contain noviose surrogates that can be easily prepared, while maintaining the ability to induce Hsp70 levels. Both sugar and sugar analogues were designed, synthesized, and evaluated in a luciferase reporter assay, which identified compound 37, a benzyl containing noviomimetic, as the most potent inducer of Hsp70.

  9. Conserved C-terminal nascent peptide binding domain of HYPK facilitates its chaperone-like activity

    Indian Academy of Sciences (India)

    Swasti Raychaudhuri; Rachana Banerjee; Subhasish Mukhopadhyay; Nitai P Bhattacharyya

    2014-09-01

    Human HYPK (Huntingtin Yeast-two-hybrid Protein K) is an intrinsically unstructured chaperone-like protein with no sequence homology to known chaperones. HYPK is also known to be a part of ribosome-associated protein complex and present in polysomes. The objective of the present study was to investigate the evolutionary influence on HYPK primary structure and its impact on the protein’s function. Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying its importance in the structural and functional evolution. NPAA domain of human HYPK has unique amino acid composition preferring glutamic acid and happens to be more stable from a conformational point of view having higher content of -helices than the rest. Cell biology studies indicate that overexpressed C-terminal human HYPK can interact with nascent proteins, co-localizes with huntingtin, increases cell viability and decreases caspase activities in Huntington’s disease (HD) cell culture model. This domain is found to be required for the chaperone-like activity of HYPK in vivo. Our study suggested that by virtue of its flexibility and nascent peptide binding activity, HYPK may play an important role in assisting protein (re)folding.

  10. Bio-physicochemical effects of gamma irradiation treatment for naphthenic acids in oil sands fluid fine tailings

    Energy Technology Data Exchange (ETDEWEB)

    Boudens, Ryan; Reid, Thomas; VanMensel, Danielle; Sabari Prakasanm, M.R. [Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON (Canada); Ciborowski, Jan J.H. [Biological Sciences, University of Windsor, Windsor, ON (Canada); Weisener, Christopher G., E-mail: weisener@uwindsor.ca [Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON (Canada)

    2016-01-01

    Naphthenic acids (NAs) are persistent compounds that are components of most petroleum, including those found in the Athabasca oil sands. Their presence in freshly processed tailings is of significant environmental concern due to their toxicity to aquatic organisms. Gamma irradiation (GI) was used to reduce the toxicity and concentration of NAs in oil sands process water (OSPW) and fluid fine tailings (FFT). This investigation systematically studied the impact of GI on the biogeochemical development and progressive reduction of toxicity using laboratory incubations of fresh and aged tailings under anoxic and oxic conditions. GI reduced NA concentrations in OSPW by up to 97% in OSPW and in FFT by 85%. The GI-treated FFT exhibited increased rates of biogeochemical change, dependent on the age of the tailings source. Dissolved oxygen (DO) flux was enhanced in GI-treated FFT from fresh and aged source materials, whereas hydrogen sulfide (HS{sup −}) flux was stimulated only in the fresh FFT. Acute toxicity to Vibrio fischeri was immediately reduced following GI treatment of fresh OSPW. GI treatment followed by 4-week incubation reduced toxicity of aged OSPW to V. fischeri. - Highlights: • Gamma irradiation substantially reduced concentrations of ecotoxic naphthenic acids • Acute toxicity was reduced in gamma irradiated process water • Gamma irradiated tailings exhibited increased rates of microbial respiration.

  11. Occurrence of C-terminal residue exclusion in peptide fragmentation by ESI and MALDI tandem mass spectrometry.

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b(n-1) + H(2)O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H(2)O and NH(3)) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment

  12. The C-terminal Region of Laminin β Chains Modulates the Integrin Binding Affinities of Laminins*S⃞

    OpenAIRE

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-01-01

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (α, β, and γ), in which three laminin globular modules in the α chain and the Glu residue in the C-terminal tail of the γ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the β chain is involved in laminin-integrin interactions. We compared the binding affinities of ...

  13. Extensive analysis of milk fatty acids in two fat-tailed sheep breeds during lactation.

    Science.gov (United States)

    Payandeh, S; Kafilzadeh, F; Juárez, M; de la Fuente, M A; Ghadimi, D; Marín, A L Martínez

    2016-12-01

    The profile of fatty acids (FA) in the milk fat of two Iranian fat-tailed sheep breeds, Sanjabi and Mehraban, was compared during lactation. Eight ewes of each breed, balanced in parity and carrying one foetus, were selected before parturition. Ewes were kept separated in individual pens during the experimental period, under the same management practices and fed the same diet, in order to eliminate any confounding effects on milk FA profile. Milk was sampled at biweekly intervals up to 10 weeks of lactation, starting 2 weeks after parturition. More than 100 FA were determined in milk fat by means of gas chromatography. The milk fat of Sanjabi ewes contained more cis-9 18:1, that of Mehraban ewes was richer in 10:0, 12:0 and 14:0, and no differences were found for 16:0 and 18:0. No breed differences were found for most branched-chain FA. Mehraban ewes showed a higher presence of vaccenic and rumenic acids in their milk fat. The milk fat of Sanjabi ewes had a lower atherogenicity index and n-6/n-3 FA ratio. The contents of several FA showed time-dependent changes, so breed differences were more apparent or disappeared as lactation progressed. The milk fat of Sanjabi ewes showed a better FA profile from the human health point of view.

  14. Interaction of limestone grains and acidic solutions from the oxidation of pyrite tailings

    Energy Technology Data Exchange (ETDEWEB)

    Simon, M. [Departamento de Edafologia, EPS-CITE IIB, Canada San Urbano, Universidad de Almeria, 04120 Almeria (Spain)]. E-mail: msimon@ual.es; Martin, F. [Departamento de Edafologia, Facultad de Ciencias, Universidad de Granada, 18002 Granada (Spain); Garcia, I. [Departamento de Edafologia, EPS-CITE IIB, Canada San Urbano, Universidad de Almeria, 04120 Almeria (Spain); Bouza, P. [Centro Nacional Patagonico, CONICEF, Boulevard Brown s/n, 9120 Puerto Madryn, Chubut (Argentina); Dorronsoro, C. [Departamento de Edafologia, Facultad de Ciencias, Universidad de Granada, 18002 Granada (Spain); Aguilar, J. [Departamento de Edafologia, Facultad de Ciencias, Universidad de Granada, 18002 Granada (Spain)

    2005-05-01

    To characterise the coatings formed and to analyse element partitioning between the aqueous and solid phase, suspensions were prepared with four grain sizes of limestone and three different amounts of acidic solution from oxidized pyrite tailings. In all cases, red coatings with three different layers covered the grain surface, sealing off the acidic solution. The inner layer was composed mainly of basaluminite, the middle layer of schwertmannite, and the outer layer of gypsum and jarosite. Zn, Cd and Tl were co-precipitated by Fe and Al; As and Pb were co-precipitated almost completely by Fe; and Cu formed mainly Cu sulphates. All trace elements reached almost total precipitation at pH 6.3, but the precipitation of As and Pb tended to decrease as the pH rose. Consequently, liming should be calculated so that the soil pH does not exceed 6.3. This calculation should take into account that the armouring of the limestone grains can cause underestimations in the amount of liming material needed. - Basaluminite, schwertmannite and jarosite armored the limestone grains, and almost all trace elements co-precipitated, but the precipitation of As and Pb tended to decrease as the pH rose.

  15. Process optimization of reaction of acid leaching residue of asbestos tailing and sodium hydroxide aqueous solution

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Silica is the major component of the acid leaching residue of asbestos tailing. The waterglass solution can be prepared by the reaction of the residue with sodium hydroxide aqueous solution. Compared to the high temperature reaction method, this process is environmental friendly and low cost. In this paper, the reaction process of the residue and the sodium hydroxide aqueous solution is optimized. The optimum reaction process parameters are as follows: the usage of sodium hydroxide is 26.4 g/100 g acid leaching residue, the reaction temperature is 90℃, the reaction time is 1 h, and the ratio of the liquid/solid is 2.0. The significance sequence of the process parameters to the alkali leaching reaction effect is the usage of sodium hydroxide > the ratio of the liquid/solid > the reaction time > the reaction temperature. The significance sequence to the leaching ratio of SiO2 is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. The significance sequence to the modulus of the sodium silicate is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. Under the optimum conditions, the leaching ratio of the SiO2 is 77.5%, and the modulus of the sodium silicate is 3.15. The XRD analysis result indicates that the major components of the alkali leaching residue are serpentine, talc, quartz and some albite.

  16. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    Science.gov (United States)

    Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  17. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    Directory of Open Access Journals (Sweden)

    Gustavo M S G Moreira

    Full Text Available Bauhinia variegata lectins (BVL-I and BVL-II are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1 the first amino acid of the excised peptide is small or hydrophobic; (2 the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3 the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  18. Epimerization-free C-terminal peptide activation, elongation and cyclization

    NARCIS (Netherlands)

    Popović, S.

    2015-01-01

    C-terminal peptide activation and cyclization reactions are generally accompanied with epimerization (partial loss of C‐terminal stereointegrity). Therefore, the focus of this thesis was to develop epimerization-free methods for C-terminal peptide activation to enable C-terminal peptide elongation a

  19. [Study on the acid hydrolysis, fiber remodeling and bionics mineralization of rat tail tendon collagen type Ⅰ].

    Science.gov (United States)

    Zhang, Zhan; Zhang, Chun; Guo, Qiaofeng

    2016-05-25

    Objective: To produce bionic bone material that is consistent with human bone in chemical composition and molecular structure using rat tail tendon collagen type Ⅰ. Methods: The type Ⅰcollagen derived from rat tail was extracted by acetic acid to form collagen fibers. The reconstructed collagen fibers were placed in the mineralized solution to mimic bone mineralization for 2-6 days. Bone mineralization was observed by transmission electron microscopy and electron diffraction.Results: Collagen fibers with characteristic D-Band structure were reconstructed by using rat tail tendon collagen type Ⅰ extracted with acid hydrolysis method. Transmission electron microscopy and electron diffraction showed that calcium hydroxyapatite precursor infiltrated into the collagen fibers, and the collagen fibers were partially mineralized after 2 days of mineralization; the collagen fibers were completely mineralized and bionic bone material of typeⅠ collagen/calcium hydroxyapatite was formed after 6 days of mineralization.Conclusion: The collagen type Ⅰ can be extracted from rat tail tendon by acid hydrolysis method, and can be reformed and mineralized to form the bionic bone material which mimics human bone in chemical composition and the molecular structure.

  20. C-terminal methylation of truncated neuropeptides: an enzyme-assisted extraction artifact involving methanol.

    Science.gov (United States)

    Stemmler, Elizabeth A; Barton, Elizabeth E; Esonu, Onyinyechi K; Polasky, Daniel A; Onderko, Laura L; Bergeron, Audrey B; Christie, Andrew E; Dickinson, Patsy S

    2013-08-01

    Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.

  1. Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I' region.

    Science.gov (United States)

    Anderson, Benjamin A; Literati, Alex; Ball, Borden; Kubelka, Jan

    2015-01-01

    Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I' band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly-Gly, Gly-Ala, Ala-Gly) in D₂O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

  2. Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I‧ region

    Science.gov (United States)

    Anderson, Benjamin A.; Literati, Alex; Ball, Borden; Kubelka, Jan

    2015-01-01

    Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I‧ band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly-Gly, Gly-Ala, Ala-Gly) in D2O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

  3. A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2 coded lysis protein dissipates the protonmotive force in Escherichia coli membrane vesicles by generating hydrophilic pores

    NARCIS (Netherlands)

    Goessens, Wil H.F.; Driessen, Arnold J.M.; Wilschut, Jan; Duin, Jan van

    1988-01-01

    The RNA phage MS2 encodes a protein, 75 amino acids long, that is necessary and sufficient for lysis of the host cell. DNA deletion analysis has shown that the lytic activity is confined to the C-terminal half of the protein. We have examined the effects of a synthetic peptide, covering the C-termin

  4. Process optimization of reaction of acid leaching residue of asbestos tailing and sodium hydroxide aqueous solution

    Institute of Scientific and Technical Information of China (English)

    DU GaoXiang; ZHENG ShuiLin; DING Hao

    2009-01-01

    Silica is the major component of the acid leaching residue of asbestos tailing. The waterglass solution can be prepared by the reaction of the residue with sodium hydroxide aqueous solution. Compared to the high temperature reaction method, this process is environmental friendly and low cost. In this paper, the reaction process of the residue and the sodium hydroxide aqueous solution is optimized. The op-timum reaction process parameters are as follows: the usage of sodium hydroxide is 26.4 g/100 g acid leaching residue, the reaction temperature is 90℃, the reaction time is 1 h, and the ratio of the liq-uid/solid is 2.0. The significance sequence of the process parameters to the alkali leaching reaction effect is the usage of sodium hydroxide > the ratio of the liquid/solid > the reaction time > the reaction temperature. The significance sequence to the leaching ratio of SiO2 is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. The significance sequence to the modulus of the sodium silicate is the ratio of the liquid/solid > the usage of sodium hydroxide > the reaction time > the reaction temperature. Under the optimum conditions, the leaching ratio of the SiO2 is 77.5%, and the modulus of the sodium silicate is 3.15. The XRD analysis result indicates that the major components of the alkali leaching residue are serpentine, talc, quartz and some albite.

  5. Recombinant production of peptide C-terminal α-amides using an engineered intein

    DEFF Research Database (Denmark)

    Albertsen, Louise; Shaw, Allan C; Norrild, Jens Chr.;

    2013-01-01

    is that they contain a C-terminal that is α-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a larger scale. Initially...... of the 198 amino acid intein with an eight amino acid linker. The optimized intein construct was used to produce the PYY derivative under high cell density cultivation conditions, generating the peptide thioester precursor in good yields and subsequent amidation provided the target peptide......., we experienced challenges with hydrolysis of the intein fusion protein, which was reduced by a T3C mutation in the intein. Subsequently, we further engineered the intein to decrease the absolute size and improve the relative yield of the PYY derivative, which was achieved by substituting 54 residues...

  6. Structure of the C-terminal domain of nsp4 from feline coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Snijder, Eric J.; Gorbalenya, Alexander E. [Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden (Netherlands); Berglind, Hanna; Nordlund, Pär [Division of Biophysics, Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institute, SE-171 77 Stockholm (Sweden); Coutard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098, AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille (France); Tucker, Paul A., E-mail: tucker@embl-hamburg.de [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  7. The C-terminal region of thermophilic tRNA (m7G46) methyltransferase (TrmB) stabilizes the dimer structure and enhances fidelity of methylation.

    Science.gov (United States)

    Tomikawa, Chie; Ochi, Anna; Hori, Hiroyuki

    2008-05-15

    Transfer RNA (m(7)G46) methyltransferase catalyzes methyl-transfer from S-adenosyl-L-methionine to N(7) atom of the semi-conserved G46 base in tRNA. Aquifex aeolicus is a hyper thermophilic eubacterium that grows at close to 95 degrees C. A. aeolicus tRNA (m(7)G46) methyltransferase [TrmB] has an elongated C-terminal region as compared with mesophilic counterparts. In this study, the authors focused on the functions of this C-terminal region. Analytic gel filtration chromatography and amino acid sequencing reveled that the start point (Glu202) of the C-terminal region is often cleaved by proteases during purification steps and the C-terminal region tightly binds to another subunit even in the presence of 6M urea. Because the C-terminal region contains abundant basic amino acid residues, the authors assumed that some of these residues might be involved in tRNA binding. To address this idea, the authors prepared eight alanine substitution mutant proteins. However, measurements of initial velocities of these mutant proteins suggested that the basic amino acid residues in the C-terminal region are not involved in tRNA binding. The authors investigated effects of the deletion of the C-terminal region. Deletion mutant protein of the C-terminal region (the core protein) was precipitated by incubation at 85 degrees C, while the wild type protein was soluble at that temperature, demonstrating that the C-terminal region contributes to the protein stability at high temperatures. The core protein had a methyl-transfer activity to yeast tRNA(Phe) transcript. Furthermore, the core protein slowly methylated tRNA transcripts, which did not contain G46 base. Moreover, the modified base was identified as m(7)G by two-dimensional thin layer chromatography. Thus, the deletion of the C-terminal region causes nonspecific methylation of N(7) atom of guanine base(s) in tRNA transcripts.

  8. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    Science.gov (United States)

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  9. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production

    Science.gov (United States)

    Poksay, Karen S.; Sheffler, Douglas J.; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E.; Cosford, Nicholas D. P.; John, Varghese

    2017-01-01

    Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds – identified here using cells and tissues expressing wt human APP – in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

  10. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production.

    Science.gov (United States)

    Poksay, Karen S; Sheffler, Douglas J; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E; Cosford, Nicholas D P; John, Varghese

    2017-01-01

    Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

  11. Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

    Science.gov (United States)

    Jones, A J; Rumsby, M G

    1977-12-01

    The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.

  12. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    Directory of Open Access Journals (Sweden)

    Rumbley Jon N

    2011-02-01

    Full Text Available Abstract Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.

  13. Geochemical characterisation of seepage and drainage water quality from two sulphide mine tailings impoundments: Acid mine drainage versus neutral mine drainage

    Science.gov (United States)

    Heikkinen, P.M.; Raisanen, M.L.; Johnson, R.H.

    2009-01-01

    Seepage water and drainage water geochemistry (pH, EC, O2, redox, alkalinity, dissolved cations and trace metals, major anions, total element concentrations) were studied at two active sulphide mine tailings impoundments in Finland (the Hitura Ni mine and Luikonlahti Cu mine/talc processing plant). The data were used to assess the factors influencing tailings seepage quality and to identify constraints for water treatment. Changes in seepage water quality after equilibration with atmospheric conditions were evaluated based on geochemical modelling. At Luikonlahti, annual and seasonal changes were also studied. Seepage quality was largely influenced by the tailings mineralogy, and the serpentine-rich, low sulphide Hitura tailings produced neutral mine drainage with high Ni. In contrast, drainage from the high sulphide, multi-metal tailings of Luikonlahti represented typical acid mine drainage with elevated contents of Zn, Ni, Cu, and Co. Other factors affecting the seepage quality included weathering of the tailings along the seepage flow path, process water input, local hydrological settings, and structural changes in the tailings impoundment. Geochemical modelling showed that pH increased and some heavy metals were adsorbed to Fe precipitates after net alkaline waters equilibrated with the atmosphere. In the net acidic waters, pH decreased and no adsorption occurred. A combination of aerobic and anaerobic treatments is proposed for Hitura seepages to decrease the sulphate and metal loading. For Luikonlahti, prolonged monitoring of the seepage quality is suggested instead of treatment, since the water quality is still adjusting to recent modifications to the tailings impoundment.

  14. Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment.

    Science.gov (United States)

    Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

    2008-09-30

    Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS.

  15. Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA

    OpenAIRE

    Jones, Denise F.; Stenzel, Rachelle A.; Donohue, Timothy J.

    2005-01-01

    The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins wer...

  16. Crystal Structure of the C-terminal Domain of Splicing Factor Prp8 Carrying Retinitis Pigmentosa Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Zhang,L.; Shen, J.; Guarnieri, M.; Heroux, A.; Yang, K.; Zhao, R.

    2007-01-01

    Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an / structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn{sup 2+} coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn{sup 2+}-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

  17. Synthesis and evaluation of novologues as C-terminal Hsp90 inhibitors with cytoprotective activity against sensory neuron glucotoxicity.

    Science.gov (United States)

    Kusuma, Bhaskar Reddy; Zhang, Liang; Sundstrom, Teather; Peterson, Laura B; Dobrowsky, Rick T; Blagg, Brian S J

    2012-06-28

    Compound 2 (KU-32) is a first-generation novologue (a novobiocin-based, C-terminal, heat shock protein 90 (Hsp90) inhibitor) that decreases glucose-induced death of primary sensory neurons and reverses numerous clinical indices of diabetic peripheral neuropathy in mice. The current study sought to exploit the C-terminal binding site of Hsp90 to determine whether the optimization of hydrogen bonding and hydrophobic interactions of second-generation novologues could enhance neuroprotective activity. Using a series of substituted phenylboronic acids to replace the coumarin lactone of 2, we identified that electronegative atoms placed at the meta-position of the B-ring exhibit improved cytoprotective activity, which is believed to result from favorable interactions with Lys539 in the Hsp90 C-terminal binding pocket. Consistent with these results, a meta-3-fluorophenyl substituted novologue (13b) exhibited a 14-fold lower ED(50) for protection against glucose-induced toxicity of primary sensory neurons compared to 2.

  18. Inhibition of acid mine drainage and immobilization of heavy metals from copper flotation tailings using a marble cutting waste

    Institute of Scientific and Technical Information of China (English)

    Gulsen Tozsin

    2016-01-01

    Acid mine drainage (AMD) with high concentrations of sulfates and metals is generated by the oxidation of sulfide bearing wastes. CaCO3-rich marble cutting waste is a residual material produced by the cutting and polishing of marble stone. In this study, the feasibility of using the marble cutting waste as an acid-neutralizing agent to inhibit AMD and immobilize heavy metals from copper flotation tailings (sul-fide-bearing wastes) was investigated. Continuous-stirring shake-flask tests were conducted for 40 d, and the pH value, sulfate content, and dissolved metal content of the leachate were analyzed every 10 d to determine the effectiveness of the marble cutting waste as an acid neu-tralizer. For comparison, CaCO3 was also used as a neutralizing agent. The average pH value of the leachate was 2.1 at the beginning of the experiment (t = 0). In the experiment employing the marble cutting waste, the pH value of the leachate changed from 6.5 to 7.8, and the sul-fate and iron concentrations decreased from 4558 to 838 mg/L and from 536 to 0.01 mg/L, respectively, after 40 d. The marble cutting waste also removed more than 80wt% of heavy metals (Cd, Cr, Cu, Ni, Pb, and Zn) from AMD generated by copper flotation tailings.

  19. Inhibition of acid mine drainage and immobilization of heavy metals from copper flotation tailings using a marble cutting waste

    Science.gov (United States)

    Tozsin, Gulsen

    2016-01-01

    Acid mine drainage (AMD) with high concentrations of sulfates and metals is generated by the oxidation of sulfide bearing wastes. CaCO3-rich marble cutting waste is a residual material produced by the cutting and polishing of marble stone. In this study, the feasibility of using the marble cutting waste as an acid-neutralizing agent to inhibit AMD and immobilize heavy metals from copper flotation tailings (sulfide- bearing wastes) was investigated. Continuous-stirring shake-flask tests were conducted for 40 d, and the pH value, sulfate content, and dissolved metal content of the leachate were analyzed every 10 d to determine the effectiveness of the marble cutting waste as an acid neutralizer. For comparison, CaCO3 was also used as a neutralizing agent. The average pH value of the leachate was 2.1 at the beginning of the experiment ( t = 0). In the experiment employing the marble cutting waste, the pH value of the leachate changed from 6.5 to 7.8, and the sulfate and iron concentrations decreased from 4558 to 838 mg/L and from 536 to 0.01 mg/L, respectively, after 40 d. The marble cutting waste also removed more than 80wt% of heavy metals (Cd, Cr, Cu, Ni, Pb, and Zn) from AMD generated by copper flotation tailings.

  20. C-terminal domain on the outer surface of the Macrobrachium rosenbergii nodavirus capsid is required for Sf9 cell binding and internalization.

    Science.gov (United States)

    Somrit, Monsicha; Watthammawut, Atthaboon; Chotwiwatthanakun, Charoonroj; Ounjai, Puey; Suntimanawong, Wanida; Weerachatyanukul, Wattana

    2017-01-02

    We have shown that Macrobrachium rosenbergii nodavirus (MrNV) was able to infect Sf9 cells and that MrNV virus-like particles (MrNV-VLPs) were capable nanocontainers for delivering nucleic acid-based materials. Here, we demonstrated that chymotryptic removal of a C-terminal peptide and its truncated variant (F344-MrNV-VLPs) exhibited a drastically reduced ability to interact and internalize into Sf9 cells. Electron microscopic observations revealed that the loss of C-terminal domain either from enzyme hydrolysis or genetic truncation did not affect the generated MrNV-VLPs' icosahedral conformation, but did drastically affect the VLPs' internalization ability into Sf9 cells. Homology-based modelling of the MrNV capsid with other icosahedral capsid models revealed that this chymotrypsin-sensitive C-terminal domain was not only exposed on the capsid surface, but also constituted the core of the viral capsid protrusion. These results therefore suggest the importance of the C-terminal domain as a structure for targeted cell interaction which is presumably localized at the protruding domain. This work thus provided the functional insights into the role of the MrNV C-terminal domain in viral entry into Sf9 cells and lead to the development of strategies in combatting MrNV infection in susceptible cells.

  1. Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides.

    Science.gov (United States)

    Lelièvre, Dominique; Terrier, Victor P; Delmas, Agnès F; Aucagne, Vincent

    2016-03-04

    The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Cα proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.

  2. Comparison of the meat quality and fatty acid composition of traditional fat-tailed (Chall) and tailed (Zel) Iranian sheep breeds.

    Science.gov (United States)

    Yousefi, Ali Reza; Kohram, Hamid; Zare Shahneh, Ahmad; Nik-khah, Ali; Campbell, Anna W

    2012-12-01

    The aim of this study was to compare the meat quality of a traditional fat-tailed breed, Chall, to a tailed Iranian sheep breed, Zel. Lambs were grazed on pasture until weaning, and then were finished until slaughter at 10-12 months. Meat quality traits were measured on the longissimus dorsi (LD) muscle. Zel lambs accumulated more intramuscular fat (IMF) (pmeat color of Zel lambs was higher for both a* (pMeat from Zel lambs was more tender (pmeat color.

  3. An essential role for the proximal but not the distal cytoplasmic tail of glycoprotein M in murid herpesvirus 4 infection.

    Directory of Open Access Journals (Sweden)

    Janet S May

    Full Text Available Murid herpesvirus-4 (MuHV-4 provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytoplasmic tail to MuHV-4 lytic replication by making recombinant viruses with varying C-terminal deletions. Removing an acidic cluster and a distal YXXPhi motif altered the capsid distribution somewhat in infected cells but had little effect on virus replication, either in vitro or in vivo. In contrast, removing a proximal YXXPhi motif as well completely prevented productive replication. gM was still expressed, but unlike its longer forms showed only limited colocalization with co-transfected gN, and in the context of whole virus appeared to support gN expression less well. We conclude that some elements of the gM cytoplasmic tail are dispensible for MuHV-4 replication, but the tail as a whole is not.

  4. A Novel Preparation Method of C-Terminal Glutamine Dipeptides

    Institute of Scientific and Technical Information of China (English)

    QIAN Shao-Song; LIU Yi; CHEN Ran; LI Jia-You; WU Xiao-Yan; JIAO Qing-Cai

    2006-01-01

    A novel synthesis method of dipeptides containing glutamine is reported. Protected L-amino acids were prepared by using inexpensive phthaloyl as the protecting group. Then the phthaloyl-L-amino acids were condensed with glutamine salts by the mixed anhydride method to afford phthaloyl dipeptides. Subsequently, the phthaloyl was removed from the dipeptides with hydrazine hydrate. As a result, optically pure glutamine-containing dipeptides were obtained in good yields.

  5. Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

    2015-02-20

    XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4{sup N326L}) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4{sup N326L} in the nucleus but only partially rescued radiosensitivity of M10-XRCC4{sup N326L}. These results collectively indicated that the functional defects of XRCC4{sup N326L} might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the

  6. TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.

    Science.gov (United States)

    Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi

    2014-04-01

    Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000.

  7. Metal speciation of historic and new copper mine tailings from Repparfjorden, Northern Norway, before and after acid, base and electrodialytic extraction

    DEFF Research Database (Denmark)

    Pedersen, Kristine B.; Jensen, Pernille Erland; Ottosen, Lisbeth M.;

    2016-01-01

    the new mine tailings. Electrodialysis, based on applying an electric field of low intensity to extract metals from polluted soils/sediments, was designed for acidic and alkaline extraction, and in both cases more Cu was extracted than in the pure acid/base extractions, while maintaining low mobilisation...

  8. Disulfide assignment of the C-terminal cysteine knot of agouti-related protein (AGRP) by direct sequencing analysis.

    Science.gov (United States)

    Young, Y; Zeni, L; Rosenfeld, R D; Stark, K L; Rohde, M F; Haniu, M

    1999-12-01

    We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].

  9. The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases.

    Science.gov (United States)

    Pilbák, Sarolta; Tomin, Anna; Rétey, János; Poppe, László

    2006-03-01

    Besides the post-translationally cyclizing catalytic Ala-Ser-Gly triad, Tyr110 and its equivalents are of the most conserved residues in the active site of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), histidine ammonia-lyase (HAL, EC 4.3.1.3) and other related enzymes. The Tyr110Phe mutation results in the most pronounced inactivation of PAL indicating the importance of this residue. The recently published X-ray structures of PAL revealed that the Tyr110-loop was either missing (for Rhodospridium toruloides) or far from the active site (for Petroselinum crispum). In bacterial HAL ( approximately 500 amino acids) and plant and fungal PALs ( approximately 710 amino acids), a core PAL/HAL domain ( approximately 480 amino acids) with >or= 30% sequence identity along the different species is common. In plant and fungal PAL a approximately 100-residue long C-terminal multi-helix domain is present. The ancestor bacterial HAL is thermostable and, in all of its known X-ray structures, a Tyr83-loop-in arrangement has been found. Based on the HAL structures, a Tyr110-loop-in conformation of the P. crispum PAL structure was constructed by partial homology modeling, and the static and dynamic behavior of the loop-in/loop-out structures were compared. To study the role of the C-terminal multi-helix domain, Tyr-loop-in/loop-out model structures of two bacterial PALs (Streptomyces maritimus, 523 amino acids and Photorhabdus luminescens, 532 amino acids) lacking this C-terminal domain were also built. Molecular dynamics studies indicated that the Tyr-loop-in conformation was more rigid without the C-terminal multi-helix domain. On this basis it is hypothesized that a role of this C-terminal extension is to decrease the lifetime of eukaryotic PAL by destabilization, which might be important for the rapid responses in the regulation of phenylpropanoid biosynthesis.

  10. C-terminal domain of hepatitis C virus core protein is essential for secretion

    Institute of Scientific and Technical Information of China (English)

    Soo-Ho Choi; Kyu-Jin Park; So-Yeon Kim; Dong-Hwa Choi; Jung-Min Park; Soon B. Hwang

    2005-01-01

    AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells.METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells.RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus,core proteins were no longer seareted into the culture medium.Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex.CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media.Since HCV replication occurs on lipid raft membrane structure,these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.

  11. Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides.

    Science.gov (United States)

    Strack, Martin; Langklotz, Sina; Bandow, Julia E; Metzler-Nolte, Nils; Albada, H Bauke

    2012-11-16

    A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

  12. Determination of acid forming potential of massive sulfide minerals and the tailings situated in lead/zinc mining district of Balya (NW Turkey)

    Science.gov (United States)

    Çelebi, E. Ender; Öncel, M. Salim

    2016-12-01

    Weathering of sulfide minerals is a major source of acid production in nature and especially in mining territories. Pyrite is not the only principal mineral that generates acid drainage: other sulfide minerals (sphalerite, galena, chalcopyrite, etc.) may also be responsible for acid production. In addition to massive sulfide minerals, sulfide-bearing mine tailings may also produce acid drainage due to oxidation and hydrolysis reactions in waste dumps. The lead/zinc (Pb/Zn) mining region in Balya and Balıkesir, in Turkey, has operated mines intensively since the 1860s; so that characterization of the sulfide minerals and tailings situated and formed around the mining site is of great importance to secure a sustainable environment. For this purpose, acid production and neutralization potentials of massive sulfide ores of the region, and in the Pb/Zn process facility mine tailings from ten different points of tailings dam, have been determined by applied conventional Acid-Base Accounting (ABA) and Net Acid Generation (NAG) static tests after chemical and mineralogical analysis. The NAG pH and net acid production potential (NAPP) values were compared on a chart in order to classify the samples as either acid generating or non-acid generating. According to the comparisons, the sulfide minerals were classified as potentially acid forming (PAF). Massive pyrite had the highest NAPP and NAG pH value of 1966.6 kg H2SO4/ton and 1.91, respectively and the galena had the lowest NAPP value of 558.9 kg H2SO4/ton. However, the sphalerite NAG leachate pH value of 4.30 was the highest in sulfide minerals so that the sphalerite plotted near the uncertainty reference border in the PAF zone. In the mine tailings, NAPP values of 105.9 kg H2SO4/ton on average and the NAG pH values of over 7.5 were determined. In addition to these tests, water leaching (agitation test) was carried out on tailings in order to generate more information. The tailings did not generate acidic leachates as

  13. C-terminal motif prediction in eukaryotic proteomes using comparative genomics and statistical over-representation across protein families

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    Cutler Sean R

    2007-06-01

    between species, among kingdoms and across eukaryotes. Motifs of note include a serine-acidic peptide (DSD* as well as several lysine enriched motifs found in nearly all eukaryotic genomes examined. Conclusion We have successfully generated a high confidence representation of eukaryotic motifs anchored at the C-terminus. A high incidence of true-positives in our results suggests that several previously unidentified tripeptide patterns are strong candidates for representing novel peptide motifs of a widely employed nature in the C-terminal biology of eukaryotes. Our application of comparative genomics, statistical over-representation and the adjustment for protein family homology has generated several hypotheses concerning the C-terminal topology as it pertains to sorting and potential protein interaction signals. This approach to background reduction could be expanded for application to protein motif prediction in the protein interior. A parallel N-terminal analysis is presented as supplementary data.

  14. C-terminal motif prediction in eukaryotic proteomes using comparative genomics and statistical over-representation across protein families

    Science.gov (United States)

    Austin, Ryan S; Provart, Nicholas J; Cutler, Sean R

    2007-01-01

    kingdoms and across eukaryotes. Motifs of note include a serine-acidic peptide (DSD*) as well as several lysine enriched motifs found in nearly all eukaryotic genomes examined. Conclusion We have successfully generated a high confidence representation of eukaryotic motifs anchored at the C-terminus. A high incidence of true-positives in our results suggests that several previously unidentified tripeptide patterns are strong candidates for representing novel peptide motifs of a widely employed nature in the C-terminal biology of eukaryotes. Our application of comparative genomics, statistical over-representation and the adjustment for protein family homology has generated several hypotheses concerning the C-terminal topology as it pertains to sorting and potential protein interaction signals. This approach to background reduction could be expanded for application to protein motif prediction in the protein interior. A parallel N-terminal analysis is presented as supplementary data. PMID:17594486

  15. Contribution of N- and C-terminal Kv4.2 channel domains to KChIP interaction [corrected].

    Science.gov (United States)

    Callsen, Britta; Isbrandt, Dirk; Sauter, Kathrin; Hartmann, L Sven; Pongs, Olaf; Bähring, Robert

    2005-10-15

    Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

  16. Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

    NARCIS (Netherlands)

    Wu, Bian; Wijma, Hein J.; Song, Lu; Rozeboom, Henriette J.; Poloni, Claudia; Tian, Yue; Arif, Muhammad I.; Nuijens, Timo; Quaedflieg, Peter J. L. M.; Szymanski, Wiktor; Feringa, Ben L.; Janssen, Dick B.

    2016-01-01

    The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explore

  17. Paracellular permeation-enhancing effect of AT1002 C-terminal amidation in nasal delivery

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    Song KH

    2015-03-01

    Full Text Available Keon-Hyoung Song,1 Sang-Bum Kim,2 Chang-Koo Shim,2 Suk-Jae Chung,2 Dae-Duk Kim,2 Sang-Ki Rhee,1 Guang J Choi,1 Chul-Hyun Kim,3 Kiyoung Kim4 1Department of Pharmaceutical Engineering, Soonchunhyang University, Asan, Republic of Korea; 2College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea; 3Department of Sports Medicine, 4Department of Medical Biotechnology, Soonchunhyang University, Asan, Republic of Korea Background: The identification of permeation enhancers has gained interest in the development of drug delivery systems. A six-mer peptide, H-FCIGRL-OH (AT1002, is a tight junction modulator with promising permeation-enhancing activity. AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. However, AT1002 is not stable in neutral pH or after incubation under physiological conditions, which is necessary to fully uncover its permeation-enhancing effect. Thus, we increased the stability or mitigated the instability of AT1002 by modifying its terminal amino acids and evaluated its subsequent biological activity.Methods: C-terminal-amidated (FCIGRL-NH2, Pep1 and N-terminal-acetylated (Ac-FCIGRL, Pep2 peptides were analyzed by liquid chromatography–mass spectrometry. We further assessed cytotoxicity on cell monolayers, as well as the permeation-enhancing activity following nasal administration of the paracellular marker mannitol.Results: Pep1 was nontoxic to cell monolayers and showed a relatively low decrease in peak area compared to AT1002. In addition, administration of mannitol with Pep1 resulted in significant increases in the area under the plasma concentration–time curve and peak plasma concentration at 3.63-fold and 2.68-fold, respectively, compared to mannitol alone. In contrast, no increase in mannitol concentration was shown with mannitol/AT1002 or mannitol/Pep2 compared to the control. Thus, Pep1 increased

  18. C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

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    Antoine Picciocchi

    Full Text Available Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+ channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

  19. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

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    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  20. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    Science.gov (United States)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

  1. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    Science.gov (United States)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-01-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring. PMID:28155880

  2. Effect of succinic acid monoethyl ester on hemoglobin glycation and tail tendon collagen properties in type 2 diabetic rats.

    Science.gov (United States)

    Saravanan, Ramalingam; Pari, Leelavinothan

    2008-06-01

    Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment of type 2 diabetes. The aim of the study was to investigate the effect of EMS and metformin administration on tail collagen content and its characteristics in streptozotocin-nicotinamide-induced type 2 diabetic rats. EMS was administered intraperitoneally for 30 days to normal and diabetic rats. In the diabetic rats, a significant increase in the levels of glucose, glycated hemoglobin, hydroxyproline, collagen content, extent glycation, fluorescence, neutral salt, acid and pepsin soluble collagen content was absorbed with a significant decrease in the level of insulin, hemoglobin in streptozotocin-nicotinamide diabetic rats. Moreover, a daily administration of nonglucidic nutrient EMS and metformin significantly decreased the levels of glucose, glycated hemoglobin, hydroxyproline, collagen content, extent glycation, fluorescence, neutral salt, acid and pepsin soluble collagen content, whereas it increased insulin, hemoglobin levels in diabetic rats. The positive influence of nonglucidic nutrient on both collagen content and its properties suggests a potential mechanism for the ability of EMS to delay diabetic complications.

  3. Crystal structure of a crustacean hyperglycemic hormone (CHH) precursor suggests structural variety in the C-terminal regions of CHH superfamily members.

    Science.gov (United States)

    Tsutsui, Naoaki; Sakamoto, Tatsuya; Arisaka, Fumio; Tanokura, Masaru; Nagasawa, Hiromichi; Nagata, Koji

    2016-12-01

    The crustacean hyperglycemic hormone (CHH) is one of the major hormones in crustaceans, and peptides belonging to the CHH superfamily have been found in diverse ecdysozoans. Although the basic function of CHH is to control energy metabolism, it also plays various roles in crustacean species, such as in molting and vitellogenesis. Here, we present the crystal structure of Pej-SGP-I-Gly, a partially active precursor of CHH from the kuruma prawn Marsupenaeus japonicus, which has an additional Gly residue in place of the C-terminal amide group of the mature Pej-SGP-I. The 1.6-angstrom crystal structure showed not only the common CHH superfamily scaffold comprising three α-helices, three disulfide bridges, and a hydrophobic core but also revealed that the C-terminal part has a variant backbone fold that is specific to Pej-SGP-I-Gly. The α-helix 4 of Pej-SGP-I-Gly was much longer than that of molt-inhibiting hormone (Pej-MIH) from the same species, and as a result, the following C-terminal helix, corresponding to α-helix 5 in MIH, was not formed. Unlike monomeric Pej-MIH, Pej-SGP-I-Gly forms a homodimer in the crystal structure via its unique α-helix 4. The unexpected dissimilar folds between Pej-SGP-I-Gly and Pej-MIH appear to be the result of their distinct C-terminal amino acid sequences. Variations in amino acid sequences and lengths and the resulting variety of backbone folds allow the C-terminal and sterically adjoining regions to confer different hormonal activities in diverse CHH superfamily members.

  4. Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

    Science.gov (United States)

    Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

    2011-02-01

    Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

  5. The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity.

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    Rui Jia

    Full Text Available The interferon-inducible transmembrane (IFITM proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1 strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1NL4-3 is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117-125, which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117-125 mutant diminishes HIV-1NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117-125 to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117-125, mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.

  6. The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

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    Mala V Rao

    Full Text Available Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M(tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M(tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M(tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

  7. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

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    Noonan James P

    2001-07-01

    Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

  8. The carboxyl-terminal tail of Noxa protein regulates the stability of Noxa and Mcl-1.

    Science.gov (United States)

    Pang, Xiaming; Zhang, Jingjing; Lopez, Hernando; Wang, Yushu; Li, Wenyang; O'Neill, Katelyn L; Evans, Jacquelynn J D; George, Nicholas M; Long, Jianhong; Chen, Yi; Luo, Xu

    2014-06-20

    The BH3-only protein Noxa is a critical mediator of apoptosis and functions primarily by sequestering/inactivating the antiapoptotic Bcl-2 family protein Mcl-1. Although Noxa is a highly labile protein, recent studies suggested that it is degraded by the proteasome in a ubiquitylation-independent manner. In the present study, we investigated the mechanism of Noxa degradation and its ability to regulate the stability of Mcl-1. We found that the ubiquitylation-independent degradation of Noxa does not require a physical association with Mcl-1. A short stretch of amino acid residues in the C-terminal tail was found to mediate the proteasome-dependent degradation of Noxa. Ectopic placement of this degron was able to render other proteins unstable. Surprisingly, mutation of this sequence not only attenuated the rapid degradation of Noxa, but also stabilized endogenous Mcl-1 through the BH3-mediated direct interaction. Together, these results suggest that the C-terminal tail of Noxa regulates the stability of both Noxa and Mcl-1.

  9. Multi-component reactive transport modeling of natural attenuation of an acid groundwater plume at a uranium mill tailings site

    Science.gov (United States)

    Zhu, Chen; Hu, Fang Q.; Burden, David S.

    2001-11-01

    Natural attenuation of an acidic plume in the aquifer underneath a uranium mill tailings pond in Wyoming, USA was simulated using the multi-component reactive transport code PHREEQC. A one-dimensional model was constructed for the site and the model included advective-dispersive transport, aqueous speciation of 11 components, and precipitation-dissolution of six minerals. Transport simulation was performed for a reclamation scenario in which the source of acidic seepage will be terminated after 5 years and the plume will then be flushed by uncontaminated upgradient groundwater. Simulations show that successive pH buffer reactions with calcite, Al(OH) 3(a), and Fe(OH) 3(a) create distinct geochemical zones and most reactions occur at the boundaries of geochemical zones. The complex interplay of physical transport processes and chemical reactions produce multiple concentration waves. For SO 42- transport, the concentration waves are related to advection-dispersion, and gypsum precipitation and dissolution. Wave speeds from numerical simulations compare well to an analytical solution for wave propagation.

  10. Order through disorder: hyper-mobile C-terminal residues stabilize the folded state of a helical peptide. a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Kalliopi K Patapati

    Full Text Available Conventional wisdom has it that the presence of disordered regions in the three-dimensional structures of polypeptides not only does not contribute significantly to the thermodynamic stability of their folded state, but, on the contrary, that the presence of disorder leads to a decrease of the corresponding proteins' stability. We have performed extensive 3.4 µs long folding simulations (in explicit solvent and with full electrostatics of an undecamer peptide of experimentally known helical structure, both with and without its disordered (four residue long C-terminal tail. Our simulations clearly indicate that the presence of the apparently disordered (in structural terms C-terminal tail, increases the thermodynamic stability of the peptide's folded (helical state. These results show that at least for the case of relatively short peptides, the interplay between thermodynamic stability and the apparent structural stability can be rather subtle, with even disordered regions contributing significantly to the stability of the folded state. Our results have clear implications for the understanding of peptide energetics and the design of foldable peptides.

  11. Contribution of Chitinase A’s C-Terminal Vacuolar Sorting Determinant to the Study of Soluble Protein Compartmentation

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    Egidio Stigliano

    2014-06-01

    Full Text Available Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin’s tetrapeptide AFVY (AlaPheValTyr and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

  12. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    Energy Technology Data Exchange (ETDEWEB)

    Serek, Robert; Forwood, Jade K. [School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Hume, David A. [School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072 (Australia); Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland 4072 (Australia); Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072 (Australia); Martin, Jennifer L.; Kobe, Bostjan, E-mail: b.kobe@uq.edu.au [School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072 (Australia); Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072 (Australia)

    2006-02-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

  13. Acid rock drainage and metal leaching from mine waste material (tailings) of a Pb-Zn-Ag skarn deposit: environmental assessment through static and kinetic laboratory tests

    OpenAIRE

    Blanca Adriana Méndez Ortiz; Alejandro Carrillo Chávez; Marcos Gustavo Monroy Fernández

    2007-01-01

    In this work, the processes and products involved in the generation of acid rock drainage – metal leaching (ARD-ML) from mine waste material (tailings) derived from the exploitation of an ore type Pb- Zn-Ag skarn were characterized. Laboratory tests (static and kinetic) of historic and recent tailings were conducted along with the mineralogical characterization of solids, and chemical analyses of solids and leachates. Pyrite (FeS2) is the most abundant sulfi de phase, and one of the main mine...

  14. Automation of C-terminal sequence analysis of 2D-PAGE separated proteins

    Directory of Open Access Journals (Sweden)

    P.P. Moerman

    2014-06-01

    Full Text Available Experimental assignment of the protein termini remains essential to define the functional protein structure. Here, we report on the improvement of a proteomic C-terminal sequence analysis method. The approach aims to discriminate the C-terminal peptide in a CNBr-digest where Met-Xxx peptide bonds are cleaved in internal peptides ending at a homoserine lactone (hsl-derivative. pH-dependent partial opening of the lactone ring results in the formation of doublets for all internal peptides. C-terminal peptides are distinguished as singlet peaks by MALDI-TOF MS and MS/MS is then used for their identification. We present a fully automated protocol established on a robotic liquid-handling station.

  15. C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin.

    Science.gov (United States)

    Zik, Moriyah; Fridmann-Sirkis, Yael; Fromm, Hillel

    2006-05-01

    Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.

  16. Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system.

    Science.gov (United States)

    Baker, Sarah J; Morris, Judy L; Gibbins, Ian L

    2003-03-17

    In order to examine the possibility that some actions of substance P may be mediated by a variant of the neurokinin-1 (NK-1) receptor, we isolated and sequenced the cDNA encoding a truncated NK-1 receptor from guinea-pig celiac ganglion and brain mRNA by two-step RT-PCR based on the 3'RACE method. The truncated NK-1 receptor sequence corresponded to a splice variant missing the final exon 5, and encoded a 311-amino acid protein that was truncated just after transmembrane domain 7, in an identical position to a truncated variant of the human NK-1 receptor. Thus, the truncated NK-1 receptor lacked the intracellular C-terminus sequence required for the phosphorylation and internalisation of the full-length NK-1 receptor. Using a sensitive one-step semi-quantitative RT-PCR assay, we detected mRNA for both the full length and truncated NK-1 receptors throughout the brain, spinal cord, sensory and autonomic ganglia, and viscera. Truncated NK-1 receptor mRNA was present in lower quantities than mRNA for the full-length NK-1R in all tissues. Highest levels of mRNA for the truncated NK-1 receptor were detected in coeliac ganglion, spinal cord, basal ganglia and hypothalamus. An antiserum to the N-terminus of the NK-1 receptor labelled dendrites of coeliac ganglion neurons that were not labelled with antisera to the C-terminus of the full length NK-1 receptor. These results show that a C-terminally truncated variant of the NK-1 receptor is likely to be widespread in central and peripheral nervous tissue. We predict that this receptor will mediate actions of substance P on neurons where immunohistochemical evidence for a full-length NK-1 receptor is lacking.

  17. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Vogel, Charlotte Katrine; Schwartz, Thue W.

    1990-01-01

    The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic...... expression vectors encoding either cystatin C, KDEL extended cystatin C, or cystatin C extended with a control sequence. It is concluded that cystatin C with the KDEL tetrapeptide as a C-terminal extension is retained intracellularly without apparent accumulation of the molecule....

  18. Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

    DEFF Research Database (Denmark)

    Cain, Stuart A; Baldwin, Andrew K; Mahalingam, Yashithra;

    2008-01-01

    in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N......-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions....

  19. Structure of the C-terminal fragment 300-320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling.

    Science.gov (United States)

    Franzoni, L; Nicastro, G; Pertinhez, T A; Tatò, M; Nakaie, C R; Paiva, A C; Schreier, S; Spisni, A

    1997-04-11

    Angiotensin II AT1A receptor is coupled to G-protein, and the molecular mechanism of signal transduction is still unclear. The solution conformation of a synthetic peptide corresponding to residues 300-320 of the rat AT1A receptor, located in the C-terminal cytoplasmic tail and indicated by mutagenesis work to be critical for the G-protein coupling, has been investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) and restrained molecular dynamics calculations. The CD data indicate that, in acidic water, at concentration below 0.8 mM, the peptide exists in a predominantly coil structure while at higher concentration it can form helical aggregates; addition of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. Using NMR-derived constraints, an ensemble of conformers for the peptide has been determined by restrained molecular dynamics calculations. Analysis of the converged three-dimensional structures indicates that a significant population of them adopts an amphipathic alpha-helical conformation that, depending upon experimental conditions, presents a variable extension in the stretch Leu6-Tyr20. An equilibrium with nonhelical structured conformers is also observed. We suggest that the capability of the peptide to modulate its secondary structure as a function of the medium dielectric constant, as well as its ability to form helical aggregates by means of intermolecular hydrophobic interactions, can play a significant role for G-protein activation.

  20. PI(4P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail.

    Directory of Open Access Journals (Sweden)

    Kai Jiang

    2016-02-01

    Full Text Available In Hedgehog (Hh signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog receptor complex relieves Ptc inhibition on Smoothened (Smo. A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4P. Increased levels of PI(4P promote, whereas decreased levels of PI(4P inhibit, Hh signaling activity. We further found that PI(4P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH domain of G protein-coupled receptor kinase 2 (Gprk2 as an essential component for enriching PI(4P and facilitating Smo activation. PI(4P also binds mouse Smo (mSmo and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4P but decreases the interaction between Ptc and PI(4P, indicating that, in addition to promoting PI(4P production, Hh regulates the pool of PI(4P associated with Ptc and Smo.

  1. REE behavior and effect factors in AMD-type acidic groundwater at sulfide tailings pond, BS nickel mine, W.A.

    Institute of Scientific and Technical Information of China (English)

    LEI Liang-qi; SONG Ci-an; XIE Xiang-li; LI Yan-hong

    2008-01-01

    AMD(Acid Mine Drainage)-type acidic groundwater (pH<4) from oxidizing sulfide tailings in BS nickel mine (Western Australia) is of higher total rare earth element(REE) contents and Ce enrichment (PAAS normalization), different from setting groundwater (pH>6.5, with lower total REE contents, Ce depletion). While the AMD contaminated groundwater (pH=4.0-6.5) around tailings pond is characterized by transition from acidic to setting groundwater in total REE content, and associated with Ce depletion (like setting groundwater). The light REE in all type groundwater shows up depletion, but its depleted extent in acidic groundwater is more remarkable. This work indicates that REE behavior in AMD-type acidic groundwater is controlled mainly by pH value and metal (Al, Mn and Fe) contents. And the critical pH value that affects REE behavior in ground acidic water would be 4, lower than the previous value (pH=5) that has been believed prevalently in surface acidic waters. The pH could affect REE behavior in groundwater by controlling the solubility of metal (Al, Mn and Fe) hydroxides and the valence of cerium. Finally, light REE depletion in acidic groundwater may due to element affinity. High content Al (affinity with heavy REE) and low content Fe (affinity with light REE) may lead to heavy REE enrichment while light REE relative depletion in water.

  2. C-terminal interactors of the AMPA receptor auxiliary subunit Shisa9.

    Directory of Open Access Journals (Sweden)

    Anna R Karataeva

    Full Text Available Shisa9 (initially named CKAMP44 has been identified as auxiliary subunit of the AMPA-type glutamate receptors and was shown to modulate its physiological properties. Shisa9 is a type-I transmembrane protein and contains a C-terminal PDZ domain that potentially interacts with cytosolic proteins. In this study, we performed a yeast two-hybrid screening that yielded eight PDZ domain-containing interactors of Shisa9, which were independently validated. The identified interactors are known scaffolding proteins residing in the neuronal postsynaptic density. To test whether C-terminal scaffolding interactions of Shisa9 affect synaptic AMPA receptor function in the hippocampus, we disrupted these interactions using a Shisa9 C-terminal mimetic peptide. In the absence of scaffolding interactions of Shisa9, glutamatergic AMPA receptor-mediated synaptic currents in the lateral perforant path of the mouse hippocampus had a faster decay time, and paired-pulse facilitation was reduced. Furthermore, disruption of the PDZ interactions between Shisa9 and its binding partners affected hippocampal network activity. Taken together, our data identifies novel interaction partners of Shisa9, and shows that the C-terminal interactions of Shisa9 through its PDZ domain interaction motif are important for AMPA receptor synaptic and network functions.

  3. Application of proteases in the C-terminal modification of peptides

    NARCIS (Netherlands)

    Gini, F.; Eggen, I.F.; Zoelen, van D.J.; Boeriu, C.G.

    2009-01-01

    The high selectivity and the mild reaction conditions of enzymatic processes prompted their application in the synthesis of peptides, where selectivity is a feature of pivotal importance. Here we report the use of the serine protease subtilisin for the selective deprotection of C-terminal tert-butyl

  4. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

    NARCIS (Netherlands)

    Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

    2013-01-01

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

  5. Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator.

    Science.gov (United States)

    Deshimaru, Shungo; Miyake, Yasuo; Ohmiya, Tadamasa; Tatsu, Yoshiro; Endo, Yasuko; Yumoto, Noboru; Toraya, Tetsuo

    2002-05-01

    The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.

  6. Effect of arbuscular mycorrhizal fungi on plant biomass and the rhizosphere microbial community structure of mesquite grown in acidic lead/zinc mine tailings.

    Science.gov (United States)

    Solís-Domínguez, Fernando A; Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M

    2011-02-15

    Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse experiment was performed to determine the effects of three AMF inocula on biomass, shoot accumulation of heavy metals, and changes in the rhizosphere microbial community structure of the native plant Prosopis juliflora (mesquite). Plants were grown in an acidic lead/zinc mine tailings amended with 10% (w/w) compost amendment, which is slightly sub-optimal for plant growth in these tailings. After two months, AMF-inoculated plants showed increased dry biomass and root length (p<0.05) and effective AMF colonization compared to controls grown in uninoculated compost-amended tailings. Mesquite shoot tissue lead and zinc concentrations did not exceed domestic animal toxicity limits regardless of whether AMF inoculation was used. The rhizosphere microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) profiles of the small subunit RNA gene for bacteria and fungi. Canonical correspondence analysis (CCA) of DGGE profiles showed that the rhizosphere fungal community structure at the end of the experiment was significantly different from the community structure in the tailings, compost, and AMF inocula prior to planting. Further, CCA showed that AMF inoculation significantly influenced the development of both the fungal and bacterial rhizosphere community structures after two months. The changes observed in the rhizosphere microbial community structure may be either a direct effect of the AMF inocula, caused by changes in plant physiology induced by

  7. Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.

    Science.gov (United States)

    Hulme, Joanne T; Konoki, Keiichi; Lin, Teddy W-C; Gritsenko, Marina A; Camp, David G; Bigelow, Diana J; Catterall, William A

    2005-04-05

    In skeletal muscle cells, voltage-dependent potentiation of Ca2+ channel activity requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15), and the most rapid sites of phosphorylation are located in the C-terminal domain. Surprisingly, the site of interaction of the complex of PKA and AKAP15 with the alpha1-subunit of Ca(V)1.1 channels lies in the distal C terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the distal C terminus is noncovalently associated with the remainder of the channel via an interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian nonmuscle cells. Deletion mapping of the C terminus of the alpha1-subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802-1841 specifically interacts with a region in the proximal C terminus containing amino acid residues 1556-1612. Analysis of the purified alpha1-subunit of Ca(V)1.1 channels from skeletal muscle by saturation sequencing of the intracellular peptides by tandem mass spectrometry identified the site of proteolytic processing as alanine 1664. Our results support the conclusion that a noncovalently associated complex of the alpha1-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of Ca(V)1.1 channels in skeletal muscle cells.

  8. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

    Directory of Open Access Journals (Sweden)

    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  9. Genome sequence of the acid-tolerant Desulfovibrio sp. DV isolated from the sediments of a Pb-Zn mine tailings dam in the Chita region, Russia

    Directory of Open Access Journals (Sweden)

    Anastasiia Kovaliova

    2017-03-01

    Full Text Available Here we report the draft genome sequence of the acid-tolerant Desulfovibrio sp. DV isolated from the sediments of a Pb-Zn mine tailings dam in the Chita region, Russia. The draft genome has a size of 4.9 Mb and encodes multiple K+-transporters and proton-consuming decarboxylases. The phylogenetic analysis based on concatenated ribosomal proteins revealed that strain DV clusters together with the acid-tolerant Desulfovibrio sp. TomC and Desulfovibrio magneticus. The draft genome sequence and annotation have been deposited at GenBank under the accession number MLBG00000000.

  10. Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation.

    Science.gov (United States)

    Clough, R C; Jordan-Beebe, E T; Lohman, K N; Marita, J M; Walker, J M; Gatz, C; Vierstra, R D

    1999-01-01

    Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr from initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.

  11. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    Energy Technology Data Exchange (ETDEWEB)

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  12. Modulation of voltage-gated potassium Kv2.1 via the cytoplasmic C terminal

    Institute of Scientific and Technical Information of China (English)

    Man Jin; Peiyuan Lu

    2011-01-01

    Voltage-gated potassium channels comprise 12 subtypes (Kv1-Kv12). Kv2.1, which is expressed in most mammalian central neurons, provides the majority of delayed-rectifier K current in cortical and hippocampal pyramidal neurons, and plays an especially prominent role in repolarizing membrane potential, as well as in facilitation of exocytosis. Kv2.1-encoded K efflux is essential for neuronal apoptosis programming. The human form of the Kv2.1 potassium channel contains large intracellular regions. The cytoplasmic C-terminal plays a key role in modulating Kv2.1 gating. The present manuscript summarized Kv2.1 structure and modulation in neurons and analyzed the roles of the cytoplasmic C-terminal.

  13. C-Terminally modified peptides via cleavage of the HMBA linker by O-, i>N>- or S-nucleophiles

    DEFF Research Database (Denmark)

    Hansen, Jonas; Diness, Frederik; Meldal, Morten Peter

    2016-01-01

    A large variety of C-terminally modified peptides was obtained by nucleophilic cleavage of the ester bond in solid phase linked peptide esters of 4-hydroxymethyl benzamide (HMBA). The developed methods provided peptides, C-terminally functionalized as esters, amides and thioesters, with high puri...

  14. Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

    Science.gov (United States)

    Qian, Zhi-Gang; Zhou, Ming-Liang; Song, Wen-Wen; Xia, Xiao-Xia

    2015-11-09

    Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

  15. Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers

    Energy Technology Data Exchange (ETDEWEB)

    Gnesa, Eric; Hsia, Yang; Yarger, Jeffery L.; Weber, Warner; Lin-Cereghino, Joan; Lin-Cereghino, Geoff; Tang, Simon; Agari, Kimiko; Vierra, Craig (AZU); (Pacific)

    2012-05-24

    Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.

  16. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  17. Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2).

    Science.gov (United States)

    Xu, Xiaoping; Mikhailova, Margarita; Chen, Zhihua; Pal, Sanjay; Robichaud, Trista K; Lafer, Eileen M; Baber, Sam; Steffensen, Bjorn

    2011-09-01

    Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell membrane-associated activation complex that involves specific binding between the hemopexin domain of proMMP-2 (PEX) and the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (C-TIMP-2). In this study, we tested the feasibility of inhibiting activation of proMMP-2 by exogenous inhibitors, which block the binding between PEX and TIMP-2. The recombinant C-TIMP-2 and synthetic peptides from C-TIMP-2 were used as inhibitors for proMMP-2 activation. Recombinant C-TIMP-2 bound specifically to both the catalytically inactive MMP-2(E404A) and the C-terminal domain of MMP-2 (PEX) in a concentration dependent manner with apparent K(d) of 3.9×10(-7)M and 1.7×10(-7)M, respectively. Moreover, C-TIMP-2 competed the binding between MMP-2(E404A) and full-length TIMP-2. Finally, activity assays showed that addition of C-TIMP-2 to HT-1080 fibrosarcoma cells inhibited proMMP-2 activation in a concentration-dependent manner. We then designed a synthetic peptide, P175L, consisting of 20 residues from the PEX-binding tail region of C-TIMP-2. P175L bound PEX and inhibited cell membrane-mediated activation of proMMP-2 in a concentration dependent manner. Deletion of the last 9 tail residues of C-TIMP-2 in P175L abrogated the inhibitory activities of the peptide showing that these residues were essential for function. Overall, these experiments have demonstrated that proMMP-2 activation can be inhibited by exogenous inhibitors which points to a potential strategy for MMP-2 specific inhibition.

  18. Probing the Impact of the EchinT C-Terminal Domain on Structure and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    S Bardaweel; J Pace; T Chou; V Cody; C Wagner

    2011-12-31

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2{sub 1} with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T{sub m} value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  19. Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

    Science.gov (United States)

    Korwar, Sudha; Morris, Benjamin L; Parikh, Hardik I; Coover, Robert A; Doughty, Tyler W; Love, Ian M; Hilbert, Brendan J; Royer, William E; Kellogg, Glen E; Grossman, Steven R; Ellis, Keith C

    2016-06-15

    C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

  20. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy.

    Science.gov (United States)

    Valieva, Maria E; Gerasimova, Nadezhda S; Kudryashova, Kseniya S; Kozlova, Anastasia L; Kirpichnikov, Mikhail P; Hu, Qi; Botuyan, Maria Victoria; Mer, Georges; Feofanov, Alexey V; Studitsky, Vasily M

    2017-01-06

    A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

  1. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

    Directory of Open Access Journals (Sweden)

    Maria E. Valieva

    2017-01-01

    Full Text Available A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD and the high mobility group (HMG domain of the structure-specific recognition protein 1 (SSRP1 subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16 and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3. Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

  2. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    Science.gov (United States)

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  3. Preparation and characterization of TiO2/acid leached serpentinite tailings composites and their photocatalytic reduction of chromium(VI).

    Science.gov (United States)

    Sun, Zhiming; Zheng, Liming; Zheng, Shuilin; Frost, Ray L

    2013-08-15

    Composite TiO2/acid leached serpentine tailings (AST) were synthesized through the hydrolysis-deposition method and characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS), Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and surface area measurement (BET). The XRD analysis showed that TiO2 coated on the surface of acid leached serpentine tailings was mixed crystal phases of rutile and anatase, the grain size of which is 10-30 nm. SEM, TEM, and EDS analysis exhibited that nano-TiO2 particles were deposited on the surface and internal cavities of acid leaching serpentine tailings. The XPS and FT-IR analysis demonstrated that the coating process of TiO2 on AST was a physical adsorption process. The large specific surface area, porous structure, and plentiful surface hydroxyl group of TiO2/AST composite resulted in the high adsorption capacity of Cr(VI). The experimental results demonstrated that initial concentration of Cr(VI), the amount of the catalyst, and pH greatly influenced the removal efficiency of Cr(VI). The removal kinetics of Cr(VI) at a relative low initial concentration was fitted well with Langmuir-Hinshelwood kinetics model with R(2) value of about unity. The as-prepared composites exhibited strong adsorption and photocatalytic capacity for the removal of Cr(VI), and the possible photocatalytic reduction mechanism was studied. The photodecomposition of Cr(VI) was as high as 95% within 2h, and the reusability of the photocatalysis was proven.

  4. Functional implications of C-terminus of TBX5 with high homology to C-terminal domain of yeast DNA-directed RNA polymerase Ⅱ largest subunit

    Institute of Scientific and Technical Information of China (English)

    ZHOU Zhu-ren; GONG Li-guo; GENG Wen-qing; QIU Guang-rong; SUN Kai-lai

    2008-01-01

    @@ TBX5, as a member of the T-box-containing transcription factor family, encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues.1 The coding region of TBX5 cDNA is 1.5 kb with eight exons including the N-terminal portion, the DNA binding domain and C-terminal region. We reported that the abnormality in transcription level of the TbX5 gene might be the mechanism underlying human simple congenital heart disease in the absence of TBX5 mutations.

  5. Characterization of the C-Terminal Propeptide Involved in Bacterial Wall Spanning of Alpha-Amylase from the Psychrophile Alteromonas Haloplanctis

    OpenAIRE

    Feller, Georges; D'Amico, Salvino; Benotmane, A. M.; Joly, F.; Van Beeumen, J.; Gerday, Charles

    1998-01-01

    The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at ...

  6. Further studies on the use of allopurinol to reduce plasma uric acid concentrations in the Red-tailed Hawk (Buteo jamaicensis) hyperuricaemic model.

    Science.gov (United States)

    Poffers, J; Lumeij, J T; Timmermans-Sprang, E P M; Redig, P T

    2002-12-01

    The present paper reports the effects of allopurinol in a raptor hyperuricaemic model. The study was performed as a follow-up to previous experiments wherein allopurinol was used in doses of 100 and 50 mg/kg, and was proved to be toxic at these higher dose rates. To investigate whether 25 mg/kg (semel in die) s.i.d. allopurinol is a safe and effective dose in Red-tailed Hawks (Buteo jamaicensis) to reduce plasma uric acid concentrations, experimental studies were performed using the physiologically occurring postprandial hyperuricaemia. Preprandial and postprandial plasma concentrations of xanthine, hypoxanthine, allopurinol, oxypurinol and uric acid were established by high-performance liquid chromatography at various time intervals after receiving allopurinol (25 mg/kg SID) or placebo. No significant differences were observed between the experimental and the control group. These results indicate that this dose is safe to administer; however, this dose failed to cause a significant effect on plasma uric acid concentrations. Because of the low therapeutic ratio of allopurinol in Red-tailed Hawks, follow-up studies have concentrated on an alternative for the treatment of hyperuricaemia, namely urate oxidase.

  7. Mouse Noxa uses only the C-terminal BH3-domain to inactivate Mcl-1.

    Science.gov (United States)

    Weber, Arnim; Ausländer, David; Häcker, Georg

    2013-09-01

    Noxa is a member of the pro-apoptotic BH3-only group of Bcl-2 proteins that is known to bind specifically to anti-apoptotic Mcl-1 and A1, antagonizing their function. Mcl-1 has been reported to have a short half-life, and Noxa up-regulation accelerates Mcl-1 degradation by the proteasome. Unlike human Noxa, mouse Noxa has two BH3-domains, which both have affinity for Mcl-1. We here investigate two aspects of the molecular function of Noxa, namely the requirements for the two BH3-domains in mouse Noxa and the role of Noxa in Mcl-1-degradation. We found that only the C-terminal BH3-domain of mouse Noxa is active in neutralizing Mcl-1. This was the result of the targeting of Noxa to the outer mitochondrial membrane through its C-terminal alpha-helix, which allowed Mcl-1-neutralization only when the BH3-domain was immediately N-terminal of the membrane anchor. However, the N-terminal BH3-domain enhanced interaction with Mcl-1 and A1. The Noxa-dependent degradation of Mcl-1 was independent of the kinase GSK3 and the deubiquitinase Usp9x in mouse embryonic fibroblasts. These data show that Noxa is targeted to the mitochondrial membrane where it neutralises Mcl-1 via its C-terminal BH3-domain and suggest that Noxa is co-degraded with Noxa, in a way independent of ubiquitin-modifying enzymes described for Mcl-1.

  8. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Directory of Open Access Journals (Sweden)

    Alison A Williams

    Full Text Available Methyl-CpG binding protein 2 (MeCP2 is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X, a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80 and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  9. BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2.

    Directory of Open Access Journals (Sweden)

    Matthew R Harter

    2016-02-01

    Full Text Available Epstein-Barr virus (EBV nuclear antigen 2 (EBNA2 plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND, in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs. Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection.

  10. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Science.gov (United States)

    Williams, Alison A; Mehler, Vera J; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  11. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

    OpenAIRE

    2015-01-01

    We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C4...

  12. The crystal structures of the synthetic C-terminal octa- and dodecapeptides of trichovirin.

    Science.gov (United States)

    Gessmann, R; Benos, P; Brückner, H; Kokkinidis, M

    1999-02-01

    The structures of two synthetic peptides with sequences corresponding to the C-terminal region of the naturally occurring 14-residue peptaibol trichovirin have been determined. The crystal structures of 8- and 12-residue segments are presented and are compared with the structures of the tetrapeptide and of the 9-residue segment, which have been reported earlier. A comparison between these segments leads to the hypothesis that the three-dimensional structure of trichovirin is to a large extent determined by the properties of a periodically repeating -Aib-Pro- pattern in the sequence of the peptide.

  13. p53 Requires an Intact C-Terminal Domain for DNA Binding and Transactivation

    OpenAIRE

    2011-01-01

    The p53 tumor suppressor plays a critical role in mediating cellular response to a wide range of environmental stresses. p53 regulates these processes mainly by acting as a short-lived DNA binding protein that stimulates transcription from numerous genes involved in cell cycle arrest, programmed cell death, and other processes. To investigate the importance of C-terminal domain of p53, we generated a series of deletion and point mutations in this region and analyzed their effects on p53 trans...

  14. A C-terminal Aldehyde Analog of the Insect Kinins Inhibits Diuresis in the Housefly

    Science.gov (United States)

    2006-09-21

    p e p t i d e s 2 8 ( 2 0 0 7 ) 1 4 6 – 1 5 2A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly Ronald J. Nachman a...secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h...to stimulate Malpighian tubule fluid secretion [2,25]. In the housefly, muscakinin has been implicated in the control of diuresis in response to

  15. C-Terminal acetylene derivatized peptides via silyl-based alkyne immobilization.

    Science.gov (United States)

    Strack, Martin; Metzler-Nolte, Nils; Albada, H Bauke

    2013-06-21

    A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)2 derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described.

  16. Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95.

    Science.gov (United States)

    Gudiukaitė, Renata; Gegeckas, Audrius; Kazlauskas, Darius; Citavicius, Donaldas

    2014-01-01

    GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80% lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.

  17. The role of N-glycans and the C-terminal loop of the subunit rBAT in the biogenesis of the cystinuria-associated transporter.

    Science.gov (United States)

    Rius, Mònica; Sala, Laura; Chillarón, Josep

    2016-02-01

    The transport system b(0,+) mediates reabsorption of dibasic amino acids and cystine in the kidney. It is made up of two disulfide-linked membrane subunits: the carrier, b(0,+)AT and the helper, rBAT (related to b(0,+) amino acid transporter). rBAT mutations that impair biogenesis of the transporter cause type I cystinuria. It has been shown that upon assembly, b(0,+)AT prevents degradation and promotes folding of rBAT; then, rBAT traffics b(0,+)AT from the endoplasmic reticulum (ER) to the plasma membrane. The role of the N-glycans of rBAT and of its C-terminal loop, which has no homology to any other sequence, in biogenesis of system b(0,+) is unknown. In the present study, we studied these points. We first identified the five N-glycans of rBAT. Elimination of the N-glycan Asn(575), but not of the others, delayed transporter maturation, as measured by pulse chase experiments and endoglycosidase H assays. Moreover, a transporter with only the N-glycan Asn(575) displayed similar maturation compared with wild-type, suggesting that this N-glycan was necessary and sufficient to achieve the maximum rate of transporter maturation. Deletion of the rBAT C-terminal disulfide loop (residues 673-685) prevented maturation and prompted degradation of the transporter. Alanine-scanning mutagenesis uncovered loop residues important for stability and/or maturation of system b(0,+). Further, double-mutant cycle analysis showed partial additivity of the effects of the Asn(679) loop residue and the N-glycan Asn(575) on transporter maturation, indicating that they may interact during system b(0,+) biogenesis. These data highlight the important role of the N-glycan Asn(575) and the C-terminal disulfide loop of rBAT in biogenesis of the rBAT-b(0,+)AT heterodimer.

  18. Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation.

    Science.gov (United States)

    Wu, Xiaobin; García-Estrada, Carlos; Vaca, Inmaculada; Martín, Juan-Francisco

    2012-02-01

    The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-valine into the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs (3371)EGHGRE(3376) (located in the putative epimerase domain) and (3629)GWSFG(3633) (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thioesterease interacts with the other ACVS domains.

  19. C-terminal of human histamine H1 receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.

    Science.gov (United States)

    Hishinuma, Shigeru; Nozawa, Hiroki; Akatsu, Chizuru; Shoji, Masaru

    2016-11-01

    It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that Gq/11 -protein-coupled human histamine H1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H1 receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H1 receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [(3) H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H1 receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [(3) H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H1 receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H1 receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively.

  20. Replacement of the C-terminal tetrapeptide (314PAPV317 to 314SSSM317) in interferon regulatory factor-2 alters its N-terminal DNA-binding activity

    Indian Academy of Sciences (India)

    Krishna Prakash; Pramod C Rath

    2010-12-01

    Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein–protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (314PAPV317) of mouse IRF-2 to be different (314SSSM317) from human IRF-2. Recombinant GST-IRF-2 with 314PAPV317 (wild type) and 314SSSM317 (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with 32P-(GAAAGT)4 by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA–protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA–protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and influence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.

  1. Growth of quailbush in acidic, metalliferous desert mine tailings: effect of Azospirillum brasilense Sp6 on biomass production and rhizosphere community structure.

    Science.gov (United States)

    de-Bashan, Luz E; Hernandez, Juan-Pablo; Nelson, Karis N; Bashan, Yoav; Maier, Raina M

    2010-11-01

    Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p < 0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development.

  2. Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses.

    Science.gov (United States)

    Baker, Laura E; Ellena, Jeffrey F; Handing, Katarzyna B; Derewenda, Urszula; Utepbergenov, Darkhan; Engel, Daniel A; Derewenda, Zygmunt S

    2016-01-01

    The Filoviridae family of negative-sense, single-stranded RNA (ssRNA) viruses is comprised of two species of Marburgvirus (MARV and RAVV) and five species of Ebolavirus, i.e. Zaire (EBOV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV) and Bundibugyo (BDBV). In each of these viruses the ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. It is tightly associated with the viral RNA in the nucleocapsid, and during the lifecycle of the virus is essential for transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. The structure of the unique C-terminal globular domain of the NP from EBOV has recently been determined and shown to be structurally unrelated to any other known protein [Dziubańska et al. (2014), Acta Cryst. D70, 2420-2429]. In this paper, a study of the C-terminal domains from the NP from the remaining four species of Ebolavirus, as well as from the MARV strain of Marburgvirus, is reported. As expected, the crystal structures of the BDBV and TAFV proteins show high structural similarity to that from EBOV, while the MARV protein behaves like a molten globule with a core residual structure that is significantly different from that of the EBOV protein.

  3. C-terminal moiety of Tudor contains its in vivo activity in Drosophila.

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    Joël Anne

    Full Text Available BACKGROUND: In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization. METHODOLOGY/PRINCIPAL FINDINGS: In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity. CONCLUSIONS/SIGNIFICANCE: I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud.

  4. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.

    Science.gov (United States)

    Tetali, Sarada D; Budamagunta, Madhu S; Voss, John C; Rutledge, John C

    2006-07-01

    Increased triglyceride-rich lipoproteins (TGRLs) in the postprandial state are associated with atherosclerosis. We investigated whether the postprandial state induced structural changes at the apolipoprotein E4 (apoE4) C terminus, its principal lipid binding domain, using electron paramagnetic resonance (EPR) spectroscopy of a site-directed spin label attached to the cysteine of apoE4-W264C. Spin coupling between labels located in the C termini was followed after mixing with preprandial and postprandial human plasma samples. Our results indicate that postprandial plasma triggers a reorganization of the protein such that the dipolar broadening is diminished, indicating a reduction in C-terminal interaction. The loss of spectral broadening was directly correlated with an increase in postprandial plasma triglycerides and was reduced with delipidated plasma. The spin-labeled apoE4 displayed a lipid preference of VLDL > LDL > HDL in the preprandial and postprandial states. The apoE4 shift to VLDL during the postprandial state was accompanied by a loss in spectral broadening of the protein. These findings suggest that apoE4 associated with LDL maintains self-association via its C terminus and that this association is diminished in VLDL-associated protein. Lipolyzed TGRL reflected a depletion of the C-terminal interaction of apoE4. Addition of palmitate to VLDL gave a similar response as lipolyzed TGRL, suggesting that lipolysis products play a major role in reorganizing apoE4 during the postprandial state.

  5. A C-terminal membrane association domain of phototropin 2 is necessary for chloroplast movement.

    Science.gov (United States)

    Kong, Sam-Geun; Kagawa, Takatoshi; Wada, Masamitsu; Nagatani, Akira

    2013-01-01

    Phototropins (phot1 and phot2), plant-specific blue light receptor kinases, mediate a range of physiological responses in Arabidopsis, including phototropism, chloroplast photorelocation movement, stomatal opening and leaf flattening. Phototropins consist of two photoreceptive domains at their N-terminus, LOV1 (light, oxygen or voltage 1) and LOV2, and a serine/threonine kinase domain at their C-terminus. Here, we determined the molecular moiety for the membrane association of phototropins using the yeast CytoTrap and Arabidopsis protoplast systems. We then examined the physiological significance of the membrane association of phototropins. This detailed study with serial deletions narrowed down the association domain to a relatively small part of the C-terminal domain of phototropin. The functional analysis of phot2 deletion mutants in the phot2-deficient Adiantum and Arabidopsis mutants revealed that the ability to mediate the chloroplast avoidance response correlated well with phot2's membrane association, especially with the Golgi apparatus. Taken together, our data suggest that a small part of the C-terminal domain of phototropins is necessary not only for membrane association but also for the physiological activities that elicit phototropin-specific responses.

  6. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene

    Science.gov (United States)

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene. PMID:28107454

  7. Structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    Science.gov (United States)

    Kim, Sun-Yong; Hakoshima, Toshio; Kitano, Ken

    2013-11-21

    Bloom syndrome is a rare genetic disorder characterized by genomic instability and cancer predisposition. The disease is caused by mutations of the Bloom syndrome protein (BLM). Here we report the crystal structure of a RecQ C-terminal (RQC) domain from human BLM. The structure reveals three novel features of BLM RQC which distinguish it from the previous structures of the Werner syndrome protein (WRN) and RECQ1. First, BLM RQC lacks an aromatic residue at the tip of the β-wing, a key element of the RecQ-family helicases used for DNA-strand separation. Second, a BLM-specific insertion between the N-terminal helices exhibits a looping-out structure that extends at right angles to the β-wing. Deletion mutagenesis of this insertion interfered with binding to Holliday junction. Third, the C-terminal region of BLM RQC adopts an extended structure running along the domain surface, which may facilitate the spatial positioning of an HRDC domain in the full-length protein.

  8. Iron-sulfur cluster biosynthesis: functional characterization of the N- and C-terminal domains of human NFU.

    Science.gov (United States)

    Liu, Yushi; Qi, Wenbin; Cowan, J A

    2009-02-10

    Human NFU (also known as HIRIP5) has been implicated in cellular iron-sulfur cluster biosynthesis. Bacterial and yeast forms are smaller than the human protein and are homologous to the C-terminal domain of the latter. This C-terminal domain contains a pair of redox active cysteines and demonstrates thioredoxin-like activity by mediating persulfide bond cleavage of sulfur-loaded NifS (an IscS-type protein), the sulfide donor for [2Fe-2S] cluster assembly on ISU-type scaffold proteins. Herein, the affinity of full-length human NFU and the individual N- and C-terminal domains for sulfide donor and cluster scaffold proteins is assessed. The influence of the N-terminal domain on C-terminal NFU binding to NifS and persulfide reductase activity is also examined. Only the C-terminal domain is required for persulfide reductase activity, while complex formation of NifS with full-length NFU is similar to that of the C-terminal domain alone (K(D) approximately 9.7 +/- 0.7 and 10.1 +/- 0.6 microM, respectively). There is negligible affinity between the isolated C- and N-terminal domains, while the N-terminal domain has negligible affinity for either sulfide donor or cluster scaffold proteins. The temperature dependence of the binding enthalpy for formation of the complex between NifS and the C-terminal domain of NFU yields a change in molar heat capacity (DeltaC(p) approximately 138 cal mol(-1) K(-1)) that suggests bonding at the protein-protein interface is dominated by electrostatic interactions. This is consistent with electrostatic potential maps for bacterial homologues of the N- and C-terminal domains of human NFU, which most likely reflect the structural characteristics expected for full-length human NFU.

  9. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

  10. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities.

    Science.gov (United States)

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm; Hermand, Damien

    2015-05-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

  11. Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.

    Science.gov (United States)

    Fan, Jin-Yuan; Means, John C; Bjes, Edward S; Price, Jeffrey L

    2015-07-01

    Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT(C/ala)) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT(C/ala) did not affect circadian behavior differently from wild-type DBT (DBT(WT)), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT(WT) protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT(C/ala) did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

  12. Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

    Energy Technology Data Exchange (ETDEWEB)

    Roujeinikova, Anna, E-mail: anna.roujeinikova@manchester.ac.uk [Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom)

    2008-04-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a putative peptidoglycan-binding domain of H. pylori MotB, a stator component of the bacterial flagellar motor, are reported. The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

  13. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D. [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, Ontario (Canada)

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  14. Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction.

    Science.gov (United States)

    Carotenuto, Marianeve; Pedone, Emilia; Diana, Donatella; de Antonellis, Pasqualino; Džeroski, Sašo; Marino, Natascia; Navas, Luigi; Di Dato, Valeria; Scoppettuolo, Maria Nunzia; Cimmino, Flora; Correale, Stefania; Pirone, Luciano; Monti, Simona Maria; Bruder, Elisabeth; Zenko, Bernard; Slavkov, Ivica; Pastorino, Fabio; Ponzoni, Mirco; Schulte, Johannes H; Schramm, Alexander; Eggert, Angelika; Westermann, Frank; Arrigoni, Gianluigi; Accordi, Benedetta; Basso, Giuseppe; Saviano, Michele; Fattorusso, Roberto; Zollo, Massimo

    2013-01-01

    Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.

  15. Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

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    Belhumeur Pierre

    2008-11-01

    Full Text Available Abstract Background HIV-1 integrase (IN is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s and/or motif(s required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype. Results Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant. Conclusion Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of

  16. Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae).

    Science.gov (United States)

    Beckwitt, R; Arcidiacono, S

    1994-03-04

    The polymerase chain reaction (PCR) has been used to amplify the portion of the Spidroin 1 gene that codes for the C-terminal part of the silk protein of the spider Nephila clavipes. Along with some substitution mutations of minor consequence, the PCR-derived sequence reveals an additional base missing from the previously published Nephila Spidroin 1 sequence. Comparison of the PCR-derived sequence with the equivalent region of Spidroin 2 indicates that the insertion of this single base results in greatly increased similarity in the resulting amino acid sequences of Spidroin 1 and Spidroin 2 (75% over 97 amino acids). The same PCR primers also amplified a fragment of the same length from Araneus bicentenarius. This sequence is also very similar to Spidroin 1 of Nephila (71% over 238 bases excluding the PCR primers, which translates into 76% over 79 amino acids).

  17. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    Science.gov (United States)

    Banerjee, Baisakhi; Banerjee, Rajat

    2015-01-01

    E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS. PMID:26617997

  18. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    Directory of Open Access Journals (Sweden)

    Baisakhi Banerjee

    2015-01-01

    Full Text Available E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700 forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS.

  19. Crystallization of the C-terminal domain of the addiction antidote CcdA in complex with its toxin CcdB

    Energy Technology Data Exchange (ETDEWEB)

    Buts, Lieven; De Jonge, Natalie; Loris, Remy, E-mail: reloris@vub.ac.be; Wyns, Lode; Dao-Thi, Minh-Hoa [Department of Molecular and Cellular Interactions, Vlaams Interinuversitair Instituut voor Biotechnologie and Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel (Belgium)

    2005-10-01

    The CcdA C-terminal domain was crystallized in complex with CcdB in two crystal forms that diffract to beyond 2.0 Å resolution. CcdA and CcdB are the antidote and toxin of the ccd addiction module of Escherichia coli plasmid F. The CcdA C-terminal domain (CcdA{sub C36}; 36 amino acids) was crystallized in complex with CcdB (dimer of 2 × 101 amino acids) in three different crystal forms, two of which diffract to high resolution. Form II belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 37.6, b = 60.5, c = 83.8 Å and diffracts to 1.8 Å resolution. Form III belongs to space group P2{sub 1}, with unit-cell parameters a = 41.0, b = 37.9, c = 69.6 Å, β = 96.9°, and diffracts to 1.9 Å resolution.

  20. C-terminal amide to alcohol conversion changes the cardiovascular effects of endomorphins in anesthetized rats.

    Science.gov (United States)

    Yu, Ye; Wang, Chang-lin; Cui, Yun; Fan, Ying-zhe; Liu, Jing; Shao, Xuan; Liu, Hong-mei; Wang, Rui

    2006-01-01

    Endomorphin1-ol (Tyr-Pro-Trp-Phe-ol, EM1-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and lower intrinsic efficacy in vitro than endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to intravenous (i.v.) or intracerebroventricular (i.c.v.) injection of EM1-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EM1-ol and EM2-ol induced dose-related decrease in SAP and HR when injected in doses of 3-100 nmol/kg, i.v. In terms of relative vasodepressor activity, it is interesting to note that EM2-ol was more potent than endomorphin2 [the dose of 25% decrease in SAP (DD25) = 6.01+/-3.19 and 13.99+/-1.56 nmol/kg, i.v., respectively] at a time when responses to EM1-ol were less potent than endomorphin1. Moreover, decreases in SAP in response to EM1-ol and EM2-ol were reduced by naloxone, atropine sulfate, L-NAME and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism. It is noteworthy that i.c.v. injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone and atropine sulfate. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphin1 and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidence that amidated C-terminus might play an important role in the regulation of the cardiovascular system.

  1. Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1 Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif.

    Directory of Open Access Journals (Sweden)

    Julia Bender

    Full Text Available The corticotropin-releasing hormone receptor type 1 (CRHR1 plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK family: postsynaptic density protein 95 (PSD95, synapse-associated protein 97 (SAP97, SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2. CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1 binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.

  2. A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response

    Institute of Scientific and Technical Information of China (English)

    Suzanne Paz; Rongtuan Lin; John Hiscott; Myriam Vilasco; Steven J Werden; Meztli Arguello; Deshanthe Joseph-Pillai; Tiejun Zhao; Thi Lien-Anh Nguyen; Qiang Sun; Eliane F Meurs

    2011-01-01

    Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-Ⅰ (RIG-Ⅰ) results in the activation of signaling cascades that culminate with the generation of the type Ⅰ interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters,kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendal or vesicular stomatitis virus (VSV) infection,mediated by Lys63-linked polyubiquitination of MAVS at Lys500,resulting in inhibition of downstream IFN signaling (Paz et al,Mol Cell Biol,2009). In this study,we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM),455-PEENEY-460,within this site is required for TRAF3 binding and activation of IFN antiviral response genes,whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS-/- mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication,whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore,recruitment of IKKε to an adjacent C-terminal site (aa 468-540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability,thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.

  3. The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain.

    Science.gov (United States)

    Barboza-Corona, J Eleazar; Park, Hyun-Woo; Bideshi, Dennis K; Federici, Brian A

    2012-03-01

    The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).

  4. The role of formin tails in actin nucleation, processive elongation, and filament bundling.

    Science.gov (United States)

    Vizcarra, Christina L; Bor, Batbileg; Quinlan, Margot E

    2014-10-31

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.

  5. The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

    Institute of Scientific and Technical Information of China (English)

    Guozhen Liu; Bo You; Ye Yin; Shuting Li; Hao Wang; Yan Ren; Jia Ji; Xiaoqian Zhao; Yongqiao Sun; Xiaowei Zhang; Jianqiu Fang; Shaohui Hu; Jian Wang; Siqi Liu; Jun Yu; Heng Zhu; Huanming Yang; Yongwu Hu; Peng Chen; Jianning Yin; Jie Wen; Jingqiang Wang; Liang Lin; Jinxiu Liu

    2003-01-01

    In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC)gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter.Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls.The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

  6. Nucleation Process of a Fibril Precursor in the C-Terminal Segment of Amyloid-β

    Science.gov (United States)

    Baftizadeh, Fahimeh; Pietrucci, Fabio; Biarnés, Xevi; Laio, Alessandro

    2013-04-01

    By extended atomistic simulations in explicit solvent and bias-exchange metadynamics, we study the aggregation process of 18 chains of the C-terminal segment of amyloid-β, an intrinsically disordered protein involved in Alzheimer’s disease and prone to form fibrils. Starting from a disordered aggregate, we are able to observe the formation of an ordered nucleus rich in beta sheets. The rate limiting step in the nucleation pathway involves crossing a barrier of approximately 40kcal/mol and is associated with the formation of a very specific interdigitation of the side chains belonging to different sheets. This structural pattern is different from the one observed experimentally in a microcrystal of the same system, indicating that the structure of a “nascent” fibril may differ from the one of an “extended” fibril.

  7. Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPas

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Khandelia, Himanshu; Morth, Jens Preben

    2010-01-01

    The Na(+)/K(+)-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume...... potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause...... severe neurological diseases. This novel model for ion transport by the Na(+)/K(+)-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely...

  8. Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Khandelia, Himanshu; Morth, J Preben

    2010-01-01

    The Na(+)/K(+)-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume...... potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause...... severe neurological diseases. This novel model for ion transport by the Na(+)/K(+)-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely...

  9. A novel system for continuous production of formic acid based on recycling of yellow phosphorous tail gas%连续化回收黄磷尾气副产甲酸的新系统

    Institute of Scientific and Technical Information of China (English)

    刘宝庆; 张义堃; 蒋家羚; 林兴华; 何锦林

    2012-01-01

    The recycling of yellow phosphorous tail gas rich in carbon monoxide is related to energy conservation and environment protection. Based on the analysis of existing technologies for the recycling of yellow phosphorous tail gas, a novel continuous process was developed by combining the resources recycling of yellow phosphorous tail gas with formic acid production, which made phosphoric acid acidification method as the core and could produce formic acid with high quality and low cost on the basis of considering the high and variable viscosity of system and the rationality of energy utilization. At the same time, a multilevel acidification reactor was also designed by integrating the reaction, heating and condensation reflux, which was divided into several reaction regions and equipped with different stirrers. The novel acidification reactor as the core equipment had the advantages of compact structure, energy conservation and wide adaptability.

  10. Structure of the nisin leader peptidase NisP revealing a C-terminal autocleavage activity.

    Science.gov (United States)

    Xu, Yueyang; Li, Xin; Li, Ruiqing; Li, Shanshan; Ni, Hongqian; Wang, Hui; Xu, Haijin; Zhou, Weihong; Saris, Per E J; Yang, Wen; Qiao, Mingqiang; Rao, Zihe

    2014-06-01

    Nisin is a widely used antibacterial lantibiotic polypeptide produced by Lactococcus lactis. NisP belongs to the subtilase family and functions in the last step of nisin maturation as the leader-peptide peptidase. Deletion of the nisP gene in LAC71 results in the production of a non-active precursor peptide with the leader peptide unremoved. Here, the 1.1 Å resolution crystal structure of NisP is reported. The structure shows similarity to other subtilases, which can bind varying numbers of Ca atoms. However, no calcium was found in this NisP structure, and the predicted calcium-chelating residues were placed so as to not allow NisP to bind a calcium ion in this conformation. Interestingly, a short peptide corresponding to its own 635-647 sequence was found to bind to the active site of NisP. Biochemical assays and native mass-spectrometric analysis confirmed that NisP possesses an auto-cleavage site between residues Arg647 and Ser648. Further, it was shown that NisP mutated at the auto-cleavage site (R647P/S648P) had full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of NisP was no longer cleaved. Expressing this mutant in L. lactis LAC71 did not affect the production of nisin but did decrease the proliferation rate of the bacteria, suggesting the biological significance of the C-terminal auto-cleavage of NisP.

  11. Modeling the human intestinal mucin (MUC2) C-terminal cystine knot dimer.

    Science.gov (United States)

    Sadasivan, Vatsala D; Narpala, Sandeep R; Budil, David E; Sacco, Albert; Carrier, Rebecca L

    2011-11-01

    Intestinal mucus, a viscous secretion that lines the mucosa, is believed to be a barrier to absorption of many therapeutic compounds and carriers, and is known to play an important physiological role in controlling pathogen invasion. Nevertheless, there is as yet no clear understanding of the barrier properties of mucus, such as the nature of the molecular interactions between drug molecules and mucus components as well as those that govern gel formation. Secretory mucins, large and complex glycoprotein molecules, are the principal determinants of the viscoelastic properties of intestinal mucus. Despite the important role that mucins play in controlling transport and in diseases such as cystic fibrosis, their structures remain poorly characterized. The major intestinal secretory mucin gene, MUC2, has been identified and fully sequenced. The present study was undertaken to determine a detailed structure of the cysteine-rich region within the C-terminal end of human intestinal mucin (MUC2) via homology modeling, and explore possible configurations of a dimer of this cysteine-rich region, which may play an important role in governing mucus gel formation. Based on sequence-structure alignments and three-dimensional modeling, a cystine knot tertiary structure homologous to that of human chorionic gonadotropin (HCG) is predicted at the C-terminus of MUC2. Dimers of this C-terminal cystine knot (CTCK) were modeled using sequence alignment based on HCG and TGF-beta, followed by molecular dynamics and simulated annealing. Results support the formation of a cystine knot dimer with a structure analogous to that of HCG.

  12. Insulin resistance uncoupled from dyslipidemia due to C-terminal PIK3R1 mutations

    Science.gov (United States)

    Huang-Doran, Isabel; Tomlinson, Patsy; Payne, Felicity; Gast, Alexandra; Sleigh, Alison; Bottomley, William; Harris, Julie; Daly, Allan; Rocha, Nuno; Rudge, Simon; Clark, Jonathan; Kwok, Albert; Romeo, Stefano; McCann, Emma; Müksch, Barbara; Dattani, Mehul; Zucchini, Stefano; Wakelam, Michael; Foukas, Lazaros C.; Savage, David B.; Murphy, Rinki; O’Rahilly, Stephen; Semple, Robert K.

    2016-01-01

    Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome. PMID:27766312

  13. The Intracellular Distal Tail of the Na+/H+ Exchanger NHE1 Is Intrinsically Disordered

    DEFF Research Database (Denmark)

    Nørholm, Ann-Beth; Hendus-Altenburger, Ruth; Bjerre, Gabriel

    2011-01-01

    dysfunction is implicated in several clinically important diseases. This study shows, for the first time for any carrier protein, that the distal part of the C-terminal intracellular tail (the cdt, residues V686-Q815) from human (h) NHE1 is intrinsically disordered. Further, we experimentally demonstrated...... disrupted the putative binding feature. When this mutant NHE1 was expressed in full length NHE1 in AP1 cells, it exhibited impaired trafficking to the plasma membrane. This study demonstrated that the distal regulatory domain of NHE1 is intrinsically disordered yet contains conserved regions of transient...... structure. We suggest that normal NHE1 function depends on a protein recognition element within the ID region that may be linked to NHE1 trafficking via an acidic ER export motif....

  14. The effect of C-terminal helix on the stability of FF domain studied by molecular dynamics simulation.

    Science.gov (United States)

    Zhao, Liling; Cao, Zanxia; Wang, Jihua

    2012-01-01

    To investigate the effect of C-terminal helix on the stability of the FF domain, we studied the native domain FF3-71 from human HYPA/FBP11 and the truncated version FF3-60 with C-terminal helix being deleted by molecular dynamics simulations with GROMACS package and GROMOS 43A1 force field. The results indicated that the structures of truncated version FF3-60 were evident different from those of native partner FF3-71. Compared with FF3-71, the FF3-60 lost some native contacts and exhibited some similar structural characters to those of intermediate state. The C-terminal helix played a major role in stabilizing the FF3-71 domain. To a certain degree, the FF domain had a tendency to form an intermediate state without the C-terminal helix. In our knowledge, this was the first study to examine the role of C-terminal helix of FF domain in detail by molecular dynamics simulations, which was useful to understand the three-state folding mechanism of the small FF domain.

  15. Trypanosoma evansi: identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain.

    Science.gov (United States)

    Jia, Yonggen; Zhao, Xinxin; Zou, Jingru; Suo, Xun

    2011-01-01

    African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts' antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.

  16. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.;

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H......(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H......(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2...

  17. C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

    Science.gov (United States)

    Jóźwiak, Jolanta; Rzhepetskyy, Yuriy; Sobczak, Magdalena; Kocik, Elżbieta; Skórzewski, Radosław; Kłopocka, Wanda; Rędowicz, Maria Jolanta

    2011-02-01

    Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

  18. Genome sequence of the copper resistant and acid-tolerant Desulfosporosinus sp. BG isolated from the tailings of a molybdenum-tungsten mine in the Transbaikal area

    Directory of Open Access Journals (Sweden)

    Olga V. Karnachuk

    2017-03-01

    Full Text Available Here, we report on the draft genome of a copper-resistant and acidophilic Desulfosporosinus sp. BG, isolated from the tailings of a molybdenum-tungsten mine in Transbaikal area. The draft genome has a size of 4.52 Mb and encodes transporters of heavy metals. The phylogenetic analysis based on concatenated ribosomal proteins revealed that strain BG clusters together with the other acidophilic copper-resistant strains Desulfosporosinus sp. OT and Desulfosporosinus sp. I2. The K+-ATPase, Na+/H+ antiporter and amino acid decarboxylases may participate in enabling growth at low pH. The draft genome sequence and annotation have been deposited at GenBank under the accession number NZ_MASS00000000.

  19. Analgesic and Anti-Inflammatory Properties of Gelsolin in Acetic Acid Induced Writhing, Tail Immersion and Carrageenan Induced Paw Edema in Mice.

    Directory of Open Access Journals (Sweden)

    Ashok Kumar Gupta

    Full Text Available Plasma gelsolin levels significantly decline in several disease conditions, since gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous gelsolin (8 mg/mouse in mice models of pain and acute inflammation. Administration of gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of gelsolin (8 mg/mouse compared with anti-inflammatory drug diclofenac sodium (10 mg/kg] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histopathological examinations of tissue revealed administration of gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of gelsolin can effectively reduce the pain and inflammation in mice model.

  20. Alkalis scrubbing vs sulphuric acid production with Claus tail gases%克劳斯尾气碱洗涤与生产硫酸的比较

    Institute of Scientific and Technical Information of China (English)

    M. RAMESHNI; 金苏闽(译)

    2012-01-01

    为使克劳斯硫回收装置的硫回收率达到100%,讨论并比较了碱洗涤与生产硫酸这2种尾气处理技术。这2种技术都可用于满足最苛刻的排放要求,生产硫酸的成本略低。但除了成本之外,还应考虑炼油厂的总体布置、原油类型及硫和氮含量、产品酸的市场需求、所需公用工程的供应等因素。%In order to reach the sulphur recovery of 100% from Claus plants, two technology options for the tail gas treating, caustic scrubbing and sulphuric acid production, are discussed and compared. Both of them can be applied to achieve the most stringent requirements and the cost of sulphuric acid production is slightly lower. Be- sides the cost, the factors of overall refinery configuration, crude type and sulphur and nitrogen contents, market demand for selling acid, availability of required utilities, and so on, should also be considered.

  1. C-terminal mutations destabilize SIL1/BAP and can cause Marinesco-Sjögren syndrome.

    Science.gov (United States)

    Howes, Jennifer; Shimizu, Yuichiro; Feige, Matthias J; Hendershot, Linda M

    2012-03-01

    Marinesco-Sjögren syndrome (MSS) is an autosomal recessive, neurodegenerative, multisystem disorder characterized by severe phenotypes developing in infancy. Recently, mutations in the endoplasmic reticulum (ER)-associated co-chaperone SIL1/BAP were identified to be the major cause of MSS. SIL1 acts as a nucleotide exchange factor for BiP, the ER Hsp70 orthologue, which plays an essential role in the folding and assembly of nascent polypeptide chains in the ER. SIL1 facilitates the release of BiP from unfolded protein substrates, enabling the subsequent folding and transport of the protein. Although most mutations leading to MSS result in deletion of the majority of the protein, three separate mutations have been identified that disrupt only the last five or six amino acids of the protein, which were assumed to encode a divergent ER retention motif. This study presents an in depth analysis of two of these mutants and reveals that the phenotype in the affected individuals is not likely to be due to depletion of SIL1 from the ER via secretion. Instead, our analyses show that the mutant proteins are particularly unstable and either form large aggregates in the ER or are rapidly degraded via the proteasome. In agreement with our findings, homology modeling suggests that the very C-terminal residues of SIL1 play a role in its structural integrity rather than its localization. These new insights might be a first step toward a possible pharmacological treatment of certain types of MSS by specifically stabilizing the mutant SIL1 protein.

  2. Novel endomorphin-1 analogs with C-terminal oligoarginine-conjugation display systemic antinociceptive activity with less gastrointestinal side effects.

    Science.gov (United States)

    Wang, Chang-lin; Qiu, Ting-ting; Diao, Yu-xiang; Zhang, Yao; Gu, Ning

    2015-09-01

    In recent study, in order to improve the bioavailability of endomorphin-1 (EM-1), we designed and synthesized a series of novel EM-1 analogs by replacement of L-Pro(2) by β-Pro, D-Ala or Sar, together with C-terminal oligoarginine-conjugation. Our results indicated that the introduction of D-Ala and β-Pro in position 2, along with oligoarginine-conjugation, didn't significantly decrease the μ-affinity and in vitro bioactivity, and the enhancement of arginine residues did not markedly influence the μ-affinity of these analogs. All analogs displayed a significant enhancement of stability, which may be due to increased resistance to proline-specific enzymatic degradation. Moreover, following intracerebroventricular (i.c.v.) administration, analogs 1, 2, 4 and 5 produced significant antinociception and increased duration of action, with the ED50 values being about 1.8- to 4.2-fold less potent than that of EM-1. In addition, our results indicated that no significant antinociceptive activity of EM-1 was seen following subcutaneous (s.c.) injection, whereas analogs 1, 2, 4 and 5 with equimolar dose induced significant and prolonged antinociception by an opioid and central mechanism. Herein, we further examined the gastrointestinal transit and colonic propulsive latencies of EM-1 and its four analogs administered centrally and peripherally. I.c.v. administration of EM-1 and analogs 1, 2, 4 and 5 significantly delayed gastrointestinal transit and colonic bead propulsion in mice, but the inhibitory effects induced by these analogs were largely attenuated. It is noteworthy that no significant gastrointestinal side effects induced by these four analogs were observed after s.c. administration. Our results demonstrated that combined modifications of EM-1 with unnatural amino acid substitutions and oligoarginine-conjugation gave an efficient strategy to improve the analgesic profile of EM-1 analogs but with less gastrointestinal side effects when administered peripherally.

  3. Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

    Science.gov (United States)

    Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

    2015-04-01

    The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

  4. Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4*

    Science.gov (United States)

    Butcher, Adrian J.; Hudson, Brian D.; Shimpukade, Bharat; Alvarez-Curto, Elisa; Prihandoko, Rudi; Ulven, Trond; Milligan, Graeme; Tobin, Andrew B.

    2014-01-01

    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr347, Thr349, Ser350, Ser357, and Ser360) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu341, Asp348, and Asp355 located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling. PMID:24817122

  5. Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment

    OpenAIRE

    Peterson, Janine H.; Tian, Pu; Ieva, Raffaele; Dautin, Nathalie; Bernstein, Harris D.

    2010-01-01

    Autotransporters are bacterial virulence factors consisting of an N-terminal “passenger domain” that is secreted in a C- to-N-terminal direction and a C-terminal “β domain” that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa pas...

  6. 以氢氟酸协同草酸精制石英砂尾矿及其作用机理%Refining quartz sand tailings and mechanisms by hydrofluoric acid associated with oxalic acid

    Institute of Scientific and Technical Information of China (English)

    张亚南; 范天博; 郭洪范; 李雪; 胡彬; 刘元元; 周永红; 刘云义

    2016-01-01

    High purity quartz sand is widely used in construction,glass manufacturing,casting and other industrial fields. In order to obtain the replacement products of high purity quartz sand,quartz sand tailings were treated by removing Fe and Al. In this work,the method of quartz tailings from Guangdong Heyuan as raw material refined with hydrofluoric acid associated with oxalate was proposed,and the optimum operating conditions were also provided. Oxalic acid dosage and reaction temperature were determined by oxalic acid complexation experiments. The acid leaching reaction time and material ratio were determined by refining experimentsvia hydrofluoric acid associated with oxalic acid. The results showed that the best operating conditions of refining the quartz sand tailings were as follows. Quartz sand tailings of 200g with the particle size≤250μm were immersed into the 100mL mixed solution of saturated oxalic acid and hydrofluoric acid in the volume ratio is 25∶1 for 4 hours. Temperature was kept at 80℃. After refining,the silica content in the refined quartz sand tailings could reach 99.97%,with the whiteness of 87.5. Experimental results demonstrated that refined solution was recycled by adding oxalic acid and hydrofluoric acid. Oxalic acid could be crystallized from the uncooled refined solution. The residual refined solution and refined quartz sand water lotion was treated as waste liquid. The impurities in the waste liquid were precipitated by the addition of a saturated lime water. The experimental results showed that the tail liquid was treated completely when the volume ratio of the tail liquid and the lime water is 1∶13 and after the end solution treatment,the tail liquid can be used as recycled water discharge.%高纯度石英砂在建筑、玻璃制造、铸造等工业领域应用广泛,将石英砂尾矿进行除Fe、Al处理,能够得到相当于高纯度石英砂的替代产品。本研究采用广东河源石英

  7. Jarosite versus Soluble Iron-Sulfate Formation and Their Role in Acid Mine Drainage Formation at the Pan de Azúcar Mine Tailings (Zn-Pb-Ag, NW Argentina

    Directory of Open Access Journals (Sweden)

    Jesica Murray

    2014-05-01

    Full Text Available Secondary jarosite and water-soluble iron-sulfate minerals control the composition of acid mine waters formed by the oxidation of sulfide in tailings impoundments at the (Zn-Pb-Ag Pan de Azúcar mine located in the Pozuelos Lagoon Basin (semi-arid climate in Northwest (NW Argentina. In the primary zone of the tailings (9.5 wt % pyrite-marcasite precipitation of anglesite (PbSO4, wupatkite ((Co,Mg,NiAl2(SO44 and gypsum retain Pb, Co and Ca, while mainly Fe2+, Zn2+, Al3+, Mg2+, As3+/5+ and Cd2+ migrate downwards, forming a sulfate and metal-rich plume. In the oxidation zone, jarosite (MFe3(TO42(OH6 is the main secondary Fe3+ phase; its most suitable composition is M = K+, Na+, and Pb2+and TO4 = SO42−; AsO42−. During the dry season, iron-sulfate salts precipitate by capillary transport on the tailings and at the foot of DC2 (tailings impoundment DC2 tailings dam where an acid, Fe2+ rich plume outcrops. The most abundant compounds in the acid mine drainage (AMD are SO42−, Fe2+, Fe3+, Zn2+, Al3+, Mg2+, Cu2+, As3+/5+, Cd2+. These show peak concentrations at the beginning of the wet season, when the soluble salts and jarosite dissolve. The formation of soluble sulfate salts during the dry season and dilution during the wet season conform an annual cycle of rapid metals and acidity transference from the tailings to the downstream environment.

  8. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

    Directory of Open Access Journals (Sweden)

    Brittany Ebersole

    Full Text Available We have used bioorthogonal click chemistry (BCC, a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R, a G protein-coupled receptor (GPCR crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443 of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH screen, we identified the palmitoyl acyltransferase (PAT zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

  9. Clinical utility of C-terminal telopeptide of type 1 collagen in multiple myeloma.

    Science.gov (United States)

    Ting, Kay R; Brady, Jennifer J; Hameed, Abdul; Le, Giao; Meiller, Justine; Verburgh, Estelle; Bayers, Christopher; Benjamin, Dalia; Anderson, Kenneth C; Richardson, Paul G; Dowling, Paul; Clynes, Martin; Fitzgibbon, Maria C; O'Gorman, Peter

    2016-04-01

    Myeloma bone disease (MBD) is a major cause of morbidity in multiple myeloma (MM). We investigated bone turnover markers (BTM) as relapse predictors and biomarkers for monitoring MBD. We measured C-terminal telopeptide of type I collagen (CTX-1), and Procollagen type 1 N Propeptide (P1NP) in 86 MM patients and 26 controls. CTX-1 was higher in newly diagnosed patients compared to control, remission and relapse (P < 0·05), and decreased following treatment. In the setting of relapse, a CTX-1 rise greater than the calculated least significant change (LSC) was observed in 26% of patients 3-6 months prior to relapse (P = 0·007), and in 60·8% up to 3 months before relapse (P = 0·015). Statistically significant changes in CTX-1 levels were also observed in patients who were with and without bisphosphonate therapy at the time of relapse. In patients with normal renal function, mean CTX-1 level was highest in the newly diagnosed group (0·771 ± 0·400 μg/l), and lowest in the remission group (0·099 ± 0·070 μg/l) (P < 0·0001). P1NP levels were not statistically different across the patient groups. We conclude that CTX-1, measured on an automated hospital laboratory platform, has a role in routine treatment monitoring and predicting relapse of MBD, even in patients on bisphosphonates.

  10. Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis.

    Directory of Open Access Journals (Sweden)

    Urša Pečar Fonović

    Full Text Available Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1-clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.

  11. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

    Science.gov (United States)

    Susor, Andrej; Liskova, Lucie; Toralova, Tereza; Pavlok, Antonin; Pivonkova, Katerina; Karabinova, Pavla; Lopatarova, Miloslava; Sutovsky, Peter; Kubelka, Michal

    2010-06-01

    The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

  12. Mutant mice lacking the p53 C-terminal domain model telomere syndromes.

    Science.gov (United States)

    Simeonova, Iva; Jaber, Sara; Draskovic, Irena; Bardot, Boris; Fang, Ming; Bouarich-Bourimi, Rachida; Lejour, Vincent; Charbonnier, Laure; Soudais, Claire; Bourdon, Jean-Christophe; Huerre, Michel; Londono-Vallejo, Arturo; Toledo, Franck

    2013-06-27

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

  13. Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast.

    Science.gov (United States)

    Sprague, E R; Redd, M J; Johnson, A D; Wolberger, C

    2000-06-15

    The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.

  14. C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function

    Directory of Open Access Journals (Sweden)

    Nancy Blaurock

    2016-01-01

    Full Text Available Systemic inflammatory response syndrome (SIRS is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis.

  15. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    Science.gov (United States)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  16. Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    Science.gov (United States)

    Park, Chin-Ju; Ko, Junsang; Ryu, Kyoung-Seok; Choi, Byong-Seok

    2014-02-01

    RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

  17. Electrodialytic remediation of copper mine tailings

    DEFF Research Database (Denmark)

    Hansen, Henrik K.; Rojo, A.; Ottpsen, Lisbeth M.

    2005-01-01

    electrodialytic remediation experiments on copper mine tailings. The results show that electric current could remove copper from watery tailing if the potential gradient was higher than 2V/cm during 21 days. With addition of sulphuric acid, the process was enhanced because the pH decreased to around 4......, and the copper by this reason was released in the solution. Furthermore, with acidic tailing the potential gradient was less than 2V/cm.The maximum copper removal reached in the anode side was 53% with addition of sulphuric acid in 21 days experiment at 20V using approximately 1.8kg mine tailing on dry basis....... In addition, experiments with acidic tailing show that the copper removal is proportional with time....

  18. A perspective of stepwise utilisation of Bayer red mud: Step two--Extracting and recovering Ti from Ti-enriched tailing with acid leaching and precipitate flotation.

    Science.gov (United States)

    Huang, Yanfang; Chai, Wencui; Han, Guihong; Wang, Wenjuan; Yang, Shuzhen; Liu, Jiongtian

    2016-04-15

    The extraction and recovery of Ti from Ti-enriched tailing with acid leaching and precipitate flotation, as one of the critical steps, was proposed for the stepwise utilization of red mud. The factors influencing acid leaching and precipitate flotation were examined by factorial design. The leaching thermodynamics, kinetics of Ti(4+), Al(3+) and Fe(3+), and the mechanism of selectively Fe(3+) removal using [Hbet][Tf2N] as precipitating reagent were discussed. The extracting of Ti(4+), Al(3+) and Fe(3+) in concentrated H2SO4 is controlled by diffusion reactions, depending mainly upon leaching time and temperature. The maximum extracting efficiency of Ti(4+) is approximately 92.3%, whereas Al(3+) and Fe(3+) leaching are respectively 75.8% and 84.2%. [Hbet][Tf2N], as a precipitating reagent, operates through a coordination mechanism in flotation. The pH value is the key factor influencing the flotation recovery of Ti(4+), whereas the dosage of precipitating reagent is that for Al(3+) recovery. The maximum flotation recovery of Ti(4+) is 92.7%, whereas the maximum Al(3+) recovery is 93.5%. The total recovery rate for extracting and recovering titanium is 85.5%. The liquor with Ti(4+) of 15.5g/L, Al(3+) of 30.4g/L and Fe(3+) of 0.48g/L was obtained for the following hydrolysis step in the integrated process for red mud utilisation.

  19. Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity

    NARCIS (Netherlands)

    Kraaij, Cindy van; Breukink, Eefjan; Rollema, Harry S.; Siezen, Roland J.; Demel, Rudy A.; Kruijff, Ben de; Kuipers, Oscar P.

    1997-01-01

    Three mutants of the lantibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR an

  20. C-terminal truncations in human 3 '-5 ' DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy

    NARCIS (Netherlands)

    Richards, Anna; van den Maagdenberg, Arn M. J. M.; Jen, Joanna C.; Kavanagh, David; Bertram, Paula; Spitzer, Dirk; Liszewski, M. Kathryn; Barilla-LaBarca, Maria-Louise; Terwindt, Gisela M.; Kasai, Yumi; McLellan, Mike; Grand, Mark Gilbert; Vanmolkot, Kaate R. J.; de Vries, Boukje; Wan, Jijun; Kane, Michael J.; Mamsa, Hafsa; Schaefer, Ruth; Stam, Anine H.; Haan, Joost; Paulus, T. V. M. de Jong; Storimans, Caroline W.; van Schooneveld, Mary J.; Oosterhuis, Jendo A.; Gschwendter, Andreas; Dichgans, Martin; Kotschet, Katya E.; Hodgkinson, Suzanne; Hardy, Todd A.; Delatycki, Martin B.; Hajj-Ali, Rula A.; Kothari, Parul H.; Nelson, Stanley F.; Frants, Rune R.; Baloh, Robert W.; Ferrari, Michel D.; Atkinson, John P.

    2007-01-01

    Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle- age onset. In nine families, we identified heterozygous C- terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activ

  1. Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

    NARCIS (Netherlands)

    Corici, L.N.; Frissen, A.E.; Zoelen, van D.J.; Eggen, I.F.; Peter, F.; Davidescu, C.M.; Boeriu, C.G.

    2011-01-01

    Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert-butanol/DMF. F

  2. One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

    Directory of Open Access Journals (Sweden)

    Merkx Maarten

    2008-10-01

    Full Text Available Abstract Background Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed. Results A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation. Conclusion An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

  3. Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

    KAUST Repository

    Quintero, Francisco J.

    2011-01-24

    The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

  4. Androgen deprivation causes truncation of the C-terminal region of androgen receptor in human prostate cancer LNCaP cells.

    Science.gov (United States)

    Harada, Naoki; Inoue, Kaoru; Yamaji, Ryoichi; Nakano, Yoshihisa; Inui, Hiroshi

    2012-06-01

    The androgen receptor (AR) acts as a ligand-dependent transcription factor, whereas mutant AR lacking the C-terminal ligand-binding domain functions in a ligand-independent manner. In the present study we report that the C-terminal truncated AR, which we named AR-NH1 (the N-terminal fragment of AR cleaved in the neighborhood of helix 1 of the ligand-binding domain), is produced in LNCaP prostatic carcinoma cells. The AR-NH1 of ~90 kDa was observed in an androgen-independent LNCaP subline and was further accumulated by the proteasome inhibitor MG132. MG132 treatment caused the accumulation of AR-NH1 even in parent LNCaP cells. AR-NH1 was produced in the absence of ligand or in the presence of the AR antagonist bicalutamide, whereas AR agonists suppressed its production. AR-NH1 was detected with different AR antibodies recognizing amino acid residues 1-20 and 300-316 and was also generated from exogenous AR. Both siRNA-mediated AR knockdown and treatment with a serine protease inhibitor (4-(2-aminoethyl)-benzenesulfonyl fluoride) reduced AR-NH1 levels. According to the predicted cleavage site (between amino acid residues 660-685) and its nuclear localization, it is assumed that AR-NH1 functions as a constitutively active transcription factor. These data suggest that AR-NH1 is produced under hormone therapy and contributes to the development of castration-resistant prostate cancer due to its ligand-independent transcriptional activity.

  5. Structural basis for the recognition of RNA polymerase II C-terminal domain by CREPT and p15RS.

    Science.gov (United States)

    Mei, Kunrong; Jin, Zhe; Ren, Fangli; Wang, Yinying; Chang, Zhijie; Wang, Xinquan

    2014-01-01

    CREPT and p15RS are two recently identified homologous proteins that regulate cell proliferation in an opposite way and are closely related to human cancer development. Both CREPT and p15RS consist of an N-terminal RPR domain and a C-terminal domain with high sequence homology. The transcription enhancement by CREPT is attributed to its interaction with RNA polymerase II (Pol II). Here we provide biochemical and structural evidence to support and extend this molecular mechanism. Through fluorescence polarization analysis, we show that the RPR domains of CREPT and p15RS (CREPT-RPR and p15RS-RPR) bind to different Pol II C-terminal domain (CTD) phosphoisoforms with similar affinity and specificity. We also determined the crystal structure of p15RS-RPR. Sequence and structural comparisons with RPR domain of Rtt103, a homolog of CREPT and p15RS in yeast, reveal structural basis for the similar binding profile of CREPT-RPR and p15RS-RPR with Pol II CTD. We also determined the crystal structure of the C-terminal domain of CREPT (CREPT-CTD), which is a long rod-like dimer and each monomer adopts a coiled-coil structure. We propose that dimerization through the C-terminal domain enhances the binding strength between CREPT or p15RS with Pol II by increasing binding avidity. Our results collectively reveal the respective roles of N-terminal RPR domain and C-terminal domain of CREPT and p15RS in recognizing RNA Pol II.

  6. Evolutionary origins of C-terminal (GPPn 3-hydroxyproline formation in vertebrate tendon collagen.

    Directory of Open Access Journals (Sweden)

    David M Hudson

    Full Text Available Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPPn in addition to the fully occupied A1 site at Pro986. The C-terminal (GPPn motif has five consecutive GPP triplets in α1(I, four in α2(I and three in α1(II, all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin and type II collagen (cartilage and notochord were examined by mass spectrometry. The (GPPn domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human, up to five 3-hydroxyproline residues per (GPPn motif were found in α1(I and four in α2(I, with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPPn site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.

  7. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    Directory of Open Access Journals (Sweden)

    Ralf eEnz

    2012-04-01

    Full Text Available Metabotropic glutamate receptors (mGluRs regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g. night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson´s disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors´ C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  8. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    Science.gov (United States)

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  9. The C-terminal binding protein (CTBP-1) regulates dorsal SMD axonal morphology in Caenorhabditis elegans.

    Science.gov (United States)

    Reid, A; Sherry, T J; Yücel, D; Llamosas, E; Nicholas, H R

    2015-12-17

    C-terminal binding proteins (CtBPs) are transcriptional co-repressors which cooperate with a variety of transcription factors to repress gene expression. Caenorhabditis elegans CTBP-1 expression has been observed in the nervous system and hypodermis. In C. elegans, CTBP-1 regulates several processes including Acute Functional Tolerance to ethanol and functions in the nervous system to modulate both lifespan and expression of a lipase gene called lips-7. Incorrect structure and/or function of the nervous system can lead to behavioral changes. Here, we demonstrate reduced exploration behavior in ctbp-1 mutants. Our examination of a subset of neurons involved in regulating locomotion revealed that the axonal morphology of dorsal SMD (SMDD) neurons is altered in ctbp-1 mutants at the fourth larval (L4) stage. Expressing CTBP-1 under the control of the endogenous ctbp-1 promoter rescued both the exploration behavior phenotype and defective SMDD axon structure in ctbp-1 mutants at the L4 stage. Interestingly, the pre-synaptic marker RAB-3 was found to localize to the mispositioned portion of SMDD axons in a ctbp-1 mutant. Further analysis of SMDD axonal morphology at days 1, 3 and 5 of adulthood revealed that the number of ctbp-1 mutants showing an SMDD axonal morphology defect increases in early adulthood and the observed defect appears to be qualitatively more severe. CTBP-1 is prominently expressed in the nervous system with weak expression detected in the hypodermis. Surprisingly, solely expressing CTBP-1a in the nervous system or hypodermis did not restore correct SMDD axonal structure in a ctbp-1 mutant. Our results demonstrate a role for CTBP-1 in exploration behavior and the regulation of SMDD axonal morphology in C. elegans.

  10. A C-terminal truncated mutation of spr-3 gene extends lifespan in Caenorhabditis elegans

    Institute of Scientific and Technical Information of China (English)

    Ping Yang; Ruilin Sun; Minghui Yao; Weidong Chen; Zhugang Wang; Jian Fei

    2013-01-01

    The lifespan of Caenorhabditis elegans is determined by various genetic and environmental factors.In this paper,spr-3,a C.elegans homologous gene of the mammalian neural restrictive silencing factor (NRSF/REST),is reported to be an important gene regulating lifespan of C.elegans.A deletion mutation ofspr-3,spr-3(ok2525),or RNAi inhibition of spr-3 expression led to the short lifespan phenotype in C.elegans.However,a nonsense mutation of spr-3,spr3(by108),increased the lifespan by 26% when compared with that of wild-type nematode.The spr-3(by108) also showed increased resistance to environmental stress.The spr-3(by108) mutated gene encodes a C-terminal truncated protein with a structure comparable with the REST4,a splice variant of the NRSF/REST in mammalian.The long lifespan phenotype of spr-3(by108) mutant is confirmed as a gain of function and dependent on normal functions of daf16 and glp-1.The lifespan of the spr-3(by108) can be synergistically enhanced by inducing a mutation in daf-2.Quantitative polymerase chain reaction results showed that the expression of daf-16 as well as its target gene sod-3,mtl1,and sip-1 was up-regulated in the spr-3(by108) mutant.These results would be helpful to further understand the complex function of NRSF/REST gene in mammalian,especially in the aging process and longevity determination.

  11. Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein.

    Directory of Open Access Journals (Sweden)

    Gerrit Volkmann

    Full Text Available BACKGROUND: Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. METHODOLOGY: A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. CONCLUSIONS: We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.

  12. Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential.

    Science.gov (United States)

    Chikumi, Hiroki; Barac, Ana; Behbahani, Babak; Gao, Yuan; Teramoto, Hidemi; Zheng, Yi; Gutkind, J Silvio

    2004-01-08

    PDZ-RhoGEF, LARG, and p115RhoGEF are members of a newly identified family of Rho-guanine nucleotide exchange factors (GEFs) exhibiting a unique structural feature consisting of the presence of an area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a functional motif by which Galpha(12) and Galpha(13) can bind and regulate the activity of these RhoGEFs, thus providing a direct link from these heterotrimeric G proteins to Rho. PDZ-RhoGEF and LARG can also be phosphorylated by tyrosine kinases, including FAK, and associate with Plexin B, a semaphorin receptor, which controls axon guidance during development, through their PDZ domain, thereby stimulating Rho. Interestingly, while characterizing a PDZ-RhoGEF antiserum, we found that a transfected PDZ-RhoGEF construct associated with the endogenous PDZ-RhoGEF. Indeed, we observed that PDZ-RhoGEF and LARG can form homo- and hetero-oligomers, whereas p115RhoGEF can only homo-oligomerize, and that this intermolecular interaction was mediated by their unique C-terminal regions. Deletion of the C-terminal tail of PDZ-RhoGEF had no significant effect on the GEF catalytic activity towards Rho in vitro, but resulted in a drastic increase in the ability to stimulate a serum response element reporter and the accumulation of the GTP-bound Rho in vivo. Furthermore, removal of the C-termini of each of the three RGL-containing GEFs unleashed their full transforming potential. Together, these findings suggest the existence of a novel mechanism controlling the activity of PDZ-RhoGEF, LARG, and p115RhoGEF, which involves homo- and hetero-oligomerization through their inhibitory C-terminal region.

  13. Experimental and theoretical proton affinities of methionine, methionine sulfoxide and their N- and C-terminal derivatives

    Science.gov (United States)

    Lioe, Hadi; O'Hair, Richard A. J.; Gronert, Scott; Austin, Allen; Reid, Gavin E.

    2007-11-01

    The proton affinities of methionine, methionine sulfoxide and their derivatives (methionine methyl ester, methionine sulfoxide methyl ester, methionine methyl amide, methionine sulfoxide methyl amide, N-acetyl methionine, N-acetyl methionine sulfoxide, N-acetyl methionine methyl ester, N-acetyl methionine sulfoxide methyl ester, N-acetyl methionine methyl amide and N-acetyl methionine sulfoxide methyl amide) were experimentally determined using the kinetic method, in which proton bound dimers formed via electrospray ionization (ESI) were subjected to collision induced dissociation (CID) in a triple quadrupole mass spectrometer. In addition, theoretical calculations carried out at the MP2/6-311 + G(2d,p)//B3LYP/6-31 + G(d,p) level of theory to determine the global minima of the neutral and protonated species of all derivatives studied, were used to predict theoretical proton affinities. The density function theory calculations not only support the experimental proton affinities, but also provide structural insights into the types of hydrogen bonding that stabilize the neutral and protonated methionine or methionine sulfoxide derivatives. Comparison of the proton affinities of the various methionine and methionine sulfoxide derivatives reveals that: (i) oxidation of methionine derivatives to methionine sulfoxide derivatives results in an increase in proton affinity due to higher intrinsic proton affinity and an increase in the ring size formed through charge complexation of the sulfoxide group, which allows more efficient hydrogen bonding compared to the sulfide group; (ii) C-terminal modification by methyl esterification or methyl amidation increases the proton affinity in the order of methyl amide > methyl ester > carboxylic acid due to improved charge stabilization; (iii) N-terminal modification by N-acetylation decreases proton affinity of the derivatives due to lower intrinsic proton affinity of the N-acetyl group as well as due to stabilization of the attached

  14. Tail gut cyst.

    Science.gov (United States)

    Rao, G Mallikarjuna; Haricharan, P; Ramanujacharyulu, S; Reddy, K Lakshmi

    2002-01-01

    The tail gut is a blind extension of the hindgut into the tail fold just distal to the cloacal membrane. Remnants of this structure may form tail gut cyst. We report a 14-year-old girl with tail gut cyst that presented as acute abdomen. The patient recovered after cyst excision.

  15. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    Energy Technology Data Exchange (ETDEWEB)

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  16. An insoluble frontotemporal lobar degeneration-associated TDP-43 C-terminal fragment causes neurodegeneration and hippocampus pathology in transgenic mice.

    Science.gov (United States)

    Walker, Adam K; Tripathy, Kalyan; Restrepo, Clark R; Ge, Guanghui; Xu, Yan; Kwong, Linda K; Trojanowski, John Q; Lee, Virginia M-Y

    2015-12-20

    Frontotemporal dementia (FTD) causes progressive personality, behavior and/or language disturbances and represents the second most common form of dementia under the age of 65. Over half of all FTD cases are classified pathologically as frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein of 43 kDa (TDP-43) pathology (FTLD-TDP). In FTLD-TDP brains, TDP-43 is phosphorylated, C-terminally cleaved, lost from the nucleus and accumulates in the cytoplasm and processes of neurons and glia. However, the contribution of TDP-43 C-terminal fragments (CTFs) to pathogenesis remains poorly understood. Here, we developed transgenic (Tg) mice with forebrain Camk2a-controlled doxycycline-suppressible expression of a TDP-43 CTF (amino acids 208-414, designated 208 TDP-43 CTF), previously identified in FTLD-TDP brains. In these 208 TDP-43 Tg mice, detergent-insoluble 208 TDP-43 CTF was present in a diffuse punctate pattern in neuronal cytoplasm and dendrites without forming large cytoplasmic inclusions. Remarkably, the hippocampus showed progressive neuron loss and astrogliosis in the dentate gyrus (DG). This was accompanied by phosphorylated TDP-43 in the CA1 subfield, and ubiquitin and mitochondria accumulations in the stratum lacunosum moleculare (SLM) layer, without loss of endogenous nuclear TDP-43. Importantly, 208 TDP-43 CTF and phosphorylated TDP-43 were rapidly cleared when CTF expression was suppressed in aged Tg mice, which ameliorated neuron loss in the DG despite persistence of ubiquitin accumulation in the SLM. Our results demonstrate that Camk2a-directed 208 TDP-43 CTF overexpression is sufficient to cause hippocampal pathology and neurodegeneration in vivo, suggesting an active role for TDP-43 CTFs in the pathogenesis of FTLD-TDP and related TDP-43 proteinopathies.

  17. Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects.

    Science.gov (United States)

    Drabikowski, W; Dalgarno, D C; Levine, B A; Gergely, J; Grabarek, Z; Leavis, P C

    1985-08-15

    Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.

  18. Solution structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae

    OpenAIRE

    Lou, Yuan-Chao; Wang, Iren; Rajasekaran, M.; Kao, Yi-Fen; Ho, Meng-Ru; Hsu, Shang-Te Danny; Chou, Shan-Ho; Wu, Shih-Hsiung; Chen, Chinpan

    2013-01-01

    Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determ...

  19. The C-terminal dimerization motif of cyclase-associated protein is essential for actin monomer regulation.

    Science.gov (United States)

    Iwase, Shohei; Ono, Shoichiro

    2016-12-01

    Cyclase-associated protein (CAP) is a conserved actin-regulatory protein that functions together with actin depolymerizing factor (ADF)/cofilin to enhance actin filament dynamics. CAP has multiple functional domains, and the function to regulate actin monomers is carried out by its C-terminal half containing a Wiskott-Aldrich Syndrome protein homology 2 (WH2) domain, a CAP and X-linked retinitis pigmentosa 2 (CARP) domain, and a dimerization motif. WH2 and CARP are implicated in binding to actin monomers and important for enhancing filament turnover. However, the role of the dimerization motif is unknown. Here, we investigated the function of the dimerization motif of CAS-2, a CAP isoform in the nematode Caenorhabditis elegans, in actin monomer regulation. CAS-2 promotes ATP-dependent recycling of ADF/cofilin-bound actin monomers for polymerization by enhancing exchange of actin-bound nucleotides. The C-terminal half of CAS-2 (CAS-2C) has nearly as strong activity as full-length CAS-2. Maltose-binding protein (MBP)-tagged CAS-2C is a dimer. However, MBP-CAS-2C with a truncation of either one or two C-terminal β-strands is monomeric. Truncations of the dimerization motif in MBP-CAS-2C nearly completely abolish its activity to sequester actin monomers from polymerization and enhance nucleotide exchange on actin monomers. As a result, these CAS-2C variants, also in the context of full-length CAS-2, fail to compete with ADF/cofilin to release actin monomers for polymerization. CAS-2C variants lacking the dimerization motif exhibit enhanced binding to actin filaments, which is mediated by WH2. Taken together, these results suggest that the evolutionarily conserved dimerization motif of CAP is essential for its C-terminal region to exert the actin monomer-specific regulatory function.

  20. The fnr Gene of Bacillus licheniformis and the Cysteine Ligands of the C-Terminal FeS Cluster

    OpenAIRE

    Klinger, Anette; Schirawski, Jan; Glaser, Philippe; Unden, Gottfried

    1998-01-01

    In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an intern...

  1. Phage Endolysin: A Way To Understand A Binding Function Of C-Terminal Domains A Mini Review

    OpenAIRE

    Jarábková Veronika; Tišáková Lenka; Godány Andrej

    2015-01-01

    Endolysins are bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in the end of phage reproduction cycle, in an infected host cell. Usually, for endolysins from phages that infect Gram-positive bacteria, a modular structure is typical. Therefore, these are composed of at least two separate functional domains: an N-terminal catalytic domain (EAD) and a C-terminal cell wall binding domain (CBD). Specific ligand recognition of CBDs and following peptidoglycan (PG) binding most...

  2. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

    Directory of Open Access Journals (Sweden)

    Salih Maysoon

    2009-06-01

    Full Text Available Abstract Background Tail anchored (TA membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP possesses two alternatively spliced tail anchors (TA1 or TA2 but their specificity of subcellular targeting remains unknown. Results TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER, whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Conclusion Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  3. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase.

    Science.gov (United States)

    Wray, Lewis V; Fisher, Susan H

    2008-04-01

    The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

  4. Synthesis, antimicrobial activity, and membrane permeabilizing properties of C-terminally modified nisin conjugates accessed by CuAAC.

    Science.gov (United States)

    Slootweg, Jack C; van der Wal, Steffen; Quarles van Ufford, H C; Breukink, Eefjan; Liskamp, Rob M J; Rijkers, Dirk T S

    2013-12-18

    Functionalization of the lantibiotic nisin with fluorescent reporter molecules is highly important for the understanding of its mode of action as a potent antimicrobial peptide. In addition to this, multimerization of nisin to obtain multivalent peptide constructs and conjugation of nisin to bioactive molecules or grafting it on surfaces can be attractive methods for interference with bacterial growth. Here, we report a convenient method for the synthesis of such nisin conjugates and show that these nisin derivatives retain both their antimicrobial activity and their membrane permeabilizing properties. The synthesis is based on the Cu(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) as a bioorthogonal ligation method for large and unprotected peptides in which nisin was C-terminally modified with propargylamine and subsequently efficiently conjugated to a series of functionalized azides. Two fluorescently labeled nisin conjugates together with a dimeric nisin construct were prepared while membrane insertion as well as antimicrobial activity were unaffected by these modifications. This study shows that C-terminal modification of nisin does not deteriorate biological activity in sharp contrast to N-terminal modification and therefore C-terminally modified nisin analogues are valuable tools to study the antibacterial mode of action of nisin. Furthermore, the ability to use stoichiometric amounts of the azide containing molecule opens up possibilities for surface tethering and more complex multivalent structures.

  5. Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

    Science.gov (United States)

    Inui, Ken; Zhao, Zongpei; Yuan, Juan; Jayaprakash, Sakthidasan; Le, Le T M; Drakulic, Srdja; Sander, Bjoern; Golas, Monika M

    2017-02-20

    In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

  6. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

    Science.gov (United States)

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-01-01

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  7. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

    Science.gov (United States)

    Bewley, Kathryn D.; Bennallack, Philip R.; Burlingame, Mark A.; Robison, Richard A.; Griffitts, Joel S.

    2016-01-01

    Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts. PMID:27791142

  8. Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

    Science.gov (United States)

    Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

    2015-11-01

    The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified.

  9. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    Directory of Open Access Journals (Sweden)

    Sebastien Lageix

    2015-02-01

    Full Text Available The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype, while other substitutions block kinase activation (Gcn- phenotype, in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD, previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

  10. Activity of the HMGB1-Derived Immunostimulatory Peptide Hp91 Resides in the Helical C-terminal Portion and is Enhanced by Dimerization

    Science.gov (United States)

    Saenz, R.; Messmer, B.; Futalan, D.; Tor, Y.; Larsson, M.; Daniels, G.; Esener, S.; Messmer, D.

    2013-01-01

    We have previously shown that an 18 amino acid long peptide, named Hp91, whose sequence corresponds to a region within the endogenous protein HMGB1, activates dendritic cells (DCs) and acts as adjuvant in vivo by potentiating Th1-type antigen-specific immune responses. We analyzed the structure-function relationship of the Hp91 peptide to investigate the amino acids and structure responsible for immune responses. We found that the cysteine at position 16 of Hp91 enabled formation of reversible peptide dimmers, monomer and dimmer were compared for DC binding and activation. Stable monomers and dimers were generated using a maleimide conjugation reaction. The dimer showed enhanced ability to bind to and activate DCs. Furthermore, the C-terminal 9 amino acids of Hp91, named UC1018 were sufficient for DC binding and Circular dichroism showed that UC1018 assumes an alpha-helical structure. The ninemer peptide UC1018 induced more potent antigen-specific CTL responses in vivo as compared to Hp91 and it protected mice from tumor development when used in a prophylactic vaccine setting. We have identified a short alpha helical peptide that acts as potent adjuvant inducing protective immune responses in vivo. PMID:24172222

  11. Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

    Institute of Scientific and Technical Information of China (English)

    Yan Cai; Jing Yang; He Huang; Fang Li; Ganqiu Wu; Jing Yang; Xuegang Luo

    2009-01-01

    BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood.OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins.DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008.MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coli (E. Coli) JM109 were purchased from Promega. E. Coil BL21 was provided by Novagen.METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. Coli BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained.MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods.RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then cloned into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass

  12. The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

    Science.gov (United States)

    Vedovato, Natascia; Gadsby, David C

    2010-07-01

    Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the

  13. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  14. Tumor-targeted delivery of a C-terminally truncated FADD (N-FADD) significantly suppresses the B16F10 melanoma via enhancing apoptosis

    Science.gov (United States)

    Yang, Yun-Wen; Zhang, Chun-Mei; Huang, Xian-Jie; Zhang, Xiao-Xin; Zhang, Lin-Kai; Li, Jia-Huang; Hua, Zi-Chun

    2016-01-01

    Fas-associated protein with death domain (FADD), a pivotal adaptor protein transmitting apoptotic signals, is indispensable for the induction of extrinsic apoptosis. However, overexpression of FADD can form large, filamentous aggregates, termed death effector filaments (DEFs) by self-association and initiate apoptosis independent of receptor cross-linking. A mutant of FADD, which is truncated of the C-terminal tail (m-FADD, 182–205 aa) named N-FADD (m-FADD, 1–181 aa), can dramatically up-regulate the strength of FADD self-association and increase apoptosis. In this study, it was found that over-expression of FADD or N-FADD caused apoptosis of B16F10 cells in vitro, even more, N-FADD showed a more potent apoptotic effect than FADD. Meanwhile, Attenuated Salmonella Typhimurium strain VNP20009 was engineered to express FADD or N-FADD under the control of a hypoxia-induced NirB promoter and each named VNP-pN-FADD and VNP-pN-N-FADD. The results showed both VNP-pN-FADD and VNP-pN-N-FADD delayed tumor growth in B16F10 mice model, while VNP-pN-N-FADD suppressed melanoma growth more significantly than VNP-pN-FADD. Additionally, VNP-pN-FADD and VNP-pN-N-FADD induced apoptosis of tumor cells by activating caspase-dependent apoptotic pathway. Our results show that N-FADD is a more potent apoptotic inducer and VNP20009-mediated targeted expression of N-FADD provides a possible cancer gene therapeutic approach for the treatment of melanoma. PMID:27767039

  15. Downregulation of 5-HT7 Serotonin Receptors by the Atypical Antipsychotics Clozapine and Olanzapine. Role of Motifs in the C-Terminal Domain and Interaction with GASP-1.

    Science.gov (United States)

    Manfra, Ornella; Van Craenenbroeck, Kathleen; Skieterska, Kamila; Frimurer, Thomas; Schwartz, Thue W; Levy, Finn Olav; Andressen, Kjetil Wessel

    2015-07-15

    The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXXΦ motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1.

  16. Does the fat tailed Damara ovine breed have a distinct lipid metabolism leading to a high concentration of branched chain fatty acids in tissues?

    OpenAIRE

    Susana P Alves; Bessa, Rui J.B.; Quaresma, Mário A. G.; Tanya Kilminster; Tim Scanlon; Chris Oldham; John Milton; Johan Greeff; Almeida, André M.

    2013-01-01

    Articles in International Journals Fat tailed sheep breeds are known for their adaptation to nutritional stress, among other harsh production conditions. Damara sheep, native to Southern Africa, have recently been exported to other areas of the world, particularly Australia, aiming to produce lamb in semi-arid regions. Damaras have a unique hanging fat tail, a fat depot able to be mobilized under nutritional stress. In this article we perform an in-depth characterization of the fa...

  17. Effect of Arbuscular Mycorrhizal Fungi on Plant Biomass and the Rhizosphere Microbial Community Structure of Mesquite Grown in Acidic Lead/Zinc Mine Tailings

    OpenAIRE

    Solís-Domínguez, Fernando A.; Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M.

    2011-01-01

    Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse exp...

  18. Evidence for Amino Acid Snorkeling from a High-Resolution, In Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane.

    Science.gov (United States)

    Keskin, Abdurrahman; Akdoğan, Emel; Dunn, Cory D

    2017-02-01

    Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. To reveal characteristics of TAs that may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. Specifically, we generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then, using next-generation sequencing, revealed which Fis1p TA mutations inhibited membrane insertion and allowed Gal4p activity in the nucleus. Prompted by our global analysis, we subsequently analyzed the ability of individual Fis1p TA mutants to localize to mitochondria. Our findings suggest that the membrane-associated domain of the Fis1p TA may be bipartite in nature, and we encountered evidence that the positively charged patch at the carboxyl terminus of Fis1p is required for both membrane insertion and organelle specificity. Furthermore, lengthening or shortening of the Fis1p TA by up to three amino acids did not inhibit mitochondrial targeting, arguing against a model in which TA length directs insertion of TAs to distinct organelles. Most importantly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings, emerging from the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning, provide strong, in vivo evidence that lysine and arginine can "snorkel," or become stably incorporated within a lipid bilayer by placing terminal charges of their side chains at the membrane interface.

  19. Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Tomoyuki; Kitano, Ken; Terawaki, Shin-ichi; Maesaki, Ryoko; Hakoshima, Toshio, E-mail: hakosima@bs.naist.jp [Structural Biology Laboratory, Nara Institute of Science and Technology, Keihanna Science City, Nara 630-0192 (Japan)

    2007-10-01

    The radixin FERM domain complexed with the CD44 cytoplasmic tail peptide has been crystallized. A diffraction data set from the complex was collected to 2.1 Å. CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell–cell and cell–matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane–cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 Å.

  20. The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation

    DEFF Research Database (Denmark)

    Schroeder, Ralf; Merten, Nicole; Mathiesen, Jesper Mosolff;

    2009-01-01

    Prostaglandin D(2) activation of the seven transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expressio...

  1. Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

    Science.gov (United States)

    Marcotte, Douglas; Rushe, Mia; M Arduini, Robert; Lukacs, Christine; Atkins, Kateri; Sun, Xin; Little, Kevin; Cullivan, Michael; Paramasivam, Murugan; Patterson, Thomas A; Hesson, Thomas; D McKee, Timothy; May-Dracka, Tricia L; Xin, Zhili; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda R; Lyssikatos, Joseph P; Silvian, Laura F

    2017-02-01

    Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

  2. Peptide-based inhibitors of the hepatitis C virus NS3 protease: structure-activity relationship at the C-terminal position.

    Science.gov (United States)

    Rancourt, Jean; Cameron, Dale R; Gorys, Vida; Lamarre, Daniel; Poirier, Martin; Thibeault, Diane; Llinàs-Brunet, Montse

    2004-05-06

    The structure-activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline residue, at the expense of a substantial drop in the enzymatic activity. The fact that an aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as 1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding norvaline derivative 2, prompted a systematic study of substituent effects on the three-membered ring. We report herein that the incorporation of a vinyl group with the proper configuration onto this small cycle produced inhibitors of the protease with much improved in vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue that produced NS3 protease inhibitors that are significantly more active than inhibitors containing a cysteine at the same position.

  3. The Role of Formin Tails in Actin Nucleation, Processive Elongation, and Filament Bundling*

    Science.gov (United States)

    Vizcarra, Christina L.; Bor, Batbileg; Quinlan, Margot E.

    2014-01-01

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements. PMID:25246531

  4. Environmental Factors Influencing the Structural Dynamics of Soil Microbial Communities During Assisted Phytostabilization of Acid-Generating Mine Tailings: a Mesocosm Experiment

    Science.gov (United States)

    Valentín-Vargas, Alexis; Root, Robert A.; Neilson, Julia W; Chorover, Jon; Maier, Raina M.

    2014-01-01

    Compost-assisted phytostabilization has recently emerged as a robust alternative for reclamation of metalliferous mine tailings. Previous studies suggest that root-associated microbes may be important for facilitating plant establishment on the tailings, yet little is known about the long-term dynamics of microbial communities during reclamation. A mechanistic understanding of microbial community dynamics in tailings ecosystems undergoing remediation is critical because these dynamics profoundly influence both the biogeochemical weathering of tailings and the sustainability of a plant cover. Here we monitor the dynamics of soil microbial communities (i.e. bacteria, fungi, archaea) during a 12-month mesocosm study that included 4 treatments: 2 unplanted controls (unamended and compost-amended tailings) and 2 compost-amended seeded tailings treatments. Bacterial, fungal and archaeal communities responded distinctively to the revegetation process and concurrent changes in environmental conditions and pore water chemistry. Compost addition significantly increased microbial diversity and had an immediate and relatively long-lasting buffering-effect on pH, allowing plants to germinate and thrive during the early stages of the experiment. However, the compost buffering capacity diminished after six months and acidification took over as the major factor affecting plant survival and microbial community structure. Immediate changes in bacterial communities were observed following plant establishment, whereas fungal communities showed a delayed response that apparently correlated with the pH decline. Fluctuations in cobalt pore water concentrations, in particular, had a significant effect on the structure of all three microbial groups, which may be linked to the role of cobalt in metal detoxification pathways. The present study represents, to our knowledge, the first documentation of the dynamics of the three major microbial groups during revegetation of compost

  5. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar [Research Laboratory for Nuclear Reactors and Department of Nuclear Engineering, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8550 (Japan); Adachi, Noritaka [Graduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027 (Japan); Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp [Research Laboratory for Nuclear Reactors and Department of Nuclear Engineering, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8550 (Japan)

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  6. C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

    Science.gov (United States)

    Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

    2009-10-12

    Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

  7. Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication.

    Directory of Open Access Journals (Sweden)

    Britta Hansmann

    2015-09-01

    Full Text Available Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2, a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin's antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials.

  8. Konsentrasi Asam Lemak Terbang dan Glukosa Darah Domba Ekor Tipis yang Diberi Bungkil Kedelai Terproteksi Tanin (VOLATILE FATTY ACID CONCENTRATION AND BLOOD GLUCOSE ON THIN-TAILED SHEEP GIVEN WITH TANINE-PROTECTED SOYBEAN MEAL

    Directory of Open Access Journals (Sweden)

    Siti Nuraliah

    2015-10-01

    Full Text Available This study aims to analyze the influence of tannin-protected protein source feed to livestock productivitybased on its influence on methane production, Volatile fatty acids (VFA production, and blood glucose inthe use of tannin-protected protein on complete feed in thin-tailed ram. The study uses thin-tailed ramaged about 8 months, as many as 16 rams with body weight of 11.81±1.65 kg. The researchusesa completerandomized design(CRD withfour treatmentsandfourreplications. The treatments areP0:15% soybeanmeals without tannin protection in complete feed, P1:15% soybean meals with 0.5% tannin protection incomplete feed, P2:15% soybean meals with 1% tannin protection in complete feed and P3:15% soybeanmeals with 1.5% tannin protection in complete feed. The results indicates that administration of tanninsin soybean meal in complete feed showed significant effect (P <0.05 on blood glucose, the production ofpropionic acid in the 3rd hour, but the VFA production at hour 0, hour 6, as well as methane productionshowed no significant effect (P> 0.05. The conclusion is that 15% protected soybean meal with 1% oftannin in the complete feed is able to contribute to the proportion of propionate and contribute to increasedblood glucose on a thin-tailed ram but can not to reduce methane production.

  9. Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

    OpenAIRE

    Kim, Jeong Hoon; Yang, Catherine K.; Stallcup, Michael R

    2006-01-01

    The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino...

  10. Differential Regulation of Two Palmitoylation Sites in the Cytoplasmic Tail of the β1-Adrenergic Receptor*

    OpenAIRE

    2011-01-01

    S-Palmitoylation of G protein-coupled receptors (GPCRs) is a prevalent modification, contributing to the regulation of receptor function. Despite its importance, the palmitoylation status of the β1-adrenergic receptor, a GPCR critical for heart function, has never been determined. We report here that the β1-adrenergic receptor is palmitoylated on three cysteine residues at two sites in the C-terminal tail. One site (proximal) is adjacent to the seventh transmembrane domain and is a consensus ...

  11. The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases.

    Science.gov (United States)

    Maresová, Helena; Palyzová, Andrea; Kyslík, Pavel

    2007-06-30

    The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 degrees C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3' end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. "In silicio" analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368-430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea.

  12. Mutations in Streptococcus pneumoniae penicillin-binding protein 2x: importance of the C-terminal penicillin-binding protein and serine/threonine kinase-associated domains for beta-lactam binding.

    Science.gov (United States)

    Maurer, Patrick; Todorova, Katya; Sauerbier, Julia; Hakenbeck, Regine

    2012-06-01

    Penicillin-binding protein 2x (PBP2x) mutations that occur during the selection with beta-lactams are located within the central penicillin-binding/transpeptidase (TP) domain, and are believed to mediate resistance by interfering with the formation of a covalent complex of the active site serine with the antibiotic. We now investigated the effect of two point mutations found in two independently obtained laboratory mutants that are located at the surface of the TP domain with their side chains facing outside (G422D respectively R426C). They have no significant effect on resistance to cefotaxime in vivo or on binding to Bocillin™FL to the active site in vitro using purified PBP2x derivatives, thus apparently do not affect the active site directly. In contrast, in silico modeling revealed that they affect van der Waal's interactions with the PASTA1 (PBP and serine/threonine kinase associated) domain of the C-terminal extension and a noncovalent cefuroxime molecule found in the X-ray structure of an acylated PBP2x, suggesting some effect of the mutations on the interaction of the TP domain with PASTA1 and/or with the antibiotic associated with PASTA1. The effect of the PASTA domains on covalent binding of PBP2x to Bocillin FL was then investigated using a series of soluble truncated PBP2x derivatives. Deletion of 127 C-terminal residues, that is, of both PASTA domains, decreased binding dramatically by ∼90%. Surprisingly, deletion of only 40 amino acids resulted in the same phenotype, whereas the absence of 30 amino acids affected binding marginally by 10%, documenting a crucial role of the C-terminal domain for beta-lactam binding.

  13. THE HYDROGENOSOMAL ENZYME HYDROGENASE FROM THE ANAEROBIC FUNGUS NEOCALLIMASTIX SP L2 IS RECOGNIZED BY ANTIBODIES, DIRECTED AGAINST THE C-TERMINAL MICROBODY PROTEIN TARGETING SIGNAL SKL

    NARCIS (Netherlands)

    MARVINSIKKEMA, FD; KRAAK, MN; VEENHUIS, M; GOTTSCHAL, JC; PRINS, RA

    1993-01-01

    The question was addressed whether antibodies directed against the general microbody C-terminal protein targeting signal SKL recognized hydrogenosomal proteins from Neocallimastix sp. L2. Immunofluorescence, immunocytochemistry and Western blotting experiments using these antibodies indicated the pr

  14. C-Terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin.

    Science.gov (United States)

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  15. Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    Directory of Open Access Journals (Sweden)

    Anja eScharinger

    2015-08-01

    Full Text Available Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM. It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA. Using these mice we provide biochemical evidence for the existence of long (CTM-containing and short (CTM-deficient Cav1.3 α1-subunits in brain. The long (HA-labeled Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It is required to stabilize gating properties of Cav1.3 channels required for normal electrical excitability.

  16. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

    Science.gov (United States)

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei

    2016-07-01

    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.

  17. Preferential recognition of isocitrate dehydrogenase by a rabbit monoclonal antibody (ab124797) against the C-terminal peptide of RANKL.

    Science.gov (United States)

    Terasawa, Kazue; Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Mishima-Tsumagari, Chiemi; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-05-01

    A rabbit monoclonal antibody (Abcam ab124797), with high affinity for a synthetic peptide corresponding to the C-terminal region of the receptor activator of nuclear factor (NF)-κB ligand (RANKL), specifically recognizes a 37 kDa protein by immunoblotting, in good agreement with the molecular mass of RANKL. However, our mass spectroscopy analysis revealed that the protein recognized by the antibody is the α-subunit of NAD(+)-dependent isocitrate dehydrogenase (ICDH), a key Krebs cycle enzyme in mitochondria. Consistently, immunocytochemical staining with the antibody revealed a network organization characteristic of mitochondria, which overlapped with staining by MitoTracker and was lost after the siRNA-mediated downregulation of ICDH. The C-terminal peptide of ICDH contains similar chemical characteristics to that of the RANKL peptide and interacts with the antibody, although the affinity is a hundred times weaker. The present study provides an example of the preferential recognition of a surrogate protein by a rabbit monoclonal antibody.

  18. Membrane tethering of APP c-terminal fragments is a prerequisite for T668 phosphorylation preventing nuclear sphere generation.

    Science.gov (United States)

    Bukhari, Hassan; Kolbe, Katharina; Leonhardt, Gregor; Loosse, Christina; Schröder, Elisabeth; Knauer, Shirley; Marcus, Katrin; Müller, Thorsten

    2016-11-01

    A central molecular hallmark of Alzheimer's disease (AD) is the β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP), which causes the generation of different c-terminal fragments like C99, AICD57, or AICD50 that fully or in part contain the APP transmembrane domain. In this study, we demonstrate that membrane-tethered C99 is phosphorylated by JNK3A at residue T668 (APP695 numbering) to a higher extent than AICD57, whereas AICD50 is not capable of being phosphorylated. The modification decreases the turnover of APP, while the blockade of APP cleavage increases APP phosphorylation. Generation of nuclear spheres, complexes consisting of the translocated AICD, FE65 and other proteins, is significantly reduced as soon as APP c-terminal fragments are accessible for phosphorylation. This APP modification, which we identified as significantly reduced in high plaque-load areas of the human brain, is linearly dependent on the level of APP expression. Accordingly, we show that APP abundance is likewise capable of modulating nuclear sphere generation. Thus, the precise and complex regulation of APP phosphorylation, abundance, and cleavage impacts the generation of nuclear spheres, which are under discussion of being of relevance in neurodegeneration and dementia. Future pharmacological manipulation of nuclear sphere generation may be a promising approach for AD treatment.

  19. Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin.

    Science.gov (United States)

    Iacovache, Ioan; Degiacomi, Matteo T; Pernot, Lucile; Ho, Sylvia; Schiltz, Marc; Dal Peraro, Matteo; van der Goot, F Gisou

    2011-07-01

    Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.

  20. Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin.

    Directory of Open Access Journals (Sweden)

    Ioan Iacovache

    2011-07-01

    Full Text Available Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs. PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP. By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.

  1. C-Terminal to Intact Fibroblast Growth Factor 23 Ratio in Relation to Estimated Glomerular Filtration Rate in Elderly Population

    Directory of Open Access Journals (Sweden)

    Maria Bożentowicz-Wikarek

    2016-08-01

    Full Text Available Background/Aims: An analytical equivalence between intact fibroblasts growth factor(iFGF23 and C-terminal(cFGF23 assays is logically expected, however, numerous studies demonstrate lack of a strong association between them. Previously, we have demonstrated the increase in cFGF23 slightly precedes the increase of iFGF23 with the impairment of kidney excretory function; without actually analyzing the ratio between both assays, which are postulated to be affected by declining kidney function. Therefore, the aim of this study was to analyze the ratio between C and iFGF23 in relation to the estimated glomerular filtration rate (eGFR in an elderly population. Methods: We analysed the variability of c/iFGF23 ratio in the population of 3264 elderly PolSenior study participants (≥ 65years in the relation to eGFR calculated according full Modification of Diet in Renal Disease, serum levels of C-reactive protein (hs-CRP, and iron. Results: The log10(c/i FGF23 ratio increased in the subsequent CKD stages. Serum iron and CRP levels reduced the log10 and increased it with age in multivariate regression analysis. Conclusions: Our results suggest impairment in the cleavage of the C-terminal FGF23 fragments with the deterioration of kidney excretory function and age in the elderly population. Inflammation and low serum iron level seems to diminish degradation capacity of FGF23 fragments.

  2. The C-Terminal Domain of Yeast PCNA Is Required for Physical And Functional Interactions With Cdc9 DNA Ligase

    Energy Technology Data Exchange (ETDEWEB)

    Vijayakumar, S.; Chapados, B.R.; Schmidt, K.H.; Kolodner, R.D.; Tainer, J.A.; Tomkinson, A.E.

    2007-07-13

    There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the {beta}-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the coordinated, sequential action of these enzymes.

  3. Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Jing Liu; Li-Xin Zhu; Yu-Ying Kong; Guang-Di Li; Yuan Wang

    2005-01-01

    AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E.coli)and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally trunczated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot.RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization.CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

  4. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

  5. Chaperone-like effect of the linker on the isolated C-terminal domain of rabbit muscle creatine kinase.

    Science.gov (United States)

    Chen, Zhe; Chen, Xiang-Jun; Xia, Mengdie; He, Hua-Wei; Wang, Sha; Liu, Huihui; Gong, Haipeng; Yan, Yong-Bin

    2012-08-01

    Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.

  6. Cloning of C-Terminal of Opioid μ-Receptor and Construction of Its Expression Plasmid for Yeast Two Hybrid System

    Institute of Scientific and Technical Information of China (English)

    YANHui; GONGZe-hui

    2004-01-01

    Aim: To obtain the C-terminal DNA and construct the expression plasmid in yeast two-hybrid. Methods: About 177bp DNA fragment was amplified from the complete sequence of ( receptor by PCR. After being sequenced, the C-terminal fragment was ligased into EcoR I-BamH I site of pGBKT7 vector to form recombinants. The recombinant plasmid

  7. A C-terminal Hydrophobic, Solvent-protected Core and a Flexible N-terminus are Potentially Required for Human Papillomavirus 18 E7 Protein Functionality

    Energy Technology Data Exchange (ETDEWEB)

    Liu, S.; Tian, Y; Greenaway, F; Sun, M

    2010-01-01

    The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.

  8. Evidence that the amyloid-β protein precursor intracellular domain, AICD, derives from β-secretase-generated C-terminal fragment.

    Science.gov (United States)

    Flammang, Brice; Pardossi-Piquard, Raphaëlle; Sevalle, Jean; Debayle, Delphine; Dabert-Gay, Anne-Sophie; Thévenet, Aurélie; Lauritzen, Inger; Checler, Frédéric

    2012-01-01

    One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is the senile plaque, an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39-43 amino acids) referred to as amyloid-β (Aβ) peptides. Aβ peptides are derived from combined proteolytic cleavages undergone on the amyloid-β protein precursor (AβPP) by a set of enzymes called secretases. Several lines of anatomical and biological evidence suggest that Aβ peptides would not account for all pathological stigmata and molecular dysfunctions taking place in AD. In amyloidogenic and non-amyloidogenic pathways, AβPP first undergoes β- or α-secretases-mediated cleavages yielding C99 and C83, respectively. These two membrane-embedded C-terminal fragments are both potential targets of subsequent γ-secretase-mediated proteolysis. The latter cleavage not only generates either p3 or Aβ peptides but similarly gives rise to an AβPP IntraCellular Domain (AICD fragment) that could modulate the transcription of several genes linked to AD pathology. It is therefore striking that AICD theoretically derives from both amyloidogenic and non-amyloidogenic AβPP processing pathways. Here we show that AICD predominantly derives from C99 by means of recombinant substrates and transiently transfected cells expressing C99. Our data suggest a preferred pathogenic pathway for AICD production and suggests that this fragment, in addition to C99 and Aβ peptides, could contribute to AD pathology.

  9. C-terminal binding protein (CtBP activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

    Directory of Open Access Journals (Sweden)

    Taichi Q Itoh

    Full Text Available In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes". Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP, a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per, vrille (vri, and PAR domain protein 1ε (Pdp1ε. Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

  10. Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal C-terminal region of chemokine receptors CCR2 and CCR5.

    Science.gov (United States)

    Toda, Etsuko; Terashima, Yuya; Esaki, Kaori; Yoshinaga, Sosuke; Sugihara, Minoru; Kofuku, Yutaka; Shimada, Ichio; Suwa, Makiko; Kanegasaki, Shiro; Terasawa, Hiroaki; Matsushima, Kouji

    2014-01-15

    Chemokine receptors mediate the migration of leucocytes during inflammation. The cytoplasmic protein FROUNT binds to chemokine receptors CCR2 [chemokine (C-C motif) receptor 2] and CCR5, and amplifies chemotactic signals in leucocytes. Although the interaction between FROUNT and chemokine receptors is important for accurate chemotaxis, the interaction mechanism has not been elucidated. In the present study we identified a 16-amino-acid sequence responsible for high-affinity binding of FROUNT at the membrane-proximal C-terminal intracellular region of CCR2 (CCR2 Pro-C) by yeast two-hybrid analysis. Synthesized peptides corresponding to the CCR2 Pro-C sequence directly interacted with FROUNT in vitro. CCR2 Pro-C was predicted to form an amphipathic helix structure. Residues on the hydrophobic side are completely conserved among FROUNT-binding receptors, suggesting that the hydrophobic side is the responsible element for FROUNT binding. The L316T mutation to the hydrophobic side of the predicted helix decreased the affinity for FROUNT. Co-immunoprecipitation assays revealed that the CCR2 L316T mutation diminished the interaction between FROUNT and full-length CCR2 in cells. Furthermore, this mutation impaired the ability of the receptor to mediate chemotaxis. These findings provide the first description of the functional binding element in helix 8 of CCR2 for the cytosolic regulator FROUNT that mediates chemotactic signalling.

  11. Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-delta1.

    Science.gov (United States)

    Grujic, Ognjen; Bhullar, Rajinder P

    2009-06-12

    Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta1, and increases its enzymatic activity. Since PLC-delta1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta1. Therefore, we created a GST-PLC-delta1 construct consisting of the first 294 amino acids of PLC-delta1 (GST-PLC-delta1(1-294)). In vitro binding experiments confirmed that PLC-delta1(1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta1, demonstrating that PLC-delta1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-delta1 were able to inhibit RalA binding to PLC-delta1(1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta1 function.

  12. The arginine residue within the C-terminal active core of Bombyx mori pheromone biosynthesis-activating neuropeptide (PBAN is essential for receptor binding and activation

    Directory of Open Access Journals (Sweden)

    Takeshi eKawai

    2012-03-01

    Full Text Available In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis activating neuropeptide (PBAN. Bombyx mori PBAN (BomPBAN consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R residue two positions from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to reveal the role of the Arg residue in the BomPBAN active core. We synthesized a ten-residue peptide corresponding to the C-terminal part of BomPBAN with a series of point mutants at the 2nd position (ie, Arg from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells concomitantly expressing a fluorescent PBAN receptor chimera (PBANR-EGFP and loaded with the fluorescent Ca2+ indicator, Fura Red-AM. PBAN analogs with the C2 position replaced with alanine (Ala, A, aspartic acid (Asp, D, serine (Ser, S or L-2-aminooctanoic acid (Aoc decreased PBAN-like activity. RC2A (SKTRYFSPALamide and RC2D (SKTRYFSPDLamide had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide. We also prepared Rhodamine Red-labeled PBAN analogs of the mutants and examined their ability to bind PBANR. In contrast to 100 nM Rhodamine Red-PBAN C10, none of the mutants at the same concentration exhibited PBANR binding. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation.

  13. Electro-remediation of copper mine tailings. Comparing copper removal efficiencies for two tailings of different age

    DEFF Research Database (Denmark)

    Hansen, Henrik K.; Lamas, Victor; Gutierrez, Claudia;

    2013-01-01

    less than 2 years ago (new tailings). Parameters analyzed were the applied voltage drop, acid concentration during pretreatment, and the use of either passive or ion exchange membranes in the experimental setup. The comparison of the results confirms that there are differences in the electroremediation...... between the two tailings, even if the pH is similar and a mineralogical analysis showed similarities between the samples with respect to composition. It was found that an electroremediation is more favorable on the old tailings. The results showed that the best experimental conditions for both tailings......This work compares and evaluates the copper removal efficiency when applying electric fields to two mine tailings originating from the same mine but of different age. Eight experiments were carried out - four on tailings deposited more than 20 years ago (old tailings) and four on tailings deposited...

  14. Structural and pharmacological characteristics of chimeric peptides derived from peptide E and beta-endorphin reveal the crucial role of the C-terminal YGGFL and YKKGE motifs in their analgesic properties.

    Science.gov (United States)

    Condamine, Eric; Courchay, Karine; Rego, Jean-Claude Do; Leprince, Jérôme; Mayer, Catherine; Davoust, Daniel; Costentin, Jean; Vaudry, Hubert

    2010-05-01

    Peptide E (a 25-amino acid peptide derived from proenkephalin A) and beta-endorphin (a 31-amino acid peptide derived from proopiomelanocortin) bind with high affinity to opioid receptors and share structural similarities but induce analgesic effects of very different intensity. Indeed, whereas they possess the same N-terminus Met-enkephalin message sequence linked to a helix by a flexible spacer and a C-terminal part in random coil conformation, in contrast with peptide E, beta-endorphin produces a profound analgesia. To determine the key structural elements explaining this very divergent opioid activity, we have compared the structural and pharmacological characteristics of several chimeric peptides derived from peptide E and beta-endorphin. Structures were obtained under the same experimental conditions using circular dichroism, computational estimation of helical content and/or nuclear magnetic resonance spectroscopy (NMR) and NMR-restrained molecular modeling. The hot-plate and writhing tests were used in mice to evaluate the antinociceptive effects of the peptides. Our results indicate that neither the length nor the physicochemical profile of the spacer plays a fundamental role in analgesia. On the other hand, while the functional importance of the helix cannot be excluded, the last 5 residues in the C-terminal part seem to be crucial for the expression or absence of the analgesic activity of these peptides. These data raise the question of the true function of peptides E in opioidergic systems.

  15. Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Kalinowski, Agnieszka; Yaron, Peter N; Qin, Zhao; Shenoy, Siddharth; Buehler, Markus J; Lösche, Mathias; Dahl, Kris Noel

    2014-12-01

    Hutchinson-Gilford progeria syndrome is a premature aging disorder associated with the expression of ∆50 lamin A (∆50LA), a mutant form of the nuclear structural protein lamin A (LA). ∆50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein-membrane association mediated by the retained farnesyl group. To better characterize the protein-membrane interface, we quantified binding of purified recombinant ∆50LA tail domain (∆50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated ∆50LA-TD binds to the membrane interface only in the presence of Ca(2+) or Mg(2+) at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of ∆50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of ∆50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association.

  16. Effects of a mixture of fatty acids from sugar cane (Saccharum officinarum L.) wax oil in two models of inflammation: zymosan-induced arthritis and mice tail test of psoriasis.

    Science.gov (United States)

    Ledón, N; Casacó, A; Remirez, D; González, A; Cruz, J; González, R; Capote, A; Tolón, Z; Rojas, E; Rodríguez, V J; Merino, N; Rodríguez, S; Ancheta, O; Cano, M C

    2007-10-01

    A mixture of fatty acids obtained from sugar cane (Saccharum officinarum L.) wax oil (FAM), in which the main constituents are palmitic, oleic, linoleic, and linolenic acids, was evaluated in two models of inflammation: zymosan-induced arthritis and in the tail test for psoriasis, both on mice. In the first model, FAM significantly reduced zymozan-induced increase of beta glucuronidase (DE(50) 90+/-7 mg/kg). Histopathological studies showed inhibition in cellular infiltration and reduction of synovial hyperplasia and synovitis, whereas in the second test, histopathological and ultrastructural studies showed that topical application of FAM induced orthokeratosis with the presence of keratohyalin granules in the previously parakeratotic adult mouse tail, and without effects on epidermal thickness. The ED(50) of FAM in this model was 155+/-10 mg. The results of our studies showed that topical application of FAM exerts an important anti-inflammatory activity in both tests without evidence of irritant effects. The anti-inflamatory effects exerted by FAM may be due to its inhibitory effects on arachidonic acid metabolism. To our knowledge, this is the first report on the anti-inflammatory effect of sugar cane by-products in experimental models of arthritis and psoriasis.

  17. NFAT1 C-terminal domains are necessary but not sufficient for inducing cell death.

    Directory of Open Access Journals (Sweden)

    Douglas V Faget

    Full Text Available The proteins belonging to the nuclear factor of activated T cells (NFAT family of transcription factors are expressed in several cell types and regulate genes involved in differentiation, cell cycle and apoptosis. NFAT proteins share two conserved domains, the NFAT-homology region (NHR and a DNA-binding domain (DBD. The N- and C-termini display two transactivation domains (TAD-N and TAD-C that have low sequence similarity. Due to the high sequence conservation in the NHR and DBD, NFAT members have some overlapping roles in gene regulation. However, several studies have shown distinct roles for NFAT proteins in the regulation of cell death. The TAD-C shows low sequence similarity among NFAT family members, but its contribution to specific NFAT1-induced phenotypes is poorly understood. Here, we described at least two regions of NFAT1 TAD-C that confer pro-apoptotic activity to NFAT1. These regions extend from amino acids 699 to 734 and 819 to 850 of NFAT1. We also showed that the NFAT1 TAD-C is unable to induce apoptosis by itself and requires a functional DBD. Furthermore, we showed that when fused to NFAT1 TAD-C, NFAT2, which is associated with cell transformation, induces apoptosis in fibroblasts. Together, these results suggest that the NFAT1 TAD-C includes NFAT death domains that confer to different NFAT members the ability to induce apoptosis.

  18. Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo.

    Science.gov (United States)

    Smith, Christopher E; Bell, Charles E

    2016-02-13

    Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a "synaptosome" complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1-177, and a flexible C-terminal "tail", residues 178-261. Here, we quantitatively compare the binding of the full-length protein (Redβ(FL)) and the N-terminal domain (Redβ(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Redβ(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ(177) binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182-261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Redβ(FL) and Redβ(177) to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo.

  19. α-Helical to β-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein

    Science.gov (United States)

    Singh, Jesse; Whitford, Paul; Hayre, Natha; Cox, Daniel; Onuchic, José.

    2011-03-01

    We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (α H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation.

  20. Graphene on C-terminated face of 4H-SiC observed by noncontact scanning nonlinear dielectric potentiometry

    Science.gov (United States)

    Yamasue, Kohei; Fukidome, Hirokazu; Tashima, Keiichiro; Suemitsu, Maki; Cho, Yasuo

    2016-08-01

    We studied graphene synthesized on the C-terminated face (C-face) of a 4H-SiC substrate by noncontact scanning nonlinear dielectric potentiometry. As already reported by other researchers, multilayer graphene sheets with moiré patterns were observed in our sample, which indicates the existence of rotational disorder between adjacent layers. We found that the potentials of graphene on the C-face are almost neutral and significantly smaller than those observed on the Si-terminated face (Si-face). In addition, the neutrality of potentials is not affected by various topographic features underlying the multilayer graphene sheets. These results indicate that graphene on the C-face of SiC is decoupled or screened from the underlying structures and substrate, unlike graphene on the Si-face.

  1. The fnr gene of Bacillus licheniformis and the cysteine ligands of the C-terminal FeS cluster.

    Science.gov (United States)

    Klinger, A; Schirawski, J; Glaser, P; Unden, G

    1998-07-01

    In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.

  2. Phage Endolysin: A Way To Understand A Binding Function Of C-Terminal Domains A Mini Review

    Directory of Open Access Journals (Sweden)

    Jarábková Veronika

    2015-12-01

    Full Text Available Endolysins are bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in the end of phage reproduction cycle, in an infected host cell. Usually, for endolysins from phages that infect Gram-positive bacteria, a modular structure is typical. Therefore, these are composed of at least two separate functional domains: an N-terminal catalytic domain (EAD and a C-terminal cell wall binding domain (CBD. Specific ligand recognition of CBDs and following peptidoglycan (PG binding mostly allows a rapid lytic activity of an EAD. Here we briefly characterize phage endolysin CBDs in conjuction with their domain architecture, (nonnecessity for the following lytic activity and a high/low specificity of their ligands as well. Such an overall assessment of CBDs may help to find new ways to widen opportunities in their protein design to create ‛designer recombinant endolysins’ with diverse applications.

  3. The effect of C-terminal fragment of JNK2 on the stability of p53 and cell proliferation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramatically inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.

  4. Erythrocytosis-associated HIF-2α Mutations Demonstrate a Critical Role for Residues C-terminal to the Hydroxylacceptor Proline*

    Science.gov (United States)

    Furlow, Paul W.; Percy, Melanie J.; Sutherland, Scott; Bierl, Charlene; McMullin, Mary Frances; Master, Stephen R.; Lappin, Terence R. J.; Lee, Frank S.

    2009-01-01

    A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an α subunit (HIF-α) and a β subunit (HIF-β). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-α in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-α for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-α and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2α. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2α in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-α. PMID:19208626

  5. The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread.

    Science.gov (United States)

    Belin, C; Schmitt, C; Gaire, F; Walter, B; Demangeat, G; Pinck, L

    1999-06-01

    The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

  6. Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence.

    Directory of Open Access Journals (Sweden)

    Peng Xue

    Full Text Available Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE. The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.

  7. NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein.

    Science.gov (United States)

    Haba, Noam Y; Gross, Renana; Novacek, Jiri; Shaked, Hadassa; Zidek, Lukas; Barda-Saad, Mira; Chill, Jordan H

    2013-07-16

    WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIP(C), a C-terminal domain fragment of WIP that includes residues 407-503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIP(C) and the high occurrence (25%) of proline residues, we employed 5D-NMR(13)C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, (15)N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446-456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468-478. The (13)C-detected approach allows chemical-shift assignment in the WIP(C) polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIP(C). Thus, we conclude that the disordered WIP(C) fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function.

  8. Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.; Eremeeva, Elena V.; Malikova, Natalia P.; Cheng, Chongyun; Liu, Zhi-Jie; Vysotski, Eugene S.

    2016-09-01

    The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

  9. Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein.

    Science.gov (United States)

    Burakova, Ludmila P; Natashin, Pavel V; Markova, Svetlana V; Eremeeva, Elena V; Malikova, Natalia P; Cheng, Chongyun; Liu, Zhi-Jie; Vysotski, Eugene S

    2016-09-01

    The full-length cDNA genes encoding five new isoforms of Ca(2+)-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473-474nm with no shoulder at 400nm). Fluorescence spectra of Ca(2+)-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca(2+)-discharged aequorin, but different from Ca(2+)-discharged obelins and clytin which fluorescence is red-shifted by 25-30nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

  10. Interaction of a C-terminal Truncated Hepatitis C Virus Core Protein with Plasmid DNA Vaccine Leads to in vitro Assembly of Heterogeneous Virus-like Particles

    Directory of Open Access Journals (Sweden)

    Nelson Acosta-Rivero

    2005-01-01

    Full Text Available Recently, it has been shown that HCV core proteins (HCcAg with C-terminal deletions assemble in vitro into virus-like particles (VLPs in the presence of structured RNA molecules. Results presented in this work showed that a truncated HCcAg variant covering the first 120 aa (HCcAg.120 with a 32 aa N-terminal fusion peptide (6xHistag-XpressTMepitope interacts with plasmid DNA vaccine. Interestingly, the buoyant density of VLPs containing HCcAg.120 in CsCl gradients changed from 1.15-1,17 g mLˉ1 to 1.30-1.34 g mLˉ1 after addition of plasmid DNA to assembly reactions. In addition, a delay in electrophoretic mobility of HCcAg.120-plasmid samples on agarose gels was observed indicating a direct interaction between VLPs and nucleic acids. Remarkably, addition of either plasmid DNA or tRNA to assembly reactions leaded to heterogeneous and larger VLPs formation than those observed in HCcAg.120 assembly reactions. VLPs containing HCcAg.120 induced a specific IgG antibodies in mice that reacted with hepatocytes from HCV-infected patients. VLPs obtained in this work would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure. Besides, the capacity of particles containing HCcAg.120 to interact with nucleic acids could be used in the development of DNA vaccines and viral vectors based on these particles.

  11. Innovations in tailings management

    Energy Technology Data Exchange (ETDEWEB)

    Beier, N. [Alberta Univ., Devon, AB (Canada). Oil Sands Tailings Research Facility; Sego, D. [Alberta Univ., Devon, AB (Canada). Oil Sands Tailings Research Facility; Alberta Univ., Edmonton, AB (Canada). Dept. of Civil and Environmental Engineering

    2010-09-15

    This article described the coordinated research efforts at the University of Alberta's Oil Sands Tailings Research Facility (OSTRF) to address oil sands tailings management issues. The facility was established in 2003 to provide the infrastructure needed for research opportunities at the intermediate scale. To date, OSTRF has been involved in 32 separate research projects. Alberta's Energy Resources Conservation Board (ERCB) recently created Directive 74 to oblige operators to improve tailings management and document their progress on tailings at each mine site. The initial OSTRF research program focused on improving the understanding and management of the massive inventories of mature fine tailings (MFT). The research focused primarily on the geotechnical and chemical behaviour of engineered tailings and the management and treatment of process affected water (PAW). The focus on water relates to its characterization, its treatment with coke and rapid removal of water from the total tailings stream. The next phase of research to be undertaken at OSTRF will include the reduction or elimination of volatile organic carbons and greenhouse gases from tailings ponds; further development and scaling of the novel in-line tailings dewatering system and alternative methods to release water from the MFT inventory; treatment technologies for re-use of PAW in utilities and for upgrading while also developing quality standards for eventual discharge of PAW off lease; and stabilization and capping of soft tailings deposits in preparation for reclamation, including methods of dewatering the upper surface of soft tailing to enhance its strength. 1 tab., 1 fig.

  12. Determination of the Content of Acrolein, Acetic Acid and Acrylic Acid in Tail Gas from Acrylic Acid Production of Propylene Oxidation by Gas Chromatography%气相色谱法测定丙烯氧化生产丙烯酸尾气中丙烯醛、乙酸、丙烯酸含量

    Institute of Scientific and Technical Information of China (English)

    陈科杰

    2013-01-01

    采用毛细管气相色谱法测定丙烯氧化生产丙烯酸尾气中丙烯醛、乙酸、丙烯酸的含量,色谱柱为FFAP(30 m×0.53 mm,1μm),检测器为FID,用外标法进行定量,尾气样品中丙烯醛、乙酸、丙烯酸的检出限分别为0.025,0.033,0.031 mg/m3,测定结果的相对偏差为0.97%~2.63%(n=5),加标回收率为98.29%~103.60%。%The content of acrolein, acetic acid and acrylic acid in tail gas from acrylic acid produection of propylene oxidation was determined by capillary gas chronmatography with FFAP column (30 m×0.53 mm,1 µm), FID as the detector and external standard as the quantitative method. The detection limits of 3 components in tail gas were 0.025, 0.033 and 0.031 mg/m3, respectively. The relative standard deviation was 0.97%-2.63%(n=5), and the recoveries were 98.29%-103.60%.

  13. Bar-tailed

    NARCIS (Netherlands)

    Duijns, S.; Hidayati, N.A.; Piersma, T.

    2013-01-01

    Capsule Across the European wintering range Bar-tailed Godwits Limosa lapponica lapponica selected polychaete worms and especially Ragworms Hediste diversicolor, with differences between areas due to variations in prey availability.Aims To determine the diet of Bar-tailed Godwits across their winter

  14. Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

    Science.gov (United States)

    Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

    2017-02-01

    Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process.

  15. 石棉尾矿酸浸渣填充改性道路沥青的研究%Research on Road Asphalt Filled and Modified with Acid-leaching Residue of Asbestos Tailings

    Institute of Scientific and Technical Information of China (English)

    孙志明; 郑水林; 文明; 吴照洋

    2009-01-01

    The acid-leaching residue of asbestos tailings is silicon slag of asbestos tailings, one kind of solid waste after acid leaching extraction of magnesium. In this experiment, using acid leaching residue of asbestos tailings after calcination as the filler of asphalt modifier, through the test of penetration, ductility, softening point of modified asphalt, the effects of the adding volume and mixing conditions (temperature, time, etc.) of the acid leaching residue on the performance of road asphalt has been studied. The results showed that the appropriate conditions of modified process is that the adding volume of acid leaching residue 6%, mixing temperature 140℃, and heating mixing time 20rain. In this condition, the performance of modified asphalt material, such as high temperature and low temperature performance, temperature sensitivity, and anti-aging property have been significantly improved.%石棉尾矿酸浸渣是石棉尾矿蛇纹石酸浸提取氧化镁后的硅质粉体材料.本实验以煅烧后的石棉尾矿酸浸渣作为道路沥青填充改性剂,通过测定改性沥青的针入度、延度、软化点等指标,研究了酸浸渣填充量、混合控温以及加热混合时间对道路沥青综合性能的影响.结果表明,石棉尾矿酸浸渣填充改性沥青适宜的填充工艺条件为填充量6%,混合控温140℃,加热混合时间20min;在适宜的填充工艺条件下,改性沥青的高温性能、低温性能、高低温稳定性以及抗老化性均得到显著改善或提高.

  16. Washery tailings from coal preparation used as aluminium feedstock

    Energy Technology Data Exchange (ETDEWEB)

    Kuehn, L.; Schieder, T.; Belsky, M.; Lotze, J.; Winkhaus, G.

    1982-06-01

    Kaolinite clays and washery tailings of coal mines are the major feedstocks of the West German aluminium industry. Statistics are given in order to assess the amount of washery tailings available and their suitability as feedstocks for aluminium production. The chemical and mineralogical composition are similar for washery tailings from different mines; accordingly, also their behaviour regarding decomposition by acids will be almost identical. Al/sub 2/O/sub 3/ extraction yields are satisfactory with strongly precalcinated material using concentrated sulphuric acid or azeotropic hydrochloric acid - in the latter case, however, only if fluoride is added to the acid.

  17. Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2

    Energy Technology Data Exchange (ETDEWEB)

    Ozorowski, Gabriel [University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697-3900 (United States); Milton, Saskia [University of California, Irvine, Irvine, CA 92697-3900 (United States); Luecke, Hartmut, E-mail: hudel@uci.edu [University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697 (United States); University of California, Irvine, Irvine, CA 92697 (United States)

    2013-01-01

    Structure of a 20-amino-acid peptide of AHNAK bound asymmetrically to the AnxA2–S100A10A heterotetramer (1:2:2 symmetry) provides insights into the atomic level interactions that govern this membrane-repair scaffolding complex. AHNAK, a large 629 kDa protein, has been implicated in membrane repair, and the annexin A2–S100A10 heterotetramer [(p11){sub 2}(AnxA2){sub 2})] has high affinity for several regions of its 1002-amino-acid C-terminal domain. (p11){sub 2}(AnxA2){sub 2} is often localized near the plasma membrane, and this C2-symmetric platform is proposed to be involved in the bridging of membrane vesicles and trafficking of proteins to the plasma membrane. All three proteins co-localize at the intracellular face of the plasma membrane in a Ca{sup 2+}-dependent manner. The binding of AHNAK to (p11){sub 2}(AnxA2){sub 2} has been studied previously, and a minimal binding motif has been mapped to a 20-amino-acid peptide corresponding to residues 5654–5673 of the AHNAK C-terminal domain. Here, the 2.5 Å resolution crystal structure of this 20-amino-acid peptide of AHNAK bound to the AnxA2–S100A10 heterotetramer (1:2:2 symmetry) is presented, which confirms the asymmetric arrangement first described by Rezvanpour and coworkers and explains why the binding motif has high affinity for (p11){sub 2}(AnxA2){sub 2}. Binding of AHNAK to the surface of (p11){sub 2}(AnxA2){sub 2} is governed by several hydrophobic interactions between side chains of AHNAK and pockets on S100A10. The pockets are large enough to accommodate a variety of hydrophobic side chains, allowing the consensus sequence to be more general. Additionally, the various hydrogen bonds formed between the AHNAK peptide and (p11){sub 2}(AnxA2){sub 2} most often involve backbone atoms of AHNAK; as a result, the side chains, particularly those that point away from S100A10/AnxA2 towards the solvent, are largely interchangeable. While the structure-based consensus sequence allows interactions with various

  18. Distributed computing and NMR constraint-based high-resolution structure determination: applied for bioactive Peptide endothelin-1 to determine C-terminal folding.

    Science.gov (United States)

    Takashima, Hiroyuki; Mimura, Norio; Ohkubo, Tadayasu; Yoshida, Takuya; Tamaoki, Haruhiko; Kobayashi, Yuji

    2004-04-14

    Distributed computing has been implemented to the solution structure determination of endothelin-1 to evaluate efficiency of the method for NMR constraint-based structure calculations. A key target of the investigation was determination of the C-terminal folding of the peptide, which had been dispersed in previous studies of NMR, despite its pharmacological significances. With use of tens of thousands of random initial structures to explore the conformational space comprehensively, we determined high-resolution structures with good convergences of C-terminal as well as previously defined N-terminal structures. The previous studies had missed the C-terminal convergence because of initial structure dependencies trapped in localized folding of the N-terminal region, which are strongly constricted by two disulfide bonds.

  19. Extrusion of the C-terminal helix in navel orangeworm moth pheromone-binding protein (AtraPBP1) controls pheromone binding.

    Science.gov (United States)

    Xu, Wei; Xu, Xianzhong; Leal, Walter S; Ames, James B

    2011-01-07

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129-142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.

  20. Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation

    Directory of Open Access Journals (Sweden)

    Dobocan Monica C

    2009-10-01

    Full Text Available Abstract Background C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells. Findings We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin. Conclusion CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.

  1. The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation.

    Science.gov (United States)

    Sun, Tong; Fu, Junjiang; Shen, Tao; Lin, Xia; Liao, Lan; Feng, Xin-Hua; Xu, Jianming

    2016-05-27

    Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells.

  2. C-terminal heat shock protein 90 inhibitor decreases hyperglycemia-induced oxidative stress and improves mitochondrial bioenergetics in sensory neurons.

    Science.gov (United States)

    Zhang, Liang; Zhao, Huiping; Blagg, Brian S J; Dobrowsky, Rick T

    2012-04-06

    Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70, and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress.

  3. A C-Terminal Heat Shock Protein 90 Inhibitor Decreases Hyperglycemia-induced Oxidative Stress and Improves Mitochondrial Bioenergetics in Sensory Neurons

    Science.gov (United States)

    Zhang, Liang; Zhao, Huiping; Blagg, Brian S.J.; Dobrowsky, Rick T.

    2012-01-01

    Summary Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70 and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress. PMID:22413817

  4. Sequence and modified group analysis on C-terminal modified analogs of endomorphin-2 using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this paper, a series of C-terminal modified analogs of endomorphin-2 is investigated using ESI-FT-ICR-MS. Some b, y″, a, and internal ions are found in the CID spectra and slight mass differ- ences between the calculated and observed results are obtained. Moreover, if the C-terminal modified group is t-butyloxy, it can lose butene through McLafferty rearrangement. FT-ICR MS shows its power in peptide sequencing successfully helping us obtain the structure of peptide analogs.

  5. Sequence and modified group analysis on C-terminal modified analogs of endomorphin-2 using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this paper,a series of C-terminal modified analogs of endomorphin-2 is investigated using ESI-FT-ICR-MS. Some b, y", a, and internal ions are found in the CID spectra and slight mass differences between the calculated and observed results are obtained. Moreover, if the C-terminal modified group is t-butyloxy, it can lose butene through McLafferty rearrangement. FT-ICR MS shows its power in peptide sequencing successfully helping us obtain the structure of peptide analogs.

  6. The Tail of BPM

    Science.gov (United States)

    Kruba, Steve; Meyer, Jim

    Business process management suites (BPMS's) represent one of the fastest growing segments in the software industry as organizations automate their key business processes. As this market matures, it is interesting to compare it to Chris Anderson's 'Long Tail.' Although the 2004 "Long Tail" article in Wired magazine was primarily about the media and entertainment industries, it has since been applied (and perhaps misapplied) to other markets. Analysts describe a "Tail of BPM" market that is, perhaps, several times larger than the traditional BPMS product market. This paper will draw comparisons between the concepts in Anderson's article (and subsequent book) and the BPM solutions market.

  7. Estimating tail probabilities

    Energy Technology Data Exchange (ETDEWEB)

    Carr, D.B.; Tolley, H.D.

    1982-12-01

    This paper investigates procedures for univariate nonparametric estimation of tail probabilities. Extrapolated values for tail probabilities beyond the data are also obtained based on the shape of the density in the tail. Several estimators which use exponential weighting are described. These are compared in a Monte Carlo study to nonweighted estimators, to the empirical cdf, to an integrated kernel, to a Fourier series estimate, to a penalized likelihood estimate and a maximum likelihood estimate. Selected weighted estimators are shown to compare favorably to many of these standard estimators for the sampling distributions investigated.

  8. Proceedings of the 15. annual British Columbia MEND ML/ARD workshop : the management of tailings and tailings impoundments

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2009-02-15

    This Mine Environment Neutral Drainage (MEND) workshop was held to promote the exchange of information and ideas on environmental protection and reclamation associated with mining. The workshop covered a broad spectrum of reclamation issues and the key environmental challenges facing the mining industry, such as acid mine drainage (AMD) control, and metal leaching and acid rock drainage (ML/ARD). The theme for the 2008 workshop was the management of tailings and tailings impoundments. Topics of discussion included hydrogeology and geochemistry; tailings disposal; mitigation; closure plans; and postclosure performance. The emphasis was on full-scale case studies, practical constraints and sustaining successful disposal strategies and remediation. The session on tailings management reviewed overarching policies and practices; methods of subaerial tailings disposal and case studies of tailings management; and detailed investigations of geochemical properties and processes. The conference featured 22 presentations, of which 4 have been catalogued separately for inclusion in this database. refs., tabs., figs.

  9. The C-terminal extension peptide of non-photoconvertible water-soluble chlorophyll-binding proteins (Class II WSCPs) affects their solubility and stability: comparative analyses of the biochemical and chlorophyll-binding properties of recombinant Brassica, Raphanus and Lepidium WSCPs with or without their C-terminal extension peptides.

    Science.gov (United States)

    Takahashi, Shigekazu; Uchida, Akira; Nakayama, Katsumi; Satoh, Hiroyuki

    2014-02-01

    Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.

  10. The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Yayoicho, Inage-ku, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2013-05-31

    Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

  11. Injurious tail biting in pigs

    DEFF Research Database (Denmark)

    D'Eath, R.B.; Amott, G.; Turner, S. P.;

    2014-01-01

    risk, it is important to detect and treat tail biting as soon as it occurs. Early warning signs before the first bloody tails appear, such as pigs holding their tails tucked under, could in future be automatically detected using precision livestock farming methods enabling earlier reaction......Tail biting is a serious animal welfare and economic problem in pig production. Tail docking, which reduces but does not eliminate tail biting, remains widespread. However, in the EU tail docking may not be used routinely, and some ‘alternative’ forms of pig production and certain countries do...... not allow tail docking at all. Against this background, using a novel approach focusing on research where tail injuries were quantified, we review the measures that can be used to control tail biting in pigs without tail docking. Using this strict criterion, there was good evidence that manipulable...

  12. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II.

    Science.gov (United States)

    Zhang, Mengmeng; Gill, Gordon N; Zhang, Yan

    2010-01-01

    In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

  13. Mass spectrometry quantification revealed accumulation of C-terminal fragment of apolipoprotein E in the Alzheimer's frontal cortex.

    Directory of Open Access Journals (Sweden)

    Meiyao Wang

    Full Text Available Polymorphic variation in the apolipoprotein E (apoE gene is the major genetic susceptibility factor for late-onset Alzheimer's disease (AD and likely contributes to neuropathology through various pathways. It is also recognized that apoE undergoes proteolytic cleavage in the brain and the resultant apoE fragments likely have a variety of bioactive properties that regulate neuronal signaling and may promote neurodegeneration. ApoE fragmentation in the human brain has been intensively studied using different immunochemical methods, but has never been analyzed in a quantitative manner to establish preferably accumulated fragments. Here we report quantification using multiple reaction monitoring mass spectrometry (MRM MS with (15N-labeled full-length apoE4 as an internal standard. Measurements were performed on frontal cortex from control and severe AD donors. Our data point to a preferable accumulation of C-terminal apoE fragment in the insoluble fraction of tissue homogenate in the severe AD group versus the control group. Further insight into the biological consequences of this accumulation may lead to a better understanding of the basic mechanism of AD pathology.

  14. The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

    Science.gov (United States)

    Nakayama, Yoshitaka; Becker, Michael; Ebrahimian, Haleh; Konishi, Tomoyuki; Kawasaki, Hisashi; Krämer, Reinhard; Martinac, Boris

    2016-01-01

    The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

  15. Expression and characterization of Kunitz domain 3 and C-terminal of human tissue factor pathway inhibitor-2

    Institute of Scientific and Technical Information of China (English)

    Lina Zhu; Jiping Wang; Jingui Mu; Huijun Wang; Chenqi Zhang; Jue Wang; Xingang Liu; Xiaomin Yan; Linsen Dai; Duan Ma

    2009-01-01

    Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and C-terminal of hTFPI-2 (bTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SP-Sepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR),Raman spectroscopy, and circular dichroism (CD)experiment results implied that hTFPI-2/KD3C con-tained small contents of or-helix and β-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauche-trans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced β-strands in hTFPI-2/KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.

  16. Synapse associated protein 102 (SAP102 binds the C-terminal part of the scaffolding protein neurobeachin.

    Directory of Open Access Journals (Sweden)

    Juliane Lauks

    Full Text Available Neurobeachin (Nbea is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  17. Synapse associated protein 102 (SAP102) binds the C-terminal part of the scaffolding protein neurobeachin.

    Science.gov (United States)

    Lauks, Juliane; Klemmer, Patricia; Farzana, Fatima; Karupothula, Ramesh; Zalm, Robbert; Cooke, Nancy E; Li, Ka Wan; Smit, August B; Toonen, Ruud; Verhage, Matthijs

    2012-01-01

    Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  18. Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization.

    Science.gov (United States)

    Kundu, Nidhi; Tichkule, Swapnil; Pandit, Shashi Bhushan; Chattopadhyay, Kausik

    2017-01-15

    Pore-forming toxins (PFTs) are typically produced as water-soluble monomers, which upon interacting with target cells assemble into transmembrane oligomeric pores. Vibrio parahaemolyticus thermostable direct hemolysin (TDH) is an atypical PFT that exists as a tetramer in solution, prior to membrane binding. The TDH structure highlights a core β-sandwich domain similar to those found in the eukaryotic actinoporin family of PFTs. However, the TDH structure harbors an extended C-terminal region (CTR) that is not documented in the actinoporins. This CTR remains tethered to the β-sandwich domain through an intra-molecular disulphide bond. Part of the CTR is positioned at the inter-protomer interface in the TDH tetramer. Here we show that the truncation, as well as mutation, of the CTR compromise tetrameric assembly, and the membrane-damaging activity of TDH. Our study also reveals that intra-protomer disulphide bond formation during the folding/assembly process of TDH restrains the CTR to mediate its participation in the formation of inter-protomer contact, thus facilitating TDH oligomerization. However, once tetramerization is achieved, disruption of the disulphide bond does not affect oligomeric assembly. Our study provides critical insights regarding the regulation of the oligomerization mechanism of TDH, which has not been previously documented in the PFT family.

  19. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II.

    Science.gov (United States)

    Voss, Kirsten; Forné, Ignasi; Descostes, Nicolas; Hintermair, Corinna; Schüller, Roland; Maqbool, Muhammad Ahmad; Heidemann, Martin; Flatley, Andrew; Imhof, Axel; Gut, Marta; Gut, Ivo; Kremmer, Elisabeth; Andrau, Jean-Christophe; Eick, Dirk

    2015-01-01

    Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.

  20. The structure of Abeta42 C-terminal fragments probed by a combined experimental and theoretical study.

    Science.gov (United States)

    Wu, Chun; Murray, Megan M; Bernstein, Summer L; Condron, Margaret M; Bitan, Gal; Shea, Joan-Emma; Bowers, Michael T

    2009-03-27

    The C-terminus of amyloid beta-protein (Abeta) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Abeta42 have been shown to disrupt oligomerization and to strongly inhibit Abeta42-induced neurotoxicity. Here we study the structures of selected CTFs [Abeta(x-42); x=29-31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable beta-structure: beta-hairpin for Abeta(x-42) (x=29-31) and extended beta-strand for Abeta(39-42). The beta-hairpin of Abeta(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the beta-hairpin in Abeta42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of beta-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.

  1. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    Energy Technology Data Exchange (ETDEWEB)

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  2. The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain

    Science.gov (United States)

    Shi, Hang; Rojas, Raul; Bonifacino, Juan S.; Hurley, James H.

    2006-01-01

    The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29, and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1Å resolution reveals two curvedβ -sandwich domains connected by a polar core and a flexible linker. Vps26 has an unexpected structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235–246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a Gly in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting. PMID:16732284

  3. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

    Science.gov (United States)

    Slubowski, Christian J; Funk, Alyssa D; Roesner, Joseph M; Paulissen, Scott M; Huang, Linda S

    2015-04-01

    Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance.

  4. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

    Science.gov (United States)

    Voss, Kirsten; Forné, Ignasi; Descostes, Nicolas; Hintermair, Corinna; Schüller, Roland; Maqbool, Muhammad Ahmad; Heidemann, Martin; Flatley, Andrew; Imhof, Axel; Gut, Marta; Gut, Ivo; Kremmer, Elisabeth; Andrau, Jean-Christophe; Eick, Dirk

    2015-01-01

    Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression. PMID:26566685

  5. The identification of putative RNA polymerase II C-terminal domain associated proteins in red and green algae.

    Science.gov (United States)

    Yang, Chunlin; Hager, Paul W; Stiller, John W

    2014-01-01

    A tandemly repeated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is functionally essential and strongly conserved in many organisms, including animal, yeast and plant models. Although present in simple, ancestral red algae, CTD tandem repeats have undergone extensive modifications and degeneration during the evolutionary transition to developmentally complex rhodophytes. In contrast, CTD repeats are conserved in both green algae and their more complex land plant relatives. Understanding the mechanistic differences that underlie these variant patterns of CTD evolution requires knowledge of CTD-associated proteins in these 2 lineages. To provide an initial baseline comparison, we bound potential phospho-CTD associated proteins (PCAPs) to artificially synthesized and phosphorylated CTD repeats from the unicellular red alga Cyanidioschyzon merolae and green alga Chlamydomonas reinhardtii. Our results indicate that red and green algae share a number of PCAPs, including kinases and proteins involved in mRNA export. There also are important taxon-specific differences, including mRNA splicing-related PCAPs recovered from Chlamydomonas but not Cyanidioschyzon, consistent with the relative intron densities in green and red algae. Our results also offer the first experimental indication that different proteins bind 2 distinct types of repeats in Cyanidioschyzon, suggesting a division of function between the proximal and distal CTD, similar to patterns identified in more developmentally complex model organisms.

  6. C-terminal functional unit of Rapana thomasiana (marine snail, gastropod) hemocyanin isoform RtH1: isolation and characterization.

    Science.gov (United States)

    Parvanova, Katja; Idakieva, Krassimira; Todinova, Svetla; Genov, Nicolay

    2003-09-23

    Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1-h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1-h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1-h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1-h. The thermal stability of the apo-RtH1-h is characterized fluorimetrically by the "melting" temperature T(m) (65 degrees C) and by the transition temperature T(m) (83 degrees C) obtained by differential scanning calorimetry for oxy-RtH1-h. The results confirm the role of the copper-dioxygen complex for the stabilization of the Hc structure in solution.

  7. Enhanced valine production in Corynebacterium glutamicum with defective H+-ATPase and C-terminal truncated acetohydroxyacid synthase.

    Science.gov (United States)

    Wada, Masaru; Hijikata, Nowaki; Aoki, Ryo; Takesue, Nobuchika; Yokota, Atsushi

    2008-11-01

    We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H(+)-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H(+)-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H(+)-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H(+)-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H(+)-ATPase defect is also effective in the fermentative production of other practical compounds.

  8. The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, Ross A.; Lee, Megan S.; Tsutakawa, Susan E.; Williams, R. Scott; Tainer, John A.; Glover, J. N. Mark

    2009-07-13

    The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF- 50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins.Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

  9. The C-terminal domain of Nrf1 negatively regulates the full-length CNC-bZIP factor and its shorter isoform LCR-F1/Nrf1β; both are also inhibited by the small dominant-negative Nrf1γ/δ isoforms that down-regulate ARE-battery gene expression.

    Directory of Open Access Journals (Sweden)

    Yiguo Zhang

    Full Text Available The C-terminal domain (CTD, aa 686-741 of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1 shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein and its shorter isoform LCR-F1/Nrf1β (55-kDa. Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112Nrf1 factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1β and Nrf2-C112Nrf1, results in an increase in their transcriptional ability to regulate antioxidant response element (ARE-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1γ and 25-kDa Nrf1δ, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1β and Nrf2. Relative to Nrf1, LCR-F1/Nrf1β is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST domain and acidic domain 2 (AD2. Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1β fused within two different chimaeric contexts to yield Gal4D:Nrf1β607 and Nrf1β:C270Nrf2, positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1β and down-regulation by Nrf1γ and Nrf1δ.

  10. The C-terminal domain of Nrf1 negatively regulates the full-length CNC-bZIP factor and its shorter isoform LCR-F1/Nrf1β; both are also inhibited by the small dominant-negative Nrf1γ/δ isoforms that down-regulate ARE-battery gene expression.

    Science.gov (United States)

    Zhang, Yiguo; Qiu, Lu; Li, Shaojun; Xiang, Yuancai; Chen, Jiayu; Ren, Yonggang

    2014-01-01

    The C-terminal domain (CTD, aa 686-741) of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1) shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L) CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein) and its shorter isoform LCR-F1/Nrf1β (55-kDa). Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112Nrf1 factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER) membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1β and Nrf2-C112Nrf1, results in an increase in their transcriptional ability to regulate antioxidant response element (ARE)-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1γ and 25-kDa Nrf1δ, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1β and Nrf2. Relative to Nrf1, LCR-F1/Nrf1β is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST) domain and acidic domain 2 (AD2). Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1β fused within two different chimaeric contexts to yield Gal4D:Nrf1β607 and Nrf1β:C270Nrf2, positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1β and down-regulation by Nrf1γ and Nrf1δ.

  11. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  12. The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten;

    2010-01-01

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocyto...

  13. Characterizing the N- and C-terminal Small Ubiquitin-like Modifier (SUMO)-interacting Motifs of the Scaffold Protein DAXX

    NARCIS (Netherlands)

    Escobar-Cabrera, E.; Okon, M.; Lau, D.K.W.; Dart, C.F.; Bonvin, A.M.J.J.; McIntosh, L.P.

    2011-01-01

    DAXX is a scaffold protein with diverse roles that often depend upon binding SUMO via its N- and/or C-terminal SUMO-interacting motifs (SIM-N and SIM-C). Using NMR spectroscopy, we characterized the in vitro binding properties of peptide models of SIM-N and SIM-C to SUMO-1 and SUMO-2. In each case,

  14. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

    DEFF Research Database (Denmark)

    van den Bremer, E. T. J.; Beurskens, F. J.; Voorhorst, M.;

    2015-01-01

    Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexameri...

  15. Importance of Reelin C-terminal region in the development and maintenance of the postnatal cerebral cortex and its regulation by specific proteolysis

    DEFF Research Database (Denmark)

    Kohno, Takao; Honda, Takao; Kubo, Ken-Ichiro

    2015-01-01

    During brain development, Reelin exerts a variety of effects in a context-dependent manner, whereas its underlying molecular mechanisms remain poorly understood. We previously showed that the C-terminal region (CTR) of Reelin is required for efficient induction of phosphorylation of Dab1, an esse...

  16. Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing.

    Science.gov (United States)

    Nakajima, Chihiro; Kuyama, Hiroki; Tanaka, Koichi

    2012-09-15

    An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.

  17. Synthesis and contractile activity of the C-terminal heptapeptide of substance P with N5-dimethyl glutamine in the 6-position. Active site studies.

    Science.gov (United States)

    Poulos, C P; Pinas, N; Theodoropoulos, D

    1980-09-15

    The synthesis and testing of [N5-dimethyl-Gln6]-SP5-11 showed 37 +/- 12% contractile activity relative to SP, and intrinsic efficacy 98 +/- 4%. This finding indicates that the carboxamide groups of the dual Gln5-Cln6 moiety are not equally related with the contractile response of the C-terminal heptapeptide of SP.

  18. Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

    Directory of Open Access Journals (Sweden)

    Fowke Keith R

    2005-10-01

    Full Text Available Abstract Background In addition to mediating the integration process, HIV-1 integrase (IN has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s and/or motif(s within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. Results Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV and sequence Q (211KELQKQITK in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal

  19. Chemical and thermal unfolding of a global staphylococcal virulence regulator with a flexible C-terminal end.

    Directory of Open Access Journals (Sweden)

    Avisek Mahapa

    Full Text Available SarA, a Staphylococcus aureus-specific dimeric protein, modulates the expression of numerous proteins including various virulence factors. Interestingly, S. aureus synthesizes multiple SarA paralogs seemingly for optimizing the expression of its virulence factors. To understand the domain structure/flexibility and the folding/unfolding mechanism of the SarA protein family, we have studied a recombinant SarA (designated rSarA using various in vitro probes. Limited proteolysis of rSarA and the subsequent analysis of the resulting protein fragments suggested it to be a single-domain protein with a long, flexible C-terminal end. rSarA was unfolded by different mechanisms in the presence of different chemical and physical denaturants. While urea-induced unfolding of rSarA occurred successively via the formation of a dimeric and a monomeric intermediate, GdnCl-induced unfolding of this protein proceeded through the production of two dimeric intermediates. The surface hydrophobicity and the structures of the intermediates were not identical and also differed significantly from those of native rSarA. Of the intermediates, the GdnCl-generated intermediates not only possessed a molten globule-like structure but also exhibited resistance to dissociation during their unfolding. Compared to the native rSarA, the intermediate that was originated at lower GdnCl concentration carried a compact shape, whereas, other intermediates owned a swelled shape. The chemical-induced unfolding, unlike thermal unfolding of rSarA, was completely reversible in nature.

  20. C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions.

    Science.gov (United States)

    Casals, Cristina; Johansson, Hanna; Saenz, Alejandra; Gustafsson, Magnus; Alfonso, Carlos; Nordling, Kerstin; Johansson, Jan

    2008-02-01

    Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.

  1. Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.

    Science.gov (United States)

    Matsumoto, Shunsuke; Igura, Mayumi; Nyirenda, James; Matsumoto, Masaki; Yuzawa, Satoru; Noda, Nobuo; Inagaki, Fuyuhiko; Kohda, Daisuke

    2012-05-22

    Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a β-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.

  2. An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

    Science.gov (United States)

    Schiele, Felix; Park, John; Redemann, Norbert; Luippold, Gerd; Nar, Herbert

    2014-02-20

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

  3. The GSTM2 C-Terminal Domain Depresses Contractility and Ca2+ Transients in Neonatal Rat Ventricular Cardiomyocytes

    Science.gov (United States)

    Hewawasam, Ruwani P.; Liu, Dan; Casarotto, Marco G.; Board, Philip G.; Dulhunty, Angela F.

    2016-01-01

    The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca2+ release from the sarcoplasmic reticulum (SR) during excitation–contraction coupling in the heart. The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 μM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 μM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 μM GSTM2C, but not 5 and 10 μM GSTM2C and was unaffected by exposure to 15 μM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 μM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart. PMID:27612301

  4. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms.

    Directory of Open Access Journals (Sweden)

    Noheon Park

    Full Text Available The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. Bmal1 encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated Bmal1 (Bmal1GTΔC allele. The homozygous mutant (Bmal1GTΔC/GTΔC mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (Bmal1+/GTΔC mice displayed a gradual loss of rhythms, in contrast to Bmal1+/- mice where rhythms were sustained. Bmal1GTΔC/GTΔC mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of Bmal1GTΔC/GTΔC mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in Bmal1+/GTΔC and Bmal1GTΔC/GTΔC mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1GTΔC was unable to activate transcription of Per1 promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of Bmal1 has pivotal roles in the regulation of circadian rhythms and the Bmal1GTΔC mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1.

  5. A constitutive effector region on the C-terminal side of switch I of the Ras protein.

    Science.gov (United States)

    Fujita-Yoshigaki, J; Shirouzu, M; Ito, Y; Hattori, S; Furuyama, S; Nishimura, S; Yokoyama, S

    1995-03-01

    The "switch I" region (Asp30-Asp38) of the Ras protein takes remarkably different conformations between the GDP- and GTP-bound forms and coincides with the so-called "effector region." As for a region on the C-terminal side of switch I, the V45E and G48C mutants of Ras failed to promote neurite outgrowth of PC12 cells (Fujita-Yoshigaki, J., Shirouzu, M., Koide, H., Nishimura, S., and Yokoyama, S. (1991) FEBS Lett. 294, 187-190). In the present study, we performed alanine-scanning mutagenesis within the region Lys42-Ile55 of Ras and found that the K42A, I46A, G48A, E49A, and L53A mutations significantly reduced the neurite-inducing activity. This is an effector region by definition, but its conformation is known to be unaffected by GDP-->GTP exchange. So, this region is referred to as a "constitutive" effector (Ec) region, distinguished from switch I, a "switch" effector (Es) region. The Ec region mutants exhibiting no neurite-inducing activity were found to be correlatably unable to activate mitogen-activated protein (MAP) kinase in PC12 cells. Therefore, the Ec region is essential for the MAP kinase activation in PC12 cells, whereas mutations in this region only negligibly affect the binding of Ras to Raf-1 (Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, H., Toyama, Y., Yamasaki, K., Fuhrman, S. A., Villafranca, E., Kaziro, Y., and Yokoyama, S. (1994) Oncogene 9, 2153-2157).

  6. Tissue distribution and safety evaluation of a claudin-targeting molecule, the C-terminal fragment of Clostridium perfringens enterotoxin.

    Science.gov (United States)

    Li, Xiangru; Saeki, Rie; Watari, Akihiro; Yagi, Kiyohito; Kondoh, Masuo

    2014-02-14

    We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3 h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6 h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10 min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.

  7. Tail-anchored membrane proteins: exploring the complex diversity of tail-anchored-protein targeting in plant cells.

    Science.gov (United States)

    Abell, Ben M; Mullen, Robert T

    2011-02-01

    Tail-anchored (TA) proteins are special class of integral membrane proteins that in recent years have received a considerable amount of attention due to their diverse cellular functions and unique targeting and insertion mechanisms. Defined by the presence of a single, hydrophobic membrane-spanning domain at or near their C terminus, TA proteins must be inserted into membranes post-translationally and are orientated such that their larger N-terminal domain (most often the functional domain) faces the cytosol, while their shorter C-terminal domain faces the interior of the organelle. The C-terminal domain of TA proteins also usually contains the information responsible for their selective targeting to the proper subcellular membrane, a process that, based primarily on studies with yeasts and mammals, appears to be highly complex due to the presence of multiple pathways. Within this context, we discuss here the biogenesis of plant TA proteins and the potential for hundreds of new TA proteins identified via bioinformatics screens to contribute to the already remarkable number of roles that this class of membrane proteins participates in throughout plant growth and development.

  8. A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation.

    Directory of Open Access Journals (Sweden)

    Eoin N Leen

    2016-01-01

    Full Text Available Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES, adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs in the host cell. For murine norovirus (MNV we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132. Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.

  9. Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

    Directory of Open Access Journals (Sweden)

    Dirlei Nico

    Full Text Available Nucleoside hydrolases (NHs show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36 responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL. Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314 and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011 that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and

  10. Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2

    Institute of Scientific and Technical Information of China (English)

    Qun LI; Yves LAUMONNIER; Tatiana SYROVETS; Thomas SIMMET

    2011-01-01

    Aim:To identify proteins that interact with the C-terminal fragment of annexin A2 (A21C),generated by plasmin cleavage of the plasmin receptor,a heterotetramer (AA2t) containing annexin A2.Methods:The gene that encodes the A21C fragment was obtained from PCR-amplifled cDNA isolated from human monocytes,and was ligated into the pBTM116 vector using a DNA ligation kit.The resultant plasmid (pBTM116-A21C) was sequenced with an ABI PRISM 310 Genetic Analyzer.The expression of an A21C bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis.The identification of proteins that interact with A21C and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening.The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit.Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov).Confirmation of the interaction between the protein LexA-A21C and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay.Results:The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach.After screening 1×107 clones,23 independent β-Gal-positive clones were identified.Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17,ProCathepsin S,RPS2,ZBTB4,OGDH,CCDC32,PAPD4,and actin which was already known to interact with annexin A2).Conclusion:A21C A21C interacts with various proteins to form protein complexes,which may contribute to the molecular mechanism of monocyte activation induced by plasmin.The yeast two-hybrid system is an efficient approach for investigating protein interactions.

  11. Highly active and stable ion-exchanged Fe-Ferrierite catalyst for N2O decomposition under nitric acid tail gas conditions

    NARCIS (Netherlands)

    Melian Cabrera, Ignacio; Mentruit, C; Pieterse, JAZ; van den Brink, RW; Mul, G; Kapteijn, F; Moulijn, JA

    2005-01-01

    This communication reports on the excellent performance and durability of a wet ion-exchanged Fe-Ferrierite catalysts for N2O decomposition under conditions of nitric acid plants, especially in the presence of water (0.5% vol) and at a high space time W/F-0(N2O) = 900 kg(cat) s mol(-1). In contrast

  12. Highly active and stable ion-exchanged Fe–Ferrierite catalyst for N2O decomposition under nitric acid tail gas conditions

    NARCIS (Netherlands)

    Melián-Cabrera, I.; Mentruit, C.; Pieterse, J.A.Z.; Brink, R.W. van den; Mul, G.; Kapteijn, F.; Moulijn, J.A.

    2005-01-01

    This communication reports on the excellent performance and durability of a wet ion-exchanged Fe–Ferrierite catalysts for N2O decomposition under conditions of nitric acid plants, especially in the presence of water (0.5% vol) and at a high space time W/F0(N2O) = 900 kgcat s mol-1. In contrast to io

  13. Estimation of Jump Tails

    DEFF Research Database (Denmark)

    Bollerslev, Tim; Todorov, Victor

    We propose a new and flexible non-parametric framework for estimating the jump tails of Itô semimartingale processes. The approach is based on a relatively simple-to-implement set of estimating equations associated with the compensator for the jump measure, or its "intensity", that only utilizes ...

  14. Managing 'tail liability'.

    Science.gov (United States)

    Frese, Richard C; Weber, Ryan J

    2013-11-01

    To reduce and control their level of tail liability, hospitals should: Utilize a self-insurance vehicle; Consider combined limits between the hospital and physicians; Communicate any program changes to the actuary, underwriter, and auditor; Continue risk management and safety practices; Ensure credit is given to the organization's own medical malpractice program.

  15. On tails of perpetuities

    CERN Document Server

    Hitczenko, Pawel

    2011-01-01

    We establish an upper bound on the tails of a random variable that arises as a solution of a stochastic difference equation. In the non--negative case our bound is similar to a lower bound obtained by Goldie and Gr\\"ubel in 1996.

  16. INCLUSION OF KAPOK SEED OIL IN THE DIET FOR GROWING OF THIN-TAILED SHEEP TO REDUCE CHOLESTEROL AND TO IMPROVE OMEGA-SIX FATTY ACID CONTENTS OF LAMB

    Directory of Open Access Journals (Sweden)

    W. Widiyanto

    2014-10-01

    Full Text Available This research was conducted to study the influence of protected kapok seed oil (PKSOsupplementation in its combination with concentrate, in this case was rice bran (RB on lipid content ofthin tailed sheep received field grass as basal feed. A number of 24 heads of male thin-tailed sheep wereused as experimental material. These sheep were divided into 8 treatment groups. There were twotreatment factors, i.e. : PKSO supplementation (S as factor I and RB supplementation (K as factor II.Factor I consisted of 2 levels, i.e. 0% (S0 and 10% (S1, whereas factor II consist of 4 levels, i.e. 0%(K0, 15% (K1, 30% (K2 and 45% (K3, respectively, based on dry matter (DM intake. Severalvariables were measured, namely content of intra muscular fat (IMF, iodine number of muscular fattyacid, content of meat cholesterol and omega 6 fatty acid (in this case linoleic acid. The collected datawere statistically analyzed by analysis of variance with factorial treatment pattern (2 x 4 in completelyrandomized design (CRD. Content of IMF in sheep supplemented with 0 and 15% KSO levels was notsignificantly different from those without kapok seed oil (KSO supplementation at the same level ofRB feeding (3.45 and 3.99% vs 3.01 and 3.75%. Supplementation of protected KSO in combinationwith 30 and 45% RP resulted in significantly increased (P < 0.05 IMF content (4.75 and 5.93% vs 4.32and 5.40% and significantly decreased (P < 0.05 meat chollesterol content (62.46 and 65.26 mg/100 g vs 89.21 and 72.63 mg/100 g also significantly increased (P < 0.05 linoleic acid proportion (15.52 and18.25% vs 3.68 and 4.04% and iodine number (12.52 and 10.25 vs 4.09 and 4.98.

  17. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    /α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type...... A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools...

  18. Native chemical ligation between asparagine and valine: application and limitations for the synthesis of tri-phosphorylated C-terminal tau.

    Science.gov (United States)

    Reimann, Oliver; Glanz, Maria; Hackenberger, Christian P R

    2015-06-15

    We present the successful native chemical ligation (NCL) at an Asn-Val site employing β-mercaptovaline and subsequent desulfurization in the synthesis of native phosphorylated C-terminal tau, relevant for Alzheimer's disease related research. Despite recent progress in the field of NCL we illustrate limitations of this ligation site that stem from thioester hydrolysis and predominantly aspartimide formation. We systematically investigated the influence of pH, temperature, peptide concentration and thiol additives on the outcome of this ligation and identified conditions under which the ligation can be driven toward complete conversion, which required the deployment of a high surplus of thioester. Application of the optimized conditions allowed us to gain access to challenging tri-phosphorylated C-terminal tau peptide in practical yields.

  19. Crystal structure of the C-terminal domain of the Salmonella type III secretion system export apparatus protein InvA.

    Science.gov (United States)

    Worrall, Liam J; Vuckovic, Marija; Strynadka, Natalie C J

    2010-05-01

    InvA is a prominent inner-membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N-terminal integral membrane domain and a C-terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C-terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V-type ATPase and a ring building motif found in other T3SS proteins respectively.

  20. Radiological impact of uranium tailings and alternatives for their management

    Energy Technology Data Exchange (ETDEWEB)

    Momeni, M. H.; Kisieleski, W. E.; Tyler, S.; Zielen, A.; Yuan, Y.; Roberts, C. J.

    1979-01-01

    Uncontrolled tailings piles are mobile sources of fugitive dust that may produce a practically uncleanable adjacent environment. A practical procedure for managing solid tailings is addition of surface moisture, mechanical and gravitational separation of slimes, and storage of slimes below solution tailings. Presently practical alternatives for tailings management are variations of two basic methods - surface below-ground disposal. Isolation of tailings by natural materials such as clay lenses and combinations of overburden, top soil, vegetation and rip-rap may provide both minimization of exposure and stability. Experimental measurement of radon flux over two inactive tailings, acid and carbonate leached tailings resulted in average specific flux values of phi infinity approx. = 0.64 and phi infinity approx. = 0.30 (pCi Rn-222/m/sup 2/ sec) / (pCi Ra-226/g), respectively. The average diffusion coefficient for these tailings were, respectively, 2.4 x 10/sup -3/ and 5.7 x 10/sup -4/ cm/sup 2//sec. Tailings covered with native soil of clay-silt-sand mixture to a depth of 225 cm resulted in attenuation of flux with diffusion coefficients of 3.69 x 10/sup -3/ and 3.60 x 10/sup -3/ cm/sup 2//sec for ACID and ALKO sites, respectively. By means of the UDAD code dose commitments were estimated for inhalation of particulates and radon and for external exposure under three degrees of surface moisture on the tailings. Based on these analyses and assumption that the dose contribution from ingestion pathway is comparable in magnitude to that of inhalation, both compliance with the 25 mrem/year limit and reduction of flux to background level is feasible. Stability of alternative decommissioned tailings over the predictable future is discussed.

  1. The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions.

    Directory of Open Access Journals (Sweden)

    Amit Bhardwaj

    Full Text Available BACKGROUND: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. METHODOLOGY: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. CONCLUSION: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DeltaF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

  2. Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.; Kelly, Charles; Mitchell, Tim J.; Brady, L. Jeannine; Deivanayagam, Champion (King); (Cornell); (UAB); (Glasgow); (Florida)

    2012-05-29

    The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

  3. NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein.

    Science.gov (United States)

    Smith, Archer D; Jameson, Guy N L; Dos Santos, Patricia C; Agar, Jeffrey N; Naik, Sunil; Krebs, Carsten; Frazzon, Jeverson; Dean, Dennis R; Huynh, Boi Hanh; Johnson, Michael K

    2005-10-04

    NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and Mössbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.

  4. Differential subcellular localization of the splice variants of the zinc transporter ZnT5 is dictated by the different C-terminal regions.

    Directory of Open Access Journals (Sweden)

    Jared K Thornton

    Full Text Available Zinc is emerging as an important intracellular signaling molecule, as well as fulfilling essential structural and catalytic functions through incorporation in a myriad of zinc metalloproteins so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the subcellular localizations of zinc transporters.Two splice variants of the human SLC30A5 Zn transporter gene (ZnT5 have been reported in the literature. These variants differ at their N- and C-terminal regions, corresponding with the use of different 5' and 3' exons. We demonstrate that full length human ZnT5 variant B is a genuine transcript in human intestinal cells and confirm expression of both variant A and variant B in a range of untreated human tissues by splice variant-specific RT-PCR. Using N- or C-terminal GFP or FLAG fusions of both isoforms of ZnT5 we identify that the differential subcellular localization to the Golgi apparatus and ER respectively is a function of their alternative C-terminal sequences. These different C-terminal regions result from the incorporation into the mature transcript of either the whole of exon 14 (variant B or only the 5' region of exon 14 plus exons 15-17 (variant A.We thus propose that exons 15 to 17 include a signal that results in trafficking of ZnT5 to the Golgi apparatus and that the 3' end of exon 14 includes a signal that leads to retention in the ER.

  5. A Fmoc-compatible Method for the Solid-Phase Synthesis of Peptide C-Terminal (alpha)-Thioesters based on the Safety-Catch Hydrazine Linker

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Hackel, B J; de Yoreo, J J; Mitchell, A R

    2003-11-22

    C-terminal peptide thioesters are key intermediates for the synthesis/semisynthesis of proteins and for the production of cyclic peptides by native chemical ligation. They can be synthetically prepared by solid-phase peptide synthesis (SPPS) methods or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal a-thioester peptides by SPPS was largely restricted to the Boc/Benzyl methodology because of the poor stability of the thioester bond to the basic conditions employed for the deprotection of the N{sup {alpha}}-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters by Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazide linker, which is totally stable to the Fmoc-SPPS conditions. Once the peptide synthesis has been completed, activation of the linker can be achieved by mild oxidation. This step transforms the hydrazide group into a highly reactive diazene intermediate which can react with different H-AA-SEt to yield the corresponding {alpha}-thioester peptide in good yields. This method has been successfully used for the generation of different thioester peptides, circular peptides and a fully functional SH3 protein domain.

  6. Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

    Energy Technology Data Exchange (ETDEWEB)

    Pazehoski, Kristina O., E-mail: pazehosk@pitt.edu [Division of Natural Sciences, University of Pittsburgh at Greensburg, Greensburg, PA 15601 (United States); Cobine, Paul A., E-mail: pac0006@auburn.edu [Department of Biological Sciences, 101 Rouse Life Science Building, Auburn University, AL 36849 (United States); Winzor, Donald J. [Department of Biochemistry, University of Queensland, Brisbane, Queensland 4072 (Australia); Dameron, Charles T., E-mail: cdameron@francis.edu [Department of Chemistry, Saint Francis University, Loretto, PA 15940 (United States)

    2011-03-11

    Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

  7. Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1.

    Science.gov (United States)

    Yagi, Hiromasa; Kajiwara, Nobumoto; Tanaka, Hideaki; Tsukihara, Tomitake; Kato-Yamada, Yasuyuki; Yoshida, Masasuke; Akutsu, Hideo

    2007-07-03

    The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.

  8. Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

    Science.gov (United States)

    Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

    2012-03-29

    The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN.

  9. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    Science.gov (United States)

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases.

  10. Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

    Science.gov (United States)

    Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo

    2014-12-17

    The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

  11. Screening in interacting protein in C terminal of MIC6 from Toxoplasma gondii%弓形虫MIC6羧基端相互作用蛋白的筛选

    Institute of Scientific and Technical Information of China (English)

    郑斌; 尹志奎; 何蔼; 詹希美

    2011-01-01

    目的 筛选与弓形虫微线体蛋白6羧基端(MIC6C)相互作用的蛋白.方法 以GST-MIC6C蛋白作为探针蛋白,利用GST pull-down技术,从弓形虫裂解液中筛选与其相互作用的蛋白,SDS-PAGE分析实验产物;将目标蛋白条带转印至PVDF膜,测蛋白序列,BLAST2在线对比搜索相似蛋白序列,初步确定目标蛋白.制备目标蛋白的抗体,Western blot分析GST pull-down实验产物.结果 GST pull-down实验筛选到的目标蛋白的序列与弓形虫醛缩酶的序列一致;制备了醛缩酶的多克隆抗体,目标蛋白条带能被该抗体特异地识别.结论 采用GST pull-down技术,初步筛选出与弓形虫MIC6C相互作用的蛋白为醛缩酶.%To screen the interacting protein from tachyzoite lysates in C terminal of microneme protein 6 (MIC6C) of Toxoplasma gondii, glutathione sepharose beads were incubated with GST-MIC6C protein,and then incubated with tachyzoite lysates. Bound proteins were eluted by using sample buffer, and eluants were resolved by SDS-PAGE and transferred onto pol-yvinylidene difluoride (PVDF) membranes. Protein band were cut from PVDF membrane, and the protein were subjected to N-terminal amino acid sequencing. BLAST2 server in Internet was used to analyse the amino acid sequences. Resutts showed that the N-terminal amino acid sequence of target protein was coincident with the amino acid sequence of aldolase, and the target protein band on NC membrane was specifically recognized by anti-aldolase antibody. The protein which interacts with C terminal of microneme protein 6 (MIC6C) of Toxoplasma gondii is aldolase.

  12. PEROXISOMAL AMINE OXIDASE OF HANSENULA-POLYMORPHA DOES NOT REQUIRE ITS SRL-CONTAINING C-TERMINAL SEQUENCE FOR TARGETING

    NARCIS (Netherlands)

    FABER, KN; HAIMA, P; DEHOOP, MJ; HARDER, W; VEENHUIS, M; AB, G

    1993-01-01

    Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C-terminus. In t

  13. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  14. Silver extraction using nitric acid from lead-zinc mine tail slag by response surface design%响应面法优化硝酸浸铅锌矿尾渣回收银工艺研究

    Institute of Scientific and Technical Information of China (English)

    杨彦松

    2011-01-01

    以硝酸浸铅锌矿尾渣回收银工艺进行了研究.以银浸出率为评价指标,探讨了硝酸质量浓度、液固比、浸出时间和浸出温度对回收银工艺的影响.在单因素实验的基础上,利用响应面法对银浸出的条件进行了优化.结果表明,当硝酸质量浓度为33.16%,液固比为16.39:1,在64.62℃的条件下浸出1.3h,该模型预测的最大银浸出率为69.73%.验证实验误差<3%,表明该模型与实际情况拟合良好.%The extraction process using nitric acid leaching silver from lead-zinc mine tail slag was studied. According to silver leaching rate using as an evaluation index, the effect of nitric acid mass concentration, the liquid-solid ratio,leaching time and leaching temperature on silver leaching rate was discussed. Based on single factor experiment, the leaching condition is further optimized by response surface method. The results showed that the largest model silver leaching rate of 69. 73% was obtained under the conditions of the concentration of 33.16% nitric acid quality and the liquid-solid ratio of 16.39:1 at 64.62 ℃ for 1.3 h. The verify error of the model was less than 3% ,and the model was fitted for the actual experiments well.

  15. Effects of alkali or acid treatment on the isomerization of amino acids.

    Science.gov (United States)

    Ohmori, Taketo; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa

    2012-10-01

    The effect of alkali treatment on the isomerization of amino acids was investigated. The 100×D/(D+L) values of amino acids from peptide increased with increase in the number of constituent amino acid residues. Furthermore, the N-terminal amino acid of a dipeptide was isomerized to a greater extent than the C-terminal residue.

  16. New technology of iron extraction and gold recovery from cyanide tailings by cyanide process after reduction roasting and acid leaching%氰化尾渣还原焙烧酸浸提铁及氰化浸金新工艺

    Institute of Scientific and Technical Information of China (English)

    郑雅杰; 龚昶; 孙召明

    2014-01-01

    The cyanide tailings were treated by reduction roasting and acid leaching. The cyanide tailings were reduced under conditions of reduction temperature 850℃, the mass ratio of pulverized coal to cyanide tailings 13%and reduction time 100 min. Fe2O3 in the cyanide tailings is turned into Fe3O4 or FeO. The reduced cyanide tailings were leached by sulfuric acid under conditions of a concentration of sulfuric acid 50%, the sulfuric acid dosage coefficient 1.2, leaching temperature 105℃and leaching time 3 h. The leaching rate of Fe is 93.66%. The leaching rate of Au is 92.4%under conditions of sodium cyanide dosage 4 kg/t, leaching time 28 h, the ratio of liquid to solid 2:1 after acidic residue is roasted at 600 ℃for 2 h to remove carbon. The mass of cyanide tailings decreases by 38.8%after reduction roasting, sulfuric acid leaching, oxidizing roasting and cyanide leaching.%以氰化尾渣为原料,采用还原焙烧酸浸工艺对其进行处理。当还原温度为850℃、加入煤粉质量为氰化尾渣质量的13%、还原时间为100 min时,对氰化尾渣进行还原,氰化尾渣中Fe2O3转化为Fe3O4或FeO。还原后采用硫酸浸出,当硫酸浓度为50%、硫酸用量系数为1.2、反应温度为105℃、反应时间为3 h时,铁的浸出率达到93.66%。还原焙烧渣在600℃氧化焙烧2 h经过脱碳后氰化浸金,当氰化钠用量为4 kg/t、反应时间为28 h、液固比为2:1时,金的浸出率达到92.4%。经过还原焙烧、硫酸浸出、氧化焙烧及氰化浸金,氰化尾渣渣量减少了38.8%。

  17. The acidic C-terminus of vaccinia virus I3 single-strand binding protein promotes proper assembly of DNA-protein complexes.

    Science.gov (United States)

    Harrison, Melissa L; Desaulniers, Megan A; Noyce, Ryan S; Evans, David H

    2016-02-01

    The vaccinia virus I3L gene encodes a single-stranded DNA binding protein (SSB) that is essential for virus DNA replication and is conserved in all Chordopoxviruses. The I3 protein contains a negatively charged C-terminal tail that is a common feature of SSBs. Such acidic tails are critical for SSB-dependent replication, recombination and repair. We cloned and purified variants of the I3 protein, along with a homolog from molluscum contagiosum virus, and tested how the acidic tail affected DNA-protein interactions. Deleting the C terminus of I3 enhanced the affinity for single-stranded DNA cellulose and gel shift analyses showed that it also altered the migration of I3-DNA complexes in agarose gels. Microinjecting an antibody against I3 into vaccinia-infected cells also selectively inhibited virus replication. We suggest that this domain promotes cooperative binding of I3 to DNA in a way that would maintain an open DNA configuration around a replication site.

  18. Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

    Science.gov (United States)

    Dufrasne, François E.; Lombard, Catherine; Goubau, Patrick; Ruelle, Jean

    2016-01-01

    BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail. PMID:27754450

  19. Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env Impairs Viral Release and Hampers BST-2 Antagonism

    Directory of Open Access Journals (Sweden)

    François E. Dufrasne

    2016-10-01

    Full Text Available BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2 relies on the envelope glycoprotein (Env to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail.

  20. Membrane Targeting and Insertion of the C-Tail Protein SciP.

    Science.gov (United States)

    Pross, Eva; Soussoula, Lavinia; Seitl, Ines; Lupo, Domenico; Kuhn, Andreas

    2016-10-09

    C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-terminal part of the SciP protein sequence are present. Two short hydrophobic sequences were identified that targeted a green fluorescent protein-SciP fusion protein to the membrane involving the signal recognition particle. After targeting, the membrane insertion of SciP is catalyzed by YidC independent of the SecYEG translocase. However, when the C-terminal tail of SciP was extended to 21 aa residues, we found that SecYEG becomes involved and makes its membrane insertion more efficient.

  1. Suspended electrodialytic extraction of toxic elements for detoxification of three different mine tailings

    DEFF Research Database (Denmark)

    Jensen, Pernille Erland; Ottosen, Lisbeth M.; Hansen, H.K.;

    2016-01-01

    investigated including pre-treatment of the tailings with acid; insertion of bipolar electrodes; and implementation of pulsed or sinusoidal electric fields. In line with these efforts, we investigated the efficiency when extracting toxic elements from a suspension of tailings, rather than from a solid matrix......, which could well be implemented as a final treatment step prior to deposition of tailings. Six electrodialytic experiments in laboratory scale with three different mine tailings (Codelco, Zinkgruvan, and Nalunaq) show that it is possible to extract residual Cu from the all the three suspended mine...... have shown that electrokinetic treatment can remove Cd, Cu, Pb, and Zn from tailings soils; As from tailings; and Cu from tailings. Still, however, a major concern is the long treatment-time required for the element-transport through the tailings matrix. Therefore several enhancement methods have been...

  2. Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure

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    Hayashi Nobuhiro

    2008-02-01

    Full Text Available Abstract Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure

  3. Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

    Directory of Open Access Journals (Sweden)

    Amandine Rovini

    Full Text Available We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+ ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs, increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+ end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length

  4. Intracellular localization of the M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization is mediated by a C-terminal tryptophan-based motif.

    Science.gov (United States)

    Uwada, Junsuke; Yoshiki, Hatsumi; Masuoka, Takayoshi; Nishio, Matomo; Muramatsu, Ikunobu

    2014-07-15

    The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W(442) or I(445) with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxΦ, which is the canonical binding motif for the μ2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 μ2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-μ2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization.

  5. The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds

    DEFF Research Database (Denmark)

    Roher, N; Miró, F; Boldyreff, B

    2001-01-01

    The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment...... with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar...

  6. Expression of the C-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum in tobacco plant

    OpenAIRE

    Olawole, O.

    2009-01-01

    Carbohydrate-binding modules have been shown to alter plant cell wall structural architecture. Hence, they have the potential application of being used to engineer the plant to produce tailor-made natural fibers in the cell wall. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The C-terminal CBM22-2 of the glycoside hydrolase (GH) 10 had been characterized to interact with xylan, a major hemicellulosic component in the secondary cell wall of ...

  7. Chemical shift assignments and secondary structure prediction of the C-terminal domain of the response regulator BfmR from Acinetobacter baumannii.

    Science.gov (United States)

    Olson, Andrew L; Thompson, Richele J; Melander, Christian; Cavanagh, John

    2014-04-01

    Acinetobacter baumannii is a Gram-negative pathogen responsible for severe nocosomial infections by forming biofilms in healthcare environments. The two-domain response regulator BfmR has been shown to be the master controller for biofilm formation. Inactivation of BfmR resulted in an abolition of pili production and consequently biofilm creation. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the C-terminal domain of BfmR (residues 130-238) from A. baumannii.

  8. Fast and catalyst-free hydrazone ligation via ortho-halo-substituted benzaldehydes for protein C-terminal labeling at neutral pH.

    Science.gov (United States)

    Xu, Yang; Xu, Ling; Xia, Yuan; Guan, Chao-Jian; Guo, Qing-Xiang; Fu, Yao; Wang, Chen; Li, Yi-Ming

    2015-08-28

    Rapid and catalyst-free hydrazone ligation reaction between ortho-halobenzaldehyde derivatives and peptide/protein hydrazides was observed at neutral pH and room temperature. 2-Chlorobenzaldehyde exhibited the fastest reaction and highest conversion rates among the series of ortho-halobenzaldehydes. The resulting hydrazone-containing bioconjugation products were also found to be fairly stable under experimental conditions. The new ligation strategy was successfully used for protein C-terminal labeling and should provide a practical approach for the modification of proteins.

  9. Synthesis of peptides containing C-terminal methyl esters using trityl side-chain anchoring: application to the synthesis of a-factor and a-factor analogs.

    Science.gov (United States)

    Diaz-Rodriguez, Veronica; Mullen, Daniel G; Ganusova, Elena; Becker, Jeffrey M; Distefano, Mark D

    2012-11-16

    A new cysteine anchoring method was developed for the synthesis of peptides containing C-terminal cysteine methyl esters. This method consists of attachment of Fmoc-Cys-OCH(3) to either 2-ClTrt-Cl or Trt-Cl resins (via the side-chain thiol) followed by preparation of the desired peptide using Fmoc-based SPPS. We applied this method to the synthesis of the mating pheromone a-factor and a 5-FAM labeled a-factor analog. The peptides were obtained with high yield and purity and were shown to be bioactive in a growth arrest assay.

  10. A Chlamydia trachomatis OmcB C-Terminal Fragment Is Released into the Host Cell Cytoplasm and Is Immunogenic in Humans ▿

    OpenAIRE

    2011-01-01

    The Chlamydia trachomatis outer membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals in C. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed tha...

  11. Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Nielsen, Morten Schallburg; Brejning, Jeanette; García, R.;

    1997-01-01

    Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C-terminal ......Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C...

  12. Preparation of polymeric aluminium ferric chloride from bauxite tailings

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    Ma D.

    2013-01-01

    Full Text Available Bauxite tailings are the main solid wastes in the ore dressing process. The Al2O3 and Fe2O3 contents in bauxite tailings can reach 50% and 13% respectively. The present study proposed a feasible method to use bauxite tailings to prepare polymeric aluminium ferric chloride (PAFC, a new composite inorganic polymer for water purification. Bauxite tailings roasted reacting with hydrochloric acid under air, pickle liquor which mainly contains Fe3+, Al3+ was generated, then calcium aluminate was used to adjust pH value and the basicity of the pickle liquor, the PAFC was subsequently prepared after the polymerization process. The optimal synthesizing parameters for the preparation of PAFC obtained were as follows: the concentration of hydrochloric acid of 24 wt%, ratio of hydrochloric acid to bauxite tailings of 6:1, temperature of 90ºC, leaching time of 2.5 hours, ration of pickle liquor to calcium aluminate of 12:1, polymerization temperature of 90ºC and polymerization time of about 3 hours. The basicity of PAFC was higher than 68%, the sum concentration of Al2O3 and Fe2O3 was beyond 12.5%. The results of flocculation tests indicate that the PAFC has a better performance of removing the turbidity of wastewater compared to PAC, and PAFC prepared by bauxite tailings is a kind of high quality flocculants.

  13. Telling tails: selective pressures acting on investment in lizard tails.

    Science.gov (United States)

    Fleming, Patricia A; Valentine, Leonie E; Bateman, Philip W

    2013-01-01

    Caudal autotomy is a common defense mechanism in lizards, where the animal may lose part or all of its tail to escape entrapment. Lizards show an immense variety in the degree of investment in a tail (i.e., length) across species, with tails of some species up to three or four times body length (snout-vent length [SVL]). Additionally, body size and form also vary dramatically, including variation in leg development and robustness and length of the body and tail. Autotomy is therefore likely to have fundamentally different effects on the overall body form and function in different species, which may be reflected directly in the incidence of lost/regenerating tails within populations or, over a longer period, in terms of relative tail length for different species. We recorded data (literature, museum specimens, field data) for relative tail length (n=350 species) and the incidence of lost/regenerating tails (n=246 species). We compared these (taking phylogeny into account) with intrinsic factors that have been proposed to influence selective pressures acting on caudal autotomy, including body form (robustness, body length, leg development, and tail specialization) and ecology (foraging behavior, physical and temporal niches), in an attempt to identify patterns that might reflect adaptive responses to these different factors. More gracile species have relatively longer tails (all 350 spp., P lizards (all 246 spp., P < 0.01; Scindidae, P < 0.05), larger skinks (P < 0.05), climbing geckos (P < 0.05), or active-foraging iguanids (P < 0.05). The selective advantage of investing in a relatively longer tail may be due to locomotor mechanics, although the patterns observed are also largely consistent with predictions based on predation pressure.

  14. Arginine residues in the C-terminal and their relationship with the analgesic activity of the toxin from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1).

    Science.gov (United States)

    Wang, Yu; Song, Yong-Bo; Yang, Guang-Zhao; Cui, Yong; Zhao, Yong-Shan; Liu, Yan-Feng; Ma, Yan; Wu, Chun-Fu; Zhang, Jing-Hai

    2012-09-01

    In this study, we investigated the functional role of arginines in the C-terminal (65-67) of BmK AGP-SYPU1, an analgesic peptide from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, arginines at the C-terminal (65-66) were deleted or added to the C-terminal (67). The genes for three mutants of BmK AGP-SYPU1 were obtained by PCR. An analgesic activity assay was used to evaluate the role of arginine residues in the analgesic activity. The three-dimensional structure of BmK AGP-SYPU1 was established by homology modeling. As a result, we showed that the arginines in the C-terminal are crucial for the analgesic activity and may be located at analgesic functional sites. Our work has implications for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity.

  15. Characterization of Mycobacterium tuberculosis EsxA membrane insertion: roles of N- and C-terminal flexible arms and central helix-turn-helix motif.

    Science.gov (United States)

    Ma, Yue; Keil, Verena; Sun, Jianjun

    2015-03-13

    EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed.

  16. Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit.

    Directory of Open Access Journals (Sweden)

    Derek S Woods

    Full Text Available DNA double strand breaks (DSBs can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK. DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ. Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.

  17. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    Energy Technology Data Exchange (ETDEWEB)

    Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Wasmer, Christian [Harvard Medical School (United States); Bousset, Luc; Sourigues, Yannick [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Schuetz, Anne [ETH Zurich, Physical Chemistry (Switzerland); Loquet, Antoine [Max Planck Institute for Biophysical Chemistry (Germany); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Melki, Ronald, E-mail: melki@lebs.cnrs-gif.fr [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France)

    2011-11-15

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly {sup 13}C, {sup 15}N labeled protein sample, sequential chemical-shift information for 74% of the N, C{alpha}, C{beta} triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  18. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation.

    Science.gov (United States)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

    2015-12-01

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells.

  19. Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs.

    Science.gov (United States)

    Jung, Chan Jin; Lee, Myoung Hui; Min, Myung Ki; Hwang, Inhwan

    2011-02-01

    Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains.

  20. Novel human mutation and CRISPR/Cas genome-edited mice reveal the importance of C-terminal domain of MSX1 in tooth and palate development.

    Science.gov (United States)

    Mitsui, Silvia Naomi; Yasue, Akihiro; Masuda, Kiyoshi; Naruto, Takuya; Minegishi, Yoshiyuki; Oyadomari, Seiichi; Noji, Sumihare; Imoto, Issei; Tanaka, Eiji

    2016-12-05

    Several mutations, located mainly in the MSX1 homeodomain, have been identified in non-syndromic tooth agenesis predominantly affecting premolars and third molars. We identified a novel frameshift mutation of the highly conserved C-terminal domain of MSX1, known as Msx homology domain 6 (MH6), in a Japanese family with non-syndromic tooth agenesis. To investigate the importance of MH6 in tooth development, Msx1 was targeted in mice with CRISPR/Cas system. Although heterozygous MH6 disruption did not alter craniofacial development, homozygous mice exhibited agenesis of lower incisors with or without cleft palate at E16.5. In addition, agenesis of the upper third molars and the lower second and third molars were observed in 4-week-old mutant mice. Although the upper second molars were present, they were abnormally small. These results suggest that the C-terminal domain of MSX1 is important for tooth and palate development, and demonstrate that that CRISPR/Cas system can be used as a tool to assess causality of human disorders in vivo and to study the importance of conserved domains in genes.

  1. Effect of pH on the structure of the recombinant C-terminal domain of Nephila clavipes dragline silk protein.

    Science.gov (United States)

    Gauthier, Martin; Leclerc, Jérémie; Lefèvre, Thierry; Gagné, Stéphane M; Auger, Michèle

    2014-12-08

    Spider silk proteins undergo a complex series of molecular events before being converted into an outstanding hierarchically organized fiber. Recent literature has underlined the crucial role of the C-terminal domain in silk protein stability and fiber formation. However, the effect of pH remains to be clarified. We have thus developed an efficient purification protocol to obtain stable native-like recombinant MaSp1 C-terminal domain of Nephila clavipes (NCCTD). Its structure was investigated as a function of pH using circular dichroism, fluorescence and solution NMR spectroscopy. The results show that the NCCTD structure is very sensitive to pH and suggest that a molten globule state occurs at pH 5.0 and below. Electronic microscopy images also indicate fiber formation at low pH and coarser globular particles at more basic pH. The results are consistent with a spinning process model where the NCCTD acts as an aggregation nucleus favoring the β-aggregation of the hydrophobic polyalanine repeats upon spinning.

  2. Membrane-embedded C-terminal segment of rat mitochondrial TOM40 constitutes protein-conducting pore with enriched beta-structure.

    Science.gov (United States)

    Suzuki, Hiroyuki; Kadowaki, Tomoko; Maeda, Maki; Sasaki, Hiroyuki; Nabekura, Junichi; Sakaguchi, Masao; Mihara, Katsuyoshi

    2004-11-26

    TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% beta-sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(DeltaN165)) as a recombinant protein with 62% beta-structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched beta-structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.

  3. α1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro

    Directory of Open Access Journals (Sweden)

    Westin Ulla

    2004-11-01

    Full Text Available Abstract Background Tumor microenvironment, which is largely affected by inflammatory cells, is a crucial participant in the neoplastic process through promotion of cell proliferation, survival and migration. We measured the effects of polymorphonuclear neutrophil (PMN conditioned medium alone, and supplemented with serine proteinase inhibitor α-1 antitrypsin (AAT or its C-terminal fragment (C-36 peptide, on cultured lung cancer cells. Methods Lung cancer HCC cells were grown in a regular medium or in a PMN-conditioned medium in the presence or absence of AAT (0.5 mg/ml or its C-36 peptide (0.06 mg/ml for 24 h. Cell proliferation, invasiveness and release of IL-8 and VEGF were analyzed by [3H]-thymidine incorporation, Matrigel invasion and ELISA methods, respectively. Results Cells exposed to PMN-conditioned medium show decreased proliferation and IL-8 release by 3.9-fold, p Conclusions Our data provide evidence that neutrophil derived factors decrease lung cancer HCC cell proliferation and IL-8 release, but increase cell invasiveness. These effects were found to be modulated by exogenously present serine proteinase inhibitor, AAT, and its C-terminal fragment, which points to a complexity of the relationships between tumor cell biological activities and local microenvironment.

  4. X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases.

    Science.gov (United States)

    Gawronski-Salerno, Joseph; Coon, John S; Focia, Pamela J; Freymann, Douglas M

    2007-03-01

    FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 A X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain "module" are regulated in the context of the two SRP GTPases.

  5. The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence.

    Science.gov (United States)

    Carroll, Ronan K; Rivera, Frances E; Cavaco, Courtney K; Johnson, Grant M; Martin, David; Shaw, Lindsey N

    2014-08-01

    Staphylococcus aureus is a versatile pathogen of humans and a continued public health concern due to the rise and spread of multidrug-resistant strains. As part of an ongoing investigation into the pathogenic mechanisms of this organism we previously demonstrated that an intracellular N-terminal processing protease is required for S. aureus virulence. Following on from this, here we examine the role of CtpA, the lone C-terminal processing protease of S. aureus. CtpA, a member of the S41 family, is a serine protease whose homologues in Gram-negative bacteria have been implicated in a range of biological functions, including pathogenesis. We demonstrate that S. aureus CtpA is localized to the bacterial cell wall and expression of the ctpA gene is maximal upon exposure to conditions encountered during infection. Disruption of the ctpA gene leads to decreased heat tolerance and increased sensitivity when exposed to components of the host immune system. Finally we demonstrate that the ctpA(-) mutant strain is attenuated for virulence in a murine model of infection. Our results represent the first characterization of a C-terminal processing protease in a pathogenic Gram-positive bacterium and show that it plays a critical role during infection.

  6. N- and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H1(0) and H1c.

    Science.gov (United States)

    Vyas, Payal; Brown, David T

    2012-04-01

    Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.

  7. Hsp90 C-terminal inhibitors exhibit antimigratory activity by disrupting the Hsp90α/Aha1 complex in PC3-MM2 cells.

    Science.gov (United States)

    Ghosh, Suman; Shinogle, Heather E; Garg, Gaurav; Vielhauer, George A; Holzbeierlein, Jeffrey M; Dobrowsky, Rick T; Blagg, Brian S J

    2015-02-20

    Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.

  8. Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

    Energy Technology Data Exchange (ETDEWEB)

    Worrall, Liam; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk [School of Biological Sciences, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom)

    2006-09-01

    Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I2{sub 1}2{sub 1}2{sub 1}. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry. Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.

  9. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain.

    Science.gov (United States)

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A; Enghild, Jan J; Thøgersen, Ida B; Gao, Jinlong; Kwan, Ann H; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-03-23

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

  10. Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

    Energy Technology Data Exchange (ETDEWEB)

    González-Páez, Gonzalo E.; Wolan, Dennis W. (Scripps)

    2012-09-05

    Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

  11. The C-terminal MIR-containing region in the Pmt1 O-mannosyltransferase restrains sporulation and is dispensable for virulence in Beauveria bassiana.

    Science.gov (United States)

    He, Zhangjiang; Luo, Linli; Keyhani, Nemat O; Yu, Xiaodong; Ying, Shenghua; Zhang, Yongjun

    2017-02-01

    Protein O-mannosyltransferases (Pmts) belong to a highly conserved protein family responsible for the initiation of O-glycosylation of many proteins. Pmts contain one dolichyl-phosphate-mannose-protein mannosyltransferases (PMT) domain and three MIR motifs (mannosyltransferase, inositol triphosphate, and ryanodine receptor) that are essential for activity in yeast. We report that in the insect fungal pathogen, Beauveria bassiana, deletion of the C-terminal Pmt1 MIR-containing region (Pmt1∆ (311-902)) does not alter O-mannosyltransferase activity, but does increase total cell wall protein O-mannosylation levels and results in phenotypic changes in fungal development and cell wall stability. B. bassiana mutants harboring the Pmt1 ∆ (311-902) mutation displayed a significant increase in conidiation with up-regulation of conidiation-associated genes and an increase in biomass accumulation as compared to the wild-type parent. However, decreased vegetative growth and blastospore production was noted, and Pmt1 ∆ (311-902) mutants were altered in cell wall composition and cell surface features. Insect bioassays revealed little effect on virulence for the Pmt1 ∆ (311-902) strain via cuticle infection or intrahemocoel injection assays, although differences in hyphal body differentiation in the host hemolymph and up-regulation of virulence-associated genes were noted. These data suggest novel roles for Pmt1 in negatively regulating conidiation and demonstrate that the C-terminal Pmt1 MIR-containing region is dispensable for enzymatic activity and organismal virulence.

  12. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Shuxia, E-mail: pengsx@ihep.ac.cn; Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-10-31

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

  13. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines

    DEFF Research Database (Denmark)

    Helin, K; Velu, T; Martin, P;

    1991-01-01

    , growth in agar and growth in low serum, mutant receptors display a similar hierarchy of activity. The lower activity is intrinsic in the mutants since they are expressed at similar level as the wild type and bind EGF with similar affinity. Deletion mutants lacking the last 19 or 63 amino acids (Velu et......The epidermal growth factor receptor (EGF-R) C-terminus contains three conserved tyrosines (Y-1068, Y-1148, Y-1173) which are phosphorylated upon EGF activation. To clarify the functional role of these tyrosines, each has been mutated to phenylalanine and studied as single, double and triple...

  14. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    Directory of Open Access Journals (Sweden)

    Belén Mezquita

    2014-02-01

    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  15. Enhanced Mobilization of Arsenic and Heavy Metals from Mine Tailings by Humic Acid%通过腐植酸增强尾矿中砷和重金属的迁移能力

    Institute of Scientific and Technical Information of China (English)

    Suiling Wang(著); Catherine N. Mulligan(著); 薄纯玉(译)

    2013-01-01

      砷和重金属从尾矿中的迁移是一个令人关注的问题,因为这可能对地下水和生态造成潜在的危害。最近越来越多的关注聚焦到天然有机物对环境中有毒物质迁移行为的影响上。本研究使用柱实验方法来评估利用腐植酸(HA)将砷和重金属(例如Cu、Pb和Zn)从一个氧化Pb-Zn矿尾矿样品中迁移出来的可行性。该样品采自加拿大新不伦瑞克省的巴瑟斯特。毛细管电泳分析表明,砷酸盐[As(V)]是尾矿中唯一可萃取出来的砷的形态,在pH值为11时引入HA不会引起砷的氧化还原反应或甲基化反应。初始pH值调整到11的一个0.1%的HA溶液被选作淋洗液,同时蒸馏水(初始pH调整至11)被用作对照来说明物理混合和pH值对砷和重金属迁移能力的影响。研究发现,H A能够显著地增强砷和重金属在尾矿中的迁移能力。经过一个70孔体积的淋洗,砷、铜、铅和锌的迁移分别达到了97、35、838和224 mg/kg。研究发现,在HA的存在下,砷和重金属的迁移与铁的迁移呈正相关。此外,砷的迁移与重金属的迁移也有很好的相关性。在HA存在的情况下,共存金属通过金属桥接作用机制帮助砷进入可溶的水合有机复合物中,在某种程度上增强了砷的迁移能力。应用HA进行砷和重金属的修复可能会被发展成为一项环保且有效的补救措施,从而降低和避免进一步的污染。%Arsenic and heavy metal mobilization from mine tailings is an issue of concern as it might pose potential groundwater or ecological risks. Increasing attention recently has been focused on the effects of natural organic mat-ter on the mobility behavior of the toxicants in the environment. Column experiments were carried out in this research study to evaluate the feasibility of using humic acid (HA) to mobilize arsenic and heavy metals (i.e., Cu, Pb and Zn) from an oxidized Pb–Zn mine tailings sample

  16. Changes of Bacterial Community Structure in Copper Mine Tailings After Colonization of Reed (Phragmites communis)

    Institute of Scientific and Technical Information of China (English)

    CHEN Yu-Qing; REN Guan-Ju; AN Shu-Qing; SUN Qing-Ye; LIU Chang-Hong; SHUANG Jing-Lei

    2008-01-01

    Soil samples were collected from both bare and vegetated mine tailings to study the changes in bacterial communities and soil chemical properties of copper mine tailings due to reed (Phragmites communis) colonization. The structures of bacterial communities were investigated using culture-independent 16S rRNA gene sequencing method. The bacterial diversity in the bare mine tailing was lower than that of the vegetated mine tailing. The former was dominated by sulfur metabolizing bacteria, whereas the latter was by nitrogen fixing bacteria. The bare mine tailing was acidic (pH = 3.78), whereas the vegetated mine tailing was near neutral (pH = 7.28). The contents of organic matter, total nitrogen, and ammonium acetate-extractable otassium in vegetated mine tailings were significantly higher than those in the bare mine tailings (P < 0.01), whereas available phosphorus and electrical conductivity were significantly lower than those in the bare mine tailings (P < 0.01). The results demonstrated that 16S rRNA gene sequencing could be successfully used to study the bacterial diversity in mine tailings. The colonization of the mine tailings by reed significantly changed the bacterial community and the chemical properties of tailings. The complex interactions between bacteria and plants deserve further investigation.

  17. Study on removal of silicon and preparation of high purity ferric oxide red from acid leaching filtrate of tailings%尾矿酸浸液除硅以及制备高纯铁红的研究

    Institute of Scientific and Technical Information of China (English)

    廖攀; 陈学安; 常新安; 肖卫强; 王少华

    2012-01-01

    Acid leaching filtrate of tailings from Jinchuan Company contains not only large amount of Fe2+ but also small amount of Si4*.Using CaO as neutralizer and polyacrylamide as flocculation reagent,the optimum technological conditions of removal of silicon from the acid leaching filtrate were obtained by orthogonal experiments.After silicon was removed,goethite was prepared under the conditions of 85 t and stirring by using MgO as neutralizer and air together with H2O2 as nxirlant.Finally the high purity ferric oxide red (a-Fe2O3) was prepared by calcining goethite at calcining temperature of 800℃ for 2 h followed by washing with 5% dilute HNO3. The mass fraction of main content of the as-prepared iron red was 99.2% .which could be classified as the qualified product according to the national standard of HG/T 2574?009.%金川公司尾矿酸浸液中除含有较高含量的铁之外,还含有一定量的杂质硅.以氧化钙为中和剂,聚丙烯酰胺为絮凝剂,通过正交设计与实验,得出了从该酸浸液中除硅的较佳工艺条件;随后,以氧化镁为中和剂,空气和双氧水为氧化剂,将除硅后滤液在85℃搅拌加热,制得针铁矿沉淀;最后将沉淀在800℃煅烧2h并经过5%稀硝酸洗涤得到了高纯铁红,其主成分质量分数为99.2%,符合HG/T 2574-2009工业氧化铁国家标准.

  18. Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

    DEFF Research Database (Denmark)

    Magni, M; Pandiella, A; Helin, K

    1991-01-01

    in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed...... mutants), intermediate in the dual mutants and almost complete in the triple mutants. Likewise, increases in intracellular Ca2+ concentrations [( Ca2+]i) induced by fibroblast growth factor were approximately the same in all of the clones, whereas those induced by EGF were decreased in the mutants, again...... a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather...

  19. Oilsand tailings : solving the issues

    Energy Technology Data Exchange (ETDEWEB)

    Grans