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Sample records for n-terminal truncated fragment

  1. Enhanced Fibril Fragmentation of N-Terminally Truncated and Pyroglutamyl-Modified Aβ Peptides.

    PubMed

    Wulff, Melanie; Baumann, Monika; Thümmler, Anka; Yadav, Jay K; Heinrich, Liesa; Knüpfer, Uwe; Schlenzig, Dagmar; Schierhorn, Angelika; Rahfeld, Jens-Ulrich; Horn, Uwe; Balbach, Jochen; Demuth, Hans-Ulrich; Fändrich, Marcus

    2016-04-11

    N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aβ peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aβ fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates. PMID:26970534

  2. Astrocytes and microglia but not neurons preferentially generate N-terminally truncated Aβ peptides.

    PubMed

    Oberstein, Timo Jan; Spitzer, Philipp; Klafki, Hans-Wolfgang; Linning, Philipp; Neff, Florian; Knölker, Hans-Joachim; Lewczuk, Piotr; Wiltfang, Jens; Kornhuber, Johannes; Maler, Juan Manuel

    2015-01-01

    The neuropathological hallmarks of Alzheimer's disease include extracellular neuritic plaques and neurofibrillary tangles. The neuritic plaques contain β-amyloid peptides (Aβ peptides) as the major proteinaceous constituent and are surrounded by activated microglia and astrocytes as well as dystrophic neurites. N-terminally truncated forms of Aβ peptides are highly prevalent in neuritic plaques, including Aβ 3-x beginning at Glu eventually modified to pyroglutamate (Aβ N3pE-x), Aβ 2-x, Aβ 4-x, and Aβ 5-x. The precise origin of the different N-terminally modified Aβ peptides currently remains unknown. To assess the contribution of specific cell types to the formation of different N-terminally truncated Aβ peptides, supernatants from serum-free primary cell cultures of chicken neurons, astrocytes, and microglia, as well as human astrocytes, were analyzed by Aβ-ELISA and one- and two-dimensional SDS-urea polyacrylamide gel electrophoresis followed by immunoblot analysis. To evaluate the contribution of β- and γ-secretase to the generation of N-terminally modified Aβ, cultured astrocytes were treated with membrane-anchored "tripartite β-secretase (BACE1) inhibitors" and the γ-secretase inhibitor DAPT. Neurons, astrocytes, and microglia each exhibited cell type-specific patterns of secreted Aβ peptides. Neurons predominantly secreted Aβ peptides that begin at Asp1, whereas those released from astrocytes and microglia included high proportions of N-terminally modified Aβ peptides, presumably including Aβ 2/3-x and 4/5-x. The inhibition of BACE1 reduced the amount of Aβ 1-x in cell culture supernatants but not the amount of Aβ 2-x. PMID:25204716

  3. Murine erythroid 5-aminolevulinate synthase: Truncation of a disordered N-terminal extension is not detrimental for catalysis.

    PubMed

    Stojanovski, Bosko M; Breydo, Leonid; Uversky, Vladimir N; Ferreira, Gloria C

    2016-05-01

    5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme, catalyzes the initial step of heme biosynthesis in non-plant eukaryotes. The precursor form of the enzyme is translated in the cytosol, and upon mitochondrial import, the N-terminal targeting presequence is proteolytically cleaved to generate mature ALAS. In bone marrow-derived erythroid cells, a mitochondrial- and site-specific endoprotease of yet unknown primary structure, produces a protein shorter than mature erythroid ALAS (ALAS2) found in peripheral blood erythroid cells. This truncated ALAS2 lacks the presequence and the N-terminal sequence (corresponding to ~7 KDa molecular mass) present in ALAS2 from peripheral blood erythroid cells. How the truncation affects the structural topology and catalytic properties of ALAS2 is presently not known. To address this question, we created a recombinant, truncated, murine ALAS2 (ΔmALAS2) devoid of the cleavable N-terminal region and examined its catalytic and biophysical properties. The N-terminal truncation of mALAS2 did not significantly affect the organization of the secondary structure, but a subtle reduction in the rigidity of the tertiary structure was noted. Furthermore, thermal denaturation studies revealed a decrease of 4.3°C in the Tm value of ΔmALAS2, implicating lower thermal stability. While the kcat of ΔmALAS2 is slightly increased over that of the wild-type enzyme, the slowest step in the ΔmALAS2-catalyzed reaction remains dominated by ALA release. Importantly, intrinsic disorder algorithms imply that the N-terminal region of mALAS2 is highly disordered, and thus susceptible to proteolysis. We propose that the N-terminal truncation offers a cell-specific ALAS2 regulatory mechanism without hindering heme synthesis. PMID:26854603

  4. [Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions].

    PubMed

    Chen, A'na; Liu, Xiuxia; Dai, Xiaofeng; Zhan, Jinling; Peng, Feng; Li, Lu; Wang, Fen; Li, Song; Yang, Yankun; Bai, Zhonghu

    2016-03-01

    We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT. PMID:27349118

  5. Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide.

    PubMed

    Coast, Geoffrey M; Zabrocki, Janusz; Nachman, Ronald J

    2002-04-01

    Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH(2)) and N-terminal truncated analogs of 4-14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn(1), but was reduced > 10-fold after the removal of Thr(2). Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys(8)) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg(10), and at 100 microM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg(10) was confirmed by comparing dose-response curves for Musdo-K [6-15] and Acheta kinin-V (AFSHWG-NH(2)) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity. PMID:11897389

  6. Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli.

    PubMed

    Akita, Keita; Hieda, Naoki; Baba, Nobuyuki; Kawaguchi, Satoshi; Sakamoto, Hirohisa; Nakanishi, Yuka; Yamanishi, Mamoru; Mori, Koichi; Toraya, Tetsuo

    2010-01-01

    The methods of homologous high-level expression and simple large-scale purification for coenzyme B(12)-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer alpha(6)beta(6). Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B(12), the binding of 1 mol of the inhibitor per mol of the alphabeta unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme. PMID:19762342

  7. Preparation and characterization of a truncated caricain lacking 41 residues from the N-terminal.

    PubMed

    Liu, Wei; Ye, Wanhui; Wang, Zhangming; Chao, Honglin; Lian, Juyu

    2005-05-01

    We purified an 18.8 kD protease from caricain solution. This protease was derived from caricain. It does not have the first 41 residues of the N-terminal sequence of caricain, and its N-terminal residue is Thr. Also, one of the disulfide bonds of caricain (cys22-cys63) was opened during the formation of the protease. We named this 18.8 kD protease caricain II. Caricain II has a wide pH range, and it is more sensitive to temperature changes than caricain. The proteolytic activity of caricain II is twice as much as that of caricain using casein as a substrate. However, caricain II has a low hydrolytic activity with N-benzoyl-L-arginine ethyl ester (BAEE) that is one of the special substrates of caricain. Our results indicate that caricain II is remarkably different from caricain and it can provide an improvement over caricain on the proteolytic activity. PMID:16283547

  8. Small N-terminal mutant huntingtin fragments, but not wild type, are mainly present in monomeric form: Implications for pathogenesis.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Roos, Raymund A C; van Ommen, Gert-Jan B; Dorsman, Josephine C

    2006-06-01

    N-terminal fragments of huntingtin containing an expanded polyglutamine stretch play an important role in the molecular pathogenesis of Huntington's disease. Their ultimate accumulation in insoluble protein aggregates constitutes an important pathological hallmark of Huntington's disease. We report on systematic biochemical comparison studies of soluble wild type and mutant N-terminal huntingtin fragments. The results show that soluble wild type exon 1 fragments are predominantly present in higher molecular weight complexes with a molecular size of approximately 300 kDa, while their mutant counterparts are mainly present in their monomeric form. In contrast, longer N-terminal fragments corresponding to peptides produced by caspase cleavage do not display these differential properties. These findings suggest that especially an increased amount of monomeric form of small N-terminal mutant huntingtin fragments may facilitate aberrant interactions both with itself via the polyglutamine stretch and with other proteins and thereby contribute to molecular pathogenesis. PMID:16380118

  9. Truncation of N-terminal regions of Digitalis lanata progesterone 5β-reductase alters catalytic efficiency and substrate preference.

    PubMed

    Rudolph, Kristin; Bauer, Peter; Schmid, Benedikt; Mueller-Uri, Frieder; Kreis, Wolfgang

    2014-06-01

    N-Terminal truncated forms of progesterone 5β-reductase (P5βR) were synthesized taking a full-length cDNA encoding for Digitalis lanata P5βR with a hexa-histidine tag attached at the C-terminus (rDlP5βRc) as the starting point. Four pETite-c-His/DlP5βR constructs coding for P5βR derivatives truncated in the N-terminal region, termed rDlP5βRcn-10, rDlP5βRcn-20, rDlP5βRcn-30, and rDlP5βRcn-40 were obtained by site-directed mutagenesis. The cDNAs coding for full-length rDlP5βRc, rDlP5βRcn-10 and rDlP5βRcn-20 were over-expressed in Escherichia coli and the respective enzymes were soluble and catalytically active (progesterone and 2-cyclohexen-1-one as substrates). GST-tagged recombinant DlP5βR (rDlP5βR-GST) and rDlP5βR-GSTr, with the GST-tag removed by protease treatment were produced as well and served as controls. The Km values and substrate preferences considerably differed between the various DlP5βR derivatives. As for the C-terminal His-tagged rDlP5βR the catalytic efficiency for progesterone was highest for the full-length rDlP5βRc whereas the N-terminal truncated forms preferred 2-cyclohexen-1-one as the substrate. Affinity tags and artifacts resulting from the cloning strategy used may alter substrate specificity. Therefore enzyme properties determined with recombinant proteins should not be used to infer in vivo scenarios and should be considered for each particular case. PMID:24370479

  10. N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor

    SciTech Connect

    Fisone, G.; Berthold, M.; Bedecs, K.; Unden, A.; Bartfai, T.; Bertorelli, R.; Consolo, S.; Crawley, J.; Martin, B.; Nilsson, S.; )

    1989-12-01

    The galanin N-terminal fragment (galanin-(1-16)) has been prepared by solid-phase synthesis and by enzymic cleavage of galanin by endoproteinase Asp-N. This peptide fragment displaced {sup 125}I-labeled galanin in receptor autoradiography experiments on rat forebrain and spinal cord and in equilibrium binding experiments from high-affinity binding sites in the ventral hippocampus with an IC50 of approximately 3 nM. In tissue slices of the same brain area, galanin-(1-16), similarly to galanin, inhibited the muscarinic agonist-stimulated breakdown of inositol phospholipids. Upon intracerebroventricular administration, galanin-(1-16) (10 micrograms/15 microliters) also inhibited the scopolamine (0.3 mg/kg, s.c.)-evoked release of acetylcholine, as studied in vivo by microdialysis. Substitution of (L-Trp2) for (D-Trp2) resulted in a 500-fold loss in affinity as compared with galanin-(1-16). It is concluded that, in the ventral hippocampus, the N-terminal galanin fragment (galanin-(1-16)) is recognized by the galanin receptors controlling acetylcholine release and muscarinic agonist-stimulated inositol phospholipid breakdown as a high-affinity agonist and that amino acid residue (Trp2) plays an important role in the receptor-ligand interactions.

  11. Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

    SciTech Connect

    Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei; Mondragon, Alfonso

    2010-09-02

    Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.

  12. Membrane effects of N-terminal fragment of apolipoprotein A-I: a fluorescent probe study.

    PubMed

    Trusova, Valeriya; Gorbenko, Galyna; Girych, Mykhailo; Adachi, Emi; Mizuguchi, Chiharu; Sood, Rohit; Kinnunen, Paavo; Saito, Hiroyuki

    2015-03-01

    The binding of monomeric and aggregated variants of 1-83 N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity. PMID:25595057

  13. Structure of the N-terminal fragment of Escherichia coli Lon protease

    SciTech Connect

    Li, Mi; Gustchina, Alla; Rasulova, Fatima S.; Melnikov, Edward E.; Maurizi, Michael R.; Rotanova, Tatyana V.; Dauter, Zbigniew; Wlodawer, Alexander

    2010-08-01

    The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix. The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.

  14. Metalloprotease Meprin β Generates Nontoxic N-terminal Amyloid Precursor Protein Fragments in Vivo*

    PubMed Central

    Jefferson, Tamara; Čaušević, Mirsada; auf dem Keller, Ulrich; Schilling, Oliver; Isbert, Simone; Geyer, Rebecca; Maier, Wladislaw; Tschickardt, Sabrina; Jumpertz, Thorsten; Weggen, Sascha; Bond, Judith S.; Overall, Christopher M.; Pietrzik, Claus U.; Becker-Pauly, Christoph

    2011-01-01

    Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β−/− mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing. PMID:21646356

  15. N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

    PubMed Central

    Schepmoes, Athena A.; Zhang, Qibin; Petritis, Brianne O.; Qian, Wei-Jun; Smith, Richard D.

    2009-01-01

    Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and ∼95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify >300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases. PMID:19949699

  16. N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

    SciTech Connect

    Schepmoes, Athena A.; Zhang, Qibin; Petritis, Brianne O.; Qian, Weijun; Smith, Richard D.

    2009-12-01

    Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and *95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify *300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases.

  17. Secondary structure, stability and tetramerisation of recombinant K(V)1.1 potassium channel cytoplasmic N-terminal fragment.

    PubMed

    Abbott, G W; Bloemendal, M; Van Stokkum, I H; Mercer, E A; Miller, R T; Sewing, S; Wolters, M; Pongs, O; Srai, S K

    1997-08-15

    The recombinant N-terminal fragment (amino acids 14-162) of a tetrameric voltage-gated potassium channel (K(V)1.1) has been studied using spectroscopic techniques. Evidence is presented that it forms a tetramer in aqueous solution, whereas when solubilised in 1% Triton X-100 it remains monomeric. The secondary structure content of both monomeric and tetrameric K(V)1.1 N-terminal fragment has been estimated from FTIR and CD spectroscopy to be 20-25% alpha-helix, 20-25% beta-sheet, 20% turns and 30-40% random coil. Solubilisation of the protein in detergent is shown by hydrogen-deuterium exchange analysis to alter tertiary structure rather than secondary structure and this may be the determining factor in tetramerisation ability. Using molecular modelling we propose a supersecondary structure consisting of two structural domains. PMID:9300810

  18. Structure and properties of a dimeric N-terminal fragment of human ubiquitin.

    PubMed

    Bolton, D; Evans, P A; Stott, K; Broadhurst, R W

    2001-12-01

    Previous peptide dissection and kinetic experiments have indicated that in vitro folding of ubiquitin may proceed via transient species in which native-like structure has been acquired in the first 45 residues. A peptide fragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was produced in order to test whether this portion has propensity for independent self-assembly. Surprisingly, the construct formed a folded symmetrical dimer that was stabilised by 0.8 M sodium sulphate at 298 K (the S state). The solution structure of the UQ(1-51) dimer was determined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal beta-hairpin followed by an alpha-helix and a final beta-strand, with orientations similar to intact ubiquitin. The dimer is formed by the third beta-strand of one subunit interleaving between the hairpin and third strand of the other to give a six-stranded beta-sheet, with the two alpha-helices sitting on top. The helix-helix and strand portions of the dimer interface also mimic related features in the structure of ubiquitin. The structural specificity of the UQ(1-51) peptide is tuneable: as the concentration of sodium sulphate is decreased, near-native alternative conformations are populated in slow chemical exchange. Magnetization transfer experiments were performed to characterize the various species present in 0.35 M sodium sulphate, namely the S state and two minor forms. Chemical shift differences suggest that one minor form is very similar to the S state, while the other experiences a significant conformational change in the third strand. A segmental rearrangement of the third strand in one subunit of the S state would render the dimer asymmetric, accounting for most of our results. Similar small-scale transitions in proteins are often invoked to explain solvent exchange at backbone amide proton sites that have an intermediate level of protection. PMID:11733996

  19. Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception.

    PubMed

    Katsuyama, Soh; Mizoguchi, Hirokazu; Komatsu, Takaaki; Sakurada, Chikai; Tsuzuki, Minoru; Sakurada, Shinobu; Sakurada, Tsukasa

    2011-07-01

    Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1-13), (1-11) and (1-7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3-1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1-13) and (1-11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1-13) and 4.75 for N/OFQ (1-11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1-7) led to be less potent than N/OFQ and its fragments, (1-13) and (1-11). Antinociception induced by N/OFQ or N/OFQ (1-13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1-13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1-11) and (1-7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1-13) still possess antinociceptive activity through NOP receptors. PMID:21672568

  20. Truncated N-terminal mutants of SV40 large T antigen as minimal immortalizing agents for CNS cells

    PubMed Central

    Freed, William J.; Zhang, Peisu; Sanchez, Joseph F.; Dillon-Carter, Ora; Coggiano, Mark; Errico, Stacie L.; Lewis, Brian D.; Truckenmiller, Mary Ellen

    2007-01-01

    Immortalized central nervous system (CNS) cell lines are useful as in vitro models for innumerable purposes such as elucidating biochemical pathways, studies of effects of drugs, and ultimately, such cells may also be useful for neural transplantation. The SV40 large T (LT) oncoprotein, commonly used for immortalization, interacts with several cell cycle regulatory factors, including binding and inactivating p53 and retinoblastoma family cell-cycle regulators. In an attempt to define the minimal requirements of SV40 T antigen for immortalizing cells of CNS origin, we constructed T155c, encoding the N-terminal 155 amino acids of LT. The p53 binding region is known to reside in the C-terminal region of LT. An additional series of mutants was produced to further narrow the molecular targets for immortalization, and plasmid vectors were constructed for each. In a p53 temperature sensitive cell line model, T64-7B, expression of T155c and all constructs having mutations outside of the first 82 amino acids were capable of overriding cell-cycle block at the non-permissive growth temperature. Several cell lines were produced from fetal rat mesencephalic and cerebral cortical cultures using the T155c construct. The E107K construct contained a mutation in the Rb binding region, but was nonetheless capable of overcoming cell cycle block in T64-7B cell and immortalizing primary cultured cells. Cells immortalized with T155c were often highly dependent on the presence of bFGF for growth. Telomerase activity, telomere length, growth rates, and integrity of the p53 gene in cells immortalized with T155c did not change over 100 population doublings in culture, indicating that cells immortalized with T155c were generally stable during long periods of continuous culture. PMID:15629761

  1. Identification of N-terminally truncated pyroglutamate amyloid-β in cholesterol-enriched diet-fed rabbit and AD brain.

    PubMed

    Perez-Garmendia, Roxanna; Hernandez-Zimbron, Luis Fernando; Morales, Miguel Angel; Luna-Muñoz, José; Mena, Raul; Nava-Catorce, Miriam; Acero, Gonzalo; Vasilevko, Vitaly; Viramontes-Pintos, Amparo; Cribbs, David H; Gevorkian, Goar

    2014-01-01

    The main amyloid-β peptide (Aβ) variants detected in the human brain are Aβ1-40 and Aβ1-42; however, a significant proportion of Aβ in Alzheimer's disease (AD) brain also consists of N-terminal truncated/modified species. AβN3(pE), Aβ peptide bearing amino-terminal pyroglutamate at position 3, has been demonstrated to be a major N-truncated/modified constituent of intracellular, extracellular, and vascular Aβ deposits in AD and Down syndrome brain tissue. It has been previously demonstrated that rabbits fed a diet enriched in cholesterol and given water containing trace copper levels developed AD-like pathology including intraneuronal and extracellular Aβ accumulation, tau hyperphosphorylation, vascular inflammation, astrocytosis, microgliosis, reduced levels of acetylcholine, as well as learning deficits and thus, may be used as a non-transgenic animal model of sporadic AD. In the present study, we have demonstrated for the first time the presence of AβN3(pE) in blood vessels in cholesterol-enriched diet-fed rabbit brain. In addition, we detected AβN3(pE) immunoreactivity in all postmortem AD brain samples studied. We believe that our results are potentially important for evaluation of novel therapeutic molecules/strategies targeting Aβ peptides in a suitable non-transgenic animal model. PMID:24240639

  2. Alzheimer's Aβ Peptides with Disease-Associated N-Terminal Modifications: Influence of Isomerisation, Truncation and Mutation on Cu2+ Coordination

    PubMed Central

    Drew, Simon C.; Masters, Colin L.; Barnham, Kevin J.

    2010-01-01

    Background The amyloid-β (Aβ) peptide is the primary component of the extracellular senile plaques characteristic of Alzheimer's disease (AD). The metals hypothesis implicates redox-active copper ions in the pathogenesis of AD and the Cu2+ coordination of various Aβ peptides has been widely studied. A number of disease-associated modifications involving the first 3 residues are known, including isomerisation, mutation, truncation and cyclisation, but are yet to be characterised in detail. In particular, Aβ in plaques contain a significant amount of truncated pyroglutamate species, which appear to correlate with disease progression. Methodology/Principal Findings We previously characterised three Cu2+/Aβ1–16 coordination modes in the physiological pH range that involve the first two residues. Based upon our finding that the carbonyl of Ala2 is a Cu2+ ligand, here we speculate on a hypothetical Cu2+-mediated intramolecular cleavage mechanism as a source of truncations beginning at residue 3. Using EPR spectroscopy and site-specific isotopic labelling, we have also examined four Aβ peptides with biologically relevant N-terminal modifications, Aβ1[isoAsp]–16, Aβ1–16(A2V), Aβ3–16 and Aβ3[pE]–16. The recessive A2V mutation preserved the first coordination sphere of Cu2+/Aβ, but altered the outer coordination sphere. Isomerisation of Asp1 produced a single dominant species involving a stable 5-membered Cu2+ chelate at the amino terminus. The Aβ3–16 and Aβ3[pE]–16 peptides both exhibited an equilibrium between two Cu2+ coordination modes between pH 6–9 with nominally the same first coordination sphere, but with a dramatically different pH dependence arising from differences in H-bonding interactions at the N-terminus. Conclusions/Significance N-terminal modifications significantly influence the Cu2+ coordination of Aβ, which may be critical for alterations in aggregation propensity, redox-activity, resistance to degradation and the generation

  3. N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells.

    PubMed

    Brown, Philip J; Gascoyne, Duncan M; Lyne, Linden; Spearman, Hayley; Felce, Suet Ling; McFadden, Nora; Chakravarty, Probir; Barrans, Sharon; Lynham, Steven; Calado, Dinis P; Ward, Malcolm; Banham, Alison H

    2016-07-01

    Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5' non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation. PMID:27056922

  4. N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells

    PubMed Central

    Brown, Philip J.; Gascoyne, Duncan M.; Lyne, Linden; Spearman, Hayley; Felce, Suet Ling; McFadden, Nora; Chakravarty, Probir; Barrans, Sharon; Lynham, Steven; Calado, Dinis P.; Ward, Malcolm; Banham, Alison H.

    2016-01-01

    Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5′ non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation. PMID:27056922

  5. The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin.

    PubMed Central

    Heaphy, S; Williams, J

    1982-01-01

    Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain. Images Fig. 2. Fig. 3. Fig. 4. PMID:6816218

  6. Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment.

    PubMed

    Li, Jie; Mitamura, Toshihide; Fox, Barbara A; Bzik, David J; Horii, Toshihiro

    2002-12-01

    The serine repeat antigen (SERA) of Plasmodium falciparum is a blood stage malaria vaccine candidate. It has been shown that 120 kDa SERA was proteolytically processed into N-terminal 47 kDa fragment (P47), central 56 kDa fragment (P56) that was further converted to 50 kDa (P50), and C-terminal 18 kDa fragment (P18). Here, we have examined the processing of SERA and the localization of its processed fragments by using mouse antibodies directed against recombinant proteins corresponding to different domains of SERA. Western blot analysis showed that all the processing events occurred inside parasitized erythrocytes at the stage just prior to the schizont rupture, that P47 was further processed into two 25 kDa fragments and that the two fragments, which were linked to P18 through disulfide bonds, were associated with the merozoite. In contrast, P50 was completely shed into culture medium and absent from the merozoite. This observation was further supported by the results of indirect immunofluorescence assay. These results could account for the findings that antibodies against P47 were inhibitory to the parasite growth in vitro but those against P50 were not. Finally, we demonstrated that the further processing of P47 is allelic type-dependent. The results of the present study would help in vaccine designing based on SERA. PMID:12421632

  7. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.

    PubMed

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  8. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

    PubMed Central

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  9. Striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo.

    PubMed

    Francelle, Laetitia; Galvan, Laurie; Gaillard, Marie-Claude; Petit, Fanny; Bernay, Benoît; Guillermier, Martine; Bonvento, Gilles; Dufour, Noëlle; Elalouf, Jean-Marc; Hantraye, Philippe; Déglon, Nicole; de Chaldée, Michel; Brouillet, Emmanuel

    2015-03-01

    A large number of gene products that are enriched in the striatum have ill-defined functions, although they may have key roles in age-dependent neurodegenerative diseases affecting the striatum, especially Huntington disease (HD). In the present study, we focused on Abhd11os, (called ABHD11-AS1 in human) which is a putative long noncoding RNA (lncRNA) whose expression is enriched in the mouse striatum. We confirm that despite the presence of 2 small open reading frames (ORFs) in its sequence, Abhd11os is not translated into a detectable peptide in living cells. We demonstrate that Abhd11os levels are markedly reduced in different mouse models of HD. We performed in vivo experiments in mice using lentiviral vectors encoding either Abhd11os or a small hairpin RNA targeting Abhd11os. Results show that Abhd11os overexpression produces neuroprotection against an N-terminal fragment of mutant huntingtin, whereas Abhd11os knockdown is protoxic. These novel results indicate that the loss lncRNA Abhd11os likely contribute to striatal vulnerability in HD. Our study emphasizes that lncRNA may play crucial roles in neurodegenerative diseases. PMID:25619660

  10. A CaV2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant.

    PubMed

    Dahimene, Shehrazade; Page, Karen M; Nieto-Rostro, Manuela; Pratt, Wendy S; D'Arco, Marianna; Dolphin, Annette C

    2016-09-01

    Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide. PMID:27260834

  11. Enhancing the secretion efficiency and thermostability of a Bacillus deramificans pullulanase mutant (D437H/D503Y) by N-terminal domain truncation.

    PubMed

    Duan, Xuguo; Wu, Jing

    2015-03-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 10(8) and 449.3 × 10(8) U · mol(-1), respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. PMID:25556190

  12. Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

    PubMed Central

    Duan, Xuguo

    2015-01-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 108 and 449.3 × 108 U · mol−1, respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. PMID:25556190

  13. Atomistic mechanisms of huntingtin N-terminal fragment insertion on a phospholipid bilayer revealed by molecular dynamics simulations.

    PubMed

    Côté, Sébastien; Wei, Guanghong; Mousseau, Normand

    2014-07-01

    The huntingtin protein is characterized by a segment of consecutive glutamines (Q(N)) that is responsible for its fibrillation. As with other amyloid proteins, misfolding of huntingtin is related to Huntington's disease through pathways that can involve interactions with phospholipid membranes. Experimental results suggest that the N-terminal 17-amino-acid sequence (htt(NT)) positioned just before the Q(N) region is important for the binding of huntingtin to membranes. Through all-atom explicit solvent molecular dynamics simulations, we unveil the structure and dynamics of the htt(NT)Q(N) fragment on a phospholipid membrane at the atomic level. We observe that the insertion dynamics of this peptide can be described by four main steps-approach, reorganization, anchoring, and insertion-that are very diverse at the atomic level. On the membrane, the htt(NT) peptide forms a stable α-helix essentially parallel to the membrane with its nonpolar side-chains-mainly Leu-4, Leu-7, Phe-11 and Leu-14-positioned in the hydrophobic core of the membrane. Salt-bridges involving Glu-5, Glu-12, Lys-6, and Lys-15, as well as hydrogen bonds involving Thr-3 and Ser-13 with the phospholipids also stabilize the structure and orientation of the htt(NT) peptide. These observations do not significantly change upon adding the Q(N) region whose role is rather to provide, through its hydrogen bonds with the phospholipids' head group, a stable scaffold facilitating the partitioning of the htt(NT) region in the membrane. Moreover, by staying accessible to the solvent, the amyloidogenic Q(N) region could also play a key role for the oligomerization of htt(NT)Q(N) on phospholipid membranes. PMID:24415136

  14. Crystallization and preliminary X-ray crystallographic analysis of a 40 kDa N-terminal fragment of the yeast prion-remodeling factor Hsp104

    SciTech Connect

    Lee, Sukyeong; Tsai, Francis T. F.

    2007-09-01

    An N-terminal fragment of S. cerevisiae Hsp104 has been crystallized. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones. A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 Å. Native 2 diffracted to 2.9 Å resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 Å. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.

  15. A Role for Galanin N-Terminal Fragment (1–15) in Anxiety- and Depression-Related Behaviors in Rats

    PubMed Central

    Millón, Carmelo; Flores-Burgess, Antonio; Narváez, Manuel; Borroto-Escuela, Dasiel O.; Santín, Luis; Parrado, Concepción; Narváez, José Angel; Fuxe, Kjell

    2015-01-01

    Background: Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1–15)] in anxiety- and depression-related behavioral tests in rats. Methods: The effect of GAL(1–15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1–15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1–15) were also studied in the cell line RN33B. Results: GAL(1–15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1–15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1–15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1–15) decreased 5-HT immunoreactivity more strongly than GAL. Conclusions: Our results indicate that GAL(1–15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety. PMID:25522404

  16. Purification and antimicrobial activity studies of the N-terminal fragment of ubiquitin from human amniotic fluid.

    PubMed

    Kim, Jin-Young; Lee, Sun Young; Park, Seong-Cheol; Shin, Song Yub; Choi, Sang Joon; Park, Yoonkyung; Hahm, Kyung-Soo

    2007-09-01

    A 4.3-kDa antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This peptide, which we named Amniotic Fluid Peptide-1 (AFP-1), possessed antimicrobial activity but lacked hemolytic activity. In addition, AFP-1 potently inhibited the growth of a variety of bacteria (Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus), filamentous fungi (Botrytis cinerea, Aspergillus fumigatus, Neurospora crassa and Fusarium oxysporum) and yeast cells (Candida albicans and Cryptococcus neoformans). Automated Edman degradation showed that the N-terminal sequence of AFP-1 was NH(2)-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-. The partial sequence had 100% homology to the N-terminal sequence of ubiquitin. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the molecular mass of AFP-1 was 4280.2 Da. Our data show an antimicrobial activity of ubiquitin N-terminal derived peptide that makes it suitable for use as an antimicrobial agent. PMID:17669700

  17. Beta amyloid-induced upregulation of death receptor 6 accelerates the toxic effect of N-terminal fragment of amyloid precursor protein.

    PubMed

    Xu, Yuxia; Wang, Dandan; Luo, Ying; Li, Wei; Shan, Ye; Tan, Xiangshi; Zhu, Cuiqing

    2015-01-01

    Amyloid precursor protein (APP) plays essential roles in the development of the Alzheimer's disease. Although full-length APP has been thoroughly studied, the role of the cleavage fragments especially the N-terminal fragments (N-APPs) in Alzheimer's disease pathogenesis was still elusive. In this study, we demonstrated that application of recombinant APP₁₈₋₂₈₆ could enhance beta amyloid (Aβ)-induced neuronal injuries which were related to the activation of apoptosis proteins. Aβ treatment could induce a slight increase of N-APPs release. In addition, expression of death receptor 6 (DR6) was increased in Aβ-treated neurons and APP transgenic mice. Moreover, the effect of APP₁₈₋₂₈₆ on Aβ-induced injuries could be suppressed by the application of recombinant DR6₄₁₋₃₄₁ and DR6 antibody. Furthermore, pull-down assay revealed that APP₁₈₋₂₈₆ could bind both exogenous and endogenous DR6. Aβ promoted APP₁₈₋₂₈₆ targeting to neuron which was accompanied with the increase of DR6 expression, whereas downregulation of DR6 by interference RNA could alleviate the binding of N-APPs to neuron and also suppressed Aβ-dependent toxic effect with N-APPs. These results suggested that APP N-terminal fragments might play neurotoxic roles in Aβ-induced neuronal injuries through cell surface DR6. PMID:25150572

  18. Expression of 4 truncated fragments of Pasteurella multocida toxin and their immunogenicity

    PubMed Central

    Seo, Jayoung; Pyo, Hyoju; Lee, Semi; Lee, Jaeil; Kim, Taejung

    2009-01-01

    Pasteurella multocida toxin (PMT) is a poor antigen that becomes more immunogenic after its native structure has been destroyed. In contrast, partially truncated PMT proteins, which are predicted to be good antigens when used as a vaccine, might be used to improve the control of atrophic rhinitis in pigs. In this study, 4 truncated PMT fragments were expressed in Escherichia coli, and those 4 fragments were inoculated into mice to produce the polyclonal antibodies. The results of an enzyme-linked immunosorbent assay (ELISA) revealed that #1 and #4 fragments were the most immunogenic. Immunized mice were subsequently challenged intraperitoneally with P. multocida type D. Five of the eight #1 fragment-immunized mice showed some protection against death and bacterial clearance. Pigs immunized with #1 fragment produced no or mild atrophic rhinitis (turbinate conchal score) after challenge, suggesting that this #1 fragment could be a good candidate for a subunit recombinant-type vaccine. PMID:19794890

  19. Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980.

    PubMed Central

    Egelseer, E M; Schocher, I; Sleytr, U B; Sára, M

    1996-01-01

    During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half

  20. N-terminal peptide sequence repetition influences the kinetics of backbone fragmentation: a manifestation of the Jahn-Teller effect?

    PubMed

    Good, David M; Yang, Hongqian; Zubarev, Roman A

    2013-11-01

    Analysis of large (>10,000 entries) databases consisting of high-resolution tandem mass spectra of peptide dications revealed with high statistical significance (P < 1[Symbol: see text]10(-3)) that peptides with non-identical first two N-terminal amino acids undergo cleavages of the second peptide bond at higher rates than repetitive sequences composed of the same amino acids (i.e., in general AB- and BA- bonds cleave more often than AA- and BB- bonds). This effect seems to depend upon the collisional energy, being stronger at lower energies. The phenomenon is likely to indicate the presence of the diketopiperazine structure for at least some b2 (+) ions. When consisting of two identical amino acids, these species should form through intermediates that have a symmetric geometry and, thus, must be subject to the Jahn-Teller effect that reduces the stability of such systems. PMID:23633015

  1. Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A

    PubMed Central

    Huang, Jui-Hsin; Shen, Zhe-Qing; Lien, Shu-Pei; Hsiao, Kuang-Nan; Leng, Chih-Hsiang; Chen, Chi-Chang; Siu, Leung-Kei; Chong, Pele Choi-Sing

    2015-01-01

    Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels

  2. Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A.

    PubMed

    Huang, Jui-Hsin; Shen, Zhe-Qing; Lien, Shu-Pei; Hsiao, Kuang-Nan; Leng, Chih-Hsiang; Chen, Chi-Chang; Siu, Leung-Kei; Chong, Pele Choi-Sing

    2015-01-01

    Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1-411), middle (residues 296-701), and C-terminal portions (residues 524-911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of

  3. Supramaximal elevation in B-type natriuretic peptide and its N-terminal fragment levels in anephric patients with heart failure: a case series

    PubMed Central

    2012-01-01

    Introduction Little is known about the responses of natriuretic peptides to developing congestive heart failure in ‘anephric’ end-stage kidney disease. Case presentation We present three consecutive cases of surgically-induced anephric patients in a critical care environment: a 28-year-old Caucasian woman (with congestive heart failure), a 42-year-old Caucasian woman (without congestive heart failure), and a 23-year-old Caucasian woman (without congestive heart failure). Our limited study data indicate that cut-off values advocated for B-type natriuretic peptide and its N-terminal fragment to ‘rule out’ congestive heart failure in two of our end-stage kidney disease patients (without congestive heart failure) are largely appropriate for anephric patients. However, our index (first) patient developed congestive heart failure accompanied by the phenomenon of massive and persistent elevation of these natriuretic levels. Conclusion Our findings suggest that patients from the anephric subclass suffering from congestive heart failure will develop supramaximal elevation of B-type natriuretic peptide and its N-terminal fragment, implying the need for dramatically higher cut-off values with respective magnitudes of the order of 50-fold (B-type natriuretic peptide ~5780pmol/L; 20,000ng/L) to 100-fold (N-terminal fragment ~11,800pmol/L; 100,000ng/L) higher than current values used to ‘rule in’ congestive heart failure. Further research will be required to delineate those cut-off values. The role of our devised ‘Blood Volume – B-type natriuretic peptide feedback control system’ on ‘anatomical’ and ‘functional’ anephric patients led to significant mathematically-enriched arguments supporting our proposal that this model provides plausible explanations for the study findings, and the model lends support to the important hypothesis that these two groups of anephric patients inflicted with congestive heart failure should effectively have similar

  4. Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 human interleukin-8 receptor.

    PubMed

    Clubb, R T; Omichinski, J G; Clore, G M; Gronenborn, A M

    1994-01-24

    Interleukin-8 and its receptors are key mediators of immune and inflammatory responses. Heteronuclear NMR spectroscopy has been utilized to map the binding surface on interleukin-8 (IL-8) for an N-terminal fragment of the human Type-1 IL-8 receptor. A peptide corresponding to residues 1-40 of the IL-8 type 1 receptor (IL8-r1) was titrated into a sample of uniformly 15N-labeled IL-8. IL8-r1 binds to IL-8 with a dissociation constant of 170 +/- 50 microM assuming the peptide binds with a stoichiometry of one peptide per IL-8 monomer, exchanges rapidly (> 900 s-1) between free and bound states, and selectively perturbs the chemical environment of several IL-8 residues. The binding surface on IL-8 suggested by our results is comprised of residues in strand beta 3 of the beta-sheet (Glu48 to Cys50), the turn preceding beta 3 (Ser44), the C-terminal alpha-helix (Val61) and the irregular N-terminal loop region (Thr12, Lys15, Phe17, His18, Lys20 and Phe21). The IL-8 dimer appears to present two symmetrical binding surfaces for the IL8-r1 peptide, suggesting two receptor peptides may bind per dimer. PMID:8307164

  5. Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104

    SciTech Connect

    Lee,S.; Tsai, F.

    2007-01-01

    A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 {angstrom} resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 {angstrom}. Native 2 diffracted to 2.9 {angstrom} resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 {angstrom}. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.

  6. Autocrine regulation of growth stimulation in human epithelial ovarian carcinoma by serine-proteinase-catalysed release of the urinary-type-plasminogen-activator N-terminal fragment.

    PubMed Central

    Fishman, D A; Kearns, A; Larsh, S; Enghild, J J; Stack, M S

    1999-01-01

    Ovarian carcinomas secrete single-chain urinary-type plasminogen activator (scuPA) and expression of uPA is up-regulated relative to normal ovarian epithelium, leading to an enhanced proteolytic capacity which may facilitate invasion. Furthermore, the uPA receptor (uPAR) is present on ovarian carcinoma cells and is occupied in tumour tissues. In the present study, incubation of scuPA with serum-free conditioned medium from ovarian carcinoma cells resulted in release of a 14 kDa polypeptide. N-terminal sequence analysis identified this fragment as the uPA N-terminal fragment (NTF), which contains a growth-factor and a kringle domain. NTF generation was abolished by serine-proteinase inhibitors, but not inhibitors of matrix metalloproteinases, and was not enhanced by the addition of plasminogen or plasmin. To determine whether ovarian carcinoma-cell growth is altered by uPA, the effect of exogenous scuPA or NTF on proliferation was analysed. Both NTF and scuPA induced a dose-dependent increase in proliferation, with maximal stimulation obtained at 10-20 nM. Furthermore, blocking the interaction of endogenous uPA with uPAR using anti-NTF antibodies significantly inhibited proliferation. Together these data indicate that, in addition to enhancing the invasive activity of ovarian carcinoma cells via increased pericellular proteolysis, uPA also acts as a mitogen for ovarian carcinoma cells, suggesting a biochemical mechanism whereby uPA may contribute to ovarian carcinoma progression by modulating both cell invasion and proliferation. PMID:10417342

  7. Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

    PubMed Central

    Magrì, Antonio; Munzone, Alessia; Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta; Hansson, Orjan; Satriano, Cristina; Rizzarelli, Enrico; La Mendola, Diego

    2016-01-01

    Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides. PMID:27490533

  8. Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

    PubMed Central

    Xu, Jun-Wei; Xu, Yi-Ning

    2012-01-01

    Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway. PMID:22941092

  9. Massive CA1/2 Neuronal Loss with Intraneuronal and N-Terminal Truncated Aβ42 Accumulation in a Novel Alzheimer Transgenic Model

    PubMed Central

    Casas, Caty; Sergeant, Nicolas; Itier, Jean-Michel; Blanchard, Véronique; Wirths, Oliver; van der Kolk, Nicolien; Vingtdeux, Valérie; van de Steeg, Evita; Ret, Gwenaëlle; Canton, Thierry; Drobecq, Hervé; Clark, Allan; Bonici, Bruno; Delacourte, André; Benavides, Jesús; Schmitz, Christoph; Tremp, Günter; Bayer, Thomas A.; Benoit, Patrick; Pradier, Laurent

    2004-01-01

    Alzheimer’s disease (AD) is characterized by a substantial degeneration of pyramidal neurons and the appearance of neuritic plaques and neurofibrillary tangles. Here we present a novel transgenic mouse model, APPSLPS1KI that closely mimics the development of AD-related neuropathological features including a significant hippocampal neuronal loss. This transgenic mouse model carries M233T/L235P knocked-in mutations in presenilin-1 and overexpresses mutated human β-amyloid (Aβ) precursor protein. Aβx-42 is the major form of Aβ species present in this model with progressive development of a complex pattern of N-truncated variants and dimers, similar to those observed in AD brain. At 10 months of age, an extensive neuronal loss (>50%) is present in the CA1/2 hippocampal pyramidal cell layer that correlates with strong accumulation of intraneuronal Aβ and thioflavine-S-positive intracellular material but not with extracellular Aβ deposits. A strong reactive astrogliosis develops together with the neuronal loss. This loss is already detectable at 6 months of age and is PS1KI gene dosage-dependent. Thus, APPSLPS1KI mice further confirm the critical role of intraneuronal Aβ42 in neuronal loss and provide an excellent tool to investigate therapeutic strategies designed to prevent AD neurodegeneration. PMID:15466394

  10. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  11. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    NASA Astrophysics Data System (ADS)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-01

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  12. Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II

    PubMed Central

    Benchoua, Alexandra; Trioulier, Yaël; Diguet, Elsa; Malgorn, Carole; Gaillard, Marie-Claude; Dufour, Noelle; Elalouf, Jean-Marc; Krajewski, Stan; Hantraye, Philippe; Déglon, Nicole; Brouillet, Emmanuel

    2008-01-01

    In neurodegenerative disorders associated with primary or secondary mitochondrial defects such as Huntington's disease (HD), cells of the striatum are particularly vulnerable to cell death, although the mechanisms by which this cell death is induced are unclear. Dopamine, found in high concentrations in the striatum, may play a role in striatal cell death. We show that in primary striatal cultures, dopamine increases the toxicity of an N-terminal fragment of mutated huntingtin (Htt-171-82Q). Mitochondrial complex II protein (mCII) levels are reduced in HD striatum, indicating that this protein may be important for dopamine-mediated striatal cell death. We found that dopamine enhances the toxicity of the selective mCII inhibitor, 3-nitropropionic acid. We also demonstrated that dopamine doses that are insufficient to produce cell loss regulate mCII expression at the mRNA, protein and catalytic activity level. We also show that dopamine-induced down-regulation of mCII levels can be blocked by several dopamine D2 receptor antagonists. Sustained overexpression of mCII subunits using lentiviral vectors abrogated the effects of dopamine, both by high dopamine concentrations alone and neuronal death induced by low dopamine concentrations together with Htt-171-82Q. This novel pathway links dopamine signaling and regulation of mCII activity and could play a key role in oxidative energy metabolism and explain the vulnerability of the striatum in neurodegenerative diseases. PMID:18267960

  13. The N-terminal fragment of the tomato torrado virus RNA1-encoded polyprotein induces a hypersensitive response (HR)-like reaction in Nicotiana benthamiana.

    PubMed

    Wieczorek, Przemysław; Obrępalska-Stęplowska, Aleksandra

    2016-07-01

    The hypersensitive response (HR) is a defence reaction observed during incompatible plant-pathogen interactions in plants infected with a wide range of fungi, bacteria and viruses. Here, we show that an N-terminal polyprotein fragment encoded by tomato torrado virus RNA1, located between the first ATG codon and the protease cofactor (ProCo) motif, induces an HR-like reaction in Nicotiana benthamiana. Agrobacterium tumefaciens-mediated transient expression of the first 105 amino acids (the calculated molecular weight of the fragment was ca. 11.33 kDa, hereafter refered to as the 11K domain) from ToTV RNA1 induced an HR-like phenotype in infiltrated leaves. To investigate whether the 11K domain could influence the virulence and pathogenicity of a recombinant virus, we created a potato virus X (PVX) with the 11K coding sequence inserted under a duplicated coat protein promoter. We found that 11K substantially increased the virulence of the recombinant virus. Disease phenotype induced in N. benthamiana by PVX-11K was characterized by strong local and systemic necrosis. This was not observed when the 11K domain was expressed from PVX in an antisense orientation. Further analyses revealed that the 11K domain could not suppress posttranscriptional gene silencing (PTGS) of green fluorescent protein (GFP) in the N. benthamiana 16c line. In silico analysis of the predicted secondary structure of the 11K domain indicated the presence of two putative helices that are highly conserved in tomato-infecting representatives of the genus Torradovirus. PMID:27072852

  14. Soluble prion protein and its N-terminal fragment prevent impairment of synaptic plasticity by Aβ oligomers: Implications for novel therapeutic strategy in Alzheimer's disease.

    PubMed

    Scott-McKean, Jonah J; Surewicz, Krystyna; Choi, Jin-Kyu; Ruffin, Vernon A; Salameh, Ahlam I; Nieznanski, Krzysztof; Costa, Alberto C S; Surewicz, Witold K

    2016-07-01

    The pathogenic process in Alzheimer's disease (AD) appears to be closely linked to the neurotoxic action of amyloid-β (Aβ) oligomers. Recent studies have shown that these oligomers bind with high affinity to the membrane-anchored cellular prion protein (PrP(C)). It has also been proposed that this binding might mediate some of the toxic effects of the oligomers. Here, we show that the soluble (membrane anchor-free) recombinant human prion protein (rPrP) and its N-terminal fragment N1 block Aβ oligomers-induced inhibition of long-term potentiation (LTP) in hippocampal slices, an important surrogate marker of cognitive deficit associated with AD. rPrP and N1 are also strikingly potent inhibitors of Aβ cytotoxicity in primary hippocampal neurons. Furthermore, experiments using hippocampal slices and neurons from wild-type and PrP(C) null mice (as well as rat neurons in which PrP(C) expression was greatly reduced by gene silencing) indicate that, in contrast to the impairment of synaptic plasticity by Aβ oligomers, the cytotoxic effects of these oligomers, and the inhibition of these effects by rPrP and N1, are independent of the presence of endogenous PrP(C). This suggests fundamentally different mechanisms by which soluble rPrP and its fragments inhibit these two toxic responses to Aβ. Overall, these findings provide strong support to recent suggestions that PrP-based compounds may offer new avenues for pharmacological intervention in AD. PMID:26949218

  15. Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative, anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights.

    PubMed

    Chamani, Reyhane; Asghari, S Mohsen; Alizadeh, Ali Mohammad; Eskandari, Sedigheh; Mansouri, Kamran; Khodarahmi, Reza; Taghdir, Majid; Heidari, Zahra; Gorji, Ali; Aliakbar, Alireza; Ranjbar, Bijan; Khajeh, Khosro

    2015-09-01

    Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and β structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to αVβ3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth. PMID:26187352

  16. Dynorphin 1-17 and Its N-Terminal Biotransformation Fragments Modulate Lipopolysaccharide-Stimulated Nuclear Factor-kappa B Nuclear Translocation, Interleukin-1beta and Tumor Necrosis Factor-alpha in Differentiated THP-1 Cells

    PubMed Central

    2016-01-01

    Dynorphin 1–17, (DYN 1–17) opioid peptide produces antinociception following binding to the kappa-opioid peptide (KOP) receptor. Upon synthesis and release in inflamed tissues by immune cells, DYN 1–17 undergoes rapid biotransformation and yields a unique set of opioid and non-opioid fragments. Some of these major fragments possess a role in immunomodulation, suggesting that opioid-targeted therapeutics may be effective in diminishing the severity of inflammatory disorders. This study aimed to examine the immunomodulatory effects of DYN 1–17 and major N-terminal fragments found in the inflammatory environment on nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) from lipopolysaccharide (LPS)-stimulated, differentiated THP-1 cells. The results demonstrate that NF-κB/p65 nuclear translocation was significantly attenuated following treatment with DYN 1–17 and a specific range of fragments, with the greatest reduction observed with DYN 1–7 at a low concentration (10 nM). Antagonism with a selective KOP receptor antagonist, ML-190, significantly reversed the inhibitory effects of DYN 1–17, DYN 1–6, DYN 1–7 and DYN 1–9, but not other DYN 1–17 N-terminal fragments (DYN 1–10 and 1–11) on NF-κB/p65 nuclear translocation. DYN 1–17 and selected fragments demonstrated differential modulation on the release of IL-1β and TNF-α with significant inhibition observed with DYN 1–7 at low concentrations (1 nM and 10 pM). These effects were blocked by ML-190, suggesting a KOP receptor-mediated pathway. The results demonstrate that DYN 1–17 and certain N-terminal fragments, produced in an inflamed environment, play an anti-inflammatory role by inhibiting NF-κB/p65 translocation and the subsequent cytokine release through KOP receptor-dependent and independent pathways. PMID:27055013

  17. N-terminal sequence tagging using reliably determined b2 ions: a useful approach to deconvolute tandem mass spectra of co-fragmented peptides in proteomics.

    PubMed

    Kryuchkov, Fedor; Verano-Braga, Thiago; Kjeldsen, Frank

    2014-05-30

    With the recent introduction of higher-energy collisional dissociation (HCD) in Orbitrap mass spectrometry, the popularity of that technique has grown tremendously in the proteomics society. HCD spectra, however, are characterized by a limited distribution of bn-type ions, which permit the generation of reliable sequence tags based on complementary b,y pairs both for de novo sequencing and sequence tagging strategies. Instead, most peptide HCD spectra (~95%) are dominated with b2 ions. In this work, we analyzed positive predictive values of b2 ions in HCD, and found that b2 ions can be determined with >97% certainty in the presence of a2 and its complementary yn-2 ions. Analytically, b2 ions provide information on the composition of the first two N-terminal amino acids in peptides. Their utilization in N-terminal sequence tagging leads to a significant decrease in false discovery rate by filtering out false positives while retaining true positive identifications. As a consequence, the number of peptide spectrum matches (PSMs) increased by 4.8% at fixed FDR (1%). This approach allows for deconvolution of mixture spectra and increased the number of PSM to 9.2% in a complex human sample and to 24% in a complex sample of synthetic peptides at 1% FDR. PMID:24726481

  18. The N-terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRS(apm)) is a monomer in solution.

    PubMed

    Choudhury, Aparajita; Banerjee, Rajat

    2013-03-18

    Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRSapm) was the first reported aminoacyl-tRNA synthetase of viral origin. The previous crystal structure of TyrRSapm showed a non-canonical orientation of the dimer conformation and the CP1 domain, responsible for dimer formation, displays a major modification of a motif structurally conserved in other TyrRS structures. An earlier study reported that Bacillus stearothermophilus N-terminal TyrRS exists as a dimer under native conditions. N-terminal TyrRSapm (ΔTyrRSapm, 1-234 aa) was constructed to remove the C-terminal anticodon-binding domain. Here we show by Ferguson plot analysis and analytical ultracentrifugation that ΔTyrRSapm exists as a monomer and contains a disulfide-bridge. The ΔTyrRSapm loses the ability to bind tRNA(Tyr), however it remains active in pyrophosphate exchange with similar ligand dissociation constants as the full-length enzyme. PMID:23384724

  19. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  20. Enhancement of HCV polytope DNA vaccine efficacy by fusion to an N-terminal fragment of heat shock protein gp96.

    PubMed

    Pishraft-Sabet, Leila; Kosinska, Anna D; Rafati, Sima; Bolhassani, Azam; Taheri, Tahereh; Memarnejadian, Arash; Alavian, Seyed-Moayed; Roggendorf, Michael; Samimi-Rad, Katayoun

    2015-01-01

    Induction of a strong hepatitis C virus (HCV)-specific immune response plays a key role in control and clearance of the virus. A polytope (PT) DNA vaccine containing B- and T-cell epitopes could be a promising vaccination strategy against HCV, but its efficacy needs to be improved. The N-terminal domain of heat shock protein gp96 (NT(gp96)) has been shown to be a potent adjuvant for enhancing immunity. We constructed a PT DNA vaccine encoding four HCV immunodominant cytotoxic T lymphocyte epitopes (two HLA-A2- and two H2-D(d)-specific motifs) from the Core, E2, NS3 and NS5B antigens in addition to a T-helper CD4+ epitope from NS3 and a B-cell epitope from E2. The NT(gp96) was fused to the C- or N-terminal end of the PT DNA (PT-NT(gp96) or NT(gp96)-PT), and their potency was compared. Cellular and humoral immune responses against the expressed peptides were evaluated in CB6F1 mice. Our results showed that immunization of mice with PT DNA vaccine fused to NT(gp96) induced significantly stronger T-cell and antibody responses than PT DNA alone. Furthermore, the adjuvant activity of NT(gp96) was more efficient in the induction of immune responses when fused to the C-terminal end of the HCV DNA polytope. In conclusion, the NT(gp96) improved the efficacy of the DNA vaccine, and this immunomodulatory effect was dependent on the position of the fusion. PMID:25348271

  1. The N-terminal half of membrane CD14 is a functional cellular lipopolysaccharide receptor.

    PubMed Central

    Viriyakosol, S; Kirkland, T N

    1996-01-01

    CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). It was recently reported that an N-terminal 152-amino-acid fragment of soluble CD14 was an active soluble lipopolysaccharide receptor (T. S. -C. Juan, M. J. Kelley, D. A. Johnson, L. A. Busse, E. Hailman, S. D. Wright, and H. S. Lichenstein, J. Biol. Chem. 270:1382-1387, 1995). To determine whether the N-terminal half of the membrane CD14 was a functional LPS receptor on the cell membrane, we engineered a chimeric gene coding for amino acids 1 to 151 of CD14 fused to the C-terminal region of decay-accelerating factor and expressed it in Chinese hamster ovary cells and 70Z/3 cells. We found that the chimeric, truncated CD14 is a fully functional LPS receptor in both cell lines. PMID:8550221

  2. Plasma Levels of Monocyte Chemoattractant Protein-1, n-Terminal Fragment of Brain Natriuretic Peptide and Calcidiol Are Independently Associated with the Complexity of Coronary Artery Disease

    PubMed Central

    Martín-Reyes, Roberto; Franco-Peláez, Juan Antonio; Lorenzo, Óscar; González-Casaus, María Luisa; Pello, Ana María; Aceña, Álvaro; Carda, Rocío; Martín-Ventura, José Luis; Blanco-Colio, Luis; Martín-Mariscal, María Luisa; Martínez-Milla, Juan; Villa-Bellosta, Ricardo; Piñero, Antonio; Navarro, Felipe; Egido, Jesús; Tuñón, José

    2016-01-01

    Background and Objectives We investigated the relationship of the Syntax Score (SS) and coronary artery calcification (CAC), with plasma levels of biomarkers related to cardiovascular damage and mineral metabolism, as there is sparse information in this field. Methods We studied 270 patients with coronary disease that had an acute coronary syndrome (ACS) six months before. Calcidiol, fibroblast growth factor-23, parathormone, phosphate and monocyte chemoattractant protein-1 [MCP-1], high-sensitivity C-reactive protein, galectin-3, and N-terminal pro-brain natriuretic peptide [NT-proBNP] levels, among other biomarkers, were determined. CAC was assessed by coronary angiogram as low-grade (0–1) and high-grade (2–3) calcification, measured with a semiquantitative scale ranging from 0 (none) to 3 (severe). For the SS study patients were divided in SS<14 and SS≥14. Multivariate linear and logistic regression analyses were performed. Results MCP-1 predicted independently the SS (RC = 1.73 [95%CI = 0.08–3.39]; p = 0.040), along with NT-proBNP (RC = 0.17 [95%CI = 0.05–0.28]; p = 0.004), male sex (RC = 4.15 [95%CI = 1.47–6.83]; p = 0.003), age (RC = 0.13 [95%CI = 0.02–0.24]; p = 0.020), hypertension (RC = 3.64, [95%CI = 0.77–6.50]; p = 0.013), hyperlipidemia (RC = 2.78, [95%CI = 0.28–5.29]; p = 0.030), and statins (RC = 6.12 [95%CI = 1.28–10.96]; p = 0.013). Low calcidiol predicted high-grade calcification independently (OR = 0.57 [95% CI = 0.36–0.90]; p = 0.013) along with ST-elevation myocardial infarction (OR = 0.38 [95%CI = 0.19–0.78]; p = 0.006), diabetes (OR = 2.35 [95%CI = 1.11–4.98]; p = 0.028) and age (OR = 1.37 [95%CI = 1.18–1.59]; p<0.001). During follow-up (1.79 [0.94–2.86] years), 27 patients developed ACS, stroke, or transient ischemic attack. A combined score using SS and CAC predicted independently the development of the outcome. Conclusions MCP-1 and NT-proBNP are independent predictors of SS, while low calcidiol plasma levels

  3. An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

    PubMed Central

    2016-01-01

    Huntington's disease (HD) is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73) or normal huntingtin (nHtt, Q23), we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington's disease. PMID:27595037

  4. An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model.

    PubMed

    Amaro, I Alexandra; Henderson, Lee A

    2016-01-01

    Huntington's disease (HD) is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73) or normal huntingtin (nHtt, Q23), we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington's disease. PMID:27595037

  5. Evaluation of the Naturally Acquired Antibody Immune Response to the Pv200L N-terminal Fragment of Plasmodium vivax Merozoite Surface Protein-1 in Four Areas of the Amazon Region of Brazil

    PubMed Central

    Storti-Melo, Luciane M.; Souza-Neiras, Wanessa C.; Cassiano, Gustavo C.; Taveira, Leonardo C.; Cordeiro, Antônio J.; Couto, Vanja S. C. A.; Póvoa, Marinete M.; Cunha, Maristela G.; Echeverry, Diana M.; Rossit, Andréa R. B.; Arévalo-Herrera, Myriam; Herrera, Sócrates; Machado, Ricardo L. D.

    2011-01-01

    Frequency and levels of IgG antibodies to an N-terminal fragment of the Plasmodium vivax MSP-1 (Pv200L) protein, in individuals naturally exposed to malaria in four endemic areas of Brazil, were evaluated by enzyme-linked immunosorbent assay. Plasma samples of 261 P. vivax-infected individuals from communities of Macapá, Novo Repartimento, Porto Velho, and Plácido de Castro in the Amazonian region with different malaria transmission intensities. A high mean number of studied individuals (89.3%) presented with antibodies to the Pv200L that correlated with the number of previous malaria infections; there were significant differences in the frequency of the responders (71.9–98.7) and in the antibody levels (1:200–1:51,200) among the four study areas. Results of this study provide evidence that Pv200L is a naturally immunogenic fragment of the PvMSP-1 and is associated with the degree of exposure to parasites. The fine specificity of antibodies to Pv200L is currently being assessed. PMID:21292879

  6. An N-terminal Fragment of the Prion Protein Binds to Amyloid-β Oligomers and Inhibits Their Neurotoxicity in Vivo*

    PubMed Central

    Fluharty, Brian R.; Biasini, Emiliano; Stravalaci, Matteo; Sclip, Alessandra; Diomede, Luisa; Balducci, Claudia; La Vitola, Pietro; Messa, Massimo; Colombo, Laura; Forloni, Gianluigi; Borsello, Tiziana; Gobbi, Marco; Harris, David A.

    2013-01-01

    A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide in the brain. Considerable evidence suggests that soluble Aβ oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aβ oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aβ polymerization into amyloid fibrils. The ability of N1 to bind Aβ oligomers is influenced by positively charged residues in two sites (positions 23–31 and 95–105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aβ oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aβ-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aβ oligomer toxicity and represent an entirely new class of therapeutic agents for AD. PMID:23362282

  7. N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy.

    PubMed

    Liu, Xudong; Wang, Chuan-En; Hong, Yan; Zhao, Ting; Wang, Guohao; Gaertig, Marta A; Sun, Miao; Li, Shihua; Li, Xiao-Jiang

    2016-05-01

    The Huntington's disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1-208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD. PMID:27203582

  8. N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

    PubMed Central

    Liu, Xudong; Wang, Chuan-En; Hong, Yan; Zhao, Ting; Wang, Guohao; Gaertig, Marta A.; Sun, Miao; Li, Shihua; Li, Xiao-Jiang

    2016-01-01

    The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD. PMID:27203582

  9. Incremental truncation of PHA synthases results in altered product specificity.

    PubMed

    Wang, Qian; Xia, Yongzhen; Chen, Quan; Qi, Qingsheng

    2012-05-10

    PHA synthase is the key enzyme involved in the biosynthesis of microbial polymers, polyhydroxyalkanoates (PHA). In this study, we created a hybrid library of PHA synthase gene with different crossover points by an incremental truncation method between the C-terminal fragments of the phaC(Cn) (phaC from Cupriavidus necator) and the N-terminal fragments of the phaC1(Pa) (phaC from Pseudomonas aeruginosa). As the truncation of the hybrid enzyme increased, the in vivo PHB synthesis ability of the hybrids declined gradually. PHA synthase PhaC(Cn) with a deletion on N-terminal up to 83 amino acid residues showed no synthase activity. While with the removal of up to 270 amino acids from the N-terminus, the activity of the truncated PhaC(Cn) could be complemented by the N-terminus of PhaC1(Pa). Three of the hybrid enzymes W188, W235 and W272 (named by the deleted nucleic acid number) were found to have altered product specificities. PMID:22500895

  10. Truncated PrP(c) in mammalian brain: interspecies variation and location in membrane rafts.

    PubMed

    Laffont-Proust, Isabelle; Hässig, Raymonde; Haïk, Stéphane; Simon, Stéphanie; Grassi, Jacques; Fonta, Caroline; Faucheux, Baptiste A; Moya, Kenneth L

    2006-03-01

    A key molecular event in prion diseases is the conversion of cellular prion protein (PrP(c)) into an abnormal misfolded conformer (PrP(sc)). The PrP(c) N-terminal domain plays a central role in PrP(c) functions and in prion propagation. Because mammalian PrP(c) is found as a full-length and N-terminally truncated form, we examined the presence and amount of PrP(c) C-terminal fragment in the brain of different species. We found important variations between primates and rodents. In addition, our data show that the PrP(c) fragment is present in detergent-resistant raft domains, a membrane domain of critical importance for PrP(c) functions and its conversion into PrP(sc). PMID:16542151

  11. Evaluation of combined B cell specific N-terminal immunogenic domains of LipL21 for diagnosis of leptospirosis.

    PubMed

    Anita, Kumari; Premlatha, Mallela Martha; Kanagavel, Murugesan; Akino Mercy, Charles Solomon; Raja, Veerapandian; Shanmughapriya, Santhanam; Natarajaseenivasan, Kalimuthusamy

    2016-10-01

    Leptospiral outer membrane protein LipL21 and its truncated N-terminal immunogenic region (I-LipL21) were evaluated for diagnosis of leptospirosis. The complete coding sequence of LipL21 nucleotide sequence was subjected to BCPred and VaxiJen analysis for determination of B cell specific immunogenic epitopes. Epitope1 ACS STD TGQ KDA TTV GDG (1.8837), Epitope2 WGG PPE QRN DGK TPR DTN (0.9483), Epitope3 VKG VGV YEC KAT GSG SDP (1.4077) and Epitope4 NEW ECQ CVI YAK FPG GKD (0.4462) were predicted. LipL21 and N-terminal fragment having B-cell specific epitopes with higher VaxiJen score >0.9 as truncated I-LipL21 were cloned independently in pET15b and expressed in Escherichia coli. IgM ELISA and dot blot assay was performed for sera samples collected from Delhi-NCR for leptospiral whole cell lysate (WCL), recombinant LipL21 and I-LipL21. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were found to be 92.5%, 92.8%, 83.3%, and 97% respectively for recombinant I-LipL21 by IgM-ELISA. 11-14.8% increased sensitivity was observed over LipL21 and WCL. The I-LipL21 dot blot assay showed a further increased sensitivity of 3.8% over the IgM-ELISA. Therefore I-LipL21 may be the ideal candidate protein for diagnosis of leptospirosis. PMID:27259643

  12. N-terminal domain of complexin independently activates calcium-triggered fusion.

    PubMed

    Lai, Ying; Choi, Ucheor B; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A; Wang, Austin L; Diao, Jiajie; Brunger, Axel T

    2016-08-01

    Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release. PMID:27444020

  13. N-terminal domain of complexin independently activates calcium-triggered fusion

    PubMed Central

    Lai, Ying; Choi, Ucheor B.; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A.; Wang, Austin L.; Brunger, Axel T.

    2016-01-01

    Complexin activates Ca2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca2+-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca2+-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca2+-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca2+-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca2+-triggered release. PMID:27444020

  14. N-terminal nesprin-2 variants regulate β-catenin signalling.

    PubMed

    Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa; Li, Chen; Porter, Lauren J; Zhou, Can; Gao, Fang; Zhang, Junyi; Rajgor, Dipen; Autore, Flavia; Shanahan, Catherine M; Warren, Derek T

    2016-07-15

    The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. PMID:27321956

  15. The charged region of Hsp90 modulates the function of the N-terminal domain

    PubMed Central

    Scheibel, Thomas; Siegmund, Heiko Ingo; Jaenicke, Rainer; Ganz, Peter; Lilie, Hauke; Buchner, Johannes

    1999-01-01

    Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90. PMID:9990018

  16. N-Terminal Region of the Catalytic Domain of Human N-Myristoyltransferase 1 Acts as an Inhibitory Module

    PubMed Central

    Kumar, Sujeet; Sharma, Rajendra K.

    2015-01-01

    N-myristoyltransferase (NMT) plays critical roles in the modulation of various signaling molecules, however, the regulation of this enzyme in diverse cellular states remains poorly understood. We provide experimental evidence to show for the first time that for the isoform 1 of human NMT (hNMT1), the regulatory roles extend into the catalytic core. In our present study, we expressed, purified, and characterized a truncation mutant devoid of 28 N-terminal amino acids from the catalytic module (Δ28-hNMT1s) and compared its properties to the full-length catalytic domain of hNMT1. The deletion of the N-terminal peptide had no effect on the enzyme stability. Our findings suggest that the N-terminal region in the catalytic module of hNMT1 functions serves as a regulatory control element. The observations of an ~3 fold increase in enzymatic efficiency following removal of the N-terminal peptide of hNMT1s indicates that N-terminal amino acids acts as an inhibitory segment and negatively regulate the enzyme activity. Our findings that the N-terminal region confers control over activity, taken together with the earlier observations that the N-terminal of hNMT1 is differentially processed in diverse cellular states, suggests that the proteolytic processing of the peptide segment containing the inhibitory region provides a molecular mechanism for physiological up-regulation of myristoyltransferase activity. PMID:26000639

  17. Oxidation of the N-terminal methionine of lens alpha-A crystallin

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Horwitz, J.; Emmons, T.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Antiserum against the N-terminal peptide of bovine alpha-A crystallin has been used to monitor purification of two different seropositive peptides (i.e. T1a and T1b) from a tryptic digest of bovine lens proteins. Both these peptides have similar amino acid compositions, but peptide T1b has a molecular weight 16 atomic mass units larger than T1a, suggesting posttranslational modification. Analysis of ionization fragments of the T1b peptide by mass spectrometry demonstrates that this difference in molecular weight is due to the in vivo oxidation of the N-terminal met residue of the alpha-A crystallin molecule.

  18. Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry.

    PubMed

    Ren, Da; Pipes, Gary D; Hambly, David; Bondarenko, Pavel V; Treuheit, Michael J; Gadgil, Himanshu S

    2009-01-01

    An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues. PMID:18834850

  19. Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

    PubMed

    Edamatsu, M

    2016-01-01

    Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro. PMID:26909973

  20. Protein N-terminal acetyltransferases in cancer.

    PubMed

    Kalvik, T V; Arnesen, T

    2013-01-17

    The human N-terminal acetyltransferases (NATs) catalyze the transfer of acetyl moieties to the N-termini of 80-90% of all human proteins. Six NAT types are present in humans, NatA-NatF, each is composed of specific subunits and each acetylates a set of substrates defined by the N-terminal amino-acid sequence. NATs have been suggested to act as oncoproteins as well as tumor suppressors in human cancers, and NAT expression may be both elevated and decreased in cancer versus non-cancer tissues. Manipulation of NATs in cancer cells induced cell-cycle arrest, apoptosis or autophagy, implying that these enzymes target a variety of pathways. Of particular interest is hNaa10p (human ARD1), the catalytic subunit of the NatA complex, which was coupled to a number of signaling molecules including hypoxia inducible factor-1α, β-catenin/cyclin D1, TSC2/mammalian target of rapamycin, myosin light chain kinase , DNA methyltransferase1/E-cadherin and p21-activated kinase-interacting exchange factors (PIX)/Cdc42/Rac1. The variety of mechanistic links where hNaa10p acts as a NAT, a lysine acetyltransferase or displaying a non-catalytic role, provide insights to how hNaa10p may act as both a tumor suppressor and oncoprotein. PMID:22391571

  1. Role of N-terminal region of Escherichia coli maltodextrin glucosidase in folding and function of the protein.

    PubMed

    Pastor, Ashutosh; Singh, Amit K; Shukla, Prakash K; Equbal, Md Javed; Malik, Shikha T; Singh, Tej P; Chaudhuri, Tapan K

    2016-09-01

    Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-Terminal region of MalZ. The crystal structure of this enzyme has been reported in the present communication. Based on the crystallographic structure, it has been interpreted that the N-terminal region of the enzyme (Met1-Phe131) might be unstructured or flexible. To understand the role of the N-terminal region of MalZ in its enzymatic activity, and overall stability, a truncated version (Ala111-His616) of MalZ was created. The truncated version failed to fold into an active enzyme both in E. coli cytosol and in vitro even with the assistance of chaperonins GroEL and GroES. Furthermore, the refolding effort of N-truncated MalZ in the presence of isolated N-terminal domain didn't succeed. Our studies suggest that while the structural rigidity or orientation of the N-terminal region of the MalZ protein may not be essential for its stability and function, but the said domain is likely to play an important role in the formation of the native structure of the protein when present as an integral part of the protein. PMID:27317979

  2. The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

    PubMed Central

    GUILLIER, MAUDE; ALLEMAND, FRÉDÉRIC; GRAFFE, MONIQUE; RAIBAUD, SOPHIE; DARDEL, FRÉDÉRIC; SPRINGER, MATHIAS; CHIARUTTINI, CLAUDE

    2005-01-01

    The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control. PMID:15840820

  3. The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal Domains*

    PubMed Central

    Dahlbäck, Madeleine; Jørgensen, Lars M.; Nielsen, Morten A.; Clausen, Thomas M.; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Arnot, David E.; Theander, Thor G.; Salanti, Ali

    2011-01-01

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. PMID:21398524

  4. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

    PubMed

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F; Wahl, Markus C

    2015-12-15

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  5. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

    PubMed Central

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F.; Wahl, Markus C.

    2015-01-01

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  6. Crystal structure of a major fragment of the salt-tolerant glutaminase from Micrococcus luteus K-3

    SciTech Connect

    Yoshimune, Kazuaki . E-mail: k.yoshimune@aist.go.jp; Shirakihara, Yasuo; Shiratori, Aya; Wakayama, Mamoru; Chantawannakul, Panuwan; Moriguchi, Mitsuaki

    2006-08-11

    Glutaminase of Micrococcus luteus K-3 (intact glutaminase; 48 kDa) is digested to a C-terminally truncated fragment (glutaminase fragment; 42 kDa) that shows higher salt tolerance than that of the intact glutaminase. The crystal structure of the glutaminase fragment was determined at 2.4 A resolution using multiple-wavelength anomalous dispersion (MAD). The glutaminase fragment is composed of N-terminal and C-terminal domains, and a putative catalytic serine-lysine dyad (S64 and K67) is located in a cleft of the N-terminal domain. Mutations of the S64 or K67 residues abolished the enzyme activity. The N-terminal domain has abundant glutamic acid residues on its surface, which may explain its salt-tolerant mechanism. A diffraction analysis of the intact glutaminase crystals (a twinning fraction of 0.43) located the glutaminase fragment in the unit cell but failed to turn up clear densities for the missing C-terminal portion of the molecule.

  7. Solid-Phase Synthesis and Characterization of N-Terminally Elongated Aβ-3-x -Peptides.

    PubMed

    Beyer, Isaak; Rezaei-Ghaleh, Nasrollah; Klafki, Hans-Wolfgang; Jahn, Olaf; Haußmann, Ute; Wiltfang, Jens; Zweckstetter, Markus; Knölker, Hans-Joachim

    2016-06-13

    In addition to the prototypic amyloid-β (Aβ) peptides Aβ1-40 and Aβ1-42 , several Aβ variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aβ variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid-phase peptide synthesis of the N-terminally elongated Aβ-peptides Aβ-3-38 , Aβ-3-40 , and Aβ-3-42 . Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aβ-3-38 and Aβ-3-40 are generated by transfected cells even in the presence of a tripartite β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aβ peptides starting at Val(-3) can be separated from N-terminally-truncated Aβ forms by high-resolution isoelectric-focusing techniques, despite virtually identical isoelectric points. The synthetic Aβ variants and the methods presented here are providing tools to advance our understanding of the potential roles of N-terminally elongated Aβ variants in Alzheimer's disease. PMID:27167300

  8. Deriving ribosomal binding site (RBS) statistical models from unannotated DNA sequences and the use of the RBS model for N-terminal prediction.

    PubMed

    Hayes, W S; Borodovsky, M

    1998-01-01

    Accurate prediction of the position of translation initiation (N-terminal prediction) is a difficult problem. N-terminal prediction from DNA sequence alone is ambiguous is several candidate start sites are close to each other. Protein similarity search is usually unable to indicate the true start of a gene as it would require a strong protein sequence similarity at the N-terminal portion of a protein where conservative regions are rarely situated. With the aid of the GeneMark program for gene identification, we extract DNA sequence fragments presumably containing ribosome binding sites (RBS) from unannotated complete genomic sequences. These DNA segments are aligned to generate the RBS model using the Gibbs' sampling method. N-terminal prediction is then performed by using the RBS model in conjunction with the GeneMark start codon prediction to aid in determining the true N-terminal site. PMID:9697189

  9. Caspase-3-mediated Cleavage of Cdc6 Induces Nuclear Localization of p49-truncated Cdc6 and Apoptosis

    PubMed Central

    Yim, Hyungshin; Jin, Ying Hua; Park, Byoung Duck; Choi, Hye Jin; Lee, Seung Ki

    2003-01-01

    We show that Cdc6, an essential initiation factor for DNA replication, undergoes caspase-3–mediated cleavage in the early stages of apoptosis in HeLa cells and SK-HEP-1 cells induced by etoposide, paclitaxel, ginsenoside Rh2, or tumor necrosis factor-related apoptosis-inducing ligand. The cleavage occurs at the SEVD442/G motif and generates an N-terminal truncated Cdc6 fragment (p49-tCdc6) that lacks the carboxy-terminal nuclear export sequence. Cdc6 is known to be phosphorylated by cyclin A-cyclin dependent kinase 2 (Cdk2), an event that promotes its exit from the nucleus and probably blocks it from initiating inappropriate DNA replication. In contrast, p49-tCdc6 translocation to the cytoplasm is markedly reduced under the up-regulated conditions of Cdk2 activity, which is possibly due to the loss of nuclear export sequence. Thus, truncation of Cdc6 results in an increased nuclear retention of p49-tCdc6 that could act as a dominant negative inhibitor of DNA replication and its accumulation in the nucleus could promote apoptosis. Supporting this is that the ectopic expression of p49-tCdc6 not only promotes apoptosis of etoposide-induced HeLa cells but also induces apoptosis in untreated cells. Thus, the caspase-mediated cleavage of Cdc6 creates a truncated Cdc6 fragment that is retained in the nucleus and induces apoptosis. PMID:14517333

  10. Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

    PubMed Central

    Sanford, J C; Pan, Y; Wessling-Resnick, M

    1995-01-01

    Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue. Images PMID:7749197

  11. Fragmentation Patterns and Mechanisms of Singly and Doubly Protonated Peptoids Studied by Collision Induced Dissociation

    NASA Astrophysics Data System (ADS)

    Ren, Jianhua; Tian, Yuan; Hossain, Ekram; Connolly, Michael D.

    2016-04-01

    Peptoids are peptide-mimicking oligomers consisting of N-alkylated glycine units. The fragmentation patterns for six singly and doubly protonated model peptoids were studied via collision-induced dissociation tandem mass spectrometry. The experiments were carried out on a triple quadrupole mass spectrometer with an electrospray ionization source. Both singly and doubly protonated peptoids were found to fragment mainly at the backbone amide bonds to produce peptoid B-type N-terminal fragment ions and Y-type C-terminal fragment ions. However, the relative abundances of B- versus Y-ions were significantly different. The singly protonated peptoids fragmented by producing highly abundant Y-ions and lesser abundant B-ions. The Y-ion formation mechanism was studied through calculating the energetics of truncated peptoid fragment ions using density functional theory and by controlled experiments. The results indicated that Y-ions were likely formed by transferring a proton from the C-H bond of the N-terminal fragments to the secondary amine of the C-terminal fragments. This proton transfer is energetically favored, and is in accord with the observation of abundant Y-ions. The calculations also indicated that doubly protonated peptoids would fragment at an amide bond close to the N-terminus to yield a high abundance of low-mass B-ions and high-mass Y-ions. The results of this study provide further understanding of the mechanisms of peptoid fragmentation and, therefore, are a valuable guide for de novo sequencing of peptoid libraries synthesized via combinatorial chemistry.

  12. Fragmentation Patterns and Mechanisms of Singly and Doubly Protonated Peptoids Studied by Collision Induced Dissociation.

    PubMed

    Ren, Jianhua; Tian, Yuan; Hossain, Ekram; Connolly, Michael D

    2016-04-01

    Peptoids are peptide-mimicking oligomers consisting of N-alkylated glycine units. The fragmentation patterns for six singly and doubly protonated model peptoids were studied via collision-induced dissociation tandem mass spectrometry. The experiments were carried out on a triple quadrupole mass spectrometer with an electrospray ionization source. Both singly and doubly protonated peptoids were found to fragment mainly at the backbone amide bonds to produce peptoid B-type N-terminal fragment ions and Y-type C-terminal fragment ions. However, the relative abundances of B- versus Y-ions were significantly different. The singly protonated peptoids fragmented by producing highly abundant Y-ions and lesser abundant B-ions. The Y-ion formation mechanism was studied through calculating the energetics of truncated peptoid fragment ions using density functional theory and by controlled experiments. The results indicated that Y-ions were likely formed by transferring a proton from the C-H bond of the N-terminal fragments to the secondary amine of the C-terminal fragments. This proton transfer is energetically favored, and is in accord with the observation of abundant Y-ions. The calculations also indicated that doubly protonated peptoids would fragment at an amide bond close to the N-terminus to yield a high abundance of low-mass B-ions and high-mass Y-ions. The results of this study provide further understanding of the mechanisms of peptoid fragmentation and, therefore, are a valuable guide for de novo sequencing of peptoid libraries synthesized via combinatorial chemistry. Graphical Abstract ᅟ. PMID:26832347

  13. Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain

    PubMed Central

    Winkelmann, A; Semtner, M; Meier, J C

    2015-01-01

    Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl− transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases. PMID:26043076

  14. Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain.

    PubMed

    Winkelmann, A; Semtner, M; Meier, J C

    2015-01-01

    Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl(-) transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases. PMID:26043076

  15. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    SciTech Connect

    Pozo-Yauner, Luis del; Wall, Jonathan S.; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L.; Pérez Carreón, Julio I.; and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  16. The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

    PubMed Central

    McGlade, J; Brunkhorst, B; Anderson, D; Mbamalu, G; Settleman, J; Dedhar, S; Rozakis-Adcock, M; Chen, L B; Pawson, T

    1993-01-01

    Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8344248

  17. N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.

    PubMed

    Chang, Elizabeth; Pourmal, Sergei; Zhou, Chun; Kumar, Rupesh; Teplova, Marianna; Pavletich, Nikola P; Marians, Kenneth J; Erdjument-Bromage, Hediye

    2016-07-01

    In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods. PMID:27006647

  18. N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis

    PubMed Central

    Chang, Elizabeth; Pourmal, Sergei; Zhou, Chun; Kumar, Rupesh; Teplova, Marianna; Pavletich, Nikola P.; Marians, Kenneth J.

    2016-01-01

    In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods. PMID:27006647

  19. Emerging Functions for N-Terminal Protein Acetylation in Plants.

    PubMed

    Gibbs, Daniel J

    2015-10-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour. PMID:26319188

  20. Oxidative Folding and N-terminal Cyclization of Onconase+

    PubMed Central

    Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

    2008-01-01

    Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243

  1. N-terminal sequence of some ribosome-inactivating proteins.

    PubMed

    Montecucchi, P C; Lazzarini, A M; Barbieri, L; Stirpe, F; Soria, M; Lappi, D

    1989-04-01

    The N-terminal portion of some type 1 ribosome-inactivating proteins (RIPs) isolated from the seeds of Gelonium multiflorum, Momordica charantia, Bryonia dioica, Saponaria officinalis and from the leaves of Saponaria officinalis are reported in the present paper. Their relationship with other RIPs is discussed. PMID:2753596

  2. Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2

    PubMed Central

    Bauerová-Hlinková, Vladena; Hostinová, Eva; Gašperík, Juraj; Beck, Konrad; Borko, Ľubomír; Lai, F. Anthony; Zahradníková, Alexandra; Ševčík, Jozef

    2010-01-01

    We report the domain analysis of the N-terminal region (residues 1–759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR21–606·His6, RyR2391–606·His6, RyR2409–606·His6, Trx·RyR2384–606·His6, Trx·RyR2391-606·His6 and Trx·RyR2409–606·His6. The folding of RyR21–606·His6 was analyzed by circular dichroism spectroscopy resulting in α-helix and β-sheet content of ∼23% and ∼29%, respectively, at temperatures up to 35 °C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR21–606·His6, resulted in the appearance of two specific subfragments of ∼40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His6·Tag antibody indicated that RyR21–606·His6 is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively. PMID:20045464

  3. N-terminal determinants of human cytomegalovirus IE1 protein in nuclear targeting and disrupting PML-associated subnuclear structures

    SciTech Connect

    Lee, Hye-Ra; Huh, Yong Ho; Kim, Young-Eui; Lee, Karim; Kim, Sunyoung; Ahn, Jin-Hyun . E-mail: jahn@med.skku.ac.kr

    2007-05-04

    The 72-kDa IE1 protein of human cytomegalovirus disrupts PML-associated subnuclear structures (PODs) by inducing PML desumoylation. This process correlates with the functions of IE1 in transcriptional regulation and efficient viral replication. Here, we defined the N-terminal regions of IE1 required for nuclear targeting and POD-disrupting activity. Although the 24 N-terminal amino acids encoded by exon 2, which were previously shown to be essential for nuclear targeting, did not appear to contain typical basic nuclear localization signals, these residues were able to efficiently convey the GFP protein into the nucleus, suggesting a role in promoting nuclear translocation. In assays using a series of N-terminal truncation IE1 mutants, which were forced to enter the nucleus, exon 2 was completely dispensable for POD disruption. However, the predicted two {alpha}-helix regions in exon 3 were identified as important structural determinants for protein stability and for the correlating activities in POD disruption and PML desumoylation.

  4. Unique N-terminal Arm of Mycobacterium tuberculosis PhoP Protein Plays an Unusual Role in Its Regulatory Function*

    PubMed Central

    Das, Arijit Kumar; Kumar, Vijjamarri Anil; Sevalkar, Ritesh Rajesh; Bansal, Roohi; Sarkar, Dibyendu

    2013-01-01

    Mycobacterium tuberculosis PhoP, a master regulator involved in complex lipid biosynthesis and expression of unknown virulence determinants, is composed of an N-terminal receiver domain and a C-terminal effector domain. The two experimentally characterized PhoP orthologs, from Escherichia coli and Salmonella enterica, display vastly different regulatory capabilities. Here, we demonstrate that the 20-residue-long N-terminal arm unique to M. tuberculosis PhoP plays an essential role in the expanded regulatory capabilities of this important regulator. Although the arm is not required for overall structural stability and/or phosphorylation of the PhoP N-domain, strikingly it is essential for phosphorylation-coupled transcription regulation of target genes. Consistent with this view, arm truncation of PhoP is accompanied by a conformational change of the effector domain, presenting a block in activation subsequent to phosphorylation. These results suggest that presence of the arm, unique to this regulator that shares an otherwise highly conserved domain structure with members of the protein family, contributes to the mechanism of inter-domain interactions. Thus, we propose that the N-terminal arm is an adaptable structural feature of M. tuberculosis PhoP, which evolved to fine-tune regulatory capabilities of the transcription factor in response to the changing physiology of the bacilli within its host. PMID:23963455

  5. Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

    PubMed Central

    Borel, F; Vincent, C; Leberman, R; Härtlein, M

    1994-01-01

    Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs. Images PMID:8065908

  6. Neutron Reflectometry Studies Define Prion Protein N-terminal Peptide Membrane Binding

    PubMed Central

    Le Brun, Anton P.; Haigh, Cathryn L.; Drew, Simon C.; James, Michael; Boland, Martin P.; Collins, Steven J.

    2014-01-01

    The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions. PMID:25418300

  7. Tumorigenic fragments of APC cause dominant defects in directional cell migration in multiple model systems.

    PubMed

    Nelson, Scott A; Li, Zhouyu; Newton, Ian P; Fraser, David; Milne, Rachel E; Martin, David M A; Schiffmann, David; Yang, Xuesong; Dormann, Dirk; Weijer, Cornelis J; Appleton, Paul L; Näthke, Inke S

    2012-11-01

    Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating β-catenin-binding sites from APC, which leads to upregulation of β-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in β-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APC(Min/+); multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APC(Min/+) intestines are overpopulated with cells, suggesting that a lack of migration might cause cell

  8. Tumorigenic fragments of APC cause dominant defects in directional cell migration in multiple model systems

    PubMed Central

    Nelson, Scott A.; Li, Zhouyu; Newton, Ian P.; Fraser, David; Milne, Rachel E.; Martin, David M. A.; Schiffmann, David; Yang, Xuesong; Dormann, Dirk; Weijer, Cornelis J.; Appleton, Paul L.; Näthke, Inke S.

    2012-01-01

    SUMMARY Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating β-catenin-binding sites from APC, which leads to upregulation of β-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in β-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APCMin/+; multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APCMin/+ intestines are overpopulated with cells, suggesting that a lack of migration might cause

  9. Regulation of limited N-terminal proteolysis of APE1 in tumor via acetylation and its role in cell proliferation

    PubMed Central

    Bhakat, Kishor K.; Sengupta, Shiladitya; Adeniyi, Victor F.; Roychoudhury, Shrabasti; Nath, Somsubhra; Bellot, Larry J.; Feng, Dan; Mantha, Anil K.; Sinha, Mala; Qiu, Suimin; Luxon, Bruce A.

    2016-01-01

    Mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a ubiquitous and multifunctional protein, plays an essential role in the repair of both endogenous and drug-induced DNA damages in the genome. Unlike its E.coli counterpart Xth, mammalian APE1 has a unique N-terminal domain and possesses both DNA damage repair and transcriptional regulatory functions. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the cellular and molecular mechanisms that alter APE1 functions during tumorigenesis are largely unknown. Here, we show the presence of full-length APE1 and N-terminal truncated isoforms of APE1 in tumor tissue samples of various cancer types. However, primary tumor tissue has higher levels of acetylated APE1 (AcAPE1) as well as full-length APE1 compared to adjacent non-tumor tissue. We found that APE1 is proteolytically cleaved by an unknown serine protease at its N-terminus following residue lysine (Lys) Lys6 and/or Lys7 and after Lys27 and Lys31 or Lys32. Acetylation of these Lys residues in APE1 prevents this proteolysis. The N-terminal domain of APE1 and its acetylation are required for modulation of the expression of hundreds of genes. Importantly, we found that AcAPE1 is essential for sustained cell proliferation. Together, our study demonstrates that increased acetylation levels of APE1 in tumor cells inhibit the limited N-terminal proteolysis of APE1 and thereby maintain the functions of APE1 to promote tumor cells' sustained proliferation and survival. PMID:26981776

  10. The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum.

    PubMed

    Mariappan, Malaiyalam; Gande, Santosh Lakshmi; Radhakrishnan, Karthikeyan; Schmidt, Bernhard; Dierks, Thomas; von Figura, Kurt

    2008-04-25

    Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER. PMID:18305113

  11. Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies.

    PubMed

    Burger, Dominique; Stihle, Martine; Sharma, Ashwani; Di Lello, Paola; Benz, Jörg; D'Arcy, Brigitte; Debulpaep, Maja; Fry, David; Huber, Walter; Kremer, Thomas; Laeremans, Toon; Matile, Hugues; Ross, Alfred; Rufer, Arne C; Schoch, Guillaume; Steinmetz, Michel O; Steyaert, Jan; Rudolph, Markus G; Thoma, Ralf; Ruf, Armin

    2016-07-29

    Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one. PMID:27226599

  12. Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes*

    PubMed Central

    Mizuguchi, Chiharu; Ogata, Fuka; Mikawa, Shiho; Tsuji, Kohei; Baba, Teruhiko; Shigenaga, Akira; Shimanouchi, Toshinori; Okuhira, Keiichiro; Otaka, Akira; Saito, Hiroyuki

    2015-01-01

    The N-terminal amino acid 1–83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1–83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8–33 and 8–33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1–83 fragment and 8–33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1–83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation. PMID:26175149

  13. Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes.

    PubMed

    Mizuguchi, Chiharu; Ogata, Fuka; Mikawa, Shiho; Tsuji, Kohei; Baba, Teruhiko; Shigenaga, Akira; Shimanouchi, Toshinori; Okuhira, Keiichiro; Otaka, Akira; Saito, Hiroyuki

    2015-08-21

    The N-terminal amino acid 1-83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1-83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8-33 and 8-33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1-83 fragment and 8-33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1-83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation. PMID:26175149

  14. The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1*♦

    PubMed Central

    Derler, Isabella; Plenk, Peter; Fahrner, Marc; Muik, Martin; Jardin, Isaac; Schindl, Rainer; Gruber, Hermann J.; Groschner, Klaus; Romanin, Christoph

    2013-01-01

    STIM1 and Orai1 represent the two molecular key components of the Ca2+ release-activated Ca2+ channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73–90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify “hot spot” residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca2+ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca2+ selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76–90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate. PMID:23943619

  15. Two truncated forms of rat insulin receptor-related receptor.

    PubMed

    Itoh, N; Jobo, K; Tsujimoto, K; Ohta, M; Kawasaki, T

    1993-08-25

    The insulin receptor-related receptor (IRR) (1271 amino acids) is expected to have unique functions as a novel member of the insulin receptor family. In this paper, we report two alternatively spliced variants of rat IRR mRNA, which are predicted to encode two truncated forms of IRR, sIRR-1 (410 amino acids) and sIRR-2 (469 amino acids). The amino acid sequence of sIRR-1 is identical to the N-terminal 410-amino acid sequence of IRR. sIRR-2 has an additional 59-amino acid insertion in the C-terminal region. Both truncated forms retain the N-terminal and cysteine-rich domains but lack the transmembrane and intracellular tyrosine kinase domains, indicating that the truncated forms are the secreted forms. The translation products of the truncated form mRNAs were detected in the stomach and kidney by Western analysis. However, the physiological significance of the secreted forms remains to be elucidated. PMID:7688734

  16. Activation of Histidine Kinase SpaK Is Mediated by the N-Terminal Portion of Subtilin-Like Lantibiotics and Is Independent of Lipid II.

    PubMed

    Spieß, Tobias; Korn, Sophie Marianne; Kötter, Peter; Entian, Karl-Dieter

    2015-08-15

    The biosynthesis of the lantibiotic subtilin is autoinduced in a quorum-sensing mechanism via histidine kinase SpaK. Subtilin-like lantibiotics, such as entianin, ericin S, and subtilin, specifically activated SpaK in a comparable manner, whereas the structurally similar nisin did not provide the signal for SpaK activation at nontoxic concentrations. Surprisingly, nevertheless, nisin if applied together with entianin partly quenched SpaK activation. The N-terminal entianin1-20 fragment (comprising N-terminal amino acids 1 to 20) was sufficient for SpaK activation, although higher concentrations were needed. The N-terminal nisin1-20 fragment also interfered with entianin-mediated activation of SpaK and, remarkably, at extremely high concentrations also activated SpaK. Our data show that the N-terminal entianin1-20 fragment is sufficient for SpaK activation. However, if present, the C-terminal part of the molecule further strongly enhances the activation, possibly by its interference with the cellular membrane. As shown by using lipid II-interfering substances and a lipid II-deficient mutant strain, lipid II is not needed for the sensing mechanism. PMID:26025904

  17. Activation of Histidine Kinase SpaK Is Mediated by the N-Terminal Portion of Subtilin-Like Lantibiotics and Is Independent of Lipid II

    PubMed Central

    Spieß, Tobias; Korn, Sophie Marianne

    2015-01-01

    The biosynthesis of the lantibiotic subtilin is autoinduced in a quorum-sensing mechanism via histidine kinase SpaK. Subtilin-like lantibiotics, such as entianin, ericin S, and subtilin, specifically activated SpaK in a comparable manner, whereas the structurally similar nisin did not provide the signal for SpaK activation at nontoxic concentrations. Surprisingly, nevertheless, nisin if applied together with entianin partly quenched SpaK activation. The N-terminal entianin1–20 fragment (comprising N-terminal amino acids 1 to 20) was sufficient for SpaK activation, although higher concentrations were needed. The N-terminal nisin1–20 fragment also interfered with entianin-mediated activation of SpaK and, remarkably, at extremely high concentrations also activated SpaK. Our data show that the N-terminal entianin1–20 fragment is sufficient for SpaK activation. However, if present, the C-terminal part of the molecule further strongly enhances the activation, possibly by its interference with the cellular membrane. As shown by using lipid II-interfering substances and a lipid II-deficient mutant strain, lipid II is not needed for the sensing mechanism. PMID:26025904

  18. N-terminal processing of proteins exported by malaria parasites

    PubMed Central

    Chang, Henry H.; Falick, Arnold M.; Carlton, Peter M.; Sedat, John W.; DeRisi, Joseph L.; Marletta, Michael A.

    2010-01-01

    Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signal-mediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this N-terminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence. PMID:18534695

  19. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    SciTech Connect

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  20. N-Terminal Protein Characterization by Mass Spectrometry Using Combined Microscale Liquid and Solid-phase Derivatization

    PubMed Central

    Nika, Heinz; Angeletti, Ruth Hogue; Hawke, David H.

    2014-01-01

    A sample-preparation method for N-terminal peptide isolation from protein proteolytic digests has been developed. Protein thiols and primary amines were protected by carboxyamidomethylation and acetylation, respectively, followed by trypsinization. The digest was bound to ZipTipC18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate. The digest was subsequently exposed to hydroxylamine for reversal of hydroxyl group acylation, followed by reductive release of the pyridine-2-thione moiety from the derivatives. The thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol sepharose leaving the N-terminal fragment free in solution. The use of the reversed-phase supports as a reaction bed enabled optimization of the serial modification steps for throughput and completeness of derivatization. The use of the sample-preparation method was demonstrated with low picomole amounts of in-solution- and in-gel-digested protein. The N-terminal peptide was selectively retrieved from the affinity support. The sample-preparation method provides for throughput, robustness, and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to proteome-wide applications. PMID:25187758

  1. Testing the Role of the N-Terminal Tail of D1 in the Maintenance of Photosystem II in Tobacco Chloroplasts.

    PubMed

    Michoux, Franck; Ahmad, Niaz; Wei, Zheng-Yi; Belgio, Erica; Ruban, Alexander V; Nixon, Peter J

    2016-01-01

    A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction center subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25% of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress. PMID:27446098

  2. Testing the Role of the N-Terminal Tail of D1 in the Maintenance of Photosystem II in Tobacco Chloroplasts

    PubMed Central

    Michoux, Franck; Ahmad, Niaz; Wei, Zheng-Yi; Belgio, Erica; Ruban, Alexander V.; Nixon, Peter J.

    2016-01-01

    A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction center subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25% of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress. PMID:27446098

  3. Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

    PubMed

    Kountz, Timothy S; Lee, Kyung-Soon; Aggarwal-Howarth, Stacey; Curran, Elizabeth; Park, Ji-Min; Harris, Dorathy-Ann; Stewart, Aaron; Hendrickson, Joseph; Camp, Nathan D; Wolf-Yadlin, Alejandro; Wang, Edith H; Scott, John D; Hague, Chris

    2016-08-26

    The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties. PMID:27382054

  4. N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes.

    PubMed

    Ajjaji, Dalila; Richard, Charles-Adrien; Mazerat, Sandra; Chevalier, Christophe; Vidic, Jasmina

    2016-08-12

    PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1-90) and C-terminal PB1-F2 domain (53-90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1-52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain. PMID:27282484

  5. UNIT 11.10 N-Terminal Sequence Analysis of Proteins and Peptides

    PubMed Central

    Speicher, Kaye D.; Gorman, Nicole; Speicher, David W.

    2009-01-01

    Automated N-terminal sequence analysis involves a series of chemical reactions that derivatize and remove one amino acid at a time from the N-terminal of purified peptides or intact proteins. At least several pmoles of a purified protein or 10 to 20 pmoles of a purified peptide with an unmodified N-terminal is required in order to obtain useful sequence information. In recent years the demand for N-terminal sequencing has decreased substantially as some applications for protein identification and characterization can now be more effectively performed using mass spectrometry. However, N-terminal sequencing remains the method of choice for verifying the N-terminal boundary of recombinant proteins, determining the N-terminal of protease-resistant domains, identifying proteins isolated from species where most of the genome has not yet been sequenced, and mapping modified or crosslinked sites in proteins that prove to be refractory to analysis by mass spectrometry. PMID:18429102

  6. Identification of a mitochondrial-binding site on the N-terminal end of hexokinase II

    PubMed Central

    Bryan, Nadezda; Raisch, Kevin P.

    2015-01-01

    Hexokinase II (HKII) is responsible for the first step in the glycolysis pathway by adding a phosphate on to the glucose molecule so it can proceed down the pathway to produce the energy for continuous cancer cell growth. Tumour cells overexpress the HKII enzyme. In fact, it is the overexpression of the HKII enzyme that makes the diagnosis of cancer possible when imaged by positron emission tomography (PET). HKII binds to the voltage-dependent anion channel (VDAC) located on the mitochondrial outer membrane (MOM). When bound to the MOM, HKII is blocking a major cell death pathway. Thus, HKII is responsible for two characteristics of cancer cells, rapid tumour growth and inability of cancer cells to undergo apoptosis. One method to identify novel compounds that may interfere with the HKII–VDAC-binding site is to create a molecular model using the crystal structure of HKII. However, the amino acid(s) responsible for HKII binding to VDAC are not known. Therefore, a series of truncations and point mutations were made to the N-terminal end of HKII to identify the binding site to VDAC. Deletions of the first 10 and 20 amino acids indicated that important amino acid(s) for binding were located within the first 10 amino acids. Next, a series of point mutations were made within the first 10 amino acids. It is clear from the immunofluorescence images and immunoblot results that mutating the fifth amino acid from histidine to proline completely abolished binding to the MOM. PMID:26182367

  7. N-terminal domain of prion protein directs its oligomeric association.

    PubMed

    Trevitt, Clare R; Hosszu, Laszlo L P; Batchelor, Mark; Panico, Silvia; Terry, Cassandra; Nicoll, Andrew J; Risse, Emmanuel; Taylor, William A; Sandberg, Malin K; Al-Doujaily, Huda; Linehan, Jacqueline M; Saibil, Helen R; Scott, David J; Collinge, John; Waltho, Jonathan P; Clarke, Anthony R

    2014-09-12

    The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar β-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined β-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23-90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91-231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein. PMID:25074940

  8. Structure of the Tropomyosin Overlap Complex from Chicken Smooth Muscle: Insight into the Diversity of N-Terminal Recognition

    SciTech Connect

    Frye, Jeremiah; Klenchin, Vadim A.; Rayment, Ivan

    2010-09-08

    Tropomyosin is a stereotypical {alpha}-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage {phi}29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal amino acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses {approx}15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.

  9. Crystallized N-terminal domain of influenza virus matrix protein M1 and method of determining and using same

    NASA Technical Reports Server (NTRS)

    Luo, Ming (Inventor); Sha, Bingdong (Inventor)

    2000-01-01

    The matrix protein, M1, of influenza virus strain A/PR/8/34 has been purified from virions and crystallized. The crystals consist of a stable fragment (18 Kd) of the M1 protein. X-ray diffraction studies indicated that the crystals have a space group of P3.sub.t 21 or P3.sub.2 21. Vm calculations showed that there are two monomers in an asymmetric unit. A crystallized N-terminal domain of M1, wherein the N-terminal domain of M1 is crystallized such that the three dimensional structure of the crystallized N-terminal domain of M1 can be determined to a resolution of about 2.1 .ANG. or better, and wherein the three dimensional structure of the uncrystallized N-terminal domain of M1 cannot be determined to a resolution of about 2.1 .ANG. or better. A method of purifying M1 and a method of crystallizing M1. A method of using the three-dimensional crystal structure of M1 to screen for antiviral, influenza virus treating or preventing compounds. A method of using the three-dimensional crystal structure of M1 to screen for improved binding to or inhibition of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the manufacture of an inhibitor of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the screening of candidates for inhibition of influenza virus M1.

  10. The N-Terminal Residues 43 to 60 Form the Interface for Dopamine Mediated α-Synuclein Dimerisation

    PubMed Central

    Leong, Su Ling; Hinds, Mark G.; Connor, Andrea R.; Smith, David P.; Illes-Toth, Eva; Pham, Chi L. L.; Barnham, Kevin J.; Cappai, Roberto

    2015-01-01

    α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson’s disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43–140) and C-terminally (1–95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1–95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43–140 protein, we analysed the structural characteristics of the DA:α-syn 43–140 dimer and α-syn 43–140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers. PMID:25679387

  11. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    SciTech Connect

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  12. Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain.

    PubMed Central

    Arsene, F; Kaminski, P A; Elmerich, C

    1996-01-01

    Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia. The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant. To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed. The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A. brasilense wild-type and mutant strains. Our results suggest that the N-terminal domain is not essential for NifA activity. This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia. The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation. Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase. We propose a model for the regulation of NifA activity in A. brasilense. PMID:8759845

  13. The N-terminal domain of the repressor of Staphylococcus aureus phage Φ11 possesses an unusual dimerization ability and DNA binding affinity.

    PubMed

    Biswas, Anindya; Mandal, Sukhendu; Sau, Subrata

    2014-01-01

    Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors. PMID:24747758

  14. Comparative evaluation of recombinant HSP70 (N & C-terminal) fragments in the detection of equine trypanosomosis.

    PubMed

    Kumar, Jaideep; Chaudhury, A; Yadav, S C

    2016-06-15

    Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefore, in the present study, an attempt was made to evaluate the in vitro production of recombinant heat shock protein 70 (HSP70) for detection of antibodies in experimentally infected ponies. The amino acid sequence analysis of HSP70 revealed that N-terminal region of the protein was highly conserved while the C-terminal region was most divergent. The four different regions of HSP70 protein viz. HSP-1, HSP-2, HSP-3 and HSP-4 were cloned and expressed, among which HSP-1 (N-terminal region) & HSP-2 (C-terminal region) were truncated while HSP-3 & HSP-4 were complete C-terminal proteins. The recombinant fragments were probed with sequentially pooled experimental serum samples where antibodies were detected in these fragments from 10(th) day post infection till the termination of the experiment. Further, these recombinant fragments were also comparatively evaluated with WCL antigen in ELISA using experimental as well as field serum samples. It was observed that after successful treatment of infected ponies, there was a sharp fall in antibodies (within 90 days) when tested with recombinant HSP's fragments, while antibodies persisted even after 469 days when tested against WCL antigen. The sensitivity and specificity of all HSP70 fragments were also estimated from field serum samples with reference to WCL antigen ELISA. The HSP-1 showed minimum sensitivity (41.03%) among all the

  15. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

    PubMed

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  16. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity

    PubMed Central

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  17. Kinetic Mechanism of Protein N-terminal Methyltransferase 1*

    PubMed Central

    Richardson, Stacie L.; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L.; Huang, Rong

    2015-01-01

    The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors. PMID:25771539

  18. Kinetic mechanism of protein N-terminal methyltransferase 1.

    PubMed

    Richardson, Stacie L; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L; Huang, Rong

    2015-05-01

    The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors. PMID:25771539

  19. N-terminal protein processing: A comparative proteogenomic analysis

    SciTech Connect

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  20. A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

    PubMed Central

    Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

    1999-01-01

    It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

  1. N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity

    PubMed Central

    Shin, Bok-Kyu; Ahn, Joong-Hoon; Han, Jaehong

    2015-01-01

    GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH1 (GbIspH1-truncated). Both wild type GbIspH1 (GbIspH1-full) and GbIspH1-truncated were catalytically active and produced IPP and DMAPP in a ratio of 15 : 1. Kinetic parameters of KM (17.3 ± 1.9 and 14.9 ± 2.3 µM) and kcat (369 ± 10 and 347 ± 12 min−1) at pH 8.0 were obtained for GbIspH1-full and GbIspH1-truncated, respectively. Interestingly, GbIspH1-full and GbIspH1-truncated showed significantly different pH-dependent activities, and the maximum enzyme activities were obtained at pH 8.0 and 7.5, respectively. However, catalytic activation energies (Ea) of GbIspH1-full and GbIspH1-truncated were almost the same with 36.5 ± 1.6 and 35.0 ± 1.9 kJ/mol, respectively. It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation of enzyme activity during photosynthesis. PMID:25892986

  2. Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor.

    PubMed

    Goy, Sebastian D; Olling, Alexandra; Neumann, Detlef; Pich, Andreas; Gerhard, Ralf

    2015-06-01

    Clostridium difficile may induce antibiotic-associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB-induced rise in intracellular-free Ca(2+) and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1. Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus, non-cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved 'non-cytotoxic' fragments and (iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal receptor binding domain of TcdB with target cells. PMID:25529763

  3. Peptide Scrambling During Collision-Induced Dissociation is Influenced by N-terminal Residue Basicity

    NASA Astrophysics Data System (ADS)

    Chawner, Ross; Holman, Stephen W.; Gaskell, Simon J.; Eyers, Claire E.

    2014-08-01

    `Bottom up' proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+H2O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID.

  4. Bacterial secretion of soluble and functional trivalent scFv-based N-terminal trimerbodies.

    PubMed

    Blanco-Toribio, Ana; Álvarez-Cienfuegos, Ana; Sainz-Pastor, Noelia; Merino, Nekane; Compte, Marta; Sanz, Laura; Blanco, Francisco J; Álvarez-Vallina, Luis

    2015-12-01

    Recombinant antibodies are used with great success in many different diagnostic and therapeutic applications. A variety of protein expression systems are available, but nowadays almost all therapeutic antibodies are produced in mammalian cell lines due to their complex structure and glycosylation requirements. However, production of clinical-grade antibodies in mammalian cells is very expensive and time-consuming. On the other hand, Escherichia coli (E. coli) is known to be the simplest, fastest and most cost-effective recombinant expression system, which usually achieves higher protein yields than mammalian cells. Indeed, it is one of the most popular host in the industry for the expression of recombinant proteins. In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for native laminin-111, was expressed in human embryonic kidney 293 cells and in E. coli. Mammalian and bacterially produced anti-laminin trimerbody molecules display comparable functional and structural properties, although importantly the yield of trimerbody expressed in E. coli was considerably higher than in human cells. These results demonstrated that E. coli is a versatile and efficient expression system for multivalent trimerbody-based molecules that is suitable for their industrial production. PMID:26239030

  5. NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase.

    PubMed

    Polshakov, Vladimir I; Petrova, Olga A; Parfenova, Yulia Yu; Efimov, Sergey V; Klochkov, Vladimir V; Zvereva, Maria I; Dontsova, Olga A

    2016-04-01

    Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the (1)H, (13)C, and (15)N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle. PMID:26721464

  6. The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

    PubMed Central

    Seemann, J; Weber, K; Osborn, M; Parton, R G; Gerke, V

    1996-01-01

    Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes. Images PMID:8885232

  7. Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

    PubMed

    Sivanandam, Venkatesh; Mathews, Deborah; Garmann, Rees; Erdemci-Tandogan, Gonca; Zandi, Roya; Rao, A L N

    2016-01-01

    Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging. PMID:27193742

  8. Lysozyme Mutants Accumulate in Cells while Associated at their N-terminal Alpha-domain with the Endoplasmic Reticulum Chaperone GRP78/BiP

    PubMed Central

    Kamada, Yoshiki; Nawata, Yusuke; Sugimoto, Yasushi

    2016-01-01

    Amyloidogenic human lysozyme variants deposit in cells and cause systemic amyloidosis. We recently observed that such lysozymes accumulate in the endoplasmic reticulum (ER) with the ER chaperone GRP78/BiP, accompanying the ER stress response. Here we investigated the region of lysozyme that is critical to its association with GRP78/BiP. In addition to the above-mentioned variants of lysozyme, we constructed lysozyme truncation or substitution mutants. These were co-expressed with GRP78/BiP (tagged with FLAG) in cultured human embryonic kidney cells, which were analyzed by western blotting and immunocytochemistry using anti-lysozyme and anti-FLAG antibodies. The amyloidogenic variants were confirmed to be strongly associated with GRP78/BiP as revealed by the co-immunoprecipitation assay, whereas N-terminal mutants pruned of 1-41 or 1-51 residues were found not to be associated with the chaperone. Single amino acid substitutions for the leucine array along the α-helices in the N-terminal region resulted in wild-type lysozyme remaining attached to GRP78/BiP. These mutations also tended to show lowered secretion ability. We conclude that the N-terminal α-helices region of the lysozyme is pivotal for its strong adhesion to GRP78/BiP. We suspect that wild-type lysozyme interacts with the GRP at this region as a step in the proper folding monitored by the ER chaperone. PMID:26884716

  9. Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging

    PubMed Central

    Sivanandam, Venkatesh; Mathews, Deborah; Garmann, Rees; Erdemci-Tandogan, Gonca; Zandi, Roya; Rao, A. L. N.

    2016-01-01

    Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3′ terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging. PMID:27193742

  10. The N-terminal adenosine triphosphate binding domain of Hsp90 is necessary and sufficient for interaction with estrogen receptor.

    PubMed

    Bouhouche-Chatelier, L; Chadli, A; Catelli, M G

    2001-10-01

    To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being more efficient when the charged region A was present in the mutant (1-334). The N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1-224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER. PMID:11795466

  11. The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.

    PubMed Central

    Greenfield, N. J.; Stafford, W. F.; Hitchcock-DeGregori, S. E.

    1994-01-01

    We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin. PMID:8019411

  12. Investigation of Truncated Waveguides

    NASA Technical Reports Server (NTRS)

    Lourie, Nathan P.; Chuss, David T.; Henry, Ross M.; Wollack, Edward J.

    2013-01-01

    The design, fabrication, and performance of truncated circular and square waveguide cross-sections are presented. An emphasis is placed upon numerical and experimental validation of simple analytical formulae that describe the propagation properties of these structures. A test component, a 90-degree phase shifter, was fabricated and tested at 30 GHz. The concepts explored can be directly applied in the design, synthesis and optimization of components in the microwave to sub-millimeter wavebands.

  13. The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP.

    PubMed

    Higgins, Christina E; Gross, Steven S

    2011-04-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  14. The N-terminal Peptide of Mammalian GTP Cyclohydrolase I Is an Autoinhibitory Control Element and Contributes to Binding the Allosteric Regulatory Protein GFRP*

    PubMed Central

    Higgins, Christina E.; Gross, Steven S.

    2011-01-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  15. The predicted N-terminal signal sequence of the human α₂C-adrenoceptor does not act as a functional cleavable signal peptide.

    PubMed

    Jahnsen, Jan Anker; Uhlén, Staffan

    2012-06-01

    The N-terminal region of the human α(2C)-adrenoceptor has a 22 amino acid sequence MASPALAAALAVAAAAGPNASG. This stretch is predicted to be a cleavable signal peptide. Signal peptides facilitate the translocation of membrane proteins from ribosomes into the endoplasmatic reticulum (ER) for further transport to the plasma membrane. However, recently it has been suggested that the hydrophobic stretch ALAAALAAAAA in the N-tail of the rat α(2C)-adrenoceptor, rather than being part of a signal peptide, is an ER retention signal (Angelotti, 2010). Here, we have investigated the functionality of the N-terminal region of the human α(2C)-adrenoceptor further. The predicted signal peptide was found to be non-cleavable, as shown for a modified α(2C)-adrenoceptor construct equipped with a FLAG epitope. The influence of the N-terminal region on receptor translocation to the plasma membrane was investigated by rebuilding the N-tail and then by analyzing the expression level of binding-competent receptors in transfected COS-7 cell membranes. Truncated α(2C)-adrenoceptor constructs showed decreased expression levels as compared to the wild type α(2C)-adrenoceptor. Addition of, or exchange for, the influenza virus hemagglutinin signal peptide to the α(2C)-adrenoceptor had no effect, respectively decreased, the expression level of binding-competent receptor in the membranes. Our analysis supports the conclusions that the predicted signal peptide in the N-terminal tail of the α(2C)-adrenoceptor does not act as a cleavable signal peptide. In addition, the results indicate that the presence of an intact N-tail is augmenting the amount of binding-competent α(2C)-adrenoceptors at the cell surface. PMID:22503931

  16. Cellular toxicity of yeast prion protein Rnq1 can be modulated by N-terminal wild type huntingtin.

    PubMed

    Sethi, Ratnika; Patel, Vishal; Saleh, Aliabbas A; Roy, Ipsita

    2016-01-15

    Aggregation of the N-terminal human mutant huntingtin and the consequent toxicity in the yeast model of Huntington's disease (HD) requires the presence of Rnq1 protein (Rnq1p) in its prion conformation [RNQ1(+)]. The understanding of interaction of wild-type huntingtin (wt-Htt) with the amyloidogenic prion has some gaps. In this work, we show that N-terminal fragment of wt-Htt (N-wt-Htt) ameliorated the toxic effect of [RNQ1(+)] depending on expression levels of both proteins. When the expression of N-wt-Htt was high, it increased the expression and delayed the aggregation of [RNQ1(+)]. As the expression of N-wt-Htt was reduced, it formed high molecular weight aggregates along with the prion. Even when sequestered by [RNQ1(+)], the beneficial effect of N-wt-Htt on expression of Rnq1p and on cell survival was evident. Huntingtin protein ameliorated toxicity due to the prion protein [RNQ1(+)] in yeast cells in a dose-dependent manner, resulting in increase in cell survival, hinting at its probable role as a component of the proteostasis network of the cell. Taking into account the earlier reports of the beneficial effect of expression of N-wt-Htt on the aggregation of mutant huntingtin, the function of wild-type huntingtin as an inhibitor of protein aggregation in the cell needs to be explored. PMID:26628321

  17. A modification of the N-terminal amino acid in the eremomycin aglycone.

    PubMed

    Miroshnikova, O V; Berdnikova, T F; Olsufyeva, E N; Pavlov, A Y; Reznikova, M I; Preobrazhenskaya, M N; Ciabatti, R; Malabarba, A; Colombo, L

    1996-11-01

    An Edman degradation of the antibiotic eremomycin aglycone produced the corresponding hexapeptide, which was aminoacylated with D-lysine, D-histidine or D-tryptophan derivatives to give new heptapeptide analogs of the eremomycin aglycone. The aminoacylation of the eremomycin aglycone produced an octapeptide analog. The substitution of D-lysine for the N-terminal N-methyl-D-leucine does not seriously affect the in vitro antibacterial properties of the eremomycin aglycone whereas the heptapeptides with the N-terminal D-tryptophan or D-histidine moieties and the octapeptide with the N-terminal D-lysine are practically devoid of the antibacterial properties. PMID:8982345

  18. Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

    SciTech Connect

    Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin; Lastuvkova, Hana; Kodicek, Milan; Sakalian, Michael; Ruml, Tomas

    2009-10-10

    The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibers is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.

  19. Allosteric stabilization of the amyloid-β peptide hairpin by the fluctuating N-terminal.

    PubMed

    Xu, Liang; Nussinov, Ruth; Ma, Buyong

    2016-01-28

    Immobilized ions modulate nearby hydrophobic interactions and influence molecular recognition and self-assembly. We simulated disulfide bond-locked double mutants (L17C/L34C) and observed allosteric modulation of the peptide's intra-molecular interactions by the N-terminal tail. We revealed that the non-contacting charged N-terminal residues help the transfer of entropy to the surrounding solvation shell and stabilizing β-hairpin. PMID:26666686

  20. The proline-rich N-terminal sequence of calcineurin Abeta determines substrate binding.

    PubMed

    Kilka, Susann; Erdmann, Frank; Migdoll, Alexander; Fischer, Gunter; Weiwad, Matthias

    2009-03-10

    Three different genes of catalytic subunit A of the Ca(2+)-dependent serine/threonine protein phosphatase calcineurin (CaN) are encoded in the human genome forming heterodimers with regulatory subunit B. Even though physiological roles of CaN have been investigated extensively, less is known about the specific functions of the different catalytic isoforms. In this study, all human CaN holoenzymes containing either the alpha, beta, or gamma isoform of the catalytic subunit (CaN alpha, beta, or gamma, respectively) were expressed for the first time. Comparative kinetic analysis of the dephosphorylation of five specific CaN substrates provided evidence that the distinct isoforms of the catalytic subunit confer substrate specificities to the holoenzymes. CaN alpha dephosphorylates the transcription factor Elk-1 with 7- and 2-fold higher catalytic efficiencies than the beta and gamma isoforms, respectively. CaN gamma exhibits the highest k(cat)/K(m) value for DARPP-32, whereas the catalytic efficiencies for the dephosphorylation of NFAT and RII peptide were 3- and 5-fold lower, respectively, when compared with the other isoforms. Elk-1 and NFAT reporter gene activity measurements revealed even more pronounced substrate preferences of CaNA isoforms. Moreover, kinetic analysis demonstrated that CaN beta exhibits for all tested protein substrates the lowest K(m) values. Enzymatic characterization of the CaN beta(P14G/P18G) variant as well as the N-terminal truncated form CaN beta(22-524) revealed that the proline-rich sequence of CaN beta is involved in substrate recognition. CaN beta(22-524) exhibits an at least 4-fold decreased substrate affinity and a 5-fold increased turnover number. Since this study demonstrates that all CaN isoforms display the same cytoplasmic subcellular distribution and are expressed in each tested cell line, differences in substrate specificities may determine specific physiological functions of the distinct isoforms. PMID:19154138

  1. Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: an importance of the N-terminal domain for an allosteric regulatory property.

    PubMed

    Sawitri, Widhi Dyah; Narita, Hirotaka; Ishizaka-Ikeda, Etsuko; Sugiharto, Bambang; Hase, Toshiharu; Nakagawa, Atsushi

    2016-06-01

    Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity. PMID:26826371

  2. An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

    PubMed

    Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

    2013-06-01

    In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies. PMID:23641718

  3. The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

    PubMed

    Guardiani, Carlo; Scorciapino, Mariano Andrea; Amodeo, Giuseppe Federico; Grdadolnik, Joze; Pappalardo, Giuseppe; De Pinto, Vito; Ceccarelli, Matteo; Casu, Mariano

    2015-09-15

    The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms. PMID:26303511

  4. Transformation of a fragment of beta-structural bacteriophage T4 adhesin to stable alpha-helical trimer.

    PubMed

    Miroshnikov, K A; Sernova, N V; Shneider, M M; Mesyanzhinov, V V

    2000-12-01

    Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a beta-helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile beta-structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant alpha-helical structure. This structure is probably similar to that of fibritin, which has a triple alpha-helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization. PMID:11173503

  5. The N-terminal segment of glyceraldehyde-3-phosphate dehydrogenase of Haemonchus contortus interacts with complements C1q and C3.

    PubMed

    Vedamurthy, G V; Sahoo, S; Devi, I K; Murugavel, S; Joshi, P

    2015-11-01

    Haemonchus contortus, an economically important blood-sucking parasite of sheep and goats, survives the harsh host gut environment by secreting a number of proteins referred as excretory/secretory (ES) products. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, is one of the components of H. contortus ES products. The parasite enzyme binds to complement C3 and inhibits its activity. In this study, the C3-binding activity of the parasite GAPDH was mapped to the N-terminal part of the enzyme by generating defined recombinant fragments of the protein. The N-terminal fragment also trapped complement C1q but not C5 and inhibited complement-mediated lysis of sensitized sheep erythrocytes. Competitive binding assay indicates different binding regions for C1q and C3 proteins. GAPDH stimulated proliferation of goat peripheral blood mononuclear cells in vitro and reacted with the sera from H. contortus-infected animals. However, the fragments of GAPDH did not stimulate cell proliferation nor reacted with the infected animal sera. Furthermore, denatured GAPDH failed to react with the infected animal sera in dot blot suggesting conformation-dependent epitope. These results demonstrate an elegant strategy of the parasite to completely shut down complement activation and identify GAPDH as a promising target for future therapeutic intervention. PMID:26332726

  6. N-terminal sequences direct the autophosphorylation states of the FER tyrosine kinases in vivo.

    PubMed

    Orlovsky, K; Ben-Dor, I; Priel-Halachmi, S; Malovany, H; Nir, U

    2000-09-12

    p94(fer) and p51(ferT) are two tyrosine kinases which share identical SH2 and kinase domains but differ in their N-terminal regions. While p94(fer) is expressed in most mammalian cells, the accumulation of p51(ferT) is restricted to meiotic spermatocytes. Here we show that the different N-terminal tails of p94(fer) and p51(ferT) direct different autophosphorylation states of these two kinases in vivo. N-terminal coiled-coil domains cooperated to drive the oligomerization and autophosphorylation in trans of p94(fer). Moreover, the ectopically expressed N-terminal tail of p94(fer) could act as a dominant negative mutant and associated with the endogenous p94(fer) protein in CHO cells. This increased significantly the percentage of cells residing in the G0/G1 phase, thus suggesting a role for p94(fer) in the regulation of G1 progression. Unlike p94(fer), overexpressed p51(ferT) was not autophosphorylated in COS1 cells. However, removal of the unique N-terminal 43 aa of p51(ferT) or the replacement of this region by a parallel segment from p94(fer) endowed the modified p51(ferT) with the ability to autophosphorylate. The unique N-terminal sequences of p51(ferT) thus interfere with its ability to autophosphorylate in vivo. These experiments indicate that the N-terminal sequences of the FER tyrosine kinases direct their different cellular autophosphorylation states, thereby dictating their different cellular functions. PMID:10998246

  7. BacM, an N-terminally processed bactofilin of Myxococcus xanthus, is crucial for proper cell shape

    PubMed Central

    Koch, Matthias K.; McHugh, Colleen A.; Hoiczyk, Egbert

    2011-01-01

    Summary Bactofilins are fibre-forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic ‘crooked’ cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637–4635 (BacN-P) has no obvious phenotype. In M. xanthus, BacM exists as a 150-amino-acid full-length version and as a version cleaved before Ser28. In the cell, native BacM forms 3 nm wide fibres, which assemble into bundles forming helix-like cytoplasmic cables throughout the cell, and in a subset of cells additionally a polarly arranged lateral rod-like structure. Isolated fibres consist almost completely of the N-terminally truncated version, suggesting that the proteolytic cleavage occurs before or during fibre formation. Fusion of BacM to mCherry perturbs BacM function and cellular fibre arrangement, resulting for example in the formation of one prominent polar corkscrew-like structure per cell. Immunofluorescence staining of BacM and MreB shows that their cellular distributions are not matching. Taken together, these data suggest that rod-shaped bacteria like M. xanthus use bactofilin fibres to achieve and maintain their characteristic cell morphology and cell wall stability. PMID:21414039

  8. BacM, an N-terminally processed bactofilin of Myxococcus xanthus, is crucial for proper cell shape.

    PubMed

    Koch, Matthias K; McHugh, Colleen A; Hoiczyk, Egbert

    2011-05-01

    Bactofilins are fibre-forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic 'crooked' cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637-4635 (BacN-P) has no obvious phenotype. In M. xanthus, BacM exists as a 150-amino-acid full-length version and as a version cleaved before Ser28. In the cell, native BacM forms 3 nm wide fibres, which assemble into bundles forming helix-like cytoplasmic cables throughout the cell, and in a subset of cells additionally a polarly arranged lateral rod-like structure. Isolated fibres consist almost completely of the N-terminally truncated version, suggesting that the proteolytic cleavage occurs before or during fibre formation. Fusion of BacM to mCherry perturbs BacM function and cellular fibre arrangement, resulting for example in the formation of one prominent polar corkscrew-like structure per cell. Immunofluorescence staining of BacM and MreB shows that their cellular distributions are not matching. Taken together, these data suggest that rod-shaped bacteria like M. xanthus use bactofilin fibres to achieve and maintain their characteristic cell morphology and cell wall stability. PMID:21414039

  9. N-terminal cleavage of the mitochondrial fusion GTPase OPA1 occurs via a caspase-independent mechanism in cerebellar granule neurons exposed to oxidative or nitrosative stress

    PubMed Central

    Gray, Josie J.; Zommer, Amelia E.; Bouchard, Ron J.; Duval, Nathan; Blackstone, Craig; Linseman, Daniel A.

    2013-01-01

    Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. Here, we demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from the N-terminal cleavage of OPA1 observed in CGNs undergoing caspase-mediated apoptosis (Loucks et al., 2009) and results in removal of a key lysine residue (K301) within the GTPase domain. OPA1 cleavage in CGNs occurs coincident with extensive mitochondrial fragmentation, disruption of the microtubule network, and cell death. In contrast to OPA1 cleavage induced in CGNs by removing depolarizing extracellular potassium (5K apoptotic conditions), oxidative or nitrosative stress-induced OPA1 cleavage caused by complex I inhibition or nitric oxide, respectively, is caspase-independent. N-terminal cleavage of OPA1 is also observed in vivo in aged rat and mouse midbrain and hippocampal tissues. We conclude that N-terminal cleavage and subsequent inactivation of OPA1 may be a contributing factor in the neuronal cell death processes underlying neurodegenerative diseases, particularly those associated with aging. Furthermore, these data suggest that OPA1 cleavage is a likely convergence point for mitochondrial dysfunction and imbalances in mitochondrial fission and fusion induced by oxidative or nitrosative stress. PMID:23220553

  10. N-terminal cleavage of the mitochondrial fusion GTPase OPA1 occurs via a caspase-independent mechanism in cerebellar granule neurons exposed to oxidative or nitrosative stress.

    PubMed

    Gray, Josie J; Zommer, Amelia E; Bouchard, Ron J; Duval, Nathan; Blackstone, Craig; Linseman, Daniel A

    2013-02-01

    Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. Here, we demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from the N-terminal cleavage of OPA1 observed in CGNs undergoing caspase-mediated apoptosis (Loucks et al., 2009) and results in removal of a key lysine residue (K301) within the GTPase domain. OPA1 cleavage in CGNs occurs coincident with extensive mitochondrial fragmentation, disruption of the microtubule network, and cell death. In contrast to OPA1 cleavage induced in CGNs by removing depolarizing extracellular potassium (5K apoptotic conditions), oxidative or nitrosative stress-induced OPA1 cleavage caused by complex I inhibition or nitric oxide, respectively, is caspase-independent. N-terminal cleavage of OPA1 is also observed in vivo in aged rat and mouse midbrain and hippocampal tissues. We conclude that N-terminal cleavage and subsequent inactivation of OPA1 may be a contributing factor in the neuronal cell death processes underlying neurodegenerative diseases, particularly those associated with aging. Furthermore, these data suggest that OPA1 cleavage is a likely convergence point for mitochondrial dysfunction and imbalances in mitochondrial fission and fusion induced by oxidative or nitrosative stress. PMID:23220553

  11. Determination of statherin N-terminal peptide conformation on hydroxyapatite crystals

    SciTech Connect

    Shaw, W.J.; Long, J.R.; Dindot, J.L.; Campbell, A.A.; Stayton, P.S.; Drobny, G.P.

    2000-03-01

    Proteins play an important role in inorganic crystal engineering during the development and growth of hard tissues such as bone and teeth. Although many of these proteins have been studied in the liquid state, there is little direct information describing molecular recognition at the protein-crystal interface. The authors have used {sup 13}C solid-state NMR (SSNMR) techniques to investigate the conformation of an N-terminal peptide of salivary statherin both free and adsorbed on hydroxyapatite (HAP) crystals. The torsion angle {var{underscore}phi} was determined at three positions along the backbone of the phosphorylated N-terminal 15 amino acid peptide fragment (DpSpSEEKFLRRIGRFG) by measuring distances between the backbone carbonyls carbons in the indicated adjacent amino acids using dipolar recoupling with a windowless sequence (DRAWS). Global secondary structure was determined by measuring the dipolar coupling between the {sup 13}C backbone carbonyl and the backbone {sup 15}N in the i {r{underscore}arrow} i + 4 residues (DpSpSEEKFLRRIGRFG) using rotational echo double resonance (REDOR). Peptides singly labeled at amino acids pS{sub 3}, L{sub 8}, and G{sub 12} were used for relaxation and line width measurements. The peptides adsorbed to the HAP surface have an average {var{underscore}phi} of {minus}85{degree} at the N-terminus (pSpS), {minus}60{degree} in the middle (FL) and {minus}73{degree} near the C-terminus (IG). The average {var{underscore}phi} angle measured at the pSpS position and the observed high conformational dispersion suggest a random coil conformation at this position. However, the FL position displays an average {var{underscore}phi} that indicates significant {alpha}-helical content, and the long time points in the DRAWS experiment fit best to a relatively narrow distribution of {var{underscore}phi} that falls within the protein data bank {alpha}-helical conformational space. REDOR measurements confirm the presence of helical content, where the

  12. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  13. Loss of N-terminal Acetylation Suppresses A Prion Phenotype By Modulating Global Protein Folding

    PubMed Central

    Holmes, William M.; Mannakee, Brian K.; Gutenkunst, Ryan N.; Serio, Tricia R.

    2014-01-01

    N-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. While loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI+], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype. PMID:25023910

  14. Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-Terminal Region of Phosphoprotein

    PubMed Central

    Guryanov, Sergey G.; Liljeroos, Lassi; Kasaragod, Prasad; Kajander, Tommi

    2015-01-01

    ABSTRACT The enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N0) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N0 assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N01-408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N0-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N0 binding peptide, P1-48, to the C terminus of an N021-408 fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N0-P complex. We solved the X-ray structure of the resulting N0-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N0-P interface and explains how P chaperones N0, preventing both self-assembly of N0 and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization. IMPORTANCE Measles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of

  15. N-terminal-mediated oligomerization of DnaA drives the occupancy-dependent rejuvenation of the protein on the membrane

    PubMed Central

    Aranovich, Alexander; Braier-Marcovitz, Shani; Ansbacher, Esti; Granek, Rony; Parola, Abraham H.; Fishov, Itzhak

    2015-01-01

    DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once per cell division cycle. Its overall activity cycle is driven by ATP hydrolysis and ADP–ATP exchange. The latter can be promoted by binding to specific sequences on the chromosome and/or to acidic phospholipids in the membrane. We have previously shown that the transition into an active form (rejuvenation) is strongly co-operative with respect to DnaA membrane occupancy. Only at low membrane occupancy is DnaA reactivation efficiently catalysed by the acidic phospholipids. The present study was aimed at unravelling the molecular mechanism underlying the occupancy-dependent DnaA rejuvenation. We found that truncation of the DnaA N-terminal completely abolishes the co-operative transformation between the high and low occupancy states (I and II respectively) without affecting the membrane binding. The environmentally sensitive fluorophore specifically attached to the N-terminal cysteines of DnaA reported on occupancy-correlated changes in its vicinity. Cross-linking of DnaA with a short homobifunctional reagent revealed that state II of the protein on the membrane corresponds to a distinct oligomeric form of DnaA. The kinetic transition of DnaA on the membrane surface is described in the present study by a generalized 2D condensation phase transition model, confirming the existence of two states of DnaA on the membrane and pointing to the possibility that membrane protein density serves as an on-off switch in vivo. We conclude that the DnaA conformation attained at low surface density drives its N-terminal-mediated oligomerization, which is presumably a pre-requisite for facilitated nt exchange. PMID:26272946

  16. A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding

    PubMed Central

    Frugier, Magali; Moulinier, Luc; Giegé, Richard

    2000-01-01

    Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20–70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNAAsp, we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence (29LSKKALKKLQK39 in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide. PMID:10811628

  17. Isolation of a novel cold-active family 11 Xylanase from the filamentous fungus Bispora antennata and deletion of its N-terminal amino acids on thermostability.

    PubMed

    Liu, Qiong; Wang, Yaru; Luo, Huiying; Wang, Liwen; Shi, Pengjun; Huang, Huoqing; Yang, Peilong; Yao, Bin

    2015-01-01

    In the present study, we first reported a cold-active xylanase of glycosyl hydrolase family 11, Xyn11, from the filamentous fungus Bispora antennata. The coding gene (xyn11) was cloned and successfully expressed in Pichia pastoris. Deduced Xyn11 exhibited the highest identity of 65 % with a family 11 endo-β-1,4-xylanase from Alternaria sp. HB186. Recombinant Xyn11 exhibited maximal activity at 35 °C and remained 21 % of the activity at 0 °C. Sequence alignment showed that the N-terminal sequence of Xyn11 is distinct from those of thermophilic xylanases of family 11. To determine its effect on enzyme properties, the Xyn11 mutant without the N-terminal sequence, t-Xyn11, was then constructed, expressed in P. pastoris, and compared with Xyn11. Both enzymes showed optimal activities at 35 °C and pH 5.5 and were stable at pH 2.0-12.0. Compared with truncated mutant t-Xyn11, Xyn11 retained more activity after 20-min incubation at 40 °C (Xyn11:28 % vs. t-Xyn11:4 %) and degraded xylan substrates more completely. Thus, a new factor affecting the thermostability of cold-active xylanase of family 11 was identified. PMID:25351632

  18. N-terminal Proteomics and Ribosome Profiling Provide a Comprehensive View of the Alternative Translation Initiation Landscape in Mice and Men*

    PubMed Central

    Van Damme, Petra; Gawron, Daria; Van Criekinge, Wim; Menschaert, Gerben

    2014-01-01

    Usage of presumed 5′UTR or downstream in-frame AUG codons, next to non-AUG codons as translation start codons contributes to the diversity of a proteome as protein isoforms harboring different N-terminal extensions or truncations can serve different functions. Recent ribosome profiling data revealed a highly underestimated occurrence of database nonannotated, and thus alternative translation initiation sites (aTIS), at the mRNA level. N-terminomics data in addition showed that in higher eukaryotes around 20% of all identified protein N termini point to such aTIS, to incorrect assignments of the translation start codon, translation initiation at near-cognate start codons, or to alternative splicing. We here report on more than 1700 unique alternative protein N termini identified at the proteome level in human and murine cellular proteomes. Customized databases, created using the translation initiation mapping obtained from ribosome profiling data, additionally demonstrate the use of initiator methionine decoded near-cognate start codons besides the existence of N-terminal extended protein variants at the level of the proteome. Various newly identified aTIS were confirmed by mutagenesis, and meta-analyses demonstrated that aTIS reside in strong Kozak-like motifs and are conserved among eukaryotes, hinting to a possible biological impact. Finally, TargetP analysis predicted that the usage of aTIS often results in altered subcellular localization patterns, providing a mechanism for functional diversification. PMID:24623590

  19. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

    PubMed Central

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A.; Meng, Jihong

    2015-01-01

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11–21 aa contain EV-71-specific antigenic sites, whereas positions 1–5 and 51–100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP16–43, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP16–43 is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP16–43 fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6–43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format. PMID:24952304

  20. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain.

    PubMed

    Meagher, Martin; Enemark, Eric J

    2016-07-01

    The crystal structure of the N-terminal domain of the Pyrococcus furiosus minichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation. PMID:27380371

  1. Role of N-terminal residues on folding and stability of C-phycoerythrin: simulation and urea-induced denaturation studies.

    PubMed

    Anwer, Khalid; Sonani, Ravi; Madamwar, Datta; Singh, Parvesh; Khan, Faez; Bisetty, Krishna; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2015-01-01

    The conformational state of biliproteins can be determined by optical properties of the covalently linked chromophores. Recently determined crystal structure of truncated form of α-subunit of cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue provides a new insight into the structure-function relationship of αC-PE. To compare their stabilities, we have measured urea-induced denaturation transitions of the full length αC-PE (FL-αC-PE) and truncated αC-PE (Tr-αC-PE) followed by observing changes in absorbance at 565 nm, fluorescence at 350 and 573 nm, and circular dichroism at 222 nm as a function of [urea], the molar concentration of urea. The transition curve of each protein was analyzed for ΔG(D)(0), the value of Gibbs free energy change on denaturation (ΔG(D)) in the absence of urea; m, the slope (=∂∆G(D)/∂[urea]), and C(m), the midpoint of the denaturation curve, i.e. [urea] at which ΔG(D) = 0. A difference of about 10% in ΔG(D)(0) observed between FL-αC-PE and Tr-αC-PE, suggests that the two proteins are almost equally stable, and the natural deletion of 31 residues from the N-terminal side of the full length protein does not alter its stability. Furthermore, normalization of probes shows that the urea-induced denaturation of both the proteins is a two-state process. Folding of both structural variants (Tr-αC-PE and FL-αC-PE) of P. tenue were also studied using molecular dynamics simulations at 300 K. The results show clearly that the stability of the proteins is evenly distributed over the whole structure indicating no significant role of N-terminal residues in the stability of both proteins. PMID:24279700

  2. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

    PubMed Central

    Gary, R; Bretscher, A

    1995-01-01

    Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking. Images PMID:7579708

  3. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  4. Cooperative binding of the yeast Spt10p activator to the histone upstream activating sequences is mediated through an N-terminal dimerization domain

    PubMed Central

    Mendiratta, Geetu; Eriksson, Peter R.; Clark, David J.

    2007-01-01

    The yeast Spt10p activator is a putative histone acetyltransferase (HAT) possessing a sequence-specific DNA-binding domain (DBD) which binds to the upstream activation sequences (UAS elements) in the histone gene promoters. Spt10p binds to a pair of histone UAS elements with extreme positive cooperativity. The molecular basis of this cooperativity was addressed. Spt10p (640 residues) is an elongated dimer, but the isolated DBD (residues 283–396) is a monomer and binds non-cooperatively to DNA. A Spt10p fragment comprising the N-terminal domain (NTD), HAT domain and DBD (residues 1–396) binds cooperatively and is a dimer, whereas an overlapping Spt10p fragment comprising the DBD and C-terminal domains (residues 283–640) binds non-cooperatively and is a monomer. These observations imply that cooperative binding requires dimerization. The isolated NTD (residues 1–98) is a dimer and is responsible for dimerization. We propose that cooperativity involves a conformational change in the Spt10p dimer which facilitates the simultaneous recognition of two UAS elements. In vivo, deletion of the NTD results in poor growth, but does not prevent the binding at the HTA1 promoter, suggesting that dimerization is biologically important. Residues 1–396 are sufficient for normal growth, indicating that the critical functions of Spt10p reside in the N-terminal domains. PMID:17202156

  5. Focusing of truncated Gaussian beams

    NASA Astrophysics Data System (ADS)

    Horváth, Zoltán L.; Bor, Zsolt

    2003-07-01

    It is shown that the focusing of truncated Gaussian beams can be treated by the same manner as uniform spherical waves, i.e., the diffraction integral can be expressed by the Lommel functions, which offers a very efficient way for the calculation of the three-dimensional light distribution near focus. All the expressions for the uniform spherical waves hold good for Gaussian beams if the first variable in the Lommel functions is extended to the complex domain. The intensity distribution depending on the Fresnel number and the truncation coefficient is calculated. The location of the first few minima and maxima of the intensity in focal plane is given for different values of the truncation coefficient. The phase behavior depending on the truncation coefficient is studied.

  6. The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding.

    PubMed Central

    Koch, C; Ninnemann, O; Fuss, H; Kahmann, R

    1991-01-01

    FIS protein is involved in several different cellular processes stimulating site-specific recombination in phages Mu and lambda as well as transcription of stable RNA operons in E.coli. We have performed a mutational analysis of fis and provide genetic and biochemical evidence that a truncated version of FIS lacking the N-terminal region is sufficient for specific DNA binding and for stimulating lambda excision. These mutants also retain their ability to autoregulate fis gene expression. Such mutant proteins, however, cannot stimulate the enhancer dependent DNA inversion reaction. Images PMID:1834996

  7. Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells

    PubMed Central

    Kuang, Yi; Gao, Yuan; Xu, Bing

    2011-01-01

    Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines. PMID:22037699

  8. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  9. Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells.

    PubMed

    Kuang, Yi; Gao, Yuan; Xu, Bing

    2011-12-21

    Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines. PMID:22037699

  10. Crystal structure of the Sec18p N-terminal domain

    PubMed Central

    Babor, S. Mariana; Fass, Deborah

    1999-01-01

    Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two β-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled. PMID:10611286

  11. Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X

    PubMed Central

    Antonios, Gregory; Borgers, Henning; Richard, Bernhard C.; Brauß, Andreas; Meißner, Julius; Weggen, Sascha; Pena, Vladimir; Pillot, Thierry; Davies, Sarah L.; Bakrania, Preeti; Matthews, David; Brownlees, Janet; Bouter, Yvonne; Bayer, Thomas A.

    2015-01-01

    Full-length Aβ1-42 and Aβ1-40, N-truncated pyroglutamate Aβ3-42 and Aβ4-42 are major variants in the Alzheimer brain. Aβ4-42 has not been considered as a therapeutic target yet. We demonstrate that the antibody NT4X and its Fab fragment reacting with both the free N-terminus of Aβ4-x and pyroglutamate Aβ3-X mitigated neuron loss in Tg4-42 mice expressing Aβ4-42 and completely rescued spatial reference memory deficits after passive immunization. NT4X and its Fab fragment also rescued working memory deficits in wild type mice induced by intraventricular injection of Aβ4-42. NT4X reduced pyroglutamate Aβ3-x, Aβx-40 and Thioflavin-S positive plaque load after passive immunization of 5XFAD mice. Aβ1-x and Aβx-42 plaque deposits were unchanged. Importantly, for the first time, we demonstrate that passive immunization using the antibody NT4X is therapeutically beneficial in Alzheimer mouse models showing that N-truncated Aβ starting with position four in addition to pyroglutamate Aβ3-x is a relevant target to fight Alzheimer’s disease. PMID:26626428

  12. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    SciTech Connect

    Couto, Sheila G.; Cristina Nonato, M.

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  13. Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

    PubMed Central

    Hamilton, VaNae; Singha, Ujjal K.; Smith, Joseph T.; Weems, Ebony

    2014-01-01

    Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria. PMID:24562910

  14. N-terminal modifications contribute to flowering time and immune response regulations

    PubMed Central

    Kapos, Paul; Xu, Fang; Meinnel, Thierry; Giglione, Carmela; Li, Xin

    2015-01-01

    A variety of N-terminal co-translational modifications play crucial roles in many cellular processes across eukaryotic organisms. Recently, N-terminal acetylation has been proposed as a regulatory mechanism for the control of plant immunity. Analysis of an N-terminal acetyltransferase complex A (NatA) mutant, naa15–1, revealed that NatA controls the stability of immune receptor Suppressor of NPR1, Constitutive 1 (SNC1) in an antagonistic fashion with NatB. Here, we further report on an antagonistic regulation of flowering time by NatA and NatB, where naa15–1 plants exhibit late flowering, opposite of the early flowering phenotype previously observed in natB mutants. In addition, we provide evidence for the involvement of another N-terminal modification, N-myristoylation, in controlling pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) through the characterization of N-myristoyltransferase 1 (NMT1) defective mutants, which express a low level of NMT1 protein. The mutant line lacks induced production of reactive oxygen species and MAP kinase phosphorylation in response to treatment with the known immune elicitor flg22. NMT1 deficient plants also exhibit increased susceptibility to Pst hrcC, a non-pathogenic Pseudomonas syringae tomato strain lacking a functional type-III secretion system. The potential for the NatA-NatB antagonistic relationship to exist outside of the regulation of SNC1 as well as the disclosing of NMT1s role in PTI further supports the significant contribution of N-terminal co-translational modifications in the regulation of biological processes in plants, and present interesting areas for further exploration. PMID:26361095

  15. Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro.

    PubMed Central

    Masuda, K.; Kamimura, T.; Watanabe, K.; Suga, T.; Kanesaki, M.; Takeuchi, A.; Imaizumi, A.; Suzuki, Y.

    1995-01-01

    1. In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half-length SLPIs, (Ser1-Pro54)SLPI and (Asn55-Ala107)SLPI, were investigated and compared with those of full-length SLPI. 2. The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C-terminal domain, (Asn55-Ala107)SLPI, was as strong as that of full-length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N-terminal domain of SLPI, (Ser1-Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study. 3. The inhibitory activity of (Asn55-Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full-SLPI (elastin, IC50 = 907 +/- 31 nM for SLPI, 767 +/- 33 nM for (Asn55-Ala107)SLPI; collagen, IC50 = 862 +/- 36 nM for SLPI, 727 +/- 47 nM for (Asn55-Ala107)SLPI). 4. The binding affinities of full- and half-length SLPIs for heparin were measured by affinity column chromatography. Full-length SLPI showed high affinity for heparin while the binding capacities of both half-length SLPIs were lower. (Concentration of NaCl for elution, 0.45 M for SLPI, 0.24 M for (Ser1-Pro54)SLPI, 0.27 M for (Asn55-Ala107)SLPI). 5. The effects of full-SLPI and (Asn55-Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582515

  16. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

    PubMed

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  17. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription

    PubMed Central

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as “shut-off”. However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  18. The S-layer proteins of two Bacillus stearothermophilus wild-type strains are bound via their N-terminal region to a secondary cell wall polymer of identical chemical composition.

    PubMed

    Egelseer, E M; Leitner, K; Jarosch, M; Hotzy, C; Zayni, S; Sleytr, U B; Sára, M

    1998-03-01

    Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1gamma chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition. PMID:9515918

  19. Truncation of domain V of the multidomain glucansucrase GTF180 of Lactobacillus reuteri 180 heavily impairs its polysaccharide-synthesizing ability.

    PubMed

    Meng, Xiangfeng; Dobruchowska, Justyna M; Pijning, Tjaard; Gerwig, Gerrit J; Kamerling, Johannis P; Dijkhuizen, Lubbert

    2015-07-01

    Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide chain follows a U shape course to form five domains, including domains A, B, and C, which resemble those of family GH13 enzymes, and two extra and novel domains (domains IV and V), which are attached to the catalytic core. To elucidate the functional roles of domain V, we have deleted the domain V fragments from both the N- and C-terminal ends (GTF180-ΔNΔV). Truncation of domain V of GTF180-ΔN yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. Instead, GTF180-ΔNΔV produced a large amount of oligosaccharides. Domain V is not involved in determining the linkage specificity, and the size of polysaccharide produced as the polysaccharide produced by GTF180-ΔNΔV was identical in size and structure with that of GTF180-ΔN. The data indicates that GTF180-ΔNΔV acts nonprocessively, frequently initiating synthesis of a new oligosaccharide from sucrose, instead of continuing the synthesis of a full size polysaccharide. Mutations L940E and L940F in GTF180-ΔNΔV, which are involved in the acceptor substrate binding, restored polysaccharide synthesis almost to the level of GTF180-ΔN. These results demonstrated that interactions of growing glucan chains with both domain V and acceptor substrate binding sites are important for polysaccharide synthesis. PMID:25586581

  20. Identification and characterization of an 18.4kDa antimicrobial truncation from shrimp Litopenaeus vannamei hemocyanin upon Vibrio parahaemolyticus infection.

    PubMed

    Wen, Ying; Zhan, Shixiong; Huang, He; Zhong, Mingqi; Chen, Jiehui; You, Cuihong; Wang, Fan; Zhang, Yueling

    2016-09-01

    Hemocyanin (HMC) is a multifunctional protein which plays many essential roles in invertebrate organism. Recently more and more immune-related functions have been discovered on this protein. Here the shrimp was infected with Vibrio parahaemolyticus and the shrimp sera were analyzed by two-dimensional gel electrophoresis. Totally 15 spots were identified as significantly up-regulated spots and further analyzed by MALDI-TOF/TOF mass spectrometry (MS). Four of them were identified as HMC derived truncations (HMCS1, HMCS3, HMCS4 and HMCS5). The HMCS4 primary sequence was further determined via Edman N terminal sequencing, MALDI-TOF MS and amino acid sequence alignment. The result indicated that the HMCS4 was a 165aa fragment from shrimp HMC small subunit C-terminal. The HMCS4 immunological activities were further analyzed by agglutination experiment and antibacterial assay in vitro. The results showed that the recombinant HMCS4 (rHMCS4) had strong agglutination and antibacterial activities against pathogenic bacteria at the optimum bacteriostasis concentration. In addition, the HMCS4 immunological activities were explored via mortality assay in vivo. The shrimp was challenged with V. parahaemolyticus and rHMCS4 V. parahaemolyticus mixture separately. The shrimp mortality rate was significantly decreased at 96 h post-infection with rHMCS4 injection. Our data showed that shrimp HMC truncation generation upon infection was an effective immune response against invaded pathogens. Moreover, these findings may have some potential applications in shrimp industry. PMID:27506277

  1. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    SciTech Connect

    Oeberg, Christine; Belikov, Sergey

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, {Delta}N-hH1.4, were compared. Black-Right-Pointing-Pointer Both histones bind to chromatin, however, {Delta}N-hH1.4 displays lower binding affinity. Black-Right-Pointing-Pointer Interaction of {Delta}N-hH1.4 with chromatin includes a significant unspecific component. Black-Right-Pointing-Pointer N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain ({Delta}N-hH1.4). The {Delta}N-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that {Delta}N-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  2. Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell- and tissue-dependent.

    PubMed

    Dron, Michel; Moudjou, Mohammed; Chapuis, Jérôme; Salamat, Muhammad Khalid Farooq; Bernard, Julie; Cronier, Sabrina; Langevin, Christelle; Laude, Hubert

    2010-04-01

    The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection. PMID:20154089

  3. An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin in zebrafish

    PubMed Central

    Zou, Jun; Tran, Diana; Baalbaki, Mai; Tang, Ling Fung; Poon, Annie; Pelonero, Angelo; Titus, Erron W; Yuan, Christiana; Shi, Chenxu; Patchava, Shruthi; Halper, Elizabeth; Garg, Jasmine; Movsesyan, Irina; Yin, Chaoying; Wu, Roland; Wilsbacher, Lisa D; Liu, Jiandong; Hager, Ronald L; Coughlin, Shaun R; Jinek, Martin; Pullinger, Clive R; Kane, John P; Hart, Daniel O; Kwok, Pui-Yan; Deo, Rahul C

    2015-01-01

    Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere. DOI: http://dx.doi.org/10.7554/eLife.09406.001 PMID:26473617

  4. Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain

    PubMed Central

    Buczek, Pawel; Horvath, Martin P.

    2010-01-01

    The Oxytricha nova telomere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (ΔH), entropy (ΔS), and dissociation constant (KD-DNA) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T2G4), d(T4G4), d(G3T4G4), and d(G4T4G4) each formed monovalent protein complexes. In the case of d(T4G4T4G4), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity “A site” has a dissociation constant, KD-DNA(A)=13(±4) nM, while the low-affinity “B site” is characterized by KD-DNA(B)=5600(±600) nM at 25 °C. Nucleotide substitution variants verified that the A site corresponds principally with the 3′-terminal portion of d(T4G4T4G4). The relative contributions of entropy (ΔS) and enthalpy (ΔH) for binding reactions were DNA length-dependent as was heat capacity (ΔCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA–protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology. PMID:16678852

  5. N-terminal mutations in the anti-estradiol Fab 57-2 modify its hapten binding properties.

    PubMed Central

    Saviranta PJauria, P.; Lamminmäki, U.; Hellman, J.; Eriksson, S.; Lövgren, T.

    2000-01-01

    Recombinant antibodies often contain N-terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restriction sites in the framework 1 regions. We studied the effects of such mutations in a recombinant anti-estradiol Fab fragment derived from the hybridoma cell line 57-2. The 5' ends of the heavy and light chain genes were originally modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However, the affinity and specificity of the recombinant Fab were lowered compared to the proteolytic Fab' fragment of the parental hybridoma IgG. Replacing the mutated sites with authentic amino acid coding sequences restored the binding properties as well as increased the bacterial production levels fivefold and 10-fold at 30 and 37 degrees C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Kd) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Kd for testosterone whereas the mutated light chain amino terminus decreased the Kd for all of the steroids to the same extent; the heavy and light chain effects were additive. Analysis of a newly determined crystal structure of the authentic Fab 57-2 in complex with estradiol suggests that mutations in the residue 2 in V(H), and 2 and 4 in the V(L) domain were those responsible for the observed effects. Their general roles as structure-determining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well. PMID:11206076

  6. Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein.

    PubMed

    Rodríguez, Esaú E; Arcos-López, Trinidad; Trujano-Ortiz, Lidia G; Fernández, Claudio O; González, Felipe J; Vela, Alberto; Quintanar, Liliana

    2016-09-01

    Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex. PMID:27422629

  7. Lytic Action of the Truncated yncE Gene in Escherichia coli.

    PubMed

    Li, Jianhua; Xiong, Kun; Zou, Lingyun; Chen, Zhijin; Wang, Yiran; Hu, Xiaomei; Rao, Xiancai; Cong, Yanguang

    2016-04-01

    We recently found lytic action of the truncated yncE gene. When the truncated yncE gene of Salmonella enterica serovar Paratyphi A was expressed in Escherichia coli DH5α under the control of the Ara promoter, bacterial growth was markedly inhibited. In the present study, we characterized this lytic action. The N-terminal 103 aa of YncE, containing a signal peptide, was demonstrated to be essential for inhibition. Microscopic observation showed that the bacterial envelope of E. coli was damaged by the expression of truncated yncE, resulting in the release of cytoplasmic content and the formation of bacterial ghosts. The addition of MgSO4 or spermine, which is the stabilizer of bacterial membrane structure, dramatically reversed the cell lysis induced by the toxic truncated YncE. In contrast, the lytic action was significantly enhanced by the addition of SDS or EDTA. Our data indicated that the toxic truncated YncE could cause cell lysis by the disruption of the bacterial membrane. PMID:26687463

  8. Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis

    PubMed Central

    Linster, Eric; Stephan, Iwona; Bienvenut, Willy V.; Maple-Grødem, Jodi; Myklebust, Line M.; Huber, Monika; Reichelt, Michael; Sticht, Carsten; Geir Møller, Simon; Meinnel, Thierry; Arnesen, Thomas; Giglione, Carmela; Hell, Rüdiger; Wirtz, Markus

    2015-01-01

    N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid. PMID:26184543

  9. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    PubMed Central

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-01-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

  10. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    NASA Astrophysics Data System (ADS)

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O'Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-12-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

  11. The basic N-terminal domain of TRF2 limits recombination endonuclease action at human telomeres.

    PubMed

    Saint-Léger, Adélaïde; Koelblen, Melanie; Civitelli, Livia; Bah, Amadou; Djerbi, Nadir; Giraud-Panis, Marie-Josèphe; Londoño-Vallejo, Arturo; Ascenzioni, Fiorentina; Gilson, Eric

    2014-01-01

    The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication. PMID:25483196

  12. Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

    NASA Technical Reports Server (NTRS)

    Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1991-01-01

    Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

  13. First Things First: Vital Protein Marks by N-Terminal Acetyltransferases.

    PubMed

    Aksnes, Henriette; Drazic, Adrian; Marie, Michaël; Arnesen, Thomas

    2016-09-01

    N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways. PMID:27498224

  14. Structure of the human histone chaperone FACT Spt16 N-terminal domain.

    PubMed

    Marcianò, G; Huang, D T

    2016-02-01

    The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding. PMID:26841762

  15. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

    PubMed Central

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J.; Martinho, Rui Gonçalo

    2016-01-01

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes. PMID:26861501

  16. In vitro phosphorylation of the N-terminal half of hordeivirus movement protein.

    PubMed

    Makarov, V V; Iconnikova, A Y; Guseinov, M A; Vishnichenko, V K; Kalinina, N O

    2012-09-01

    The N-terminal half of TGB1 movement protein of poa semilatent hordeivirus, which forms a ribonucleoprotein complex involved in movement of the viral genome in the plant, and its two domains, NTD and ID, are phosphorylated in vitro by a fraction enriched in cell walls from Nicotiana benthamiana. Using a set of protein kinase inhibitors with different specificities, it was found that enzymes possessing activities of casein kinase 1, protein kinase A, and protein kinase C are involved in phosphorylation. Commercial preparations of protein kinases A and C are able to phosphorylate in vitro recombinant proteins corresponding to the N-terminal half of the protein and its domains NTD and ID. Phosphorylation of the NTD has no effect on the efficiency and character of its binding to RNA. However, phosphorylation of the ID leads to a decrease in its RNA-binding activity and in the ability for homological protein-protein interactions. PMID:23157268

  17. Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

    SciTech Connect

    Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.; Moore, Ronald J.; Camp, David G.; Baker, Scott E.; Smith, Richard D.; Qian, Weijun

    2013-06-17

    Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

  18. Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures

    PubMed Central

    Lin, Haijiang; Keriel, Anne; Morales, Carlos R.; Bedard, Nathalie; Zhao, Qing; Hingamp, Pascal; Lefrançois, Stephane; Combaret, Lydie; Wing, Simon S.

    2000-01-01

    Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action. PMID:10938131

  19. Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

    PubMed Central

    Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

    2008-01-01

    Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

  20. Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.

    PubMed

    Feng, Jinlin; Li, Ruiqi; Yu, Junya; Ma, Shuangshuang; Wu, Chunyan; Li, Yan; Cao, Ying; Ma, Ligeng

    2016-08-01

    Early embryonic development generates precursors of all major cell types in Arabidopsis. Among these precursors, the hypophysis divides asymmetrically to form the progenitors of the quiescent center and columella stem cells. A great deal has been learnt about the mechanisms that control the asymmetric division of the hypophysis and embryogenesis at the transcriptional level; however, no evidence of regulation at the co- or post-translational level has been reported. Here, we show that mutation of the catalytic subunit (Naa10) or auxiliary subunit (Naa15) of NatA, an N-terminal acetyltransferase that catalyzes protein N-terminal acetylation, produces an embryo-lethal phenotype. In addition, Naa10 and Naa15 were found to interact physically in planta Further analysis revealed that the observed embryonic patterning defects started at the early globular stage and that the asymmetric division of the hypophysis was irregular; thus, no quiescent center progenitor cells were generated in naa10 and naa15 embryos. We further observed that the polar distributions of auxin and its efflux carrier PIN1 were disturbed in naa10 embryos. Our results suggest that NatA is required for asymmetric division of the hypophysis and early embryonic patterning in Arabidopsis, and provides a link between protein N-terminal acetylation and embryogenesis in plants. PMID:27385766

  1. Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface

    PubMed Central

    Kimlicka, Lynn; Lau, Kelvin; Tung, Ching-Chieh; Van Petegem, Filip

    2013-01-01

    Ryanodine receptors are large channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain–domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region. PMID:23422674

  2. Intrinsic disorder and multiple phosphorylations constrain the evolution of the flightin N-terminal region.

    PubMed

    Lemas, Dominick; Lekkas, Panagiotis; Ballif, Bryan A; Vigoreaux, Jim O

    2016-03-01

    Flightin is a myosin binding phosphoprotein that originated in the ancestor to Pancrustacea ~500 MYA. In Drosophila melanogaster, flightin is essential for length determination and flexural rigidity of thick filaments. Here, we show that among 12 Drosophila species, the N-terminal region is characterized by low sequence conservation, low pI, a cluster of phosphorylation sites, and a high propensity to intrinsic disorder (ID) that is augmented by phosphorylation. Using mass spectrometry, we identified eight phosphorylation sites within a 29 amino acid segment in the N-terminal region of D. melanogaster flightin. We show that phosphorylation of D. melanogaster flightin is modulated during flight and, through a comparative analysis to orthologs from other Drosophila species, we found phosphorylation sites that remain invariant, sites that retain the charge character, and sites that are clade-specific. While the number of predicted phosphorylation sites differs across species, we uncovered a conserved pattern that relates the number of phosphorylation sites to pI and ID. Extending the analysis to orthologs of other insects, we found additional conserved features in flightin despite the near absence of sequence identity. Collectively, our results demonstrate that structural constraints demarcate the evolution of the highly variable N-terminal region. PMID:26691840

  3. Protein N-terminal acetylation is required for embryogenesis in Arabidopsis

    PubMed Central

    Feng, Jinlin; Li, Ruiqi; Yu, Junya; Ma, Shuangshuang; Wu, Chunyan; Li, Yan; Cao, Ying; Ma, Ligeng

    2016-01-01

    Early embryonic development generates precursors of all major cell types in Arabidopsis. Among these precursors, the hypophysis divides asymmetrically to form the progenitors of the quiescent center and columella stem cells. A great deal has been learnt about the mechanisms that control the asymmetric division of the hypophysis and embryogenesis at the transcriptional level; however, no evidence of regulation at the co- or post-translational level has been reported. Here, we show that mutation of the catalytic subunit (Naa10) or auxiliary subunit (Naa15) of NatA, an N-terminal acetyltransferase that catalyzes protein N-terminal acetylation, produces an embryo-lethal phenotype. In addition, Naa10 and Naa15 were found to interact physically in planta. Further analysis revealed that the observed embryonic patterning defects started at the early globular stage and that the asymmetric division of the hypophysis was irregular; thus, no quiescent center progenitor cells were generated in naa10 and naa15 embryos. We further observed that the polar distributions of auxin and its efflux carrier PIN1 were disturbed in naa10 embryos. Our results suggest that NatA is required for asymmetric division of the hypophysis and early embryonic patterning in Arabidopsis, and provides a link between protein N-terminal acetylation and embryogenesis in plants. PMID:27385766

  4. Expanding the Phenotype Associated with NAA10-Related N-Terminal Acetylation Deficiency.

    PubMed

    Saunier, Chloé; Støve, Svein Isungset; Popp, Bernt; Gérard, Bénédicte; Blenski, Marina; AhMew, Nicholas; de Bie, Charlotte; Goldenberg, Paula; Isidor, Bertrand; Keren, Boris; Leheup, Bruno; Lampert, Laetitia; Mignot, Cyril; Tezcan, Kamer; Mancini, Grazia M S; Nava, Caroline; Wasserstein, Melissa; Bruel, Ange-Line; Thevenon, Julien; Masurel, Alice; Duffourd, Yannis; Kuentz, Paul; Huet, Frédéric; Rivière, Jean-Baptiste; van Slegtenhorst, Marjon; Faivre, Laurence; Piton, Amélie; Reis, André; Arnesen, Thomas; Thauvin-Robinet, Christel; Zweier, Christiane

    2016-08-01

    N-terminal acetylation is a common protein modification in eukaryotes associated with numerous cellular processes. Inherited mutations in NAA10, encoding the catalytic subunit of the major N-terminal acetylation complex NatA have been associated with diverse, syndromic X-linked recessive disorders, whereas de novo missense mutations have been reported in one male and one female individual with severe intellectual disability but otherwise unspecific phenotypes. Thus, the full genetic and clinical spectrum of NAA10 deficiency is yet to be delineated. We identified three different novel and one known missense mutation in NAA10, de novo in 11 females, and due to maternal germ line mosaicism in another girl and her more severely affected and deceased brother. In vitro enzymatic assays for the novel, recurrent mutations p.(Arg83Cys) and p.(Phe128Leu) revealed reduced catalytic activity. X-inactivation was random in five females. The core phenotype of X-linked NAA10-related N-terminal-acetyltransferase deficiency in both males and females includes developmental delay, severe intellectual disability, postnatal growth failure with severe microcephaly, and skeletal or cardiac anomalies. Genotype-phenotype correlations within and between both genders are complex and may include various factors such as location and nature of mutations, enzymatic stability and activity, and X-inactivation in females. PMID:27094817

  5. A Truncated Waveguide Phase Shifter

    NASA Technical Reports Server (NTRS)

    Lourie, Nathan P.; Chuss, D. T.; Henry, R.; Wollack, E. J.

    2011-01-01

    The design, fabrication and performance of a simple phase shifter based upon truncated circular and square waveguides is presented. An emphasis is placed upon validation of simple analytical formulae that describe the propagation properties of the structure. A test device is prototyped at approximately 40GHz; however, the concepts explored can be directly extended to millimeter and submillimeter applications.

  6. Fragmentation reactions of deprotonated peptides containing proline. The proline effect.

    PubMed

    Harrison, Alex G; Young, Alex B

    2005-09-01

    The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS(2) and MS(3) experiments, deuterium labelling and accurate mass measurements when necessary. The [M--H--CO(2)](-) (a(2)) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C(2)H(4); this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a(3) and y(1) ions. When proline is in the central position of tripeptides, a(3), y(2) and y(1) ions are the primary fragmentation products of [M--H](-), while when the proline is in the C-terminal position, a(3)and y(1) ions are the major primary products. In the latter case, the a(3) ion fragments primarily to the ''b(2) ion; further evidence is presented that the ''b(2) ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline. PMID:16041740

  7. Signaling by the Engulfment Receptor Draper: A Screen in Drosophila melanogaster Implicates Cytoskeletal Regulators, Jun N-Terminal Kinase, and Yorkie

    PubMed Central

    Fullard, John F.; Baker, Nicholas E.

    2015-01-01

    Draper, the Drosophila melanogaster homolog of the Ced-1 protein of Caenorhabditis elegans, is a cell-surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, we screened chromosomal deficiencies to identify loci that dominantly modify the phenotype of overexpression of Draper isoform II (suppressed differentiation of the posterior crossvein in the wing). We found evidence for 43 genetic modifiers of Draper II. Twenty-four of the 37 suppressor loci and 3 of the 6 enhancer loci were identified. An additional 5 suppressors and 2 enhancers were identified among mutations in functionally related genes. These studies reveal positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott–Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in Draper signaling. Relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador/Warts/Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are also discussed. PMID:25395664

  8. Thermodynamics of the protonation equilibria of two fragments of N-terminal β-hairpin of FPB28 WW domain.

    PubMed

    Makowska, Joanna; Uber, Dorota; Chmurzyński, Lech

    2012-01-12

    The pK(a) values of two peptides derived from the formin-binding protein 28 WW domain [Ac-Lys-Thr-Ala-Asp-Gly-Lys-Thr-NH(2) (D7), Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH(2) (D9)] were determined by potentiometric titration in the temperature range from 25 to 60 °C, and their heat capacities were determined, by differential scanning calorimetry, in the temperature range from 10 to 90 °C. For both peptides, heat capacity has a maximum at t ≈ 50 °C, with height about 0.1 kcal/(mol × deg), suggesting that a modest unfolding transition occurs. The first two pK(a)'s are low at temperatures below 50 °C, suggesting that the two lysine residues are close to each other and the peptides have bent shapes at lower temperatures; this effect is greater for D7 compared with D9. With increasing temperature beyond 50 °C (i.e., that of the thermodynamic unfolding transition), pK(a1) and pK(a2) increase rapidly for D9, whereas their temperature variation is less significant for D7. This observation, and the fact that the enthalpies and entropies of the dissociation of the two first protons (determined from the temperature dependence of the respective pK(a)'s) decrease significantly near the transition temperature, suggest that the peptide undergoes a transition from a bent to an amorphous shape and that the presence of charged lysine residues stabilizes the folded state. PMID:22128840

  9. Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase (hNTAN1)

    PubMed Central

    Cantor, Jason R.; Stone, Everett M.; Georgiou, George

    2011-01-01

    The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein we report the bacterial expression, purification, and biochemical characterization of the human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn, but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the point mutation Cys75Ala. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway. PMID:21375249

  10. Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

    PubMed Central

    Li, Lin; Gannon, Patrick; Linster, Eric; Huber, Monika; Kapos, Paul; Bienvenut, Willy; Giglione, Carmela; Zhang, Yuelin; Chen, She

    2015-01-01

    Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis. PMID:25966763

  11. Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia.

    PubMed

    Muñoz-Bertomeu, Jesús; Sales, Ester; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2007-11-01

    Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, beta-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T1 plants derived from self-pollinated seeds of T0 lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T0 plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species. PMID:17714440

  12. Truncated States Obtained by Iteration

    NASA Astrophysics Data System (ADS)

    Cardoso B., W.; Almeida G. de, N.

    2008-02-01

    We introduce the concept of truncated states obtained via iterative processes (TSI) and study its statistical features, making an analogy with dynamical systems theory (DST). As a specific example, we have studied TSI for the doubling and the logistic functions, which are standard functions in studying chaos. TSI for both the doubling and logistic functions exhibit certain similar patterns when their statistical features are compared from the point of view of DST.

  13. The gaf Fimbrial Gene Cluster of Escherichia coli Expresses a Full-Size and a Truncated Soluble Adhesin Protein

    PubMed Central

    Tanskanen, Jarna; Saarela, Sirkku; Tankka, Sanna; Kalkkinen, Nisse; Rhen, Mikael; Korhonen, Timo K.; Westerlund-Wikström, Benita

    2001-01-01

    The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified from the periplasm of E. coli by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named ΔGafD. Expression of gafD from the cloned gaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of ΔGafD. The peptide was also detected in the periplasm of the wild-type E. coli strain from which the gaf gene cluster originally was cloned. We expressed gafD fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of ΔGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using gafD indicated that ΔGafD was processed from GafD and is not a primary translation product. The ΔGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [14C]ΔGafD to GlcNAc-agarose. ΔGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when

  14. An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway.

    PubMed

    Serero, Alexandre; Giglione, Carmela; Sardini, Alessandro; Martinez-Sanz, Juan; Meinnel, Thierry

    2003-12-26

    Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. PMID:14532271

  15. Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.

    PubMed

    van Veen, Harrie A; Geerts, Marlieke E J; van Berkel, Patrick H C; Nuijens, Jan H

    2002-10-01

    Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants. PMID:12381362

  16. N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

    PubMed Central

    Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

    2014-01-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

  17. The Functional Study of the N-Terminal Region of Influenza B Virus Nucleoprotein

    PubMed Central

    Liu, Ming; Lam, Mandy Ka-Han; Zhang, Qinfen; Elderfield, Ruth; Barclay, Wendy S.; Shaw, Pang-Chui

    2015-01-01

    Influenza nucleoprotein (NP) is a major component of the ribonucleoprotein (vRNP) in influenza virus, which functions for the transcription and replication of viral genome. Compared to the nucleoprotein of influenza A (ANP), the N-terminal region of influenza B nucleoprotein (BNP) is much extended. By virus reconstitution, we found that the first 38 residues are essential for viral growth. We further illustrated the function of BNP by mini-genome reconstitution, fluorescence microscopy, electron microscopy, light scattering and gel shift. Results show that the N terminus is involved in the formation of both higher homo-oligomers of BNP and BNP-RNA complex. PMID:26368391

  18. The C-terminus of CaMKII is truncated when expressed in E. coli.

    PubMed

    Praseeda, M; Beena, Mary K; Asha, Sarah John; Omkumar, R V

    2004-04-01

    The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system. PMID:15078206

  19. Structure-Activity Relationships of the Antimicrobial Peptide Arasin 1 — And Mode of Action Studies of the N-Terminal, Proline-Rich Region

    PubMed Central

    Paulsen, Victoria S.; Blencke, Hans-Matti; Benincasa, Monica; Haug, Tor; Eksteen, Jacobus J.; Styrvold, Olaf B.; Scocchi, Marco; Stensvåg, Klara

    2013-01-01

    Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH2 terminus of the peptide and the fragment arasin 1(1–23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1–23) were shown to be non-toxic to human red blood cells and arasin 1(1–23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1–23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1–23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1–23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1–23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC. PMID:23326415

  20. Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking.

    PubMed

    Martincuks, Antons; Fahrenkamp, Dirk; Haan, Serge; Herrmann, Andreas; Küster, Andrea; Müller-Newen, Gerhard

    2016-08-01

    Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the

  1. Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate.

    PubMed

    Bondulich, Marie K; Guo, Tong; Meehan, Christopher; Manion, John; Rodriguez Martin, Teresa; Mitchell, Jacqueline C; Hortobagyi, Tibor; Yankova, Natalia; Stygelbout, Virginie; Brion, Jean-Pierre; Noble, Wendy; Hanger, Diane P

    2016-08-01

    Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies. PMID:27297240

  2. Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate

    PubMed Central

    Bondulich, Marie K.; Guo, Tong; Meehan, Christopher; Manion, John; Rodriguez Martin, Teresa; Mitchell, Jacqueline C.; Hortobagyi, Tibor; Yankova, Natalia; Stygelbout, Virginie; Brion, Jean-Pierre; Noble, Wendy

    2016-01-01

    Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies. PMID:27297240

  3. Immune challenge induces N-terminal cleavage of the Drosophila serpin Necrotic

    PubMed Central

    Pelte, Nadège; Robertson, Andrew S.; Zou, Zhen; Belorgey, Didier; Dafforn, Timothy R.; Jiang, Haobo; Lomas, David; Reichhart, Jean-Marc; Gubb, David

    2007-01-01

    The Drosophila Necrotic protein is a serine proteinase inhibitor, which regulates the Toll-mediated innate immune response. Necrotic specifically inhibits an extracellular serine proteinase cascade leading to activation of the Toll ligand, Spätzle. Necrotic carries a polyglutamine extension amino-terminal to the core serpin structure. We show here that cleavage of this N-terminal extension occurs following immune challenge. This modification is blocked in PGRP-SAsemmelweiss mutants after Gram-positive bacterial challenge and in persephone mutants after fungal or Gram-positive bacterial challenge, indicating that activation of either of the Toll pathway upstream branches induces N-terminal cleavage of the serpin. The absolute requirement of persephone gene product for this cleavage indicates that Gram-positive bacteria activate a redundant set of proteinases upstream of Toll. Both full-length Necrotic and the core serpin are active inhibitors of a range of serine proteinases: the highest affinity being for cathepsin G and elastases. We found a 13-fold increase in the specificity of the core serpin over that of full-length Necrotic for one of the tested proteinases (porcine pancreatic elastase). This finding indicates that cleavage of the Necrotic amino-terminal extension might modulate Toll activation following the initial immune response. PMID:16360948

  4. N-terminal domain-mediated homodimerization is required for photoreceptor activity of Arabidopsis CRYPTOCHROME 1.

    PubMed

    Sang, Yi; Li, Qing-Hua; Rubio, Vicente; Zhang, Yan-Chun; Mao, Jian; Deng, Xing-Wang; Yang, Hong-Quan

    2005-05-01

    Cryptochromes (CRY) are blue light receptors that share sequence similarity with photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA. Transgenic Arabidopsis thaliana seedlings expressing the C-terminal domains of the Arabidopsis CRY fused to beta-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype, indicating that the signaling mechanism of Arabidopsis CRY is mediated through the C-terminal domain. The role of the Arabidopsis CRY N-terminal photolyase-like domain in CRY action remains poorly understood. Here, we report the essential role of the Arabidopsis CRY1 N-terminal domain (CNT1) in the light activation of CRY1 photoreceptor activity. Yeast two-hybrid assay, in vitro binding, in vivo chemical cross-linking, gel filtration, and coimmunoprecipitation studies indicate that CRY1 homodimerizes in a light-independent manner. Mutagenesis and transgenic studies demonstrate that CNT1-mediated dimerization is required for light activation of the C-terminal domain of CRY1 (CCT1). Transgenic data and native gel electrophoresis studies suggest that multimerization of GUS is both responsible and required for mediating a COP phenotype on fusion to CCT1. These results indicate that the properties of the GUS multimer are analogous to those of the light-modified CNT1 dimer. Irradiation with blue light modifies the properties of the CNT1 dimer, resulting in a change in CCT1, activating CCT1, and eventually triggering the CRY1 signaling pathway. PMID:15805487

  5. Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

    PubMed

    Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

    2016-04-19

    Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. PMID:26954624

  6. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

    SciTech Connect

    Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine; Drummer, Heidi E.; Poumbourios, Pantelis . E-mail: apoumbourios@burnet.edu.au

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.

  7. Evidence for N-Terminal Myristoylation of Tetrahymena Arginine Kinase Using Peptide Mass Fingerprinting Analysis.

    PubMed

    Motomura, Shou; Suzuki, Tomohiko

    2016-06-01

    In this study, we confirmed N-terminal myristoylation of Tetrahymena pyriformis arginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H(+)) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesized T. pyriformis AK1 is myristoylated. The fixed specimens of T. pyriformis were reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate that T. pyriformis AK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups. PMID:27129461

  8. Selection and characterization of llama single domain antibodies against N-terminal huntingtin.

    PubMed

    Schut, Menno H; Pepers, Barry A; Klooster, Rinse; van der Maarel, Silvère M; El Khatabi, Mohamed; Verrips, Theo; den Dunnen, Johan T; van Ommen, Gert-Jan B; van Roon-Mom, Willeke M C

    2015-03-01

    Huntington disease is caused by expansion of a CAG repeat in the huntingtin gene that is translated into an elongated polyglutamine stretch within the N-terminal domain of the huntingtin protein. The mutation is thought to introduce a gain-of-toxic function in the mutant huntingtin protein, and blocking this toxicity by antibody binding could alleviate Huntington disease pathology. Llama single domain antibodies (VHH) directed against mutant huntingtin are interesting candidates as therapeutic agents or research tools in Huntington disease because of their small size, high thermostability, low cost of production, possibility of intracellular expression, and potency of blood-brain barrier passage. We have selected VHH from llama phage display libraries that specifically target the N-terminal domain of the huntingtin protein. Our VHH are capable of binding wild-type and mutant human huntingtin under native and denatured conditions and can be used in Huntington disease studies as a novel antibody that is easy to produce and manipulate. PMID:25294428

  9. Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms

    PubMed Central

    Giglione, Carmela; Serero, Alexandre; Pierre, Michèle; Boisson, Bertrand; Meinnel, Thierry

    2000-01-01

    The N-terminal protein processing pathway is an essential mechanism found in all organisms. However, it is widely believed that deformylase, a key enzyme involved in this process in bacteria, does not exist in eukaryotes, thus making it a target for antibacterial agents such as actinonin. In an attempt to define this process in higher eukaryotes we have used Arabidopsis thaliana as a model organism. Two deformylase cDNAs, the first identified in any eukaryotic system, and six distinct methionine aminopeptidase cDNAs were cloned. The corresponding proteins were characterized in vivo and in vitro. Methionine aminopeptidases were found in the cytoplasm and in the organelles, while deformylases were localized in the organelles only. Our work shows that higher plants have a much more complex machinery for methionine removal than previously suspected. We were also able to identify deformylase homologues from several animals and clone the corresponding cDNA from human cells. Our data provide the first evidence that lower and higher eukaryotes, as well as bacteria, share a similar N-terminal protein processing machinery, indicating universality of this system. PMID:11060042

  10. Plasmodium vivax: N-terminal diversity in the blood stage SERA genes from Indian isolates.

    PubMed

    Rahul, C N; Shiva Krishna, K; Meera, M; Phadke, Sandhya; Rajesh, Vidya

    2015-06-01

    Worldwide malaria risk due to Plasmodium vivax makes development of vaccine against P. vivax, a high priority. Serine Repeat Antigen of P. vivax (PvSERA) is a multigene family of blood stage proteins with 12 homologues. Sequence diversity studies are important for understanding them as potential vaccine candidates. No information on N-terminal diversity of these genes is available in literature. In this paper, we evaluate the genetic polymorphism of N-terminal regions of the highly expressed member PvSERA4 and PvSERA5 genes from Indian field isolates. Our results show that PvSERA4 has deletions and insertions in Glutamine rich tetrameric repeat units contributing to its diversity. PvSERA5 also exhibits high genetic diversity with non-synonymous substitutions leading to identification of novel haplotypes from India. Our first report helps in elucidating the allelic variants of PvSERA genes in this region and contributes to evaluating their efficacy as vaccine candidates. PMID:25976464

  11. Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

    PubMed Central

    Lai, Chun-Liang; Jao, Christine C.; Lyman, Edward; Gallop, Jennifer L.; Peter, Brian J.; McMahon, Harvey T.; Langen, Ralf; Voth, Gregory A.

    2012-01-01

    Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H0) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed. PMID:22922484

  12. A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method

    PubMed Central

    Huang, Junfeng; Qin, Hongqiang; Sun, Zhen; Huang, Guang; Mao, Jiawei; Cheng, Kai; Zhang, Zhang; Wan, Hao; Yao, Yating; Dong, Jing; Zhu, Jun; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa

    2015-01-01

    Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome. PMID:25959593

  13. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.

    PubMed

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

    2015-06-01

    The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH2. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH2 (LC50=0.045mmol/L) was similar to the lead compound (LC50=0.048mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides. PMID:25116632

  14. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability.

    PubMed

    Wilson, Kirilee A; Maerz, Anne L; Bär, Séverine; Drummer, Heidi E; Poumbourios, Pantelis

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Bär, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function. PMID:17577584

  15. X-ray crystal structure of the trimeric N-terminal domain of gephyrin.

    PubMed

    Sola, M; Kneussel, M; Heck, I S; Betz, H; Weissenhorn, W

    2001-07-01

    Gephyrin is a ubiquitously expressed protein that, in the central nervous system, forms a submembraneous scaffold for anchoring inhibitory neurotransmitter receptors in the postsynaptic membrane. The N- and C-terminal domains of gephyrin are homologous to the Escherichia coli enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis. This enzymatic pathway is highly conserved from bacteria to mammals, as underlined by the ability of gephyrin to rescue molybdenum cofactor deficiencies in different organisms. Here we report the x-ray crystal structure of the N-terminal domain (amino acids 2-188) of rat gephyrin at 1.9-A resolution. Gephyrin-(2-188) forms trimers in solution, and a sequence motif thought to be involved in molybdopterin binding is highly conserved between gephyrin and the E. coli protein. The atomic structure of gephyrin-(2-188) resembles MogA, albeit with two major differences. The path of the C-terminal ends of gephyrin-(2-188) indicates that the central and C-terminal domains, absent in this structure, should follow a similar 3-fold arrangement as the N-terminal region. In addition, a central beta-hairpin loop found in MogA is lacking in gephyrin-(2-188). Despite these differences, both structures show a high degree of surface charge conservation, which is consistent with their common catalytic function. PMID:11325967

  16. Identification of an N-terminal formylated, two-peptide bacteriocin from Enterococcus faecalis 710C.

    PubMed

    Liu, Xiaoji; Vederas, John C; Whittal, Randy M; Zheng, Jing; Stiles, Michael E; Carlson, Denise; Franz, Charles M A P; McMullen, Lynn M; van Belkum, Marco J

    2011-05-25

    Enterococcus faecalis 710C, isolated from beef product, has a broad antimicrobial activity spectrum against foodborne pathogens. Two bacteriocins, enterocin 7A (Ent7A) and enterocin 7B (Ent7B), were purified from the culture supernatant of E. faecalis 710C and characterized using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and electrospray infusion tandem mass spectrometry analyses. These data and subsequent genetic analysis showed that Ent7A and Ent7B are produced without N-terminal leader sequences and have amino acid sequences that are identical to those of enterocins MR10A and MR10B, respectively. However, the observed masses for Ent7A and Ent7B are 5200.80 and 5206.65 Da (monoisotopic mass), respectively, which are higher than the theoretical molecular masses of MR10A and MR10B, respectively. This study provides evidence that both Ent7A and Ent7B are formylated on the N-terminal methionine residue. Purified Ent7A and Ent7B are active against spoilage microorganisms and foodborne pathogens, including Clostridium sporogenes , Listeria monocytogenes , and Staphylococcus aureus as well as Brevundimonas diminuta , which has been associated with infections among immune-suppressed cancer patients. PMID:21469734

  17. Specific N-terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria

    PubMed Central

    Wall, Erin A.; Caufield, J. Harry; Lyons, Charles E.; Manning, Keith A.; Dokland, Terje; Christie, Gail E.

    2015-01-01

    Summary Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report we demonstrate that L27 in Staphylococcus aureus and other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram negative bacterium Escherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of the E. coli ribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway. PMID:25388641

  18. Crystallization and Preliminary Diffraction Analysis of Truncated Human Pleckstrin

    SciTech Connect

    S Jackson; S Sugiman-Marangos; K Cheung; M Junop

    2011-12-31

    Pleckstrin is a major substrate of protein kinase C in platelets and leukocytes and appears to play an important role in exocytosis through a currently unknown mechanism. Pleckstrin function is regulated by phosphorylation, which is thought to cause dissociation of pleckstrin dimers, thereby facilitating phosphoinositide interactions and membrane localization. Evidence also exists suggesting that phosphorylation causes a subtle conformational change in pleckstrin. Structural studies of pleckstrin have been initiated in order to characterize these structural changes and ultimately advance understanding of pleckstrin function. Here, the crystallization and preliminary X-ray diffraction analysis of a truncated version of pleckstrin consisting of the N-terminal PH domain, the protein kinase C phosphorylation sites and the DEP domain (NPHDEP) are reported. In addition, the oligomeric state and phospholipid-binding properties of NPHDEP were analyzed. This work demonstrates that NPHDEP behaves as a monomer in solution and suggests that all three pleckstrin domains contribute to the dimerization interface. Furthermore, based on the binding properties of NPHDEP, the C-terminal PH domain appears to increase the specificity of pleckstrin for phosphoinositides. This work represents a significant step towards determining the structure of pleckstrin.

  19. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-01-01

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD. PMID:24619194

  20. Structure of N-Terminal Sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-Peptide with Phospholipase A2 from Venom of Andaman Cobra Sub-Species Naja naja sagittifera at 2.0 Å Resolution

    PubMed Central

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-01-01

    Alzheimer’s disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD’s neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer’s Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ–Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD. PMID:24619194

  1. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. PMID:26500262

  2. Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain

    PubMed Central

    Moparthi, Lavanya; Survery, Sabeen; Kreir, Mohamed; Simonsen, Charlotte; Kjellbom, Per; Högestätt, Edward D.; Johanson, Urban; Zygmunt, Peter M.

    2014-01-01

    We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ9-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca2+, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease). PMID:25389312

  3. HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal {alpha} helix

    SciTech Connect

    Mallick, Jaideep; Datta, Kasturi . E-mail: kdatta@mail.jnu.ac.in

    2005-10-01

    Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domain of HABP1, its N- and C-terminal truncated variants ({delta}N.HABP1 and {delta}C.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, {delta}C.HABP1 too directly interacts with CDC 25 while {delta}N.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal {alpha}-helix.

  4. Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I.

    PubMed

    Monchois, V; Vignon, M; Russell, R R

    1999-11-01

    Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation. PMID:10570812

  5. Copper(I) Forms a Redox-Stable 1:2 Complex with α-Synuclein N-Terminal Peptide in a Membrane-Like Environment.

    PubMed

    Dell'Acqua, Simone; Pirota, Valentina; Monzani, Enrico; Camponeschi, Francesca; De Ricco, Riccardo; Valensin, Daniela; Casella, Luigi

    2016-06-20

    α-Synuclein (αS) is the main protein component of Lewy bodies, characterizing the pathogenesis of Parkinson's disease. αS is unstructured in solution but adopts a helical structure in its extended N-terminal segment upon association with membranes. In vitro the protein binds avidly Cu(II), but in vivo the protein is N-acetylated, and Cu(II) binding is lost. We have now clarified the binding characteristics of the Cu(I) complex with the truncated αS peptide 1-15, both in N-acetylated and free amine forms, in a membrane mimetic environment and found that complexation occurs with a 1:2 Cu(I)-αS stoichiometry, where Cu(I) is bound to Met1 and Met5 residues of two helical peptide chains. The resulting tetrahedral Cu(I) center is redox-stable, does not form reactive oxygen species, and is unreactive against dopamine in the presence of O2. This suggests that, unlike cytosolic Cu(I)-αS, which retains the capacity to activate O2 and promote oxidative reactions, membrane-bound Cu(I)-αS may serve as a sink for unreactive copper. PMID:27259006

  6. Identification of a human ABCC10 orthologue in Catharanthus roseus reveals a U12-type intron determinant for the N-terminal domain feature.

    PubMed

    El-Guizani, Taissir; Guibert, Clotilde; Triki, Saida; St-Pierre, Benoit; Ducos, Eric

    2014-04-01

    ABC (ATP-binding cassette) transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrates across biological membranes. In general, members of C subfamily (ABCC) are structurally characterized by an additional (N-terminal) transmembrane domain (TMD0). Phylogenetic analysis of plant ABCCs separates their protein sequences into three distinct clusters: I and II are plant specific whereas cluster III contains both human and plant ABCCs. Screening of the Plant Medicinal Genomics Resource database allowed us to identify 16 ABCCs partial sequences in Catharanthus roseus; two of which belong to the unique CrABCC1 transcript that we identified in cluster III. Genomic organization of CrABCC1 TMD0 coding sequence displays an AT-AC U12-type intron that is conserved in higher plant orthologues. We showed that CrABCC1, like its human orthologue ABCC10, produces alternative transcripts that encode protein sequences with a truncated form of TMD0 without the first transmembrane span (TM1). Subcellular localization of CrABCC1 TMD0 variants using yellow fluorescent protein fusions reveals that the TM1 is required for a correct routing of the TMD0 to the tonoplast. Finally, the specific repartition of CrABCC1 orthologues in some species suggests that this gene was lost several times during evolution and that its physiological function may, rely on a common feature of multicellular eukaryotes. PMID:24840820

  7. A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris.

    PubMed

    Dagher, Suzanne F; Bruno-Bárcena, José M

    2016-01-01

    The β-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1-110), which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the α mating factor (MFα) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MFα (MFα-rBht(23-594)), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53- and 14-fold, respectively. Leader interference was demonstrated when MFα preceded the putative classical rBHT(1-22) leader (amino acids 1-22), explaining the limited secretion of soluble protein by P. pastoris (GS115 : : MFα-rBht(1-594)). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-β-galactosidase single-chain variable antibody fragment scFv13R4. The recombinants carrying chimeras of the N-terminal 1-110 regions of rBHT preceding scFv13R4 correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : MFα-rBht(23-594)-HIS and GS115 : : MFα-rBht(23-594)) showed comparable activity rates of GOS generation. PMID:26552922

  8. The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

    PubMed

    Pedone, P V; Omichinski, J G; Nony, P; Trainor, C; Gronenborn, A M; Clore, G M; Felsenfeld, G

    1997-05-15

    The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo. PMID:9184231

  9. The N-terminal sequence of the extrinsic PsbP protein modulates the redox potential of Cyt b559 in photosystem II

    PubMed Central

    Nishimura, Taishi; Nagao, Ryo; Noguchi, Takumi; Nield, Jon; Sato, Fumihiko; Ifuku, Kentaro

    2016-01-01

    The PsbP protein, an extrinsic subunit of photosystem II (PSII) in green plants, is known to induce a conformational change around the catalytic Mn4CaO5 cluster securing the binding of Ca2+ and Cl– in PSII. PsbP has multiple interactions with the membrane subunits of PSII, but how these affect the structure and function of PSII requires clarification. Here, we focus on the interactions between the N-terminal residues of PsbP and the α subunit of Cytochrome (Cyt) b559 (PsbE). A key observation was that a peptide fragment formed of the first N-terminal 15 residues of PsbP, ‘pN15’, was able to convert Cyt b559 into its HP form. Interestingly, addition of pN15 to NaCl-washed PSII membranes decreased PSII’s oxygen-evolving activity, even in the presence of saturating Ca2+ and Cl– ions. In fact, pN15 reversibly inhibited the S1 to S2 transition of the OEC in PSII. These data suggest that pN15 can modulate the redox property of Cyt b559 involved in the side-electron pathway in PSII. This potential change of Cyt b559, in the absence of the C-terminal domain of PsbP, however, would interfere with any electron donation from the Mn4CaO5 cluster, leading to the possibility that multiple interactions of PsbP, binding to PSII, have distinct roles in regulating electron transfer within PSII. PMID:26887804

  10. The N-terminal sequence of the extrinsic PsbP protein modulates the redox potential of Cyt b559 in photosystem II.

    PubMed

    Nishimura, Taishi; Nagao, Ryo; Noguchi, Takumi; Nield, Jon; Sato, Fumihiko; Ifuku, Kentaro

    2016-01-01

    The PsbP protein, an extrinsic subunit of photosystem II (PSII) in green plants, is known to induce a conformational change around the catalytic Mn4CaO5 cluster securing the binding of Ca(2+) and Cl(-) in PSII. PsbP has multiple interactions with the membrane subunits of PSII, but how these affect the structure and function of PSII requires clarification. Here, we focus on the interactions between the N-terminal residues of PsbP and the α subunit of Cytochrome (Cyt) b559 (PsbE). A key observation was that a peptide fragment formed of the first N-terminal 15 residues of PsbP, 'pN15', was able to convert Cyt b559 into its HP form. Interestingly, addition of pN15 to NaCl-washed PSII membranes decreased PSII's oxygen-evolving activity, even in the presence of saturating Ca(2+) and Cl(-) ions. In fact, pN15 reversibly inhibited the S1 to S2 transition of the OEC in PSII. These data suggest that pN15 can modulate the redox property of Cyt b559 involved in the side-electron pathway in PSII. This potential change of Cyt b559, in the absence of the C-terminal domain of PsbP, however, would interfere with any electron donation from the Mn4CaO5 cluster, leading to the possibility that multiple interactions of PsbP, binding to PSII, have distinct roles in regulating electron transfer within PSII. PMID:26887804

  11. Regulation of stress response signaling by the N-terminal dishevelled/EGL-10/pleckstrin domain of Sst2, a regulator of G protein signaling in Saccharomyces cerevisiae.

    PubMed

    Burchett, Scott A; Flanary, Paul; Aston, Christopher; Jiang, Lixin; Young, Kathleen H; Uetz, Peter; Fields, Stanley; Dohlman, Henrik G

    2002-06-21

    All members of the regulator of G protein signaling (RGS) family contain a conserved core domain that can accelerate G protein GTPase activity. The RGS in yeast, Sst2, can inhibit a G protein signal leading to mating. In addition, some RGS proteins contain an N-terminal domain of unknown function. Here we use complementary whole genome analysis methods to investigate the function of the N-terminal Sst2 domain. To identify a signaling pathway regulated by N-Sst2, we performed genome-wide transcription profiling of cells expressing this fragment alone and found differences in 53 transcripts. Of these, 40 are induced by N-Sst2, and nearly all contain a stress response element (STRE) in the promoter region. To identify components of a signaling pathway leading from N-Sst2 to STREs, we performed a genome-wide two-hybrid analysis using N-Sst2 as bait and found 17 interacting proteins. To identify the functionally relevant interacting proteins, we analyzed all of the available gene deletion mutants and found three (vps36 Delta, pep12 Delta, and tlg2 Delta) that induce STRE and also repress pheromone-dependent transcription. We selected VPS36 for further characterization. A vps36 Delta mutation diminishes signaling by pheromone as well as by downstream components including the G protein, effector kinase (Ste11), and transcription factor (Ste12). Conversely, overexpression of Vps36 enhances the pheromone response in sst2 Delta cells but not in wild type. These findings indicate that Vps36 and Sst2 have opposite and opposing effects on the pheromone and stress response pathways, with Vps36 acting downstream of the G protein and independently of Sst2 RGS activity. PMID:11940600

  12. Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

    PubMed Central

    Gong, Ting; Liao, Yuan; He, Fei; Yang, Yang; Yang, Dan-Dan; Chen, Xiang-Dong

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3Δ rho4Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4. PMID:23264647

  13. Expression, purification, crystallization and crystallographic analysis of the N-terminal domain of translocated intimin receptor.

    PubMed

    Huang, Bing Yang; Gu, Jiang; Zhang, Yan Fang; Zhou, Jun Jun; Song, Xiao Yong; Lin, Yi; Li, Xin Min; Li, Lu

    2016-01-01

    Translocated intimin receptor (Tir) is an Escherichia coli-encoded protein that is transported into the host cell through a sophisticated bacterial type III secretion system (T3SS). Tir anchors the infected cell membrane twice using both its N- and C-termini from inside the host cytoplasm for signalling. It plays a key role in enterohemorrhagic Escherichia coli (EHEC) infection, attaching and effacing (A/E) lesions and intracellular signal transduction. Here, the overexpression, purification and crystallization of its N-terminal intracellular domain are reported. The crystal belonged to the orthorhombic space group I4122, with unit-cell parameters a = b = 59.79, c = 183.11 Å. The asymmetric unit contained one molecule, with a solvent content of 51% and a VM of 2.55 Å(3) Da(-1). PMID:26750484

  14. Oxidation State of the XRCC1 N-terminal Domain Regulates DNA Polymerase Beta Binding Affinity

    SciTech Connect

    Cuneo, M.; London, R

    2010-01-01

    Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase {beta} (Pol {beta}) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol {beta} have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the interface, the oxidized XRCC1-NTD forms additional interactions with Pol {beta}, enhancing affinity by an order of magnitude. Transient disulfide bond formation is increasingly recognized as an important molecular regulatory mechanism. The results presented here suggest a paradigm in DNA repair in which the redox state of a scaffolding protein plays an active role in organizing the repair complex.

  15. Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase

    SciTech Connect

    Biswas, Tapan; Tsodikov, Oleg V.

    2009-01-15

    Hexameric DnaB helicase unwinds the DNA double helix during replication of genetic material in bacteria. DnaB is an essential bacterial protein; therefore, it is an important potential target for antibacterial drug discovery. We report a crystal structure of the N-terminal region of DnaB from the pathogen Mycobacterium tuberculosis (MtDnaBn), determined at 2.0 {angstrom} resolution. This structure provides atomic resolution details of formation of the hexameric ring of DnaB by two distinct interfaces. An extensive hydrophobic interface stabilizes a dimer of MtDnaBn by forming a four-helix bundle. The other, less extensive, interface is formed between the dimers, connecting three of them into a hexameric ring. On the basis of crystal packing interactions between MtDnaBn rings, we suggest a model of a helicase-primase complex that explains previously observed effects of DnaB mutations on DNA priming.

  16. Retroviral retargeting by envelopes expressing an N-terminal binding domain.

    PubMed Central

    Cosset, F L; Morling, F J; Takeuchi, Y; Weiss, R A; Collins, M K; Russell, S J

    1995-01-01

    We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment. PMID:7666532

  17. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    PubMed

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  18. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    PubMed

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  19. Partial N-terminal sequence analysis of human class II molecules expressing the DQw3 determinant.

    PubMed

    Obata, F; Endo, T; Yoshii, M; Otani, F; Igarashi, M; Takenouchi, T; Ikeda, H; Ogasawara, K; Kasahara, M; Wakisaka, A

    1985-09-01

    HLA-DQ molecules were isolated from DRw9-homozygous and DR4-homozygous cell lines by using a monoclonal antibody HU-18, which recognizes class II molecules carrying the conventional DQw3 determinant. The partial N-terminal sequence analysis of the DQw3 molecules revealed that they have sequences homologous to those of murine I-A molecules. Within the limits of our sequence analysis, the DQw3 molecules from the two cell lines are identical to each other in both the alpha and beta chains. The DQ alpha as well as DQ beta chains were found to have amino acid substitutions when compared to other I-A-like molecules whose sequences have been reported. These differences may contribute to the DQw supertypic specificity. The polymorphic nature of DQ molecules is in marked contrast to that of DR molecules where DR alpha chains are highly conserved while DR beta chains have easily detectable amino acid substitutions. PMID:2411700

  20. Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

    PubMed Central

    Cope, Stephanie M.; Shinde, Sandip; Best, Robert B.; Ghirlanda, Giovanna; Vaiana, Sara M.

    2013-01-01

    We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family. PMID:24094407

  1. 60 YEARS OF POMC: N-terminal POMC peptides and adrenal growth.

    PubMed

    Bicknell, Andrew B

    2016-05-01

    The peptide hormones contained within the sequence of proopiomelanocortin (POMC) have diverse roles ranging from pigmentation to regulation of adrenal function to control of our appetite. It is generally acknowledged to be the archetypal hormone precursor, and as its biology has been unravelled, so too have many of the basic principles of hormone biosynthesis and processing. This short review focuses on one group of its peptide products, namely, those derived from the N-terminal of POMC and their role in the regulation of adrenal growth. From a historical and a personal perspective, it describes how their role in regulating proliferation of the adrenal cortex was identified and also highlights the key questions that remain to be answered. PMID:26759392

  2. Molecular basis for histone N-terminal methylation by NRMT1

    PubMed Central

    Wu, Ruoxi; Yue, Yuan; Zheng, Xiangdong; Li, Haitao

    2015-01-01

    NRMT1 is an N-terminal methyltransferase that methylates histone CENP-A as well as nonhistone substrates. Here, we report the crystal structure of human NRMT1 bound to CENP-A peptide at 1.3 Å. NRMT1 adopts a core methyltransferase fold that resembles DOT1L and PRMT but not SET domain family histone methyltransferases. Key substrate recognition and catalytic residues were identified by mutagenesis studies. Histone peptide profiling revealed that human NRMT1 is highly selective to human CENP-A and fruit fly H2B, which share a common “Xaa-Pro–Lys/Arg” motif. These results, along with a 1.5 Å costructure of human NRMT1 bound to the fruit fly H2B peptide, underscore the importance of the NRMT1 recognition motif. PMID:26543159

  3. Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release

    PubMed Central

    Nisticò, Robert; Florenzano, Fulvio; Mango, Dalila; Ferraina, Caterina; Grilli, Massimo; Di Prisco, Silvia; Nobili, Annalisa; Saccucci, Stefania; D'Amelio, Marcello; Morbin, Michela; Marchi, Mario; Mercuri, Nicola B.; Davis, Roger J.; Pittaluga, Anna; Feligioni, Marco

    2015-01-01

    Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a critical step for neuronal death occurring in several neurological conditions. JNKs can be activated via receptor tyrosine kinases, cytokine receptors, G-protein coupled receptors and ligand-gated ion channels, including the NMDA glutamate receptors. While JNK has been generally associated with postsynaptic NMDA receptors, its presynaptic role remains largely unexplored. Here, by means of biochemical, morphological and functional approaches, we demonstrate that JNK and its scaffold protein JIP1 are also expressed at the presynaptic level and that the NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Moreover, using knockout mice for single JNK isoforms, we proved that JNK2 is the essential isoform in mediating this presynaptic event. Overall the present findings unveil a novel JNK2 localization and function, which is likely to play a role in different physiological and pathological conditions. PMID:25762148

  4. Role of the N-Terminal Seven Residues of Surfactant Protein B (SP-B)

    PubMed Central

    Sharifahmadian, Mahzad; Sarker, Muzaddid; Palleboina, Dharamaraju; Waring, Alan J.; Walther, Frans J.; Morrow, Michael R.; Booth, Valerie

    2013-01-01

    Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the “insertion sequence”. These studies employed a construct of SP-B, SP-B (1–25,63–78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state 2H NMR were used to study the structure of SP-B (1–25,63–78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8–25,63–78) and SP-B (1–25,63–78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics. PMID:24023779

  5. N-terminal tagging of the dopamine transporter impairs protein expression and trafficking in vivo

    PubMed Central

    Vecchio, Laura M.; Bermejo, M. Kristel; Beerepoot, Pieter; Ramsey, Amy J.

    2014-01-01

    The dopamine transporter (DAT) is the primary protein responsible for the uptake of dopamine from the extracellular space back into presynaptic neurons. As such, it plays an important role in the cessation of dopaminergic neurotransmission and in the maintenance of extracellular dopamine homeostasis. Here, we report the development of a new BAC transgenic mouse line that expresses DAT with an N-terminal HA-epitope (HAD-Tg). In this line, two copies of the HA-DAT BAC are incorporated into the genome, increasing DAT mRNA levels by 47%. Despite the increase in mRNA levels, HAD-Tg mice show no significant increase in the level of DAT protein in the striatum, indicating a defect in protein trafficking or stability. By crossing HAD-Tg mice with DAT knockout mice (DAT-KO), we engineered mice that exclusively express HA-tagged DAT in the absence of endogenous DAT (DAT-KO/HAD-Tg). We show that DAT-KO/HAD-Tg mice express only 8.5% of WT DAT levels in the striatum. Importantly, the HA-tagged DAT that is present in DAT-KO/HAD-Tg mice is functional, as it is able to partially rescue the DAT-KO hyperactive phenotype. Finally, we provide evidence that the HA-tagged DAT is retained in the cell body based on a reduction in the striatum:midbrain protein ratio. These results demonstrate that the presence of the N-terminal tag leads to impaired DAT protein expression in vivo due in part to improper trafficking of the tagged transporter, and highlight the importance of the N-terminus in the transport of DAT to striatal terminals. PMID:24886986

  6. Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

    PubMed

    Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

    2016-07-01

    Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

  7. PACSIN 1 forms tetramers via its N-terminal F-BAR domain.

    PubMed

    Halbach, Arndt; Mörgelin, Matthias; Baumgarten, Maria; Milbrandt, Mark; Paulsson, Mats; Plomann, Markus

    2007-02-01

    The ability of protein kinase C and casein kinase 2 substrate in neurons (PACSIN)/syndapin proteins to self-polymerize is crucial for the simultaneous interactions with more than one Src homology 3 domain-binding partner or with lipid membranes. The assembly of this network has profound effects on the neural Wiskott-Aldrich syndrome protein-mediated attachment of the actin polymerization machinery to vesicle membranes as well as on the movement of the corresponding vesicles. Also, the sensing of vesicle membranes and/or the induction of membrane curvature are more easily facilitated in the presence of larger PACSIN complexes. The N-terminal Fes-CIP homology and Bin-Amphiphysin-Rvs (F-BAR) domains of several PACSIN-related proteins have been shown to mediate self-interactions, whereas studies using deletion mutants derived from closely related proteins led to the view that oligomerization depends on the formation of a trimeric complex via a coiled-coil region present in these molecules. To address whether the model of trimeric complex formation is applicable to PACSIN 1, the protein was recombinantly expressed and tested in four different assays for homologous interactions. The results showed that PACSIN 1 forms tetramers of about 240 kDa, with the self-interaction having a K(D) of 6.4 x 10(-8) M. Ultrastructural analysis of these oligomers after negative staining showed that laterally arranged PACSIN molecules bind to each other via a large globular domain and form a barrel-like structure. Together, these results demonstrate that the N-terminal F-BAR domain of PACSIN 1 forms the contact site for a tetrameric structure, which is able to simultaneously interact with multiple Src homology 3 binding partners. PMID:17288557

  8. N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis.

    PubMed

    Rada, Petr; Makki, Abhijith Radhakrishna; Zimorski, Verena; Garg, Sriram; Hampl, Vladimír; Hrdý, Ivan; Gould, Sven B; Tachezy, Jan

    2015-12-01

    Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution. PMID:26475173

  9. N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

    PubMed Central

    Rada, Petr; Makki, Abhijith Radhakrishna; Zimorski, Verena; Garg, Sriram; Hampl, Vladimír; Hrdý, Ivan; Gould, Sven B.

    2015-01-01

    Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution. PMID:26475173

  10. Lamp with a truncated reflector cup

    DOEpatents

    Li, Ming; Allen, Steven C.; Bazydola, Sarah; Ghiu, Camil-Daniel

    2013-10-15

    A lamp assembly, and method for making same. The lamp assembly includes first and second truncated reflector cups. The lamp assembly also includes at least one base plate disposed between the first and second truncated reflector cups, and a light engine disposed on a top surface of the at least one base plate. The light engine is configured to emit light to be reflected by one of the first and second truncated reflector cups.

  11. Truncations of xyloglucan xylosyltransferase 2 provide insights into the roles of the N- and C-terminus.

    PubMed

    Culbertson, Alan T; Smith, Adrienne L; Cook, Matthew D; Zabotina, Olga A

    2016-08-01

    Xyloglucan is the most abundant hemicellulose in the primary cell wall of dicotyledonous plants. In Arabidopsis, three xyloglucan xylosyltransferases, XXT1, XXT2, and XXT5, participate in xylosylation of the xyloglucan backbone. Despite the importance of these enzymes, there is a lack of information on their structure and the critical residues required for substrate binding and transferase activity. In this study, the roles of different domains of XX2 in protein expression and catalytic activity were investigated by constructing a series of N- and C-terminal truncations. XXT2 with an N-terminal truncation of 31 amino acids after the predicted transmembrane domain showed the highest protein expression, but truncations of more than 31 residues decreased protein expression and catalytic activity. XXT2 constructs with C-terminal truncations showed increased protein expression but decreased activity, particularly for truncations of 44 or more amino acids. Site-directed mutagenesis was also used to investigate six positively charged residues near the C-terminus and found that four of the mutants showed decreased enzymatic activity. We conclude that the N- and C-termini of XXT2 have important roles in protein folding and enzymatic activity: the stem region (particularly the N-terminus of the catalytic domain) is critical for protein folding and the C-terminus is essential for enzymatic activity but not for protein folding. PMID:27193738

  12. Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin.

    PubMed

    Mukhi, Nitika; Dhindwal, Sonali; Uppal, Sheetal; Kapoor, Abhijeet; Arya, Richa; Kumar, Pravindra; Kaur, Jagreet; Kundu, Suman

    2016-03-29

    Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein-ligand and protein-protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site

  13. Modifications in the purification protocol of Celosia cristata antiviral proteins lead to protein that can be N-terminally sequenced.

    PubMed

    Gholizadeh, Ashraf; Kapoor, H C

    2004-12-01

    Plants antiviral proteins are being used as anticancer agents and inhibit other viral diseases in humans. We modified the purification protocol of the two N-terminally blocked antiviral glycoproteins, CCP-25 and CCP-27, purified from the leaves of Celosia cristata. This not only gave rise to single pure samples with few steps of purification but also resulted in N-terminally free proteins. The extra purity of the samples was analyzed by reverse phase HPLC. Deglycosylation studies of CCP-25 with PNGase F enzyme revealed that its asparagine or asparagine-linked glycon contents are negligible. Partial N-terminal sequence of the CCP-25 showed the sequence (ANDIS), which seems to be conserved among plant antiviral proteins. PMID:15579125

  14. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling★

    PubMed Central

    Ma, Yinghuan; Bao, Yongxin; Li, Chenghao; Jiao, Fubin; Xin, Hongjie; Yuan, Zhengwei

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway. PMID:25337099

  15. N-Terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199.

    PubMed

    Yoshida, Hiroyuki; Nagaoka, Aya; Nakamura, Sachiko; Tobiishi, Megumi; Sugiyama, Yoshinori; Inoue, Shintaro

    2014-01-01

    Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization. PMID:24269685

  16. Reducing Truncation Error In Integer Processing

    NASA Technical Reports Server (NTRS)

    Thomas, J. Brooks; Berner, Jeffrey B.; Graham, J. Scott

    1995-01-01

    Improved method of rounding off (truncation of least-significant bits) in integer processing of data devised. Provides for reduction, to extremely low value, of numerical bias otherwise generated by accumulation of truncation errors from many arithmetic operations. Devised for use in integer signal processing, in which rescaling and truncation usually performed to reduce number of bits, which typically builds up in sequence of operations. Essence of method to alternate direction of roundoff (plus, then minus) on alternate occurrences of truncated values contributing to bias.

  17. The N-terminal region of mature mitochondrial aspartate aminotransferase can direct cytosolic dihydrofolate reductase into mitochondria in vitro.

    PubMed

    Giannattasio, S; Azzariti, A; Marra, E; Quagliariello, E

    1994-06-30

    Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria. PMID:8024546

  18. Nonsyndromic hearing loss DFNA10 and a novel mutation of EYA4: evidence for correlation of normal cardiac phenotype with truncating mutations of the Eya domain.

    PubMed

    Makishima, Tomoko; Madeo, Anne C; Brewer, Carmen C; Zalewski, Christopher K; Butman, John A; Sachdev, Vandana; Arai, Andrew E; Holbrook, Brenda M; Rosing, Douglas R; Griffith, Andrew J

    2007-07-15

    Dominant, truncating mutations of eyes absent 4 (EYA4) on chromosome 6q23 can cause either nonsyndromic hearing loss DFNA10 or hearing loss with dilated cardiomyopathy (DCM). It has been proposed that truncations of the C-terminal Eya domain cause DFNA10 whereas upstream truncations of the N-terminal variable region cause hearing loss with DCM. Here we report an extended family co-segregating autosomal dominant, postlingual-onset, progressive, sensorineural hearing loss (SNHL) with a novel frameshift mutation, 1,490insAA, of EYA4. The 1,490insAA allele is predicted to encode a truncated protein with an intact N-terminal variable region, but lacking the entire C-terminal Eya domain. Clinical studies including electrocardiography, echocardiography, and magnetic resonance imaging (MRI) of the heart in nine affected family members revealed no DCM or associated abnormalities and confirmed their nonsyndromic phenotype. These are the first definitive cardiac evaluations of DFNA10 hearing loss to support a correlation of EYA4 mutation position with the presence or absence of DCM. These results will facilitate the counseling of patients with these phenotypes and EYA4 mutations. PMID:17567890

  19. Characteristics of thermostable amylopullulanase of Geobacillus thermoleovorans and its truncated variants.

    PubMed

    Nisha, M; Satyanarayana, T

    2015-05-01

    The far-UV CD spectroscopic analysis of the secondary structure in the temperature range between 30 and 90°C revealed a compact and thermally stable structure of C-terminal truncated amylopullulanase of Geobacillus thermoleovorans NP33 (gt-apuΔC) with a higher melting temperature [58°C] than G. thermoleovorans NP33 amylopullulanase (gt-apu) [50°C] and the N-terminal truncated amylopullulanase from G. thermoleovorans NP33 (gt-apuΔN) [55°C]. A significant decline in random coils in gt-apuΔC and gt-apuΔN suggested an improvement in conformational stability, and thus, an enhancement in their thermal stability. The improvement in the thermostability of gt-apuΔC was corroborated by the thermodynamic parameters for enzyme inactivation. The Trp fluorescence emission (335 nm) and the acrylamide quenching constant (22.69 M(-1)) of gt-apuΔC indicated that the C-terminal truncation increases the conformational stability of the protein with the deeply buried tryptophan residues. The 8-Anilino Naphthalene Sulfonic acid (ANS) fluorescence experiments indicated the unfolding of gt-apu to expose its hydrophobic surface to a greater extent than the gt-apuΔC and gt-apuΔN. PMID:25748845

  20. N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast.

    PubMed

    Cha, Joon-Yung; Lee, Sang-Hoon; Seo, Kyung Hye; Choi, Young Jin; Cheong, Mi Sun; Son, Daeyoung

    2016-02-01

    Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance. PMID:26724757

  1. Truncation of C-terminal 20 amino acids in PA-X contributes to adaptation of swine influenza virus in pigs.

    PubMed

    Xu, Guanlong; Zhang, Xuxiao; Sun, Yipeng; Liu, Qinfang; Sun, Honglei; Xiong, Xin; Jiang, Ming; He, Qiming; Wang, Yu; Pu, Juan; Guo, Xin; Yang, Hanchun; Liu, Jinhua

    2016-01-01

    The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting, and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a "triple-reassortment" H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs. PMID:26912401

  2. Truncation of C-terminal 20 amino acids in PA-X contributes to adaptation of swine influenza virus in pigs

    PubMed Central

    Xu, Guanlong; Zhang, Xuxiao; Sun, Yipeng; Liu, Qinfang; Sun, Honglei; Xiong, Xin; Jiang, Ming; He, Qiming; Wang, Yu; Pu, Juan; Guo, Xin; Yang, Hanchun; Liu, Jinhua

    2016-01-01

    The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting, and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a “triple-reassortment” H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs. PMID:26912401

  3. Perspective on rainbow-ladder truncation

    NASA Astrophysics Data System (ADS)

    Eichmann, G.; Alkofer, R.; Cloët, I. C.; Krassnigg, A.; Roberts, C. D.

    2008-04-01

    Prima facie the systematic implementation of corrections to the rainbow-ladder truncation of QCD's Dyson-Schwinger equations will uniformly reduce in magnitude those calculated mass-dimensioned results for pseudoscalar and vector meson properties that are not tightly constrained by symmetries. The aim and interpretation of studies employing rainbow-ladder truncation are reconsidered in this light.

  4. Perspective on rainbow-ladder truncation

    SciTech Connect

    Eichmann, G.; Alkofer, R.; Krassnigg, A.; Cloeet, I. C.; Roberts, C. D.

    2008-04-15

    Prima facie the systematic implementation of corrections to the rainbow-ladder truncation of QCD's Dyson-Schwinger equations will uniformly reduce in magnitude those calculated mass-dimensioned results for pseudoscalar and vector meson properties that are not tightly constrained by symmetries. The aim and interpretation of studies employing rainbow-ladder truncation are reconsidered in this light.

  5. Structure of the EMMPRIN N-terminal domain 1: Dimerization via [beta]-strand swapping

    SciTech Connect

    Luo, Jinquan; Teplyakov, Alexey; Obmolova, Galina; Malia, Thomas; Wu, Sheng-Jiun; Beil, Eric; Baker, Audrey; Swencki-Underwood, Bethany; Zhao, Yonghong; Sprenkle, Justin; Dixon, Ken; Sweet, Raymond; Gilliland, Gary L.

    2010-09-27

    Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which has a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length

  6. Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

    PubMed

    Song, Letian; Tsang, Adrian; Sylvestre, Michel

    2015-06-01

    Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG. PMID:25640404

  7. Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

    PubMed Central

    Qiu, Shihong; Ogino, Minako; Luo, Ming

    2015-01-01

    ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the

  8. An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility.

    PubMed

    Hofmann, Irmgard; Munro, Sean

    2006-04-15

    Small GTPases of the Arf and Rab families play key roles in the function of subcellular organelles. Each GTPase is usually found on only one compartment and, hence, they confer organelle specificity to many intracellular processes. However, there has so far been little evidence for specific GTPases present on lysosomes. Here, we report that two closely related human Arf-like GTPases, Arl8a and Arl8b (also known as Arl10b/c and Gie1/2), localise to lysosomes in mammalian cells, with the single homologue in Drosophila cells having a similar location. Conventionally, membrane binding of Arf and Arl proteins is mediated by both an N-terminal myristoyl group and an N-terminal amphipathic helix that is inserted into the lipid bilayer upon activation of the GTPase. Arl8a and Arl8b do not have N-terminal myristoylation sites, and we find that Arl8b is instead N-terminally acetylated, and an acetylated methionine is necessary for its lysosomal localization. Overexpression of Arl8a or Arl8b results in a microtubule-dependent redistribution of lysosomes towards the cell periphery. Live cell imaging shows that lysosomes move more frequently both toward and away from the cell periphery, suggesting a role for Arl8a and Arl8b as positive regulators of lysosomal transport. PMID:16537643

  9. Molecular evolution of troponin I and a role of its N-terminal extension in nematode locomotion.

    PubMed

    Barnes, Dawn E; Hwang, Hyundoo; Ono, Kanako; Lu, Hang; Ono, Shoichiro

    2016-03-01

    The troponin complex, composed of troponin T (TnT), troponin I (TnI), and troponin C (TnC), is the major calcium-dependent regulator of muscle contraction, which is present widely in both vertebrates and invertebrates. Little is known about evolutionary aspects of troponin in the animal kingdom. Using a combination of data mining and functional analysis of TnI, we report evidence that an N-terminal extension of TnI is present in most of bilaterian animals as a functionally important domain. Troponin components have been reported in species in most of representative bilaterian phyla. Comparison of TnI sequences shows that the core domains are conserved in all examined TnIs, and that N- and C-terminal extensions are variable among isoforms and species. In particular, N-terminal extensions are present in all protostome TnIs and chordate cardiac TnIs but lost in a subset of chordate TnIs including vertebrate skeletal-muscle isoforms. Transgenic rescue experiments in Caenorhabditis elegans striated muscle show that the N-terminal extension of TnI (UNC-27) is required for coordinated worm locomotion but not in sarcomere assembly and single muscle-contractility kinetics. These results suggest that N-terminal extensions of TnIs are retained from a TnI ancestor as a functional domain. © 2016 Wiley Periodicals, Inc. PMID:26849746

  10. Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

    PubMed Central

    Chotewutmontri, Prakitchai; Bruce, Barry D.

    2015-01-01

    Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

  11. Molecular Insights into the Dynamics of Pharmacogenetically Important N-Terminal Variants of the Human β2-Adrenergic Receptor

    PubMed Central

    Sengupta, Durba; Joshi, Manali

    2014-01-01

    The human β2-adrenergic receptor (β2AR), a member of the G-protein coupled receptor (GPCR) family, is expressed in bronchial smooth muscle cells. Upon activation by agonists, β2AR causes bronchodilation and relief in asthma patients. The N-terminal polymorphism of β2AR at the 16th position, Arg16Gly, has warranted a lot of attention since it is linked to variations in response to albuterol (agonist) treatment. Although the β2AR is one of the well-studied GPCRs, the N-terminus which harbors this mutation, is absent in all available experimental structures. The goal of this work was to study the molecular level differences between the N-terminal variants using structural modeling and atomistic molecular dynamics simulations. Our simulations reveal that the N-terminal region of the Arg variant shows greater dynamics than the Gly variant, leading to differential placement. Further, the position and dynamics of the N-terminal region, further, affects the ligand binding-site accessibility. Interestingly, long-range effects are also seen at the ligand binding site, which is marginally larger in the Gly as compared to the Arg variant resulting in the preferential docking of albuterol to the Gly variant. This study thus reveals key differences between the variants providing a molecular framework towards understanding the variable drug response in asthma patients. PMID:25501358

  12. N-terminal propeptide of type III procollagen as a biomarker of anabolic response to recombinant human GH and testosterone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Context: Biomarkers that predict musculoskeletal response to anabolic therapies should expedite drug development. During collagen synthesis in soft lean tissue, N-terminal propeptide of type III procollagen (P3NP) is released into circulation. We investigated P3NP as a biomarker of lean body mass (L...

  13. The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication

    PubMed Central

    Zhang, Jie; Guo, Hong; Chen, Qingxiu; Zhang, Fuxian; Fang, Qin

    2016-01-01

    Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1–471) of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection. PMID:26871941

  14. Facile synthesis of SAM–peptide conjugates through alkyl linkers targeting protein N-terminal methyltransferase 1†

    PubMed Central

    Zhang, Gang

    2016-01-01

    We report the first chemical synthesis of SAM–peptide conjugates through alkyl linkers to prepare bisubstrate analogs for protein methyltransferases. We demonstrate its application by developing a series of bisubstrate inhibitors for protein N-terminal methyltransferase 1 and the most potent one exhibits a Ki value of 310 ± 55 nM.

  15. A Truncated Progesterone Receptor (PR-M) Localizes to the Mitochondrion and Controls Cellular Respiration

    PubMed Central

    Dai, Qunsheng; Shah, Anish A.; Garde, Rachana V.; Yonish, Bryan A.; Zhang, Li; Medvitz, Neil A.; Miller, Sara E.; Hansen, Elizabeth L.; Dunn, Carrie N.

    2013-01-01

    The cDNA for a novel truncated progesterone receptor (PR-M) was previously cloned from human adipose and aortic cDNA libraries. The predicted protein sequence contains 16 unique N-terminal amino acids, encoded by a sequence in the distal third intron of the progesterone receptor PR gene, followed by the same amino acid sequence encoded by exons 4 through 8 of the nuclear PR. Thus, PR-M lacks the N terminus A/B domains and the C domain for DNA binding, whereas containing the hinge and hormone-binding domains. In this report, we have localized PR-M to mitochondria using immunofluorescent localization of a PR-M-green fluorescent protein (GFP) fusion protein and in Western blot analyses of purified human heart mitochondrial protein. Removal of the putative N-terminal mitochondrial localization signal obviated association of PR-M with mitochondria, whereas addition of the mitochondrial localization signal to green fluorescent protein resulted in mitochondrial localization. Immunoelectron microscopy and Western blot analysis after mitochondrial fractionation identified PR-M in the outer mitochondrial membrane. Antibody specificity was shown by mass spectrometry identification of a PR peptide in a mitochondrial membrane protein isolation. Cell models of overexpression and gene silencing of PR-M demonstrated a progestin-induced increase in mitochondrial membrane potential and an increase in oxygen consumption consistent with an increase in cellular respiration. This is the first example of a truncated steroid receptor, lacking a DNA-binding domain that localizes to the mitochondrion and initiates direct non-nuclear progesterone action. We hypothesize that progesterone may directly affect cellular energy production to meet the increased metabolic demands of pregnancy. PMID:23518922

  16. A truncated progesterone receptor (PR-M) localizes to the mitochondrion and controls cellular respiration.

    PubMed

    Dai, Qunsheng; Shah, Anish A; Garde, Rachana V; Yonish, Bryan A; Zhang, Li; Medvitz, Neil A; Miller, Sara E; Hansen, Elizabeth L; Dunn, Carrie N; Price, Thomas M

    2013-05-01

    The cDNA for a novel truncated progesterone receptor (PR-M) was previously cloned from human adipose and aortic cDNA libraries. The predicted protein sequence contains 16 unique N-terminal amino acids, encoded by a sequence in the distal third intron of the progesterone receptor PR gene, followed by the same amino acid sequence encoded by exons 4 through 8 of the nuclear PR. Thus, PR-M lacks the N terminus A/B domains and the C domain for DNA binding, whereas containing the hinge and hormone-binding domains. In this report, we have localized PR-M to mitochondria using immunofluorescent localization of a PR-M-green fluorescent protein (GFP) fusion protein and in Western blot analyses of purified human heart mitochondrial protein. Removal of the putative N-terminal mitochondrial localization signal obviated association of PR-M with mitochondria, whereas addition of the mitochondrial localization signal to green fluorescent protein resulted in mitochondrial localization. Immunoelectron microscopy and Western blot analysis after mitochondrial fractionation identified PR-M in the outer mitochondrial membrane. Antibody specificity was shown by mass spectrometry identification of a PR peptide in a mitochondrial membrane protein isolation. Cell models of overexpression and gene silencing of PR-M demonstrated a progestin-induced increase in mitochondrial membrane potential and an increase in oxygen consumption consistent with an increase in cellular respiration. This is the first example of a truncated steroid receptor, lacking a DNA-binding domain that localizes to the mitochondrion and initiates direct non-nuclear progesterone action. We hypothesize that progesterone may directly affect cellular energy production to meet the increased metabolic demands of pregnancy. PMID:23518922

  17. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    PubMed

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. PMID:25727088

  18. c-Jun N-terminal Kinase Phosphorylation of Stathmin Confers Protection against Cellular Stress*

    PubMed Central

    Ng, Dominic C. H.; Zhao, Teresa T.; Yeap, Yvonne Y. C.; Ngoei, Kevin R.; Bogoyevitch, Marie A.

    2010-01-01

    The cell stress response encompasses the range of intracellular events required for adaptation to stimuli detrimental to cell survival. Although the c-Jun N-terminal kinase (JNK) is a stress-activated kinase that can promote either cell survival or death in response to detrimental stimuli, the JNK-regulated mechanisms involved in survival are not fully characterized. Here we show that in response to hyperosmotic stress, JNK phosphorylates a key cytoplasmic microtubule regulatory protein, stathmin (STMN), on conserved Ser-25 and Ser-38 residues. In in vitro biochemical studies, we identified STMN Ser-38 as the critical residue required for efficient phosphorylation by JNK and identified a novel kinase interaction domain in STMN required for recognition by JNK. We revealed that JNK was required for microtubule stabilization in response to hyperosmotic stress. Importantly, we also demonstrated a novel cytoprotective function for STMN, as the knockdown of STMN levels by siRNA was sufficient to augment viability in response to hyperosmotic stress. Our findings show that JNK targeting of STMN represents a novel stress-activated cytoprotective mechanism involving microtubule network changes. PMID:20630875

  19. Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

    SciTech Connect

    Schwarten, Melanie; Stoldt, Matthias; Mohrlueder, Jeannine; Willbold, Dieter

    2010-05-07

    During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8{sup G116} shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

  20. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    SciTech Connect

    Lin, Yi-Tzu; Wen, Wan-Ching; Yen, Pauline H.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  1. Serum type III procollagen N-terminal peptide in coal miners.

    PubMed

    Janssen, Y M; Engelen, J J; Giancola, M S; Low, R B; Vacek, P; Borm, P J

    1992-01-01

    Health surveillance of workers exposed to fibrogenic agents ideally should identify individuals at risk or detect pulmonary fibrosis in preclinical stages. We investigated serum procollagen type III N-terminal peptide (PIIIP) in several groups of active miners and in a nondust-exposed control group. The purpose of this study was to determine the applicability of PIIIP as an early noninvasive marker of pulmonary fibrosis in workers exposed to coal mine dust. PIIIP levels were significantly elevated in miners without radiological signs of coal workers pneumoconiosis (CWP) as compared with the nonexposed controls. However, in coal miners with CWP beyond ILO classification 1/0, PIIIP levels were not significantly different from nondust-exposed controls. Trend analysis within the miners group indicated a decrease in PIIIP levels with progression of the fibrosis. Our data suggest that detection of early lung fibrosis by measuring serum PIIIP values may be more sensitive than radiological diagnosis of CWP. However, follow-up of the control miners with respect to serum PIIIP and chest radiography is essential to validate PIIIP as a biological marker for CWP. PMID:1572317

  2. Pyridopyrimidinone Derivatives as Potent and Selective c-Jun N-Terminal Kinase (JNK) Inhibitors

    PubMed Central

    2015-01-01

    A novel series of 2-aminopyridopyrimidinone based JNK (c-jun N-terminal kinase) inhibitors were discovered and developed. Structure–activity relationships (SARs) were systematically developed utilizing biochemical and cell based assays and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies. Through the optimization of lead compound 1, several potent and selective JNK inhibitors with high oral bioavailability were developed. Inhibitor 13 was a potent JNK3 inhibitor (IC50 = 15 nM), had high selectivity against p38 (IC50 > 10 μM), had high potency in functional cell based assays, and had high stability in human liver microsome (t1/2 = 76 min), a clean CYP-450 inhibition profile, and excellent oral bioavailability (%F = 87). Moreover, cocrystal structures of compounds 13 and 22 in JNK3 were solved at 2.0 Å. These structures elucidated the binding mode (Type-I binding) and can pave the way for further inhibitor design of this pyridopyrimidinone scaffold for JNK inhibition. PMID:25893042

  3. Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal.

    PubMed

    Morgado, Isabel; Melo, Eduardo P; Lundberg, Erik; Estrela, Nídia L; Sauer-Eriksson, A Elisabeth; Power, Deborah M

    2008-11-25

    Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR. PMID:18620020

  4. Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

    PubMed Central

    Krieger, James; Bahar, Ivet; Greger, Ingo H.

    2015-01-01

    Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

  5. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    SciTech Connect

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  6. Highly heterologous region in the N-terminal extracellular domain of reptilian follitropin receptors.

    PubMed

    Akazome, Y; Ogasawara, O; Park, M K; Mori, T

    1996-12-01

    The primary structure of the N-terminal extracellular region of the follitropin receptor (FSH-R), which is thought to be responsible for hormone binding specificity, was determined in three reptilian species (tortoise, gecko, and lizard). Remarkably low sequence homologies were detected in the C-terminal part of the extracellular domain. This region was estimated to be a part of exon 10, which is the last exon of the FSH-R gene. In this region, not only were low homologies detected among the three reptilian species, but also specific deletions and/or insertions were found. In particular, large deletions were detected in squamate (gecko and lizard) FSH-Rs. Phylogenetic analysis indicated that these large deletions occurred recently, i.e., after the Triassic period. In another region characterized, sequence homologies were high, with tortoise-rat homology 78.4%, gecko-rat 64.7%, and lizard-rat 69.1%. In this highly conserved region, however, some reptile-specific alterations were detected, such as the loss of a cysteine residue in putative exon 7 and the existence of potential N-linked glycosylation sites in putative exon 9. PMID:8954771

  7. Calcium-controlled conformational choreography in the N-terminal half of adseverin

    NASA Astrophysics Data System (ADS)

    Chumnarnsilpa, Sakesit; Robinson, Robert C.; Grimes, Jonathan M.; Leyrat, Cedric

    2015-09-01

    Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1-A3) and the Ca2+-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca2+-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1-A3 provided insights into Ca2+-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca2+-independent F-actin severing by A1-A3, albeit at a lower efficiency than observed for gelsolin domains G1-G3. Together, these data address the calcium dependency of A1-A3 activity in relation to the calcium-independent activity of G1-G3.

  8. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    NASA Astrophysics Data System (ADS)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-04-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended `railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

  9. Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti.

    PubMed

    Pieprzyk, Joanna; Zbela, Agnieszka; Jakób, Michał; Ożyhar, Andrzej; Orłowski, Marek

    2014-06-01

    The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD-NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors. PMID:24704038

  10. N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

    PubMed Central

    Sekar, Karthik; Gentile, Andrew M.; Bostick, John W.; Tyo, Keith E. J.

    2016-01-01

    Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits. PMID:26900850

  11. Calcium-controlled conformational choreography in the N-terminal half of adseverin

    PubMed Central

    Chumnarnsilpa, Sakesit; Robinson, Robert C.; Grimes, Jonathan M.; Leyrat, Cedric

    2015-01-01

    Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1–A3) and the Ca2+-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca2+-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1–A3 provided insights into Ca2+-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca2+-independent F-actin severing by A1–A3, albeit at a lower efficiency than observed for gelsolin domains G1–G3. Together, these data address the calcium dependency of A1–A3 activity in relation to the calcium-independent activity of G1–G3. PMID:26365202

  12. Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

    SciTech Connect

    M Mitchell; J Smith; M Mason; S Harper; D Speicher; F Johnson; E Skordalakes

    2011-12-31

    The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

  13. Ozone exposure triggers insulin resistance through muscle c-Jun N-terminal kinase activation.

    PubMed

    Vella, Roxane E; Pillon, Nicolas J; Zarrouki, Bader; Croze, Marine L; Koppe, Laetitia; Guichardant, Michel; Pesenti, Sandra; Chauvin, Marie-Agnès; Rieusset, Jennifer; Géloën, Alain; Soulage, Christophe O

    2015-03-01

    A growing body of evidence suggests that exposure to traffic-related air pollution is a risk factor for type 2 diabetes. Ozone, a major photochemical pollutant in urban areas, is negatively associated with fasting glucose and insulin levels, but most aspects of this association remain to be elucidated. Using an environmentally realistic concentration (0.8 parts per million), we demonstrated that exposure of rats to ozone induced whole-body insulin resistance and oxidative stress, with associated endoplasmic reticulum (ER) stress, c-Jun N-terminal kinase (JNK) activation, and disruption of insulin signaling in skeletal muscle. Bronchoalveolar lavage fluids from ozone-treated rats reproduced this effect in C2C12 myotubes, suggesting that toxic lung mediators were responsible for the phenotype. Pretreatment with the chemical chaperone 4-phenylbutyric acid, the JNK inhibitor SP600125, or the antioxidant N-acetylcysteine alleviated insulin resistance, demonstrating that ozone sequentially triggered oxidative stress, ER stress, and JNK activation to impair insulin signaling in muscle. This study is the first to report that ozone plays a causative role in the development of insulin resistance, suggesting that it could boost the development of diabetes. We therefore provide a potential mechanism linking pollutant exposure and the increased incidence of metabolic diseases. PMID:25277399

  14. The C-terminus of p53 binds the N-terminal domain of MDM2

    PubMed Central

    Poyurovsky, Masha V.; Katz, Chen; Laptenko, Oleg; Beckerman, Rachel; Lokshin, Maria; Ahn, Jinwoo; Byeon, In-Ja L.; Gabizon, Ronen; Mattia, Melissa; Zupnick, Andrew; Brown, Lewis M.; Friedler, Assaf; Prives, Carol

    2010-01-01

    The p53 tumor suppressor interacts with its negative regulator Mdm2 via the former’s N-terminal region and core domain. Yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2–p53 association. We show that, the Mdm2–p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C-terminus. Mdm2 decreases the association of full-length but not C-terminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches we demonstrate that a peptide from p53 C-terminus directly binds Mdm2 N-terminus in vitro. We also show that p300-acetylated p53 binds inefficiently to Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro. PMID:20639885

  15. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    SciTech Connect

    Magzoub, Mazin; Sandgren, Staffan; Lundberg, Pontus; Oglecka, Kamila; Lilja, Johanna; Wittrup, Anders; Goeran Eriksson, L.E.; Langel, Ulo; Belting, Mattias . E-mail: mattias.belting@med.lu.se; Graeslund, Astrid . E-mail: astrid@dbb.su.se

    2006-09-22

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.

  16. Functional Residues on the Surface of the N-terminal domain of Yeast Pms1

    PubMed Central

    Arana, Mercedes E.; Holmes, Shannon F.; Fortune, John M.; Moon, Andrea F.; Pedersen, Lars C.; Kunkel, Thomas A.

    2010-01-01

    Saccharomyces cerevisiae MutLα is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTD), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTD) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5Å crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to thehomologous NTDs of E. coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins. PMID:20138591

  17. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    PubMed Central

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-01-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor–Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor–Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor–Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended ‘railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit. PMID:27072897

  18. The N-terminal half of talin2 is sufficient for mouse development and survival

    SciTech Connect

    Chen, N.-T.; Lo, S.H. . E-mail: shlo@ucdavis.edu

    2005-11-18

    Using a talin2 gene-trapped embryonic stem cell clone, we have developed a talin2 mutant mouse line that expresses the N-terminal half (1-1295) of talin2 fused with {beta}-galactosidase. The homozygous mutant mice appear to be normal and healthy. In the testis, talin2 expresses as a shorter form with a unique 30 residues at N-terminus linking to a common C-terminus from 1122 to 2453 of the long form. The resulting talin2 in the mutant testis only contains 204 residues of the wild-type testis talin2. However, it did not seem to affect the morphology of testis or reproduction of male mice. In fact, male and female mutant mice are fertile. Utilizing the expression of talin2(1-1295)/{beta}-galactosidase fusion protein, we have examined the distribution of talin2 in tissues. In contrast to talin1, talin2 expression is more restricted in tissues and cell types.

  19. N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies.

    PubMed

    Liu, Y Diana; Goetze, Andrew M; Bass, Randal B; Flynn, Gregory C

    2011-04-01

    Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute. PMID:21282104

  20. Intrinsic disorder drives N-terminal ubiquitination by Ube2w.

    PubMed

    Vittal, Vinayak; Shi, Lei; Wenzel, Dawn M; Scaglione, K Matthew; Duncan, Emily D; Basrur, Venkatesha; Elenitoba-Johnson, Kojo S J; Baker, David; Paulson, Henry L; Brzovic, Peter S; Klevit, Rachel E

    2015-01-01

    Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells. PMID:25436519

  1. Tor forms a dimer through an N-terminal helical solenoid with a complex topology.

    PubMed

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M; Williams, Roger L

    2016-01-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit. PMID:27072897

  2. Epsin N-terminal homology domains bind on opposite sides of two SNAREs

    PubMed Central

    Wang, Jing; Gossing, Michael; Fang, Pengfei; Zimmermann, Jana; Li, Xu; von Mollard, Gabriele Fischer; Niu, Liwen; Teng, Maikun

    2011-01-01

    SNARE proteins are crucial for membrane fusion in vesicular transport. To ensure efficient and accurate fusion, SNAREs need to be sorted into different budding vesicles. This process is usually regulated by specific recognition between SNAREs and their adaptor proteins. How different pairs of SNAREs and adaptors achieve their recognition is unclear. Here, we report the recognition between yeast SNARE Vti1p and its adaptor Ent3p derived from three crystal structures. Surprisingly, this yeast pair Vti1p/Ent3p interacts through a distinct binding site compared to their homologues vti1b/epsinR in mammals. An opposite surface on Vti1p_Habc domain binds to a conserved area on the epsin N-terminal homology (ENTH) domain of Ent3p. Two-hybrid, in vitro pull-down and in vivo experiments indicate this binding interface is important for correct localization of Vti1p in the cell. This previously undescribed discovery that a cargo and adaptor pair uses different binding sites across species suggests the diversity of SNARE-adaptor recognition in vesicular transport. PMID:21746902

  3. Allostery and folding of the N-terminal receiver domain of protein NtrC.

    PubMed

    Tripathi, Swarnendu; Portman, John J

    2013-10-24

    The N-terminal receiver domain of protein NtrC (NtrC(r)) exhibits allosteric transitions between the inactive (unphosphorylated) and active (phosphorylated) state on the microsecond time scale. Using a coarse-grained variational model with coupled energy basins, we illustrate that significant loss of conformational flexibility is the key determinant of the inactive (I) → active (A) state transition mechanism of NtrC(r). In particular, our results reveal that the rearrangements of the native contacts involving the regulatory helix-α4 and the flexible β3-α3 loop upon activation play a crucial role in the activation mechanism. Interestingly, we find that the β3-α3 loop exhibits a gradual decrease in flexibility throughout the activation transition, while helix-α4, in contrast, becomes more rigid abruptly near the free energy barrier separating the two states. To gain further insight into role these flexible regions play in the transition mechanism, we consider folding of NtrC(r) to both states using a similar model. Our calculated folding routes suggest that helix-α4 becomes structured later when folding to the I state compared to folding of the A state, a result consistent with it is relative conformational flexibility in the two states. Finally, we find a good qualitative agreement between our predicted I → A transition mechanism and the measured backbone dynamics from nuclear magnetic resonance experiments. PMID:23961720

  4. Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

    PubMed

    Lee, Youngjin; Min, Choon Kee; Kim, Tae Gyun; Song, Hong Ki; Lim, Yunki; Kim, Dongwook; Shin, Kahee; Kang, Moonkyung; Kang, Jung Youn; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lim, Jia Jia; Kim, Ji Hun; Kim, Ji Hye; Park, Zee Yong; Kim, Yeon-Soo; Wang, Jimin; Kim, Do Han; Eom, Soo Hyun

    2015-10-01

    The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation. PMID:26341627

  5. Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity.

    PubMed

    Hu, Tiancen; Yeh, Jennifer E; Pinello, Luca; Jacob, Jaison; Chakravarthy, Srinivas; Yuan, Guo-Cheng; Chopra, Rajiv; Frank, David A

    2015-10-01

    The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni(2+)-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni(2+)-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value. PMID:26169829

  6. Procollagen III N-terminal Propeptide and Desmosine are Released by Matrix Destruction in Pulmonary Tuberculosis

    PubMed Central

    Seddon, Jo; Kasprowicz, Victoria; Walker, Naomi F.; Yuen, Ho Ming; Sunpath, Henry; Tezera, Liku; Meintjes, Graeme; Wilkinson, Robert J.; Bishai, William R.; Friedland, Jon S.; Elkington, Paul T.

    2013-01-01

    Background. Tuberculosis is transmitted by patients with pulmonary disease. Matrix metalloproteinases (MMPs) drive lung destruction in tuberculosis but the resulting matrix degradation products (MDPs) have not been studied. We investigate the hypothesis that MMP activity generates matrix turnover products as correlates of lung pathology. Methods. Induced sputum and plasma were collected prospectively from human immunodeficiency virus (HIV) positive and negative patients with pulmonary tuberculosis and controls. Concentrations of MDPs and MMPs were analyzed by ELISA and Luminex array in 2 patient cohorts. Results. Procollagen III N-terminal propeptide (PIIINP) was 3.8-fold higher in induced sputum of HIV-uninfected tuberculosis patients compared to controls and desmosine, released during elastin degradation, was 2.4-fold higher. PIIINP was elevated in plasma of tuberculosis patients. Plasma PIIINP correlated with induced sputum MMP-1 concentrations and radiological scores, demonstrating that circulating MDPs reflect lung destruction. In a second patient cohort of mixed HIV seroprevalence, plasma PIIINP concentration was increased 3.0-fold above controls (P < .001). Plasma matrix metalloproteinase-8 concentrations were also higher in tuberculosis patients (P = .001). Receiver operating characteristic analysis utilizing these 2 variables demonstrated an area under the curve of 0.832 (P < .001). Conclusions. In pulmonary tuberculosis, MMP-driven immunopathology generates matrix degradation products. PMID:23922364

  7. The N-terminal Set-β Protein Isoform Induces Neuronal Death.

    PubMed

    Trakhtenberg, Ephraim F; Morkin, Melina I; Patel, Karan H; Fernandez, Stephanie G; Sang, Alan; Shaw, Peter; Liu, Xiongfei; Wang, Yan; Mlacker, Gregory M; Gao, Han; Velmeshev, Dmitry; Dombrowski, Susan M; Vitek, Michael P; Goldberg, Jeffrey L

    2015-05-22

    Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death. PMID:25833944

  8. Intrinsic disorder drives N-terminal ubiquitination by Ube2w

    PubMed Central

    Vittal, Vinayak; Shi, Lei; Wenzel, Dawn M.; Scaglione, K. Matthew; Duncan, Emily D.; Basrur, Venkatesha; Elenitoba-Johnson, Kojo S. J.; Baker, David; Paulson, Henry L.; Brzovic, Peter S.; Klevit, Rachel E.

    2014-01-01

    Ubiquitination of the αN-terminus of protein substrates has been reported sporadically over the past twenty years. However the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the ubiquitin-conjugating enzyme (E2) Ube2w employs a novel mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N-termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike any other E2, in which its C-terminus is partly disordered and flexible to accommodate variable substrate N-termini. Flexibility of the substrate is critical for recognition by Ube2w and point mutations in, or removal of, the flexible C-terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal-Ubiquitome in cells. PMID:25436519

  9. Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

    PubMed Central

    Shaiu, Wen-Ling; Hsieh, Tao-shih

    1998-01-01

    DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against β-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression. PMID:9632819

  10. Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution.

    PubMed

    Jayakumar, Arumugam; Kang, Ya'an; Henderson, Ying; Mitsudo, Kenji; Liu, Xiaoling; Briggs, Katrina; Wang, Mary; Frederick, Mitchell J; El-Naggar, Adel K; Bebök, Zsuzsa; Clayman, Gary L

    2005-03-01

    The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion. PMID:15680911

  11. The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.

    PubMed

    Kuo, Shiu-Ming; Wang, Li-Yuan; Yu, Siyuan; Campbell, Christine E; Valiyaparambil, Sujith A; Rance, Mark; Blumenthal, Kenneth M

    2013-07-30

    We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated. PMID:23837633

  12. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio

    SciTech Connect

    Liebschner, Dorothee; Brzezinski, Krzysztof; Dauter, Miroslawa; Dauter, Zbigniew; Nowak, Marta; Kur, Józef; Olszewski, Marcin

    2012-12-01

    The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods. PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  13. Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

    SciTech Connect

    Borko, Ľubomír; Bauerová-Hlinková, Vladena Hostinová, Eva; Gašperík, Juraj; Beck, Konrad; Lai, F. Anthony; Zahradníková, Alexandra; Ševčík, Jozef

    2014-11-01

    X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

  14. Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry.

    PubMed

    Rehder, Douglas S; Gundberg, Caren M; Booth, Sarah L; Borges, Chad R

    2015-06-01

    Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un

  15. Gamma-Carboxylation and Fragmentation of Osteocalcin in Human Serum Defined by Mass Spectrometry*

    PubMed Central

    Rehder, Douglas S.; Gundberg, Caren M.; Booth, Sarah L.; Borges, Chad R.

    2015-01-01

    Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0–3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that

  16. Domain organization of phytochelatin synthase: functional properties of truncated enzyme species identified by limited proteolysis.

    PubMed

    Ruotolo, Roberta; Peracchi, Alessio; Bolchi, Angelo; Infusini, Giuseppe; Amoresano, Angela; Ottonello, Simone

    2004-04-01

    Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. No structural information on this enzyme is as yet available. It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region. To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments. Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments. As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1. The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g. mercury and zinc, but not cadmium or copper). The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme. AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region. The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals

  17. Removing N-terminal sequences in pre-S1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine.

    PubMed

    Ge, Guohong; Wang, Shixia; Han, Yaping; Zhang, Chunhua; Lu, Shan; Huang, Zuhu

    2012-01-01

    Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens. PMID:22844502

  18. Blood-brain barrier permeability to leucine-enkephalin, D-alanine2-D-leucine5-enkephalin and their N-terminal amino acid (tyrosine).

    PubMed

    Zlokovic, B V; Begley, D J; Chain-Eliash, D G

    1985-06-10

    The permeability of the blood-brain barrier to [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) (abbreviated to Leu-Enk) and of its synthetic analogue D-alanine2-[tyrosyl-3,5-3H]enkephalin-(5-D-leucine) (abbreviated to D-Ala2-D-Leu5-Enk) was studied, in the adult rat, by means of Oldendorf's27 intracarotid injection technique. The brain uptake index (BUI) corrected for residual vascular radioactivity was about the same for both peptides, indicating a low extraction from the blood during a 5- or 15-s period of exposure to the peptides. Transport of Leu-Enk was not saturated by unlabelled Enk at a concentration as high as 5 mM but was completely abolished by 5mM tyrosine and by the inhibitor of aminopeptidase activity, bacitracin (2 mM). Also the typical L-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2 carboxylic acid (BCH)9 at 10 mM concentration markedly reduced (by 80%) Leu-Enk uptake by the brain. In contrast, brain uptake of D-Ala2-D-Leu5-Enk was reduced only to about one-half of its control value by bacitracin or by 25% by BCH. Brain uptake for L-tyrosine was typically large and markedly inhibited by BCH but not inhibited by 5 mM unlabelled Leu-Enk. These results show that the measurable but low first-pass extractions for enkephalins are not representative of the uptake of these peptides into the brain, but rather reflect their extreme sensitivity to enzymatic degradation with a release of the N-terminal tyrosine residue. The results also suggest that small amounts of D-Ala2-D-Leu5-Enk might cross the blood-brain barrier in an intact form.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3891014

  19. The loss of c-Jun N-terminal protein kinase activity prevents the amyloidogenic cleavage of amyloid precursor protein and the formation of amyloid plaques in vivo.

    PubMed

    Mazzitelli, Sonia; Xu, Ping; Ferrer, Isidre; Davis, Roger J; Tournier, Cathy

    2011-11-23

    Phosphorylation plays a central role in the dynamic regulation of the processing of the amyloid precursor protein (APP) and the production of amyloid-β (Aβ), one of the clinically most important factors that determine the onset of Alzheimer's disease (AD). This has led to the hypothesis that aberrant Aβ production associated with AD results from regulatory defects in signal transduction. However, conflicting findings have raised a debate over the identity of the signaling pathway that controls APP metabolism. Here, we demonstrate that activation of the c-Jun N-terminal protein kinase (JNK) is essential for mediating the apoptotic response of neurons to Aβ. Furthermore, we discovered that the functional loss of JNK signaling in neurons significantly decreased the number of amyloid plaques present in the brain of mice carrying familial AD-linked mutant genes. This correlated with a reduction in Aβ production. Biochemical analyses indicate that the phosphorylation of APP at threonine 668 by JNK is required for γ-mediated cleavage of the C-terminal fragment of APP produced by β-secretase. Overall, this study provides genetic evidence that JNK signaling is required for the formation of amyloid plaques in vivo. Therefore, inhibition of increased JNK activity associated with aging or with a pathological condition constitutes a potential strategy for the treatment of AD. PMID:22114267

  20. Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection.

    PubMed

    Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

    2016-01-01

    Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection. PMID:27562370

  1. Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection

    PubMed Central

    Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

    2016-01-01

    Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection. PMID:27562370

  2. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase

    PubMed Central

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C.; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M.

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1–50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  3. Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases

    PubMed Central

    Marino, Francesca; Mojumdar, Aditya; Zucchelli, Chiara; Bhardwaj, Amit; Buratti, Emanuele; Vindigni, Alessandro; Musco, Giovanna; Onesti, Silvia

    2016-01-01

    The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability. PMID:26888063

  4. Inhibition of Apoptosis in Prostate Cancer Cells by Androgens Is Mediated through Downregulation of c-Jun N-terminal Kinase Activation1

    PubMed Central

    Lorenzo, Petra Isabel; Saatcioglu, Fahri

    2008-01-01

    Androgen deprivation induces the regression of prostate tumors mainly due to an increase in the apoptosis rate; however, the molecular mechanisms underlying the antiapoptotic actions of androgens are not completely understood. We have studied the antiapoptotic effects of androgens in prostate cancer cells exposed to different proapoptotic stimuli. Terminal deoxynucleotidyl transferase-mediated nick-end labeling and nuclear fragmentation analyses demonstrated that androgens protect LNCaP prostate cancer cells from apoptosis induced by thapsigargin, the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate, or UV irradiation. These three stimuli require the activation of the c-Jun N-terminal kinase (JNK) pathway to induce apoptosis and in all three cases, androgen treatment blocks JNK activation. Interestingly, okadaic acid, a phosphatase inhibitor that causes apoptosis in LNCaP cells, induces JNK activation that is also inhibited by androgens. Actinomycin D, the antiandrogen bicalutamide or specific androgen receptor (AR) knockdown by small interfering RNA all blocked the inhibition of JNK activation mediated by androgens indicating that this activity requires AR-dependent transcriptional activation. These data suggest that the crosstalk between AR and JNK pathways may have important implications in prostate cancer progression and may provide targets for the development of new therapies. PMID:18472959

  5. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase.

    PubMed

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  6. Transient stability of the helical pattern of region F19-L22 of the N-terminal domain of p53: a molecular dynamics simulation study.

    PubMed

    Espinoza-Fonseca, L Michel; Trujillo-Ferrara, José G

    2006-04-28

    Two molecular dynamics simulations of the region E17-N29 of p53 (p53(17-29)) at different temperatures were performed for a total time of 0.2 micros, to study the conformational landscape of this region. Previous studies have suggested that this region displays different structural motifs, such as helix of a double beta-turn, and that its secondary structure might be transiently stable. Interestingly, in this study it was found that the region F19-L25, and particularly its fragment F19-L22, display a stable, transient helical pattern at sub-microsecond periods. The region F19-L22, which contains one of the most important residues needed for the interaction of p53 with MDM2, seems to be formed and stabilized by the existence of one hydrophobic and one aromatic cluster. The main function of these clusters is to help their surrounding area to desolvate, to allow the hydrogen bond network, therefore favoring the formation of a stable helix. This preliminary study would be useful for a better understanding of the structure and function of the N-terminal domain of p53 and its implications for the control of different types of cancer. PMID:16530164

  7. Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues.

    PubMed

    Arndt, James R; Kondalaji, Samaneh Ghassabi; Maurer, Megan M; Parker, Arlo; Legleiter, Justin; Valentine, Stephen J

    2015-07-21

    Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation. PMID:26098795

  8. Structural modeling of the N-terminal signal–receiving domain of IκBα

    PubMed Central

    Yazdi, Samira; Durdagi, Serdar; Naumann, Michael; Stein, Matthias

    2015-01-01

    The transcription factor nuclear factor-κB (NF-κB) exerts essential roles in many biological processes including cell growth, apoptosis and innate and adaptive immunity. The NF-κB inhibitor (IκBα) retains NF-κB in the cytoplasm and thus inhibits nuclear localization of NF-κB and its association with DNA. Recent protein crystal structures of the C-terminal part of IκBα in complex with NF-κB provided insights into the protein-protein interactions but could not reveal structural details about the N-terminal signal receiving domain (SRD). The SRD of IκBα contains a degron, formed following phosphorylation by IκB kinases (IKK). In current protein X-ray structures, however, the SRD is not resolved and assumed to be disordered. Here, we combined secondary structure annotation and domain threading followed by long molecular dynamics (MD) simulations and showed that the SRD possesses well-defined secondary structure elements. We show that the SRD contains 3 additional stable α-helices supplementing the six ARDs present in crystallized IκBα. The IκBα/NF-κB protein-protein complex remained intact and stable during the entire simulations. Also in solution, free IκBα retains its structural integrity. Differences in structural topology and dynamics were observed by comparing the structures of NF-κB free and NF-κB bound IκBα-complex. This study paves the way for investigating the signaling properties of the SRD in the IκBα degron. A detailed atomic scale understanding of molecular mechanism of NF-κB activation, regulation and the protein-protein interactions may assist to design and develop novel chronic inflammation modulators. PMID:26157801

  9. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  10. The N-terminal CEBPA mutant in acute myeloid leukemia impairs CXCR4 expression.

    PubMed

    Kuo, Yuan-Yeh; Hou, Hsin-An; Chen, Yin-Kai; Li, Li-Yu; Chen, Po-Hsuen; Tseng, Mei-Hsuan; Huang, Chi-Fei; Lee, Fen-Yu; Liu, Ming-Chih; Liu, Chia-Wen; Chou, Wen-Chien; Liu, Chieh-Yu; Tang, Jih-Luh; Yao, Ming; Tien, Hwei-Fang

    2014-12-01

    CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical implications of bone marrow CXCR4 expression in patients with acute myeloid leukemia showed not only higher CXCR4 expression was an independent poor prognostic factor, irrespective of age, white blood cell counts, cytogenetics, and mutation status of NPM1/FLT3-ITD and CEBPA, but also showed CXCR4 expression was inversely associated with mutations of CEBPA, a gene encoding transcription factor C/EBPα. Patients with wild-type CEBPA had significantly higher CXCR4 expression than those with mutated CEBPA. We hypothesized that CEBPA might influence the expression of CXCR4. To test this hypothesis, we first examined endogenous CXCR4 expression in 293T and K562 cells over-expressing wild-type C/EBPα p42 and demonstrated that CXCR4 levels were increased in these cells, whilst the expression of the N-terminal mutant, C/EBPα p30, diminished CXCR4 transcription. We further showed p42 was bound to the CXCR4 promoter by the chromatin immunoprecipitation assays. Induction of p42 in the inducible K562-C/EBPα cell lines increased the chemotactic migration. Moreover, decreased expression of C/EBPα by RNA interference decreased levels of CXCR4 protein expression in U937 cells, thereby abrogating CXCR4-mediated chemotaxis. Our results provide, for the first time, evidence that C/EBPα indeed regulates the activation of CXCR4, which is critical for the homing and engraftment of acute myeloid leukemia cells, while p30 mutant impairs CXCR4 expression. PMID:25193961

  11. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS. PMID:25500996

  12. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    PubMed Central

    Marshall, Alexandra H.; Brooks, Natasha C.; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−/−) to wildtype mice after inducing a 30% total body surface area thermal injury. Animals were sacrificed after 1, 3 and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western Blotting and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by TUNEL staining. Liver function was quantified by AST and ALT activity assays. ER stress increased after burn in both JNK2−/− and wildtype mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in IL-6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2−/− group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2−/−. As expected, apoptosis in the liver increased after burn injury in wildtype mice but not in JNK2−/−. AST/ALT activity revealed that liver function recovered more quickly in JNK2−/−. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn. PMID:23324888

  13. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    SciTech Connect

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  14. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation

    PubMed Central

    2016-01-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic

  15. Stability Enhancing N-Terminal PEGylation of Oxytocin Exploiting Different Polymer Architectures and Conjugation Approaches.

    PubMed

    Collins, Jennifer; Kempe, Kristian; Wilson, Paul; Blindauer, Claudia A; McIntosh, Michelle P; Davis, Thomas P; Whittaker, Michael R; Haddleton, David M

    2016-08-01

    Oxytocin, a cyclic nine amino acid neurohypophyseal hormone therapeutic, is effectively used in the control of postpartum hemorrhaging (PPH) and is on the WHO List of Essential Medicines. However, oxytocin has limited shelf life stability in aqueous solutions, particularly at temperatures in excess of 25 °C and injectable aqueous oxytocin formulations require refrigeration (<8 °C). This is particularly problematic in the hot climates often found in many developing countries where daytime temperatures can exceed 40 °C and where reliable cold-chain storage is not always achievable. The purpose of this study was to develop N-terminal amine targeted PEGylation strategies utilizing both linear PEG and polyPEG "comb" polymers as an effective method for stabilizing solution formulations of this peptide for prolonged storage in the absence of efficient cold-chain storage. The conjugation chemistries investigated herein include irreversible amine targeted conjugation methods utilizing NHS ester and aldehyde reductive amination chemistry. Additionally, one reversible conjugation method using a Schiff base approach was explored to allow for the release of the native peptide, thus, ensuring that biological activity remains unaffected. The reversibility of this approach was investigated for the different polymer architectures, alongside a nonpolymer oxytocin analogue to monitor how pH can tune native peptide release. Elevated temperature degradation studies of the polymer conjugates were evaluated to assess the stability of the PEGylated analogues in comparison to the native peptide in aqueous formulations to mimic storage conditions in developing nations and regions where storage under appropriate conditions is challenging. PMID:27419537

  16. Huntingtin N-terminal monomeric and multimeric structures destabilized by covalent modification of heteroatomic residues

    PubMed Central

    Arndt, James R.; Kondalaji, Samaneh G.; Maurer, Megan M.; Parker, Arlo; Legleiter, Justin

    2015-01-01

    Early-stage oligomer formation of the huntingtin protein may be driven by self-association of the seventeen-residue amphipathic α-helix at the protein’s N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington’s disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS), have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures are comprised of a helix-turn-coil configuration and a helix-extended coil region. Elongated dimer species are comprised of partially-helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and possibly, the early stages of huntingtin exon 1 aggregation. PMID:26098795

  17. Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality.

    PubMed

    Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

    2015-10-01

    Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

  18. Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality

    PubMed Central

    Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

    2015-01-01

    Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

  19. Mechanochemical tuning of myosin-I by the N-terminal region

    PubMed Central

    Greenberg, Michael J.; Lin, Tianming; Shuman, Henry; Ostap, E. Michael

    2015-01-01

    Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post–power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms. PMID:26056287

  20. The vasorelaxant effect of adrenomedullin, proadrenomedullin N-terminal 20 peptide and amylin in human skin.

    PubMed

    Hasbak, Philip; Eskesen, Karen; Lind, Henrik; Holst, Jan; Edvinsson, Lars

    2006-08-01

    In this study we aimed to assess in vivo, the vasodilator effects of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and amylin in human skin vasculature and compare the responses to the effects mediated by the endogenous neuropeptides calcitonin gene-related peptide (CGRP) and substance P and to examine the mRNA expression of calcitonin receptor-like receptor (CL-R) and receptor-activity modifying proteins, RAMP1, RAMP 2 and RAMP3 in human subcutaneous arteries. Changes in skin blood flow of the forearm were measured using a Laser Doppler Imager after intradermal injection of the peptides. The mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction (real-time PCR). CGRP, adrenomedullin and amylin induced concentration-dependent, long-lasting increases in skin blood flow. The response to PAMP was shorter in duration appearing similar to the transient response induced by substance P. PAMP (10(-6)-10(-5) M) caused distinct itch sensation and local erythema. This effect could be abolished when combining the histamine H1-receptor antagonist mepyramin and PAMP. Real-time PCR data showed a higher level of mRNA for RAMP2 than CL-R, RAMP1 and RAMP3 in the tissue. Though the PCR data demonstrated the presence of mRNA for both CGRP1 and adrenomedullin receptors the rank order of potency (CGRP>adrenomedullin>amylin) for the blood flow increase indicated vasodilatation for these peptides was induced by activation of CGRP1 receptors. Intradermal injection of CGRP, adrenomedullin and amylin induces long lasting dilatation of human skin vasculature by activation of CGRP1 receptors. PAMP induces transient vasodilatation. PAMP but not CGRP, adrenomedullin and amylin causes itch sensation and local erythema. The transient effect on vasodilatation as response to PAMP is discussed. PMID:16918718

  1. Molecular insights into the recognition of N-terminal histone modifications by the BRPF1 bromodomain.

    PubMed

    Poplawski, Amanda; Hu, Kaifeng; Lee, Woonghee; Natesan, Senthil; Peng, Danni; Carlson, Samuel; Shi, Xiaobing; Balaz, Stefan; Markley, John L; Glass, Karen C

    2014-04-17

    The monocytic leukemic zinc finger (MOZ) histone acetyltransferase (HAT) acetylates free histones H3, H4, H2A, and H2B in vitro and is associated with up-regulation of gene transcription. The MOZ HAT functions as a quaternary complex with the bromodomain-PHD finger protein 1 (BRPF1), inhibitor of growth 5 (ING5), and hEaf6 subunits. BRPF1 links the MOZ catalytic subunit to the ING5 and hEaf6 subunits, thereby promoting MOZ HAT activity. Human BRPF1 contains multiple effector domains with known roles in gene transcription, as well as chromatin binding and remodeling. However, the biological function of the BRPF1 bromodomain remains unknown. Our findings reveal novel interactions of the BRPF1 bromodomain with multiple acetyllysine residues on the N-terminus of histones and show that it preferentially selects for H2AK5ac, H4K12ac, and H3K14ac. We used chemical shift perturbation data from NMR titration experiments to map the BRPF1 bromodomain ligand binding pocket and identified key residues responsible for coordination of the post-translationally modified histones. Extensive molecular dynamics simulations were used to generate structural models of bromodomain-histone ligand complexes, to analyze hydrogen bonding and other interactions, and to calculate the binding free energies. Our results outline the molecular mechanism driving binding specificity of the BRPF1 bromodomain for discrete acetyllysine residues on the N-terminal histone tails. Together, these data provide insights into how histone recognition by the bromodomain directs the biological function of BRPF1, ultimately targeting the MOZ HAT complex to chromatin substrates. PMID:24333487

  2. Age-adjusted plasma N-terminal pro-brain natriuretic peptide level in Kawasaki disease

    PubMed Central

    Jun, Heul; Ko, Kyung Ok; Lim, Jae Woo; Yoon, Jung Min; Lee, Gyung Min

    2016-01-01

    Purpose Recent reports showed that plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) could be a useful biomarker of intravenous immunoglobulin (IVIG) unresponsiveness and coronary artery lesion (CAL) development in Kawasaki disease (KD). The levels of these peptides are critically influenced by age; hence, the normal range and upper limits for infants and children are different. We performed an age-adjusted analysis of plasma NT-proBNP level to validate its clinical use in the diagnosis of KD. Methods The data of 131 patients with KD were retrospectively analyzed. The patients were divided into 2 groups—group I (high NT-proBNP group) and group II (normal NT-proBNP group)—comprising patients with NT-proBNP concentrations higher and lower than the 95th percentile of the reference value, respectively. We compared the laboratory data, responsiveness to IVIG, and the risk of CAL in both groups. Results Group I showed significantly higher white blood cell count, absolute neutrophil count, C-reactive protein level, aspartate aminotransferase level, and troponin-I level than group II (P<0.05). The risk of CAL was also significantly higher in group I (odds ratio, 5.78; P=0.012). IVIG unresponsiveness in group I was three times that in group II (odds ratio, 3.35; P= 0.005). Conclusion Age-adjusted analysis of plasma NT-proBNP level could be helpful in predicting IVIG unresponsiveness and risk of CAL development in patients with KD. PMID:27588030

  3. Galactinol synthase from kidney bean cotyledon and zucchini leaf. Purification and N-terminal sequences.

    PubMed Central

    Liu, J J; Odegard, W; de Lumen, B O

    1995-01-01

    Galactinol synthase (GS) was purified 1591-fold with a 3.9% recovery from the cotyledon of kidney bean (Phaseolus vulgaris) by a novel scheme consisting of ammonium sulfate fractionation followed by diethylaminoethyl, Affi-Gel Blue, and UDP-hexanolamine affinity chromatography. The purified enzyme had a specific activity of 8.75 mumol mg-1 min-1, a pH optimum of 7.0, and requirements for manganese ion and DTT. The enzyme exhibited a Km = 0.4 mM for UDP-galactose and a Km = 4.5 mM for myo-inositol. It was identified as a 38-kD peptide that co-purified with a 41- and a 43-kD peptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purification to homogeneity was achieved by isolating the 38-kD peptide from the SDS-PAGE gel. To clarify conflicting reports in the literature about the relative molecular mass of purified GS from zucchini leaf (Cucurbita pepo), a similar scheme with modified eluting conditions was used to purify GS from this source. Zucchini leaf GS was purified to homogeneity and identified as a 36-kD peptide on SDS-PAGE. Partial N-terminal sequences of the 38-kD peptide from kidney bean cotyledon and the 36-kD peptide from zucchini leaf were obtained. To facilitate identification of GS during the purification, an assay utilizing thin-layer chromatography and an isotopic analytic imaging scanner was developed. PMID:7480343

  4. Molecular insights into the recognition of N-terminal histone modifications by the BRPF1 bromodomain

    PubMed Central

    Poplawski, Amanda; Hu, Kaifeng; Lee, Woonghee; Natesan, Senthil; Peng, Danni; Carlson, Samuel; Shi, Xiaobing; Balaz, Stefan; Markley, John L.; Glass, Karen C.

    2014-01-01

    The monocytic leukemic zinc-finger (MOZ) histone acetyltransferase (HAT) acetylates free histones H3, H4, H2A, and H2B in vitro and is associated with up-regulation of gene transcription. The MOZ HAT functions as a quaternary complex with the bromodomain-PHD finger protein 1 (BRPF1), inhibitor of growth 5 (ING5), and hEaf6 subunits. BRPF1 links the MOZ catalytic subunit to the ING5 and hEaf6 subunits, thereby promoting MOZ HAT activity. Human BRPF1 contains multiple effector domains with known roles in gene transcription, and chromatin binding and remodeling. However, the biological function of the BRPF1 bromodomain remains unknown. Our findings reveal novel interactions of the BRPF1 bromodomain with multiple acetyllysine residues on the N-terminus of histones, and show it preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We used chemical shift perturbation data from NMR titration experiments to map the BRPF1 bromodomain ligand binding pocket and identified key residues responsible for coordination of the post-translationally modified histones. Extensive molecular dynamics simulations were used to generate structural models of bromodomain-histone ligand complexes, to analyze H-bonding and other interactions, and to calculate the binding free energies. Our results outline the molecular mechanism driving binding specificity of the BRPF1 bromodomain for discrete acetyllysine residues on the N-terminal histone tails. Together these data provide insights on how histone recognition by the bromodomain directs the biological function of BRPF1, ultimately targeting the MOZ HAT complex to chromatin substrates. PMID:24333487

  5. ClpB N-terminal domain plays a regulatory role in protein disaggregation

    PubMed Central

    Rosenzweig, Rina; Farber, Patrick; Velyvis, Algirdas; Rennella, Enrico; Latham, Michael P.; Kay, Lewis E.

    2015-01-01

    ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation. PMID:26621746

  6. N-Terminal Mutations Modulate Yeast Snf1 Protein Kinase Function

    PubMed Central

    Estruch, F.; Treitel, M. A.; Yang, X.; Carlson, M.

    1992-01-01

    The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-ΔN kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-ΔN proteins showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell. PMID:1468623

  7. Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.

    PubMed

    Subramanyam, Shyamal; Jones, William T; Spies, Maria; Spies, M Ashley

    2013-10-01

    RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction. PMID:23935068

  8. Truncated Moment Analysis of Nucleon Structure Functions

    SciTech Connect

    A. Psaker; W. Melnitchouk; M. E. Christy; C. E. Keppel

    2007-11-16

    We employ a novel new approach using "truncated" moments, or integrals of structure functions over restricted regions of x, to study local quark-hadron duality, and the degree to which individual resonance regions are dominated by leading twists. Because truncated moments obey the same Q^2 evolution equations as the leading twist parton distributions, this approach makes possible for the first time a description of resonance region data and the phenomenon of quark-hadron duality directly from QCD.

  9. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. PMID:22796565

  10. N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF.

    PubMed

    Mealman, Tiffany D; Zhou, Mowei; Affandi, Trisiani; Chacón, Kelly N; Aranguren, Mariana E; Blackburn, Ninian J; Wysocki, Vicki H; McEvoy, Megan M

    2012-08-28

    Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux. PMID:22812620

  11. The N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF

    PubMed Central

    Mealman, Tiffany D.; Zhou, Mowei; Affandi, Trisiani; Chacón, Kelly N.; Aranguren, Mariana E.; Blackburn, Ninian J.; Wysocki, Vicki H.; McEvoy, Megan M.

    2012-01-01

    Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N and C termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump, CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein’s function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal, but also to play an active role in efflux. PMID:22812620

  12. Role of Akt and c-Jun N-terminal Kinase 2 in Apoptosis Induced by Interleukin-4 Deprivation

    PubMed Central

    Cerezo, Ana; Martínez-A, Carlos; Lanzarot, Diego; Fischer, Siegmund; Franke, Thomas F.; Rebollo, Angelita

    1998-01-01

    We have shown previously that interleukin-4 (IL-4) protects TS1αβ cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1αβ cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1αβ survival is independent of Bcl-2, Bcl-x, or Bax. PMID:9802900

  13. The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels

    SciTech Connect

    Hwang, Mi-Na; Min, Chan-Hee; Kim, Hyung Sik; Lee, Ho; Yoon, Kyong-Ah; Park, Sung Yong; Lee, Eun Sook; Yoon, Sungpil . E-mail: yoons@ncc.re.kr

    2007-08-24

    We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.

  14. The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription

    PubMed Central

    Morin, Benjamin; Coutard, Bruno; Lelke, Michaela; Ferron, François; Kerber, Romy; Jamal, Saïd; Frangeul, Antoine; Baronti, Cécile; Charrel, Rémi; de Lamballerie, Xavier; Vonrhein, Clemens; Lescar, Julien; Bricogne, Gérard; Günther, Stephan; Canard, Bruno

    2010-01-01

    Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease. PMID:20862324

  15. Copper induces conformational changes in the N-terminal part of cell-surface PrPC.

    PubMed

    Leclerc, E; Serban, H; Prusiner, S B; Burton, D R; Williamson, R A

    2006-11-01

    Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC. PMID:16791441

  16. Importin α1 Mediates Yorkie Nuclear Import via an N-terminal Non-canonical Nuclear Localization Signal.

    PubMed

    Wang, Shimin; Lu, Yi; Yin, Meng-Xin; Wang, Chao; Wu, Wei; Li, Jinhui; Wu, Wenqing; Ge, Ling; Hu, Lianxin; Zhao, Yun; Zhang, Lei

    2016-04-01

    The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway was inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. Here, we provide evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, we found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, we identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin α1 in Yorkie nuclear import. PMID:26887950

  17. Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis.

    PubMed

    Palladino, L O; Tung, J S; Dunwiddie, C; Alves, K; Lenny, A B; Przysiecki, C; Lehman, D; Nutt, E; Cuca, G C; Law, S W

    1991-02-01

    Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM. PMID:1821771

  18. Magma Fragmentation

    NASA Astrophysics Data System (ADS)

    Gonnermann, Helge M.

    2015-05-01

    Magma fragmentation is the breakup of a continuous volume of molten rock into discrete pieces, called pyroclasts. Because magma contains bubbles of compressible magmatic volatiles, decompression of low-viscosity magma leads to rapid expansion. The magma is torn into fragments, as it is stretched into hydrodynamically unstable sheets and filaments. If the magma is highly viscous, resistance to bubble growth will instead lead to excess gas pressure and the magma will deform viscoelastically by fracturing like a glassy solid, resulting in the formation of a violently expanding gas-pyroclast mixture. In either case, fragmentation represents the conversion of potential energy into the surface energy of the newly created fragments and the kinetic energy of the expanding gas-pyroclast mixture. If magma comes into contact with external water, the conversion of thermal energy will vaporize water and quench magma at the melt-water interface, thus creating dynamic stresses that cause fragmentation and the release of kinetic energy. Lastly, shear deformation of highly viscous magma may cause brittle fractures and release seismic energy.

  19. Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors.

    PubMed

    Humphries, Paul S; Lafontaine, Jennifer A; Agree, Charles S; Alexander, David; Chen, Ping; Do, Quyen-Quyen T; Li, Lilian Y; Lunney, Elizabeth A; Rajapakse, Ranjan J; Siegel, Karen; Timofeevski, Sergei L; Wang, Tianlun; Wilhite, David M

    2009-04-15

    The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported. PMID:19327989

  20. Substrate recognition of holocytochrome c synthase: N-terminal region and CXXCH motif of mitochondrial cytochrome c

    PubMed Central

    Zhang, Yulin; Stevens, Julie M.; Ferguson, Stuart J.

    2014-01-01

    Holocytochrome c synthase (HCCS) attaches heme covalently to mitochondrial respiratory cytochromes c. Little is known about the reaction of heme attachment to apocytochromes c by HCCS, although recently it has been established that the CXXCH motif and the N-terminus of the apocytochrome polypeptide are important protein–protein recognition motifs. Here, we explore further the important features of the N-terminal sequence and investigate what variations in the CXXCH residues are productively recognised by HCCS in its substrate. PMID:25084480

  1. The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin☆

    PubMed Central

    Faraj, Santiago E.; Venturutti, Leandro; Roman, Ernesto A.; Marino-Buslje, Cristina B.; Mignone, Astor; Tosatto, Silvio C.E.; Delfino, José M.; Santos, Javier

    2013-01-01

    The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42–80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56–210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81–210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56–81, the moiety putatively responsible for promoting protein–protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or β-strand structure. In this context, we explored the conformation and stability of hFXN56–210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90–210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81–210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function. PMID:23951553

  2. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    SciTech Connect

    Kurooka, Hisanori; Sugai, Manabu; Mori, Kentaro; Yokota, Yoshifumi

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  3. Magnetic immunoaffinity enrichment for selective capture and MS/MS analysis of N-terminal-TMPP-labeled peptides.

    PubMed

    Bland, Céline; Bellanger, Laurent; Armengaud, Jean

    2014-02-01

    Proteogenomics is the alliance of proteomics and genomics with the aim of better annotating structural genes based on experimental, protein-based data items established by tandem mass spectrometry. While, on average, more than one-tenth of protein N-termini are incorrectly annotated, there is a crucial need for methodological approaches to systematically establish the translational starts of polypeptides, and their maturations, such as N-terminal methionine processing and peptide signal excision. Refinement of genome annotation through correction of wrongly annotation initiation start site and detection of unannotated genes can be achieved after enrichment and detection of protein N-termini by mass spectrometry. Here we describe a straightforward strategy to specifically label protein N-termini with a positively charged TMPP label to selectively capture these entities with in-house-developed anti-TMPP antibodies coupled to magnetic beads and to analyze them by nanoLC-MS/MS. While most N-terminomics-oriented approaches are based on the depletion of internal peptides to retrieve N-terminal peptides, this enrichment approach is fast and the results are highly specific for improved, ionizable, TMPP-labeled peptides. The whole proteome of the model marine bacterium, Roseobacter denitrificans, was analyzed, leading to the identification of more than twice the number of N-terminal peptides compared with the nonenriched fraction. A total of 269 proteins were characterized in terms of their N-termini. In addition, three unannotated genes were identified based on multiple, redundant N-terminal peptides. Our strategy greatly simplifies the systematic and automatic proteogenomic annotation of genomes as well as degradomics-oriented approaches, focusing the mass spectrometric efforts on the most crucial enriched fractions. PMID:24313271

  4. Simulation of Different Truncated p16INK4a Forms and In Silico Study of Interaction with Cdk4

    PubMed Central

    Fahham, Najmeh; Ghahremani, Mohammad Hossein; Sardari, Soroush; Vaziri, Behrouz; Ostad, Seyed Nasser

    2008-01-01

    Protein-protein interactions studies can greatly increase the amount of structural and functional information pertaining to biologically active molecules and processes. The information obtained from such studies can lead to design and application of new modification in order to obtain a desired bioactivity. Many application packages and servers performing docking, such as HEX, DOT, AUTODOCK, and ZDOCK are now available for predicting the lowest free energy state of a protein complex. In this study, we have focused on cyclin-dependent kinase 4 (Cdk4), a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point and p16INK4a, a tumor suppressor which inhibits Cdk4 activity. Truncated structures were created to find the more critical regions of p16 for interaction. The tertiary structures were determined by ProSAL, GENO3D Web Server. We evaluated their interactions with Cdk4 using two docking systems, HEX 4.5 and DOT 1. Calculations were performed on a high-speed computer. Minimizations and visualizations were carried out by PdbViewer 3.7. Considering shape and shape/electrostatic total energy, structures containing ANK II, III and IV motifs that lack the N-terminal region of the full length p16 molecule showed the best fit complexes among the p16 truncated forms. The free energies were compatible with that of p16 full length original form, the full length. It seems that the N-terminal of the molecule is not crucial for the interaction since the truncated structure containing only this region did not show a good total energy. PMID:19352455

  5. Non-manifesting AHI1 truncations indicate localized loss-of-function tolerance in a severe Mendelian disease gene

    PubMed Central

    Elsayed, Solaf M.; Phillips, Jennifer B.; Heller, Raoul; Thoenes, Michaela; Elsobky, Ezzat; Nürnberg, Gudrun; Nürnberg, Peter; Seland, Saskia; Ebermann, Inga; Altmüller, Janine; Thiele, Holger; Toliat, Mohammad; Körber, Friederike; Hu, Xue-Jia; Wu, Yun-Dong; Zaki, Maha S.; Abdel-Salam, Ghada; Gleeson, Joseph; Boltshauser, Eugen; Westerfield, Monte; Bolz, Hanno J.

    2015-01-01

    Determination of variant pathogenicity represents a major challenge in the era of high-throughput sequencing. Erroneous categorization may result if variants affect genes that are in fact dispensable. We demonstrate that this also applies to rare, apparently unambiguous truncating mutations of an established disease gene. By whole-exome sequencing (WES) in a consanguineous family with congenital non-syndromic deafness, we unexpectedly identified a homozygous nonsense variant, p.Arg1066*, in AHI1, a gene associated with Joubert syndrome (JBTS), a severe recessive ciliopathy. None of four homozygotes expressed any signs of JBTS, and one of them had normal hearing, which also ruled out p.Arg1066* as the cause of deafness. Homozygosity mapping and WES in the only other reported JBTS family with a homozygous C-terminal truncation (p.Trp1088Leufs*16) confirmed AHI1 as disease gene, but based on a more N-terminal missense mutation impairing WD40-repeat formation. Morpholinos against N-terminal zebrafish Ahi1, orthologous to where human mutations cluster, produced a ciliopathy, but targeting near human p.Arg1066 and p.Trp1088 did not. Most AHI1 mutations in JBTS patients result in truncated protein lacking WD40-repeats and the SH3 domain; disease was hitherto attributed to loss of these protein interaction modules. Our findings indicate that normal development does not require the C-terminal SH3 domain. This has far-reaching implications, considering that variants like p.Glu984* identified by preconception screening (‘Kingsmore panel’) do not necessarily indicate JBTS carriership. Genomes of individuals with consanguineous background are enriched for homozygous variants that may unmask dispensable regions of disease genes and unrecognized false positives in diagnostic large-scale sequencing and preconception carrier screening. PMID:25616960

  6. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

    PubMed Central

    Dwivedi, Gajendradhar R.; Srikanth, Kolluru D.; Anand, Praveen; Naikoo, Javed; Srilatha, N. S.; Rao, Desirazu N.

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  7. Antigenic modules in the N-terminal S1 region of the transmissible gastroenteritis virus spike protein

    PubMed Central

    Reguera, Juan; Ordoño, Desiderio; Santiago, César; Enjuanes, Luis

    2011-01-01

    The N-terminal S1 region of the transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein contains four antigenic sites (C, B, D and A, from the N- to the C-terminal end) and is engaged in host-cell receptor recognition. The most N-terminal portion of the S1 region, which comprises antigenic sites C and B, is needed for the enteric tropism of TGEV, whereas the major antigenic site A at the C-terminal moiety is required for both respiratory and enteric cell tropism, and is engaged in recognition of the aminopeptidase N (APN) receptor. This study determined the kinetics for binding of a soluble S1 protein to the APN protein. Moreover, the S1 region of the TGEV S protein was dissected, with the aim of identifying discrete modules displaying unique antigenic sites and receptor-binding functions. Following protease treatments and mammalian cell expression methods, four modules or domains (D1–D4) were defined at the S1 region. Papain treatment identified an N-terminal domain (D1) resistant to proteolysis, whereas receptor binding defined a soluble and functional APN receptor-binding domain (D3). This domain was recognized by neutralizing antibodies belonging to the antigenic site A and therefore could be used as an immunogen for the prevention of viral infection. The organization of the four modules in the S1 region of the TGEV S glycoprotein is discussed. PMID:21228126

  8. Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix

    SciTech Connect

    Wand, A.J.; Roder, H.; Englander, S.W.

    1986-03-11

    The hydrogen exchange behavior of the N-terminal helical segment in horse heart cytochrome c was studied in both the reduced and the oxidized forms by use of two-dimensional nuclear magnetic resonance methods. The amide protons of the first six residues are not H bonded and exchange rapidly with solvent protons. The most N-terminal H-bonded groups--the amide NH of Lys-7 to Phe-10--exhibit a sharp gradient in exchange rate indicative of dynamic fraying behavior, consistent with statistical-mechanical principles. This occurs identically in both reduced and oxidized cytochrome c. In the oxidized form, residues 11-14, which form the last helical turn, all exchange with a similar rate, about one million times slower than the rate characteristic of freely exposed peptide NH, even though some are on the aqueous face of the helix and others are fully buried. These and similar observations in several other proteins appear to document local cooperative unfolding reactions as determinants of protein H exchange reactions. The N-terminal segment of cytochrome c is insensitive to the heme redox state, as in the crystallographic model, except for residues closest to the heme (Cys-14 and Ala-15), which exchange about 15-fold more slowly in the reduced form. The cytochrome c H exchange results can be further considered in terms of the conformation of the native and the transiently unfolded forms and their free energy relationships in both the reduced and the oxidized states.

  9. The Pitx2c N-terminal domain is a critical interaction domain required for asymmetric morphogenesis

    PubMed Central

    Simard, Annie; Di Giorgio, Luciano; Amen, Melanie; Westwood, Ashley; Amendt, Brad A.; Ryan, Aimee K.

    2010-01-01

    The paired-like homeodomain transcription factor Pitx2c has an essential role in patterning the left-right axis. However, neither its transcriptional targets nor the molecular mechanisms through which it exerts its patterning function are known. Here we provide evidence that the N-terminal domain of Pitx2c is important for this activity. Overexpression of the Pitx2c N-terminus in ovo randomizes the direction of heart looping, the first morphological asymmetry conserved in vertebrate embryos. In addition, the Pitx2c N-terminal domain blocks the ability of Pitx2c to synergize with Nkx2.5 to transactivate the procollagen lysyl hydroxylase (Plod-1) promoter in transient transfection assays. A five amino acid region containing leucine-41 is required for both of these effects. Our data suggest that the Pitx2c N-terminal domain competes with endogenous Pitx2c for binding to a protein interaction partner that is required for the activation of genes that direct asymmetric morphogenesis along the left-right axis. PMID:19681163

  10. N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function

    PubMed Central

    Inobe, Tomonao; Genmei, Reiko

    2015-01-01

    The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes. PMID:26208326

  11. The role of the N-terminal leucine residue in snake venom cardiotoxin II (Naja naja atra).

    PubMed

    Wu, C Y; Chen, W C; Ho, C L; Chen, S T; Wang, K T

    1997-04-28

    The N-terminal leucine residue of snake venom cardiotoxin II (CTX II) (Naja naja atra) was systematically replaced with D-leucine (CTXII-L1-D-L), glycine (CTXII-L1G) or deleted [CTXII-(2-60)] to study the role of leucine residue in CTX II molecule. CTX II, CTXL1-D-L, CTXL1G and CTX(2-60) were produced by chemical synthesis method and purified by high performance liquid chromatography. Owing to folding problem in CTXII-(2-60), only CTX II, CTXII-L1-D-L and CTXII-L1G were produced in a pure form and characterized by amino acid analysis, mass spectrometry and peptide mapping. In the structural aspect, changing the Leu-1 by D-Leu or Gly causes a drastic alteration in the whole CTX II structure as detected by circular dichroism, 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence assay. In the functional aspect, both CTXII-L1-D-L and CTXII-L1G are still retained substantial biological activity of CTX II. Therefore, the results indicate that both the chirality and the side-chain of the N-terminal leucine residue of CTX II are important elements in maintaining the whole CTX II structure. In addition, this study is the first report in elucidating the reason why the first N-terminal residue of most CTXs (90.3%) is leucine residue. PMID:9168920

  12. The autolysis of human HtrA1 is governed by the redox state of its N-terminal domain.

    PubMed

    Risør, Michael W; Poulsen, Ebbe Toftgaard; Thomsen, Line R; Dyrlund, Thomas F; Nielsen, Tania A; Nielsen, Niels Chr; Sanggaard, Kristian W; Enghild, Jan J

    2014-06-17

    Human HtrA1 (high-temperature requirement protein A1) belongs to a conserved family of serine proteases involved in protein quality control and cell fate. The homotrimeric ubiquitously expressed protease has chymotrypsin-like specificity and primarily targets hydrophobic stretches in selected or misfolded substrate proteins. In addition, the enzyme is capable of exerting autolytic activity by removing the N-terminal insulin-like growth factor binding protein (IGFBP)/Kazal-like tandem motif without affecting the protease activity. In this study, we have addressed the mechanism governing the autolytic activity and find that it depends on the integrity of the disulfide bonds in the N-terminal IGFBP/Kazal-like domain. The specificity of the autolytic cleavage reveals a strong preference for cysteine in the P1 position of HtrA1, explaining the lack of autolysis prior to disulfide reduction. Significantly, the disulfides were reduced by thioredoxin, suggesting that autolysis of HtrA1 in vivo is linked to the endogenous redox balance and that the N-terminal domain acts as a redox-sensing switch. PMID:24846539

  13. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    PubMed

    Dwivedi, Gajendradhar R; Srikanth, Kolluru D; Anand, Praveen; Naikoo, Javed; Srilatha, N S; Rao, Desirazu N

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  14. Site-Specific Labeling of Protein Lysine Residues and N-Terminal Amino Groups with Indoles and Indole-Derivatives.

    PubMed

    Larda, Sacha Thierry; Pichugin, Dmitry; Prosser, Robert Scott

    2015-12-16

    Indoles and indole-derivatives can be used to site-specifically label proteins on lysine and N-terminal amino groups under mild, nondenaturing reaction conditions. Hen egg white lysozyme (HEWL) and α-lactalbumin were labeled with indole, fluoroindole, or fluoroindole-2-carboxylate via electrophilic aromatic substitutions to lysine side chain Nε- and N-terminal amino imines, formed in situ in the presence of formaldehyde. The reaction is highly site-selective, easily controlled by temperature, and does not eliminate the native charge of the protein, unlike many other common lysine-specific labeling strategies. (19)F NMR was used to monitor reaction progression, and in the case of HEWL, unique resonances for each labeled side chain could be resolved. We demonstrate that the indole tags are highly selective for primary amino groups. (19)F NMR demonstrates that each lysine exhibits a different rate of conjugation to indoles making it possible to employ these tags as a means of probing surface topology by NMR or mass spectrometry. Given the site-specificity of this tagging method, the mildness of the reaction conditions (aqueous, buffered, or unbuffered) and the low stoichiometry required for the reaction, indole-derivatives should serve as a valuable addition to the bioconjugation toolkit. We propose that labeling lysine side chains and N-terminal amino groups with indoles is a versatile and general strategy for bioconjugations with substituted indoles having broad implications for protein functionalization. PMID:26587689

  15. Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

    PubMed Central

    Aregger, Michael; Cowling, Victoria H.

    2013-01-01

    Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation. PMID:23863084

  16. Membrane insertion of the N-terminal α-helix of equinatoxin II, a sea anemone cytolytic toxin

    PubMed Central

    2004-01-01

    Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic α-helix. In the present study, we have investigated the transfer of the N-terminal α-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic α-helix (Ile18→Trp, Val22→Trp and Ala25→Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22→Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction. PMID:15317486

  17. The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

    PubMed Central

    Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A; Fromme, J Christopher

    2016-01-01

    The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth. DOI: http://dx.doi.org/10.7554/eLife.12411.001 PMID:26765562

  18. The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

    PubMed

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2015-12-01

    Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues. PMID:26632841

  19. N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells.

    PubMed

    Kisiela, Dagmara I; Aulik, Nicole A; Atapattu, Dhammika N; Czuprynski, Charles J

    2010-07-01

    Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase-9-dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagic Escherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N-terminal region in addressing certain RTX toxins to mitochondria. PMID:20109159

  20. Specific binding sites for proadrenomedullin N-terminal 20 peptide (PAMP) in the rat.

    PubMed

    Iwasaki, H; Hirata, Y; Iwashina, M; Sato, K; Marumo, F

    1996-07-01

    Adrenomedullin (AM), a potent and novel vasodilator 52-residue peptide originally isolated from pheochromocytoma, is processed from a precursor molecule (preproAM) in which another unique 20-residue sequence, termed proadrenomedullin N-terminal 20 peptide (PAMP), exists. Using [125I Tyr0] rat PAMP as a radioligand, we have examined PAMP binding sites in various rat tissues and cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding sites for rat PAMP, although very low, were widely distributed in various rat tissues examined. The relatively more abundant sites were present in aorta and adrenal glands, followed by lung, kidney, brain, spleen, and heart. An equilibrium binding study using cultured rat VSMC revealed the presence of a single class of high-affinity [dissociation constant (Kd): 3.5 x 10(-8) M] binding sites for rat PAMP with a maximal binding capacity of 4.5 x 10(6) sites per cell. Binding studies revealed that synthetic rat PAMP(1-19)-NH2 was about 10-fold less potent, and rat PAMP(1-20)-OH and human PAMP were about 20-fold less potent than rat PAMP(1-20)-NH2. SDS-polyacylamide gel electrophoresis after affinity-labeling of membranes from various rat tissues (aorta, adrenal glands, lung) and VSMC revealed a distinct labeled band with the apparent molecular mass of 90 kDa, which was diminished by excess unlabeled rat PAMP. A nonhydrolyzable GTP analog (GTP-gammaS) dose-dependently reduced binding of [125I] rat PAMP to VSMC membranes, while ATP-gammaS had no effect. Neither cyclic AMP nor inositol-1,4,5-triphosphate formation was affected by rat PAMP in rat VSMC. The present study demonstrates for the first time that PAMP receptors are widely distributed in various rat tissues, among which aorta and adrenal glands have the most abundant sites. Our data suggest that PAMP receptors are functionally coupled to G-proteins, although its signal transduction remains obscure. The present study also shows that amidation of C-terminal residue

  1. Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Hanks, Tracey S.; Kochetkova, Irina; Pascual, David W.; Jutila, Mark A.

    2012-01-01

    In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs. PMID:22434859

  2. N-Terminal deletions modify the Cu2+ binding site in amyloid-beta.

    PubMed

    Karr, Jesse W; Akintoye, Henrietta; Kaupp, Lauren J; Szalai, Veronika A

    2005-04-12

    Copper is implicated in the in vitro formation and toxicity of Alzheimer's disease amyloid plaques containing the beta-amyloid (Abeta) peptide (Bush, A. I., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11934). By low temperature electron paramagnetic resonance (EPR) spectroscopy, the importance of the N-terminus in creating the Cu(2+) binding site in native Abeta has been examined. Peptides that contain the proposed binding site for Cu(2+)-three histidines (H6, H13, and H14) and a tyrosine (Y10)-but lack one to three N-terminal amino acids, do not bind Cu(2+) in the same coordination environment as the native peptide. EPR spectra of soluble Abeta with stoichiometric amounts of Cu(2+) show type 2 Cu(2+) EPR spectra for all peptides. The ligand donor atoms to Cu(2+) are 3N1O when Cu(2+) is bound to any of the Abetapeptides (Abeta16, Abeta28, Abeta40, and Abeta42) that contain the first 16 amino acids of full-length Abeta. When a Y10F mutant of Abeta is used, the coordination environment for Cu(2+) remains 3N1O and Cu(2+) EPR spectra of this mutant are identical to the wild-type spectra. Isotopic labeling experiments show that water is not the O-atom donor to Cu(2+) in Abeta fibrils or in the Y10F mutant. Further, we find that Cu(2+) cannot be removed from Cu(2+)-containing fibrils by washing with buffer, but that Cu(2+) binds to fibrils initially assembled without Cu(2+) in the same coordination environment as in fibrils assembled with Cu(2+). Together, these results indicate (1) that the O-atom donor ligand to Cu(2+) in Abeta is not tyrosine, (2) that the native Cu(2+) binding site in Abeta is sensitive to small changes at the N-terminus, and (3) that Cu(2+) binds to Abetafibrils in a manner that permits exchange of Cu(2+) into and out of the fibrillar architecture. PMID:15807541

  3. Purification and N-terminal analysis of urease from Helicobacter pylori.

    PubMed

    Hu, L T; Mobley, H L

    1990-04-01

    Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureases, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera

  4. Identification and Analysis of the Acetylated Status of Poplar Proteins Reveals Analogous N-Terminal Protein Processing Mechanisms with Other Eukaryotes

    PubMed Central

    Liu, Chang-Cai; Zhu, Hang-Yong; Dong, Xiu-Mei; Ning, De-Li; Wang, Hong-Xia; Li, Wei-Hua; Yang, Chuan-Ping; Wang, Bai-Chen

    2013-01-01

    Background The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (Nα-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. Methodology/Principal Findings To reveal the NPM in poplar, we investigated the Nα-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (Nα-acetylated) proteins. Most proteins (47, >81%) are subjected to Nα-acetylation following the N-terminal removal of Met, indicating that Nα-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and Nα-acetylation (NPM) to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs) and N-terminal acetyltransferase (Nat) enzymes in poplar. The Nα-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. Conclusions/Significance This study represents the first extensive investigation of Nα-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of Nα-acetylation of proteins in poplar. PMID:23536812

  5. N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers

    PubMed Central

    Kim, Chul Woo; Han, Kyoung Sim; Ryu, Ki-Sun; Kim, Byung Hee; Kim, Kyun-Hwan; Choi, Seong Il; Seong, Baik L.

    2007-01-01

    The fusion of soluble partner to the N terminus of aggregation-prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol. The chaperone-like functions of the upstream fusion partner in the artificial multidomain proteins could occur in de novo folding of native multidomain proteins. Here, we show that the N-terminal domains of three E. coli multidomain proteins such as lysyl-tRNA synthetase, threonyl-tRNA synthetase, and aconitase are potent solubility enhancers for various C-terminal heterologous proteins. The results suggest that the N-terminal domains could act as solubility enhancers for the folding of their authentic C-terminal domains in vivo. Tandem repeat of N-terminal domain or insertion of aspartic residues at the C terminus of the N-terminal domain also increased the solubility of fusion proteins, suggesting that the solubilizing ability correlates with the size and charge of N-terminal domains. The solubilizing ability of N-terminal domains would contribute to the autonomous folding of multidomain proteins in vivo, and based on these results, we propose a model of how N-terminal domains solubilize their downstream domains. PMID:17384228

  6. Forest Fragmentation

    EPA Science Inventory

    This indicator describes forest fragmentation in the contiguous United States circa 2001. This information provides a broad, recent picture of the spatial pattern of the nation’s forests and the extent to which they are being broken into smaller patches and pierced or interspe...

  7. Dualising consistent IIA/IIB truncations

    NASA Astrophysics Data System (ADS)

    Malek, Emanuel; Samtleben, Henning

    2015-12-01

    We use exceptional field theory to establish a duality between certain consistent 7-dimensional truncations with maximal SUSY from IIA to IIB. We use this technique to obtain new consistent truncations of IIB on S 3 and H p,q and work out the explicit reduction formulas in the internal sector. We also present uplifts for other gaugings of 7-d maximal SUGRA, including theories with a trombone gauging. Some of the latter can only be obtained by a non-geometric compactification.

  8. A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny.

    PubMed

    Sabino Cunha, Marcela; Lima Sampaio, Thatiane; Peterlin, B Matija; Jesus da Costa, Luciana

    2016-01-01

    Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. PMID:27399760

  9. A Novel Truncated Form of Serum Amyloid A in Kawasaki Disease

    PubMed Central

    Yu, Tom To-Sang; Ling, Xuefeng Bruce; Kanegaye, John T.; Burns, Jane C.; Cohen, Harvey J.

    2016-01-01

    Background Kawasaki disease (KD) is an acute vasculitis in children that can cause coronary artery abnormalities. Its diagnosis is challenging, and many cytokines, chemokines, acute phase reactants, and growth factors have failed evaluation as specific biomarkers to distinguish KD from other febrile illnesses. We performed protein profiling, comparing plasma from children with KD with febrile control (FC) subjects to determine if there were specific proteins or peptides that could distinguish the two clinical states. Materials and Methods Plasma from three independent cohorts from the blood of 68 KD and 61 FC subjects was fractionated by anion exchange chromatography, followed by surface-enhanced laser desorption ionization (SELDI) mass spectrometry of the fractions. The mass spectra of KD and FC plasma samples were analyzed for peaks that were statistically significantly different. Results A mass spectrometry peak with a mass of 7,860 Da had high intensity in acute KD subjects compared to subacute KD (p = 0.0003) and FC (p = 7.9 x 10−10) subjects. We identified this peak as a novel truncated form of serum amyloid A with N-terminal at Lys-34 of the circulating form and validated its identity using a hybrid mass spectrum immunoassay technique. The truncated form of serum amyloid A was present in plasma of KD subjects when blood was collected in tubes containing protease inhibitors. This peak disappeared when the patients were examined after their symptoms resolved. Intensities of this peptide did not correlate with KD-associated laboratory values or with other mass spectrum peaks from the plasma of these KD subjects. Conclusions Using SELDI mass spectrometry, we have discovered a novel truncated form of serum amyloid A that is elevated in the plasma of KD when compared with FC subjects. Future studies will evaluate its relevance as a diagnostic biomarker and its potential role in the pathophysiology of KD. PMID:27271757

  10. A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny

    PubMed Central

    Sabino Cunha, Marcela; Lima Sampaio, Thatiane; Peterlin, B. Matija; Jesus da Costa, Luciana

    2016-01-01

    Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. PMID:27399760

  11. The relationship between truncation and phosphorylation at the C-terminus of tau protein in the paired helical filaments of Alzheimer's disease

    PubMed Central

    Flores-Rodríguez, Paola; Ontiveros-Torres, Miguel A.; Cárdenas-Aguayo, María C.; Luna-Arias, Juan P.; Meraz-Ríos, Marco A.; Viramontes-Pintos, Amparo; Harrington, Charles R.; Wischik, Claude M.; Mena, Raúl; Florán-Garduño, Benjamin; Luna-Muñoz, José

    2015-01-01

    We previously demonstrated that, in the early stages of tau processing in Alzheimer's disease, the N-terminal part of the molecule undergoes a characteristic cascade of phosphorylation and progressive misfolding of the proteins resulting in a structural conformation detected by Alz-50. In this immunohistochemical study of AD brain tissue, we have found that C-terminal truncation of tau at Asp-421 was an early event in tau aggregation and analyzed the relationship between phospho-dependent tau epitopes located at the C-terminus with truncation at Glu-391. The aim of this study was to determine whether C-terminal truncation may trigger events leading to the assembly of insoluble PHFs from soluble tau aggregates present in pre-tangle cells. Our findings suggest that there is a complex interaction between phosphorylated and truncated tau species. A model is presented here in which truncated tau protein represents an early neurotoxic species while phosphorylated tau species may provide a neuroprotective role in Alzheimer's disease. PMID:25717290

  12. Characterization of the native form and the carboxy-terminally truncated halotolerant form of α-amylases from Bacillus subtilis strain FP-133.

    PubMed

    Takenaka, Shinji; Miyatake, Ayaka; Tanaka, Kosei; Kuntiya, Ampin; Techapun, Charin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Watanabe, Masanori; Yoshida, Ken-ichi

    2015-06-01

    Two amylases, amylase I and amylase II from Bacillus subtilis strain FP-133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N-terminal amino acid sequence, genomic southern blot analysis, and MALDI-TOFF-MS analysis indicated that the halotolerant amylase I was produced by limited carboxy-terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis α-amylases, but their carboxy-terminal truncation points differed. Three recombinant amylases--full-length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C-terminal truncation--were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance. PMID:25689045

  13. HIGHLY CONSERVED N-TERMINAL SEQUENCE FOR TELEOST VITELLOGENIN WITH POTENTIAL VALUE TO THE BIOCHEMISTRY, MOLECULAR BIOLOGY AND PATHOLOGY OF VITELLOGENESIS

    EPA Science Inventory

    N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish: striped bass, Morone saxatillus; mummichog, Fundulus heteroclitus; pinfish, Lagodon rhomboides; brown bullhead, Ameiurus nebulosus; medaka, Oryzias latipes; yellow perch, Percaflavescens and ...

  14. Structural characterization of the N-terminal part of the MERS-CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering.

    PubMed

    Papageorgiou, Nicolas; Lichière, Julie; Baklouti, Amal; Ferron, François; Sévajol, Marion; Canard, Bruno; Coutard, Bruno

    2016-02-01

    The N protein of coronaviruses is a multifunctional protein that is organized into several domains. The N-terminal part is composed of an intrinsically disordered region (IDR) followed by a structured domain called the N-terminal domain (NTD). In this study, the structure determination of the N-terminal region of the MERS-CoV N protein via X-ray diffraction measurements is reported at a resolution of 2.4 Å. Since the first 30 amino acids were not resolved by X-ray diffraction, the structural study was completed by a SAXS experiment to propose a structural model including the IDR. This model presents the N-terminal region of the MERS-CoV as a monomer that displays structural features in common with other coronavirus NTDs. PMID:26894667

  15. Canine MPV17 truncation without clinical manifestations

    PubMed Central

    Hänninen, Reetta L.; Ahonen, Saija; Màrquez, Merce; Myöhänen, Maarit J.; Hytönen, Marjo K.; Lohi, Hannes

    2015-01-01

    ABSTRACT Mitochondrial DNA depletion syndromes (MDS) are often serious autosomal recessively inherited disorders characterized by tissue-specific mtDNA copy number reduction. Many genes, including MPV17, are associated with the hepatocerebral form of MDS. MPV17 encodes for a mitochondrial inner membrane protein with a poorly characterized function. Several MPV17 mutations have been reported in association with a heterogeneous group of early-onset manifestations, including liver disease and neurological problems. Mpv17-deficient mice present renal and hearing defects. We describe here a MPV17 truncation mutation in dogs. We found a 1-bp insertion in exon 4 of the MPV17 gene, resulting in a frameshift and early truncation of the encoded protein. The mutation halves MPV17 expression in the lymphocytes of the homozygous dogs and the truncated protein is not translated in transfected cells. The insertion mutation is recurrent and exists in many unrelated breeds, although is highly enriched in the Boxer breed. Unexpectedly, despite the truncation of MPV17, we could not find any common phenotypes in the genetically affected dogs. The lack of observable phenotype could be due to a late onset, mild symptoms or potential tissue-specific compensatory mechanisms. This study suggests species-specific differences in the manifestation of the MPV17 defects and establishes a novel large animal model to further study MPV17 function and role in mitochondrial biology. PMID:26353863

  16. Family Therapy for the "Truncated" Nuclear Family.

    ERIC Educational Resources Information Center

    Zuk, Gerald H.

    1980-01-01

    The truncated nuclear family consists of a two-generation group in which conflict has produced a polarization of values. The single-parent family is at special risk. Go-between process enables the therapist to depolarize sharply conflicted values and reduce pathogenic relating. (Author)

  17. Irregularly Shaped Space-Filling Truncated Octahedra

    ERIC Educational Resources Information Center

    Hanson, John Robert

    2008-01-01

    For any parent tetrahedron ABCD, centroids of selected sub-tetrahedra form the vertices of an irregularly shaped space-filling truncated octahedron. To reflect these properties, such a figure will be called an ISTO. Each edge of the ISTO is parallel to and one-eighth the length of one of the edges of tetrahedron ABCD and the volume of the ISTO is…

  18. The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

    PubMed Central

    Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

    2011-01-01

    Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

  19. Microscopy-based Saccharomyces cerevisiae complementation model reveals functional conservation and redundancy of N-terminal acetyltransferases.

    PubMed

    Osberg, Camilla; Aksnes, Henriette; Ninzima, Sandra; Marie, Michaël; Arnesen, Thomas

    2016-01-01

    N-terminal acetylation is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) NatA-NatG. The Saccharomyces cerevisiae protein Arl3 depends on interaction with Sys1 for its localization to the Golgi and this targeting strictly requires NatC-mediated N-terminal acetylation of Arl3. We utilized the Arl3 acetylation-dependent localization phenotype as a model system for assessing the functional conservation and in vivo redundancy of several human NATs. The catalytic subunit of human NatC, hNaa30 (Mak3), restored Arl3 localization in the absence of yNaa30, but only in the presence of either yeast or human Naa35 subunit (Mak10). In contrast, hNaa35 was not able to replace its yeast orthologue without the co-expression of hNaa30, suggesting co-evolution of the two NatC subunits. The most recently discovered and organellar human NAT, NatF/Naa60, restored the Golgi localization of Arl3 in the absence of yNaa30. Interestingly, this was also true for hNaa60 lacking its membrane-binding domain whereas hNaa50 did not complement NatC function. This in vivo redundancy reflects NatC and NatF´s overlapping in vitro substrate specificities. The yeast model presented here provides a robust and rapid readout of NatC and NatF activity in vivo, and revealed evolutionary conservation of the NatC complex and redundancy between NatC and NatF. PMID:27555049

  20. N-Terminal Modification with Pseudo-Bifunctional PEG-Hexadecane Markedly Improves the Pharmacological Profile of Human Growth Hormone.

    PubMed

    Wu, Ling; Ji, Shaoyang; Hu, Tao

    2015-05-01

    Human growth hormone (hGH) has been used to treat children with short stature, renal failure, and Turner's syndrome. However, clinical application of hGH suffers from its short plasma half-life and low bioavailability. PEGylation and albumin binding are two of the most effective approaches to prolong the plasma half-life of hGH. However, the steric shielding effects of polyethylene glycol (PEG) and albumin can drastically decrease the bioactivity of hGH, which is opposite to the increased pharmacokinetics (PK). In the present study, a long-acting hGH with markedly improved pharmacological profile was rationally designed and prepared by N-terminal modification of hGH with pseudo-bifunctional PEG-hexadecane by using PEG (3.5 kDa or 10 kDa) as the linker. PEGylation and albumin binding with hexadecane can increase the hydrodynamic volume and decrease the immunogenicity of hGH, which thereby markedly increases the PK of hGH. Since N-terminus is far from the bioactive domain of hGH, N-terminal modification of hGH can minimize the steric shielding effects on the bioactive domain of hGH. Hexadecane-bound albumin can be slowly released from hGH during the in vivo circulation, which can slowly restore the bioactivity of hGH. Thus, the high bioactivity of PEG-hexadecane modified hGH (hGH-PEG-HD) was synergistically achieved by N-terminal modification with pseudo-bifunctional PEG-hexadecane and slow-release of albumin. The high pharmacodynamics (PD) of hGH-PEG-HD was due to the synergistic effect of the high bioactivity and the overall increased PK. PMID:25849255

  1. N-Terminal Extensions Retard Aβ42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aβ42.

    PubMed

    Szczepankiewicz, Olga; Linse, Björn; Meisl, Georg; Thulin, Eva; Frohm, Birgitta; Sala Frigerio, Carlo; Colvin, Michael T; Jacavone, Angela C; Griffin, Robert G; Knowles, Tuomas; Walsh, Dominic M; Linse, Sara

    2015-11-25

    Amyloid β-protein (Aβ) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid β-protein (Aβ) variants were believed to begin at or after the canonical β-secretase cleavage site within the amyloid β-protein precursor. However, N-terminally extended forms of Aβ (NTE-Aβ) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aβ42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aβ42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aβ42s form fibrils via the same mechanism as Aβ42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aβ42 and NTE-Aβ42 coaggregate to form mixed fibrils and fibrils of either Aβ42 or NTE-Aβ42 catalyze aggregation of all monomers. NTE-Aβ42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease. PMID:26535489

  2. Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A.

    PubMed

    Seo, Min-Duk; Park, Sung Jean; Kim, Hyun-Jung; Lee, Bong Jin

    2007-01-01

    Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase. PMID:17174309

  3. The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation

    PubMed Central

    Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D.; Sattler, Michael; Kempkes, Bettina

    2015-01-01

    Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics. PMID:26024477

  4. Microscopy-based Saccharomyces cerevisiae complementation model reveals functional conservation and redundancy of N-terminal acetyltransferases

    PubMed Central

    Osberg, Camilla; Aksnes, Henriette; Ninzima, Sandra; Marie, Michaël; Arnesen, Thomas

    2016-01-01

    N-terminal acetylation is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) NatA-NatG. The Saccharomyces cerevisiae protein Arl3 depends on interaction with Sys1 for its localization to the Golgi and this targeting strictly requires NatC-mediated N-terminal acetylation of Arl3. We utilized the Arl3 acetylation-dependent localization phenotype as a model system for assessing the functional conservation and in vivo redundancy of several human NATs. The catalytic subunit of human NatC, hNaa30 (Mak3), restored Arl3 localization in the absence of yNaa30, but only in the presence of either yeast or human Naa35 subunit (Mak10). In contrast, hNaa35 was not able to replace its yeast orthologue without the co-expression of hNaa30, suggesting co-evolution of the two NatC subunits. The most recently discovered and organellar human NAT, NatF/Naa60, restored the Golgi localization of Arl3 in the absence of yNaa30. Interestingly, this was also true for hNaa60 lacking its membrane-binding domain whereas hNaa50 did not complement NatC function. This in vivo redundancy reflects NatC and NatF´s overlapping in vitro substrate specificities. The yeast model presented here provides a robust and rapid readout of NatC and NatF activity in vivo, and revealed evolutionary conservation of the NatC complex and redundancy between NatC and NatF. PMID:27555049

  5. c-Jun N-terminal kinase modulates oxidant stress and peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen hepatotoxicity

    SciTech Connect

    Saito, Chieko; Lemasters, John J.; Jaeschke, Hartmut

    2010-07-15

    Acetaminophen (APAP) overdose, which causes liver injury in animals and humans, activates c-jun N-terminal kinase (JNK). Although it was shown that the JNK inhibitor SP600125 effectively reduced APAP hepatotoxicity, the mechanisms of protection remain unclear. C57Bl/6 mice were treated with 10 mg/kg SP600125 or vehicle (8% dimethylsulfoxide) 1 h before 600 mg/kg APAP administration. APAP time-dependently induced JNK activation (detected by JNK phosphorylation). SP600125, but not the vehicle, reduced JNK activation, attenuated mitochondrial Bax translocation and prevented the mitochondrial release of apoptosis-inducing factor at 4-12 h. Nuclear DNA fragmentation, nitrotyrosine staining, tissue GSSG levels and liver injury (plasma ALT release and necrosis) were partially attenuated by the vehicle (- 65%) and completely eliminated by SP600125 (- 98%) at 6 and 12 h. Furthermore, SP600125 attenuated the increase of inducible nitric oxide synthase (iNOS) mRNA and protein. However, APAP did not enhance plasma nitrite + nitrate levels (NO formation); SP600125 had no effect on this parameter. The iNOS inhibitor L-NIL did not reduce NO formation or injury after APAP but prevented NO formation caused by endotoxin. Since SP600125 completely eliminated the increase in hepatic GSSG levels, an indicator of mitochondrial oxidant stress, it is concluded that the inhibition of peroxynitrite was mainly caused by reduced superoxide formation. Our data suggest that the JNK inhibitor SP600125 protects against APAP-induced liver injury in part by attenuation of mitochondrial Bax translocation but mainly by preventing mitochondrial oxidant stress and peroxynitrite formation and thereby preventing the mitochondrial permeability transition pore opening, a key event in APAP-induced cell necrosis.

  6. Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

    PubMed

    Ries, W; Hotzy, C; Schocher, I; Sleytr, U B; Sára, M

    1997-06-01

    The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi. PMID:9190804

  7. Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells*

    PubMed Central

    Wang, Zili; Weitzmann, M. Neale; Sangadala, Sreedhara; Hutton, William C.; Yoon, S. Tim

    2013-01-01

    Intervertebral disc (IVD) degeneration and associated spinal disorders are leading sources of morbidity, and they can be responsible for chronic low back pain. Treatments for degenerative disc diseases continue to be a challenge. Intensive research is now focusing on promoting regeneration of degenerated discs by stimulating production of the disc matrix. Link protein N-terminal peptide (LPP) is a proteolytic fragment of link protein, an important cross-linker and stabilizer of the major structural components of cartilage, aggrecan and hyaluronan. In this study we investigated LPP action in rabbit primary intervertebral disc cells cultured ex vivo in a three-dimensional alginate matrix. Our data reveal that LPP promotes disc matrix production, which was evidenced by increased expression of the chondrocyte-specific transcription factor SOX9 and the extracellular matrix macromolecules aggrecan and collagen II. Using colocalization and pulldown studies we further document a noggin-insensitive direct peptide-protein association between LPP and BMP-RII. This association mediated Smad signaling that converges on BMP genes leading to expression of BMP-4 and BMP-7. Furthermore, through a cell-autonomous loop BMP-4 and BMP-7 intensified Smad1/5 signaling though a feedforward circuit involving BMP-RI, ultimately promoting expression of SOX9 and downstream aggrecan and collagen II genes. Our data define a complex regulatory signaling cascade initiated by LPP and suggest that LPP may be a useful therapeutic substitute for direct BMP administration to treat IVD degeneration and to ameliorate IVD-associated chronic low back pain. PMID:23940040

  8. Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

    PubMed Central

    D'Antuono, Alejandra; Loureiro, Maria Eugenia; Foscaldi, Sabrina; Marino-Buslje, Cristina

    2014-01-01

    ABSTRACT The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be

  9. Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase.

    PubMed

    Wiegand, Thomas; Gardiennet, Carole; Ravotti, Francesco; Bazin, Alexandre; Kunert, Britta; Lacabanne, Denis; Cadalbert, Riccardo; Güntert, Peter; Terradot, Laurent; Böckmann, Anja; Meier, Beat H

    2016-04-01

    We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein. PMID:26280528

  10. Structure and Dynamics of the N-terminal Domain of the Cu(I) Binding Protein CusB

    PubMed Central

    Ucisik, Melek N.; Chakravorty, Dhruva K.; Merz, Kenneth M.

    2013-01-01

    CusCFBA is one of the metal efflux systems in Escherichia coli, which is highly specific for its substrates Cu(I) and Ag(I). It serves to protect the bacteria in environments that have lethal concentrations of these metals. The membrane fusion protein CusB is the periplasmic piece of CusCFBA, which has not been fully characterized by crystallography due to its extremely disordered N-terminal region. This region has both structural and functional importance as it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. Understanding metal uptake from the periplasm is critical to gain an insight to the mechanism of the whole CusCFBA pump, which makes resolving a structure for the N-terminal region necessary as it contains the metal binding site. We ran extensive molecular dynamics simulations to reveal the structural and dynamic properties of both apo and Cu(I)-bound versions of the CusB N-terminal region. In contrast to its functional companion CusF, Cu(I)-binding to the N-terminal of CusB causes only a slight, local stabilization around the metal site. The trajectories were analyzed in detail revealing extensive structural disorder in both the apo and holo forms of the protein. CusB was further analyzed by breaking the protein up into three subdomains according to the extent of the observed disorder: the N- and C-terminal tails, the central beta strand motif, and the M21–M36 loop connecting the two metal-coordinating methionine residues. Most of the observed disorder was traced back to the tail regions leading us to hypothesize that the latter two subdomains (residues 13–45) may form a functionally competent metal binding domain as the tail regions appear to play no role in metal binding. PMID:23988152

  11. Removal of an N-terminal peptide from mitochondrial aspartate aminotransferase abolishes its interactions with mitochondria in vitro.

    PubMed Central

    O'Donovan, K M; Doonan, S; Marra, E; Passarella, S; Quagliariello, E

    1985-01-01

    Treatment of mitochondrial aspartate aminotransferase from rat liver with trypsin leads to specific cleavage of the bonds between residues 26 and 27, and residues 31 and 32. The proteolysed enzyme has only a small residual catalytic activity, but retains a conformation similar to that of the native form as judged by accessibility and reactivity of cysteine residues. Proteolysis abolishes the ability of the enzyme either to bind to mitochondria or to be imported into the organelles. This suggests that the N-terminal segment of the native enzyme is essential for both of these functions, at least in the model system used to study the import process. PMID:4026799

  12. Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

    PubMed

    Shih, Yan-Ping; Chou, Chi-Chi; Chen, Yi-Ling; Huang, Kai-Fa; Wang, Andrew H-J

    2014-01-01

    Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities. PMID:24733552

  13. Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide.

    PubMed

    Fang, Nan; Zhong, Chuan-Qi; Liang, Xiaoliang; Tang, Xiao-Feng; Tang, Bing

    2010-02-01

    Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBM(MT) with improved capacity for extracellular production when expressed in E. coli. Two mutations, L(-57)Q and E(-10)D, were identified within the N-terminal propeptide. The amount of WBM(MT) in the culture medium was found to be about three times higher than that of wild type. Besides, the introduction of mutations L(-57)Q/E(-10)D into the N-terminal propeptide also accelerated the maturation of the enzyme. Biochemical analysis indicated that the thermostability and the catalytic activity of mature WBM(MT) were similar to those of wild type. Far-UV CD spectra analysis and limited proteolysis experiments suggested that the mutations L(-57)Q/E(-10)D resulted in a structural change in the N-terminal propeptide of the proform, and the N-terminal propeptide became more flexible, which might be beneficial for the proform to keep in a translocation-competent state. Our result indicates that N-terminal propeptide engineering may be a valuable approach for improving extracellular production of recombinant subtilases expressed in E. coli. PMID:19697018

  14. Extended string-like binding of the phosphorylated HP1α N-terminal tail to the lysine 9-methylated histone H3 tail

    PubMed Central

    Shimojo, Hideaki; Kawaguchi, Ayumi; Oda, Takashi; Hashiguchi, Nobuto; Omori, Satoshi; Moritsugu, Kei; Kidera, Akinori; Hiragami-Hamada, Kyoko; Nakayama, Jun-ichi; Sato, Mamoru; Nishimura, Yoshifumi

    2016-01-01

    The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α’s N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α’s chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α’s N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α’s N-terminal tail underlies the enhancement in H3 binding due to phosphorylation. PMID:26934956

  15. Cellular HIV-1 Inhibition by Truncated Old World Primate APOBEC3A Proteins Lacking a Complete Deaminase Domain

    PubMed Central

    Katuwal, Miki; Wang, Yaqiong; Schmitt, Kimberly; Guo, Kejun; Halemano, Kalani; Santiago, Mario L.; Stephens, Edward B.

    2014-01-01

    The APOBEC3 (A3) deaminases are retrovirus restriction factors that were proposed as inhibitory components of HIV-1 gene therapy vectors. However, A3 mutational activity may induce undesired genomic damage and enable HIV-1 to evade drugs and immune responses. Here, we show that A3A protein from Colobus guereza (colA3A) can restrict HIV-1 replication in producer cells in a deaminase-independent manner without inducing DNA damage. Neither HIV-1 reverse transcription nor integration were significantly affected by colA3A, but capsid protein synthesis was inhibited. The determinants for colA3A restriction mapped to the N-terminal region. These properties extend to A3A from mandrills and De Brazza’s monkeys. Surprisingly, truncated colA3A proteins expressing only the N-terminal 100 amino acids effectively exclude critical catalytic regions but retained potent cellular restriction activity. These highlight a unique mechanism of cellular HIV-1 restriction by several Old World monkey A3A proteins that may be exploited for functional HIV-1 cure strategies. PMID:25262471

  16. Perturbative fragmentation

    SciTech Connect

    Kopeliovich, B. Z.; Pirner, H.-J.; Potashnikova, I. K.; Schmidt, Ivan; Tarasov, A. V.

    2008-03-01

    The Berger model of perturbative fragmentation of quarks to pions is improved by providing an absolute normalization and keeping all terms in a (1-z) expansion, which makes the calculation valid at all values of fractional pion momentum z. We also replace the nonrelativistic wave function of a loosely bound pion by the more realistic procedure of projecting to the light-cone pion wave function, which in turn is taken from well known models. The full calculation does not confirm the (1-z){sup 2} behavior of the fragmentation function (FF) predicted in [E. L. Berger, Z. Phys. C 4, 289 (1980); Phys. Lett. 89B, 241 (1980] for z>0.5, and only works at very large z>0.95, where it is in reasonable agreement with phenomenological FFs. Otherwise, we observe quite a different z-dependence which grossly underestimates data at smaller z. The disagreement is reduced after the addition of pions from decays of light vector mesons, but still remains considerable. The process dependent higher twist terms are also calculated exactly and found to be important at large z and/or p{sub T}.

  17. N-terminally myristoylated feline foamy virus Gag allows Env-independent budding of sub-viral particles.

    PubMed

    Liu, Yang; Kim, Yong-Boum; Löchelt, Martin

    2011-11-01

    Foamy viruses (FVs) are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP) release. In primate and feline FVs (PFV and FFV), particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal) overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N-terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation. PMID:22163342

  18. Improvement of the catalytic performance of a Bispora antennata cellulase by replacing the N-terminal semi-barrel structure.

    PubMed

    Zheng, Fei; Huang, Huoqing; Wang, Xiaoyu; Tu, Tao; Liu, Qiong; Meng, Kun; Wang, Yuan; Su, Xiaoyun; Xie, Xiangming; Luo, Huiying

    2016-10-01

    The aim of this work was to study the contribution of the N-terminal structure to cellulase catalytic performance. A wild-type cellulase (BaCel5) of glycosyl hydrolase (GH) family 5 from Bispora antennata and two hybrid enzymes (BaCel5(127) and BaCel5(167)) with replacement of the N-terminal (βα)3 (127 residues) or (βα)4 (167 residues)-barrel with the corresponding sequences of TeEgl5A from Talaromyces emersonii were produced in Pichia pastoris and biochemically characterized. BaCel5 exhibited optimal activity at pH 5.0 and 50°C but had low catalytic efficiency (25.4±0.8mLs(-1)mg(-1)). In contrast, BaCel5(127) and BaCel5(167) showed similar enzymatic properties but improved catalytic performance. When using CMC-Na, barley β-glucan, lichenan, and cellooligosaccharides as substrates, BaCel5(127) and BaCel5(167) had increased specific activities and catalytic efficiencies by ∼1.8-6.7-fold and ∼1.0-4.7-fold, respectively. The catalytic efficiency of BaCel5(167) was even higher than that of parental proteins. The underlying mechanism was analyzed by molecular docking and molecular dynamic simulation. PMID:27372007

  19. Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain.

    PubMed

    Raman, Swetha; Suguna, Kaza

    2015-06-01

    Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source. PMID:26057797

  20. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    PubMed Central

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  1. The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen*

    PubMed Central

    Kobayashi, Yuki; Shiga, Takafumi; Shibata, Toshio; Sako, Miyuki; Maenaka, Katsumi; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2014-01-01

    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. PMID:25077965

  2. Structural and Functional Analysis of the N-terminal Domain of the Streptococcus gordonii Adhesin Sgo0707

    PubMed Central

    Nylander, Åsa; Svensäter, Gunnel; Senadheera, Dilani B.; Cvitkovitch, Dennis G.; Davies, Julia R.; Persson, Karina

    2013-01-01

    The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes. PMID:23691093

  3. The N-Terminal Intrinsically Disordered Domain of Mgm101p Is Localized to the Mitochondrial Nucleoid

    PubMed Central

    Hayward, David C.; Dosztányi, Zsuzsanna; Clark-Walker, George Desmond

    2013-01-01

    The mitochondrial genome maintenance gene, MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted. PMID:23418572

  4. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.

    PubMed

    Kobayashi, Yuki; Shiga, Takafumi; Shibata, Toshio; Sako, Miyuki; Maenaka, Katsumi; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2014-09-12

    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. PMID:25077965

  5. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    SciTech Connect

    Shiheido, Hirokazu Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND3{sub 56–58}, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs.

  6. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    PubMed

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. PMID:27307584

  7. c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea.

    PubMed

    Anttonen, Tommi; Herranen, Anni; Virkkala, Jussi; Kirjavainen, Anna; Elomaa, Pinja; Laos, Maarja; Liang, Xingqun; Ylikoski, Jukka; Behrens, Axel; Pirvola, Ulla

    2016-01-01

    Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death. PMID:27257624

  8. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA.

    PubMed

    Doritchamou, Justin; Sabbagh, Audrey; Jespersen, Jakob S; Renard, Emmanuelle; Salanti, Ali; Nielsen, Morten A; Deloron, Philippe; Tuikue Ndam, Nicaise

    2015-01-01

    The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development. PMID:26393516

  9. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA

    PubMed Central

    Doritchamou, Justin; Sabbagh, Audrey; Jespersen, Jakob S.; Renard, Emmanuelle; Salanti, Ali; Nielsen, Morten A.; Deloron, Philippe; Tuikue Ndam, Nicaise

    2015-01-01

    The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development. PMID:26393516

  10. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    PubMed

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. PMID:26969163

  11. N-Terminally Myristoylated Feline Foamy Virus Gag Allows Env-Independent Budding of Sub-Viral Particles

    PubMed Central

    Liu, Yang; Kim, Yong-Boum; Löchelt, Martin

    2011-01-01

    Foamy viruses (FVs) are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP) release. In primate and feline FVs (PFV and FFV), particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal) overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N-terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation. PMID:22163342

  12. c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea123

    PubMed Central

    Anttonen, Tommi; Herranen, Anni; Virkkala, Jussi; Kirjavainen, Anna; Elomaa, Pinja; Laos, Maarja; Liang, Xingqun; Ylikoski, Jukka; Behrens, Axel

    2016-01-01

    Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death. PMID:27257624

  13. Impact of the N-terminal amino acid on the formation of pyrazines from peptides in Maillard model systems.

    PubMed

    Van Lancker, Fien; Adams, An; De Kimpe, Norbert

    2012-05-01

    Only a minor part of Maillard reaction studies in the literature focused on the reaction between carbohydrates and peptides. Therefore, in continuation of a previous study in which the influence of the peptide C-terminal amino acid was investigated, this study focused on the influence of the peptide N-terminal amino acid on the production of pyrazines in model reactions of glucose, methylglyoxal, or glyoxal. Nine different dipeptides and three tripeptides were selected. It was shown that the structure of the N-terminal amino acid is determinative for the overall pyrazine production. Especially, the production of 2,5(6)-dimethylpyrazine and trimethylpyrazine was low in the case of proline, valine, or leucine at the N-terminus, whereas it was very high for glycine, alanine, or serine. In contrast to the alkyl-substituted pyrazines, unsubstituted pyrazine was always produced more in the case of experiments with free amino acids. It is clear that different mechanisms must be responsible for this observation. This study clearly illustrates the capability of peptides to produce flavor compounds such as pyrazines. PMID:22463717

  14. Multiple organelle-targeting signals in the N-terminal portion of peroxisomal membrane protein PMP70.

    PubMed

    Iwashita, Shohei; Tsuchida, Masashi; Tsukuda, Miwa; Yamashita, Yukari; Emi, Yoshikazu; Kida, Yuichiro; Komori, Masayuki; Kashiwayama, Yoshinori; Imanaka, Tsuneo; Sakaguchi, Masao

    2010-04-01

    Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes. PMID:20007743

  15. The similarity between N-terminal targeting signals for protein import into different organelles and its evolutionary relevance

    PubMed Central

    Kunze, Markus; Berger, Johannes

    2015-01-01

    The proper distribution of proteins between the cytosol and various membrane-bound compartments is crucial for the functionality of eukaryotic cells. This requires the cooperation between protein transport machineries that translocate diverse proteins from the cytosol into these compartments and targeting signal(s) encoded within the primary sequence of these proteins that define their cellular destination. The mechanisms exerting protein translocation differ remarkably between the compartments, but the predominant targeting signals for mitochondria, chloroplasts and the ER share the N-terminal position, an α-helical structural element and the removal from the core protein by intraorganellar cleavage. Interestingly, similar properties have been described for the peroxisomal targeting signal type 2 mediating the import of a fraction of soluble peroxisomal proteins, whereas other peroxisomal matrix proteins encode the type 1 targeting signal residing at the extreme C-terminus. The structural similarity of N-terminal targeting signals poses a challenge to the specificity of protein transport, but allows the generation of ambiguous targeting signals that mediate dual targeting of proteins into different compartments. Dual targeting might represent an advantage for adaptation processes that involve a redistribution of proteins, because it circumvents the hierarchy of targeting signals. Thus, the co-existence of two equally functional import pathways into peroxisomes might reflect a balance between evolutionary constant and flexible transport routes. PMID:26441678

  16. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    NASA Astrophysics Data System (ADS)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  17. Structural and functional insight into the N-terminal domain of the clathrin adaptor Ent5 from Saccharomyces cerevi