Science.gov

Sample records for high circulating n-terminal

  1. 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.

  2. Nested N-terminal megalin fragments induce high-titer autoantibody and attenuated Heymann nephritis.

    PubMed

    Tramontano, Alfonso; Knight, Thomas; Vizzuso, Domenica; Makker, Sudesh P

    2006-07-01

    It was shown previously that an N-terminal fragment (nM60) that encompasses amino acid residues 1 to 563 of megalin could induce active Heymann nephritis (AHN) as efficiently as the native protein. For delineation of a minimal structure within this fragment that is sufficient to induce AHN, smaller protein fragments that encompass residues 1 to 236 (L6), 1 to 195 (L5), 1 to 156 (L4), and 1 to 120 (L3), representing successive C-terminal truncations within ligand-binding repeats of nM60, were cloned and produced in a baculovirus insect cell expression system. Protein fragments L4, L5, and L6 clearly were glycosylated. All four fragments stimulated proliferation of megalin-sensitized lymph node cells and induced high-titer anti-megalin autoantibodies in Lewis rats. A full-blown disease, as assessed by severity of proteinuria, was observed in rats that were immunized with L6 and L5, whereas animals that were immunized with L4 and L3 developed only mild disease. The proteinuria levels correlated with staining for complement (C3, C5b-9) and IgG1 isotype in glomerular immune deposits. The results suggest that one or more molecular determinants on the region that comprises amino acid residues 157 to 236 contribute to the induction of a full-blown form of AHN. Study of the structure, conformation, and posttranslational modifications of these determinants could provide greater insight into the molecular correlates of immunopathogenesis in this disease model.

  3. Circulating N-terminal brain natriuretic peptide and cardiac function in response to acute systemic hypoxia in healthy humans

    PubMed Central

    2014-01-01

    Background As it remains unclear whether hypoxia of cardiomyocytes could trigger the release of brain natriuretic peptide (BNP) in humans, we investigated whether breathing normobaric hypoxic gas mixture increases the circulating NT-proBNP in healthy male subjects. Methods Ten healthy young men (age 29 ± 5 yrs, BMI 24.7 ± 2.8 kg/m2) breathed normobaric hypoxic gas mixture (11% O2/89% N2) for one hour. Venous blood samples were obtained immediately before, during, and 2 and 24 hours after hypoxic exposure. Cardiac function and flow velocity profile in the middle left anterior descending coronary artery (LAD) were measured by Doppler echocardiography. Results Arterial oxygen saturation decreased steadily from baseline value of 99 ± 1% after the initiation hypoxia challenge and reached steady-state level of 73 ± 6% within 20–30 minutes. Cardiac output increased from 6.0 ± 1.2 to 8.1 ± 1.6 L/min and ejection fraction from 67 ± 4% to 75 ± 6% (both p < 0.001). Peak diastolic flow velocity in the LAD increased from 0.16 ± 0.04 to 0.28 ± 0.07 m/s, while its diameter remained unchanged. In the whole study group, NT-proBNP was similar to baseline (60 ± 32 pmol/ml) at all time points. However, at 24 h, concentration of NT-proBNP was higher (34 ± 18%) in five subjects and lower (17 ± 17%), p = 0.002 between the groups) in five subjects than at baseline. Conclusion In conclusion, there is no consistent increase in circulating NT-proBNP in response to breathing severely hypoxic normobaric gas mixture in healthy humans, a possible reason being that the oxygen flux to cardiac myocytes does not decrease because of increased coronary blood flow. However, the divergent individual responses as well as responses in different cardiac diseases warrant further investigations. PMID:24989366

  4. The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism

    PubMed Central

    Abeyama, Kazuhiro; Stern, David M.; Ito, Yuji; Kawahara, Ko-ichi; Yoshimoto, Yasushi; Tanaka, Motoyuki; Uchimura, Tomonori; Ida, Nobuo; Yamazaki, Yoshiaki; Yamada, Shingo; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Iino, Satoshi; Taniguchi, Noboru; Maruyama, Ikuro

    2005-01-01

    Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation–induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature. PMID:15841214

  5. Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

    PubMed

    Wang, Jiafu; Meng, Jianghui; Nugent, Marc; Tang, Minhong; Dolly, J Oliver

    2017-03-15

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) and showed that the lethality of this mutant is reduced 3.3 × 10(4)-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔHCN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with (125)I-BoNT/A (Kd = 0.46 nM). At a higher concentration, rAΔHCN did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within HCN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCN makes additional contributions to productive internalization/translocation steps beyond binding to neurons.

  6. Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain

    PubMed Central

    Wang, Jiafu; Meng, Jianghui; Nugent, Marc; Tang, Minhong; Dolly, J. Oliver

    2017-01-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) and showed that the lethality of this mutant is reduced 3.3 × 104-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔHCN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with 125I-BoNT/A (Kd = 0.46 nM). At a higher concentration, rAΔHCN did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within HCN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCN makes additional contributions to productive internalization/translocation steps beyond binding to neurons. PMID:28295026

  7. N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation.

    PubMed

    Kitanaka, Atsushi; Miyashita, Masahiro; Kubo, Ayumi; Satoh, Takaya; Toyoda, Michisato; Miyagawa, Hisashi

    2016-01-01

    De novo sequencing is still essential in the identification of peptides and proteins from unexplored organisms whose sequence information is not available. One of the remaining problems in de novo sequencing is discrimination between Leu and Ile residues. The discrimination is possible based on differences in side chain fragmentation between Leu and Ile under high-energy collision-induced dissociation (HE-CID) conditions. However, this is observed only when basic residues, such as Arg and Lys, are present near the N- or C-terminal end. It has been shown that the charge derivatization at the N-terminal end by a quarternary ammonium or phosphonium moiety facilitates the side chain fragmentation by HE-CID. However, the effective backbone fragmentation by low-energy CID (LE-CID) is often hampered in those derivatives with a fixed charge. Previously, we demonstrated that the N-terminal charge derivatization with the structures having high proton affinity induced the preferential formation of b-ions under LE-CID conditions, allowing straightforward interpretation of product ion spectra. In the present study, we further investigated whether the same derivatization approach is also effective for discrimination between Leu and Ile under HE-CID conditions. Consequently, the side chain fragmentation of Leu and Ile residues was most effectively enhanced by the N-terminal derivatization with 4-(guanidinomethyl)benzoic acid among the tested structures. This derivatization approach, which is compatible with both HE- and LE-CID analysis, offers a straightforward and unambiguous de novo peptide sequencing method.

  8. N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

    PubMed Central

    Kitanaka, Atsushi; Miyashita, Masahiro; Kubo, Ayumi; Satoh, Takaya; Toyoda, Michisato; Miyagawa, Hisashi

    2016-01-01

    De novo sequencing is still essential in the identification of peptides and proteins from unexplored organisms whose sequence information is not available. One of the remaining problems in de novo sequencing is discrimination between Leu and Ile residues. The discrimination is possible based on differences in side chain fragmentation between Leu and Ile under high-energy collision-induced dissociation (HE-CID) conditions. However, this is observed only when basic residues, such as Arg and Lys, are present near the N- or C-terminal end. It has been shown that the charge derivatization at the N-terminal end by a quarternary ammonium or phosphonium moiety facilitates the side chain fragmentation by HE-CID. However, the effective backbone fragmentation by low-energy CID (LE-CID) is often hampered in those derivatives with a fixed charge. Previously, we demonstrated that the N-terminal charge derivatization with the structures having high proton affinity induced the preferential formation of b-ions under LE-CID conditions, allowing straightforward interpretation of product ion spectra. In the present study, we further investigated whether the same derivatization approach is also effective for discrimination between Leu and Ile under HE-CID conditions. Consequently, the side chain fragmentation of Leu and Ile residues was most effectively enhanced by the N-terminal derivatization with 4-(guanidinomethyl)benzoic acid among the tested structures. This derivatization approach, which is compatible with both HE- and LE-CID analysis, offers a straightforward and unambiguous de novo peptide sequencing method. PMID:27900234

  9. Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine

    PubMed Central

    Maruyama, Nobuhiro; Asai, Tsuyoshi; Abe, Chiaki; Inada, Akari; Kawauchi, Takeshi; Miyashita, Kazuya; Maeda, Masahiro; Matsuo, Masafumi; Nabeshima, Yo-ichi

    2016-01-01

    Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes. PMID:27991570

  10. Crystal structures reveal N-terminal Domain of Arabidopsis thaliana ClpD to be highly divergent from that of ClpC1

    PubMed Central

    Mohapatra, Chinmayee; Kumar Jagdev, Manas; Vasudevan, Dileep

    2017-01-01

    The caseinolytic protease machinery associated chaperone protein ClpC is known to be present in bacteria, plants and other eukaryotes, whereas ClpD is unique to plants. Plant ClpC and ClpD proteins get localized into chloroplast stroma. Herein, we report high resolution crystal structures of the N-terminal domain of Arabidopsis thaliana ClpC1 and ClpD. Surprisingly, AtClpD, but not AtClpC1, deviates from the typical N-terminal repeat domain organization of known Clp chaperones and have only seven α-helices, instead of eight. In addition, the loop connecting the two halves of AtClpD NTD is longer and covers the region which in case of AtClpC1 is thought to contribute to adaptor protein interaction. Taken together, the N-terminal domain of AtClpD has a divergent structural organization compared to any known Clp chaperones which hints towards its specific role during plant stress conditions, as opposed to that in the maintenance of chloroplastic homeostasis by AtClpC1. Conservation of residues in the NTD that are responsible for the binding of the cyclic peptide activator - Cyclomarin A, as reported for mycobacterial ClpC1 suggests that the peptide could be used as an activator to both AtClpC1 and AtClpD, which could be useful in their detailed in vitro functional characterization. PMID:28287170

  11. Crystal structures reveal N-terminal Domain of Arabidopsis thaliana ClpD to be highly divergent from that of ClpC1.

    PubMed

    Mohapatra, Chinmayee; Kumar Jagdev, Manas; Vasudevan, Dileep

    2017-03-13

    The caseinolytic protease machinery associated chaperone protein ClpC is known to be present in bacteria, plants and other eukaryotes, whereas ClpD is unique to plants. Plant ClpC and ClpD proteins get localized into chloroplast stroma. Herein, we report high resolution crystal structures of the N-terminal domain of Arabidopsis thaliana ClpC1 and ClpD. Surprisingly, AtClpD, but not AtClpC1, deviates from the typical N-terminal repeat domain organization of known Clp chaperones and have only seven α-helices, instead of eight. In addition, the loop connecting the two halves of AtClpD NTD is longer and covers the region which in case of AtClpC1 is thought to contribute to adaptor protein interaction. Taken together, the N-terminal domain of AtClpD has a divergent structural organization compared to any known Clp chaperones which hints towards its specific role during plant stress conditions, as opposed to that in the maintenance of chloroplastic homeostasis by AtClpC1. Conservation of residues in the NTD that are responsible for the binding of the cyclic peptide activator - Cyclomarin A, as reported for mycobacterial ClpC1 suggests that the peptide could be used as an activator to both AtClpC1 and AtClpD, which could be useful in their detailed in vitro functional characterization.

  12. Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB

    PubMed Central

    Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

    2017-01-01

    Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing. PMID:28230084

  13. Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

    PubMed

    Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

    2017-02-23

    Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

  14. "Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry.

    PubMed

    Simeonova, Diliana Dancheva; Susnea, Iuliana; Moise, Adrian; Schink, Bernhard; Przybylski, Michael

    2009-01-01

    We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

  15. High-resolution structure of the N-terminal endonuclease domain of the Lassa virus L polymerase in complex with magnesium ions.

    PubMed

    Wallat, Gregor D; Huang, Qinfeng; Wang, Wenjian; Dong, Haohao; Ly, Hinh; Liang, Yuying; Dong, Changjiang

    2014-01-01

    Lassa virus (LASV) causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP) motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV), which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173) in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1), orthomyxo- (influenza virus PA), and bunyaviruses (La Crosse virus NL1). Although the catalytic residues (D89, E102 and K122) are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.

  16. Remote His50 Acts as a Coordination Switch in the High-Affinity N-Terminal Centered Copper(II) Site of α-Synuclein.

    PubMed

    De Ricco, Riccardo; Valensin, Daniela; Dell'Acqua, Simone; Casella, Luigi; Dorlet, Pierre; Faller, Peter; Hureau, Christelle

    2015-05-18

    Parkinson's disease (PD) etiology is closely linked to the aggregation of α-synuclein (αS). Copper(II) ions can bind to αS and may impact its aggregation propensity. As a consequence, deciphering the exact mode of Cu(II) binding to αS is important in the PD context. Several previous reports have shown some discrepancies in the description of the main Cu(II) site in αS, which are resolved here by a new scenario. Three Cu(II) species can be encountered, depending on the pH and the Cu:αS ratio. At low pH, Cu(II) is bound to the N-terminal part of the protein by the N-terminal amine, the adjacent deprotonated amide group of the Asp2 residue, and the carboxylate group from the side chain of the same Asp2. At pH 7.4, the imidazole group of remote His50 occupies the fourth labile equatorial position of the previous site. At high Cu(II):αS ratio (>1), His50 leaves the coordination sphere of the first Cu site centered at the N-terminus, because a second weak affinity site centered on His50 is now filled with Cu(II). In this new scheme, the remote His plays the role of a molecular switch and it can be anticipated that the binding of the remote His to the Cu(II) ion can induce different folding of the αS protein, having various aggregation propensity.

  17. Assay Development and High-Throughput Screening for Inhibitors of Kaposi's Sarcoma-Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes.

    PubMed

    Beauchemin, Chantal; Moerke, Nathan J; Faloon, Patrick; Kaye, Kenneth M

    2014-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.

  18. Acute effects of N-terminal progastrin fragments on gastric acid secretion in man.

    PubMed

    Goetze, Jens P; Hansen, Carsten P; Rehfeld, Jens F

    2017-03-01

    We previously identified an N-terminal fragment of progastrin in human antrum and plasma, where it circulates in high concentrations. In this study, we examined the effects of N-terminal progastrin fragments on gastric acid secretion by infusion in healthy individuals. Increasing doses of progastrin fragment 1-35 were infused intravenously during constant gastric acid stimulation by gastrin-17. In addition, the effects of progastrin fragment 1-35, fragment 6-35, and fragment 1-19 on gastrin-17 stimulated acid secretion were tested. The gastrin-17 stimulated acid secretion decreased 30% after administration of a high dose of progastrin fragment 1-35 (P < 0.05). In extension, a 1-h infusion of fragment 1-35 also decreased gastric acid output. In contrast, fragment 6-35 did not affect acid secretion, and a single infusion of gastrin-17 alone did not reveal fading of gastric acid output during the time course of the experiments. The results show that N-terminal fragments of progastrin may acutely affect gastrin-stimulated gastric acid secretion in vivo. Structure-function analysis suggests that the N-terminal pentapeptide of progastrin is required for the effect.

  19. Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein.

    PubMed

    Abaitua, F; O'Hare, P

    2008-06-01

    VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.

  20. N-Terminal Methionine Processing.

    PubMed

    Wingfield, Paul T

    2017-04-03

    Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-methionine. In general, if the side chains of the penultimate residues have a radius of gyration of 1.29 Å or less, methionine is cleaved. © 2017 by John Wiley & Sons, Inc.

  1. A highly conserved N-terminal sequence for teleost vitellogenin with potential value to the biochemistry, molecular biology and pathology of vitellogenesis

    USGS Publications Warehouse

    Folmar, L.D.; Denslow, N.D.; Wallace, R.A.; LaFleur, G.; Gross, T.S.; Bonomelli, S.; Sullivan, C.V.

    1995-01-01

    N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish (striped bass, mummichog, pinfish, brown bullhead, medaka, yellow perch and the sturgeon) are compared with published N-terminal Vtg sequences for the lamprey, clawed frog and domestic chicken. Striped bass and mummichog had 100% identical amino acids between positions 7 and 21, while pinfish, brown bullhead, sturgeon, lamprey, Xenopus and chicken had 87%, 93%, 60%, 47%, 47-60%) for four transcripts and had 40% identical, respectively, with striped bass for the same positions. Partial sequences obtained for medaka and yellow perch were 100% identical between positions 5 to 10. The potential utility of this conserved sequence for studies on the biochemistry, molecular biology and pathology of vitellogenesis is discussed.

  2. The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose)*

    PubMed Central

    Silva, Ana P. G.; Ryan, Daniel P.; Galanty, Yaron; Low, Jason K. K.; Vandevenne, Marylene; Jackson, Stephen P.; Mackay, Joel P.

    2016-01-01

    Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response. PMID:26565020

  3. Genomic analysis of 16 Colorado human NL63 coronaviruses identifies a new genotype, high sequence diversity in the N-terminal domain of the spike gene and evidence of recombination.

    PubMed

    Dominguez, Samuel R; Sims, Gregory E; Wentworth, David E; Halpin, Rebecca A; Robinson, Christine C; Town, Christopher D; Holmes, Kathryn V

    2012-11-01

    This study compared the complete genome sequences of 16 NL63 strain human coronaviruses (hCoVs) from respiratory specimens of paediatric patients with respiratory disease in Colorado, USA, and characterized the epidemiology and clinical characteristics associated with circulating NL63 viruses over a 3-year period. From 1 January 2009 to 31 December 2011, 92 of 9380 respiratory specimens were found to be positive for NL63 RNA by PCR, an overall prevalence of 1 %. NL63 viruses were circulating during all 3 years, but there was considerable yearly variation in prevalence and the month of peak incidence. Phylogenetic analysis comparing the genome sequences of the 16 Colorado NL63 viruses with those of the prototypical hCoV-NL63 and three other NL63 viruses from the Netherlands demonstrated that there were three genotypes (A, B and C) circulating in Colorado from 2005 to 2010, and evidence of recombination between virus strains was found. Genotypes B and C co-circulated in Colorado in 2005, 2009 and 2010, but genotype A circulated only in 2005 when it was the predominant NL63 strain. Genotype C represents a new lineage that has not been described previously. The greatest variability in the NL63 virus genomes was found in the N-terminal domain (NTD) of the spike gene (nt 1-600, aa 1-200). Ten different amino acid sequences were found in the NTD of the spike protein among these NL63 strains and the 75 partial published sequences of NTDs from strains found at different times throughout the world.

  4. 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

  5. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

    SciTech Connect

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G.

    2011-09-16

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

  6. Combination of high-sensitivity troponin I and N-terminal pro-B-type natriuretic peptide predicts future hospital admission for heart failure in high-risk hypertensive patients with preserved left ventricular ejection fraction.

    PubMed

    Okuyama, Ryunosuke; Ishii, Junnichi; Takahashi, Hiroshi; Kawai, Hideki; Muramatsu, Takashi; Harada, Masahide; Yamada, Akira; Motoyama, Sadako; Matsui, Shigeru; Naruse, Hiroyuki; Sarai, Masayoshi; Hasegawa, Midori; Watanabe, Eiichi; Suzuki, Atsushi; Hayashi, Mutsuharu; Izawa, Hideo; Yuzawa, Yukio; Ozaki, Yukio

    2017-02-02

    Additional risk stratification may provide more aggressive and focalized preventive treatment to high-risk hypertensive patients according to the Japanese hypertension guidelines. We prospectively investigated the predictive value of high-sensitivity troponin I (hsTnI), both independently and in combination with N-terminal pro-B-type natriuretic peptide (NT-proBNP), for incident heart failure (HF) in high-risk hypertensive patients with preserved left ventricular ejection fraction (LVEF). Baseline hsTnI and NT-proBNP levels and echocardiography data were obtained for 493 Japanese hypertensive outpatients (mean age, 68.5 years) with LVEF ≥ 50%, no symptomatic HF, and at least one of the following comorbidities: stage 3-4 chronic kidney disease, diabetes mellitus, and stable coronary artery disease. During a mean follow-up period of 86.1 months, 44 HF admissions occurred, including 31 for HF with preserved ejection fraction (HFpEF) and 13 for HF with reduced ejection fraction (HFrEF; LVEF <50%). Both hsTnI (p < 0.01) and NT-proBNP (p < 0.005) levels were significant independent predictors of HF admission. Furthermore, when the patients were stratified into 4 groups according to increased hsTnI (≥highest tertile value of 10.6 pg/ml) and/or increased NT-proBNP (≥highest tertile value of 239.7 pg/ml), the adjusted relative risks for patients with increased levels of both biomarkers versus neither biomarker were 13.5 for HF admission (p < 0.0001), 9.45 for HFpEF (p = 0.0009), and 23.2 for HFrEF (p = 0.003). Finally, the combined use of hsTnI and NT-proBNP enhanced the C-index (p < 0.05), net reclassification improvement (p = 0.0001), and integrated discrimination improvement (p < 0.05) to a greater extent than that of any single biomarker. The combination of hsTnI and NT-proBNP, which are individually independently predictive of HF admission, could improve predictions of incident HF in high-risk hypertensive patients but could

  7. A highly basic sequence at the N-terminal region is essential for targeting the DNA replication protein ORC1 to the nucleus in Leishmania donovani.

    PubMed

    Kumar, Devanand; Kumar, Diwakar; Saha, Swati

    2012-07-01

    The conserved eukaryotic DNA replication protein ORC1 is one of the constituents of pre-replication complexes that assemble at or very near origins prior to replication initiation. ORC1 has been shown to be constitutively nuclear in Leishmania major. This study investigates the sequences involved in nuclear localization of ORC1 in Leishmania donovani, the causative agent of visceral leishmaniasis. Nuclear localization signals (NLSs) have been reported in only a few Leishmania proteins. Functional analyses have delineated NLSs to regions of ~60 amino acids in length in the tyrosyl DNA phosphodiesterase I and type II DNA topoisomerase of L. donovani, and in the L. major kinesin KIN13-1. Using a panel of site-directed mutations we have identified a sequence essential for nuclear import of LdORC1. This sequence at the N terminus of the protein comprises residues 2-5 (KRSR), with K2, R3 and R5 being crucial. Independent mutation of the K2 residue causes exclusion of the protein from the nucleus, while mutating the R5 residue leads to diffusion of the protein throughout the cell. This sequence, however, is insufficient for targeting a heterologous protein (β-galactosidase) to the nucleus. Analysis of additional ORC1 mutations and reporter constructs reveals that while the highly basic tetra-amino acid sequence at the N terminus is essential for nuclear localization, the ORC1 NLS in its entirety is more complex, and of a distributive character. Our results suggest that nuclear localization signalling sequences in Leishmania nuclear proteins are more complex than what is typically seen in higher eukaryotes.

  8. Different N-terminal isoforms of Oct-1 control expression of distinct sets of genes and their high levels in Namalwa Burkitt's lymphoma cells affect a wide range of cellular processes

    PubMed Central

    Pankratova, Elizaveta V.; Stepchenko, Alexander G.; Portseva, Tatiana; Mogila, Vladic A.; Georgieva, Sofia G.

    2016-01-01

    Oct-1 transcription factor has various functions in gene regulation. Its expression level is increased in several types of cancer and is associated with poor survival prognosis. Here we identified distinct Oct-1 protein isoforms in human cells and compared gene expression patterns and functions for Oct-1A, Oct-1L, and Oct-1X isoforms that differ by their N-terminal sequences. The longest isoform, Oct-1A, is abundantly expressed and is the main Oct-1 isoform in most of human tissues. The Oct-1L and the weakly expressed Oct-1X regulate the majority of Oct-1A targets as well as additional sets of genes. Oct-1X controls genes involved in DNA replication, DNA repair, RNA processing, and cellular response to stress. The high level of Oct-1 isoforms upregulates genes related to cell cycle progression and activates proliferation both in Namalwa Burkitt's lymphoma cells and primary human fibroblasts. It downregulates expression of genes related to antigen processing and presentation, cytokine-cytokine receptor interaction, oxidative metabolism, and cell adhesion, thus facilitating pro-oncogenic processes. PMID:27407111

  9. N-Terminal Hypothesis for Alzheimer's Disease.

    PubMed

    Murray, Brian; Sharma, Bhanushee; Belfort, Georges

    2017-03-15

    Although the amyloid (abeta peptide, Aβ) hypothesis is 25 years old, is the dominant model of Alzheimer's disease (AD) pathogenesis, and guides the development of potential treatments, it is still controversial. One possible reason is a lack of a mechanistic path from the cleavage products of the amyloid precursor protein (APP) such as soluble Aβ monomer and soluble molecular fragments to the deleterious effects on synaptic form and function. From a review of the recent literature and our own published work including aggregation kinetics and structural morphology, Aβ clearance, molecular simulations, long-term potentiation measurements with inhibition binding, and the binding of a commercial monoclonal antibody, aducanumab, we hypothesize that the N-terminal domains of neurotoxic Aβ oligomers are implicated in causing the disease.

  10. Protease Substrate Profiling by N-Terminal COFRADIC.

    PubMed

    Staes, An; Van Damme, Petra; Timmerman, Evy; Ruttens, Bart; Stes, Elisabeth; Gevaert, Kris; Impens, Francis

    2017-01-01

    Detection of (neo-)N-terminal peptides is essential for identifying protease cleavage sites . We here present an update of a well-established and efficient selection method for enriching N-terminal peptides out of peptide mixtures: N-terminal COFRADIC (COmbined FRActional DIagonal Chromatography). This method is based on the old concept of diagonal chromatography, which involves a peptide modification step in between otherwise identical chromatographic separations, with this modification step finally allowing for the isolation of N-terminal peptides by longer retention of non-N-terminal peptides on the resin. N-terminal COFRADIC has been successfully applied in many protease-centric studies, as well as for studies on protein alpha-N-acetylation and on characterizing alternative translation initiation events.

  11. High performance millimeter-wave microstrip circulators and isolators

    NASA Astrophysics Data System (ADS)

    Shih, Ming; Pan, J. J.

    1990-02-01

    Millimeter wave systems, phased array antennas, and high performance components all require wideband circulators (and isolators) to perform diplexing and switching, to improve isolation and Voltage Standing Wave Ratio (VSWR), and to construct IMPATT diode reflection amplifiers. Presently, most of the millimeter-wave circulators and isolators are available in the configurations of waveguide or stripline, both of which suffer from the shortcomings of bulky size/weight, narrow bandwidth, and poor compatibility with monolithic millimeter-wave integrated circuits (MMIC). MMW microstrip circulators/isolators can eliminate or improve these shortcomings. Stub-tuned microstrip circulator configuration were developed utilizing the electromagnetic fields perturbation technique, the adhesion problems of microstrip metallization on new ferrite substrate were overcome, the fabrication, assembly, packaging techniques were improved, and then successfully designed, fabricated a Ka band circulator which has isolation and return loss of greater than 16dB, insertion loss less than 0.7dB. To assess the steady and reliable performance of the circulator, a temperature cycling test was done over the range of -20 to +50 C for 3 continuous cycles and found no significant impact or variation of circulator performance.

  12. High performance millimeter-wave microstrip circulators and isolators

    NASA Technical Reports Server (NTRS)

    Shih, Ming; Pan, J. J.

    1990-01-01

    Millimeter wave systems, phased array antennas, and high performance components all require wideband circulators (and isolators) to perform diplexing and switching, to improve isolation and Voltage Standing Wave Ratio (VSWR), and to construct IMPATT diode reflection amplifiers. Presently, most of the millimeter-wave circulators and isolators are available in the configurations of waveguide or stripline, both of which suffer from the shortcomings of bulky size/weight, narrow bandwidth, and poor compatibility with monolithic millimeter-wave integrated circuits (MMIC). MMW microstrip circulators/isolators can eliminate or improve these shortcomings. Stub-tuned microstrip circulator configuration were developed utilizing the electromagnetic fields perturbation technique, the adhesion problems of microstrip metallization on new ferrite substrate were overcome, the fabrication, assembly, packaging techniques were improved, and then successfully designed, fabricated a Ka band circulator which has isolation and return loss of greater than 16dB, insertion loss less than 0.7dB. To assess the steady and reliable performance of the circulator, a temperature cycling test was done over the range of -20 to +50 C for 3 continuous cycles and found no significant impact or variation of circulator performance.

  13. Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

    PubMed

    Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

    2014-10-22

    Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

  14. 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...

  15. Doublet N-Terminal Oriented Proteomics for N-Terminomics and Proteolytic Processing Identification.

    PubMed

    Westermann, Benoit; Jacome, Alvaro Sebastian Vaca; Rompais, Magali; Carapito, Christine; Schaeffer-Reiss, Christine

    2017-01-01

    The study of the N-terminome and the precise identification of proteolytic processing events are key in biology. Dedicated methodologies have been developed as the comprehensive characterization of the N-terminome can hardly be achieved by standard proteomics methods. In this context, we have set up a trimethoxyphenyl phosphonium (TMPP) labeling approach that allows the characterization of both N-terminal and internal digestion peptides in a single experiment. This latter point is a major advantage of our strategy as most N-terminomics methods rely on the enrichment of N-terminal peptides and thus exclude internal peptides.We have implemented a double heavy/light TMPP labeling and an automated data validation workflow that make our doublet N-terminal oriented proteomics (dN-TOP) strategy efficient for high-throughput N-terminome analysis.

  16. Engineered recombinant enteropeptidase catalytic subunit: effect of N-terminal modification.

    PubMed

    Song, Hye-Won; Choi, Sung-Il; Seong, Baik L

    2002-04-01

    Enteropeptidase (enterokinase) is a serine protease highly specific for recognition and cleavage of the target sequence of Asp-Asp-Asp-Asp-Lys (D4K). The three-dimensional structure of the enteropeptidase shows that the N-terminal amino acid is buried inside the protein providing molecular interactions necessary to maintain the conformation of the active site. To determine the influence of the N-terminal amino acid of enteropeptidase light chain (EK(L)) on the enzymatic activity, we constructed various mutants including 17 different single amino acid substitutions and three different extensions at the N-terminal end. The mutants of recombinant enteropeptidase (rEK(L)) were expressed in Saccharomyces cerevisiae and secreted into culture medium. Among 20 different mutants tested, the only mutant with the Ile --> Val substitution exhibited significant activity. The kinetic properties of the mutant protein were very similar to those of the wild-type rEK(L). Based on the three-dimensional structure where the N-terminal Ile is oriented into hydrophobic pocket, the results suggest that Val could substitute Ile without affecting the active conformation of the enzyme. The results also explain why all trypsin-like serine proteases carry either Ile or Val at the N-termini and none other amino acid residues are found. Moreover, this finding provides a mental framework for expressing the N-terminally engineered enteropeptidase in Escherichia coli, utilizing the known property of the methionine aminopeptidase that exhibits poor activity toward the N-terminal Met-Ile bond, but offers efficient cleavage of the Met-Val bond.

  17. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco.

    PubMed

    Sayanova, O; Smith, M A; Lapinskas, P; Stobart, A K; Dobson, G; Christie, W W; Shewry, P R; Napier, J A

    1997-04-15

    gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

  18. Pigment epithelium-derived factor (PEDF) suppresses IL-1β-mediated c-Jun N-terminal kinase (JNK) activation to improve hepatocyte insulin signaling.

    PubMed

    Gattu, Arijeet K; Birkenfeld, Andreas L; Iwakiri, Yasuko; Jay, Steven; Saltzman, Mark; Doll, Jennifer; Protiva, Petr; Samuel, Varman T; Crawford, Susan E; Chung, Chuhan

    2014-04-01

    Pigment epithelium-derived factor (PEDF) is an antiinflammatory protein that circulates at high levels in the metabolic syndrome. Metabolic studies of PEDF knockout (KO) mice were conducted to investigate the relationship between PEDF, inflammatory markers, and metabolic homeostasis. Male PEDF KO mice demonstrated a phenotype consisting of increased adiposity, glucose intolerance, and elevated serum levels of metabolites associated with the metabolic syndrome. Genome expression analysis revealed an increase in IL-1β signaling in the livers of PEDF KO mice that was accompanied by impaired IRS and Akt signaling. In human hepatocytes, PEDF blocked the effects of an IL-1β challenge by suppressing activation of the inflammatory mediator c-Jun N-terminal kinase while restoring Akt signaling. RNA interference of PEDF in human hepatocytes was permissive for c-Jun N-terminal kinase activation and decreased Akt signaling. A metabolomics profile identified elevated circulating levels of tricarboxyclic acid cycle intermediates including succinate, an inducer of IL-1β, in PEDF KO mice. Succinate-dependent IL-1β expression was blocked by PEDF in PEDF KO, but not wild-type hepatocytes. In vivo, PEDF restoration reduced hyperglycemia and improved hepatic insulin signaling in PEDF KO mice. These findings identify elevated PEDF as a homeostatic mechanism in the human metabolic syndrome.

  19. Thermal unfolding of the N-terminal region of p53 monitored by circular dichroism spectroscopy.

    PubMed

    Schaub, Leasha J; Campbell, James C; Whitten, Steven T

    2012-11-01

    It has been estimated that 30% of eukaryotic protein and 70% of transcription factors are intrinsically disordered (ID). The biochemical significance of proteins that lack stable tertiary structure, however, is not clearly understood, largely owing to an inability to assign well-defined structures to specific biological tasks. In an attempt to investigate the structural character of ID protein, we have measured the circular dichroism spectrum of the N-terminal region of p53 over a range of temperatures and solution conditions. p53 is a well-studied transcription factor that has a proline-rich N-terminal ID region containing two activation domains. High proline content is a property commonly associated with ID, and thus p53 may be a good model system for investigating the biochemical importance of ID. The spectra presented here suggest that the N-terminal region of p53 may adopt an ordered structure under physiological conditions and that this structure can be thermally unfolded in an apparent two-state manner. The midpoint temperature for this thermal unfolding of the N-terminal region of p53 was at the near-physiological temperature of 39°C, suggesting the possibility of a physiological role for the observed structural equilibrium.

  20. 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

  1. 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.

  2. PURIFICATION AND N-TERMINAL ANALYSES OF ALGAL BILIPROTEINS,

    DTIC Science & Technology

    R-, B- and C-phycoerythrins and R- and C- phycocyanins were isolated and purified on a preparative scale by calcium phosphate chromatography, ammonium...of C-phycoerythrin (mol.wt. 226 000). Threonine (1 residue) is N-terminal in C- phycocyanin (mol.wt. 138 000), and both threonine (about 1.3 residues...and methionine (5 residues) are N-terminal in R- phycocyanin (mol.wt. 273 000). Results suggest that the apoproteins of the various phycoerythrins are

  3. 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.

  4. Highly Compact Circulators in Square-Lattice Photonic Crystal Waveguides

    PubMed Central

    Jin, Xin; Ouyang, Zhengbiao; Wang, Qiong; Lin, Mi; Wen, Guohua; Wang, Jingjing

    2014-01-01

    We propose, demonstrate and investigate highly compact circulators with ultra-low insertion loss in square-lattice- square-rod-photonic-crystal waveguides. Only a single magneto- optical square rod is required to be inserted into the cross center of waveguides, making the structure very compact and ultra efficient. The square rods around the center defect rod are replaced by several right-angled-triangle rods, reducing the insertion loss further and promoting the isolations as well. By choosing a linear-dispersion region and considering the mode patterns in the square magneto-optical rod, the operating mechanism of the circulator is analyzed. By applying the finite-element method together with the Nelder-Mead optimization method, an extremely low insertion loss of 0.02 dB for the transmitted wave and ultra high isolation of 46 dB∼48 dB for the isolated port are obtained. The idea presented can be applied to build circulators in different wavebands, e.g., microwave or Tera-Hertz. PMID:25415417

  5. N-terminal modification of proteins with o-aminophenols.

    PubMed

    Obermeyer, Allie C; Jarman, John B; Francis, Matthew B

    2014-07-09

    The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

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

    PubMed

    del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

    2014-01-10

    It has been suggested that the N-terminal strand of the light chain variable domain (V(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(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.

  7. High-power circulator test results at 350 and 700 MHz

    SciTech Connect

    Roybal, W.

    2000-08-01

    The high-power RF systems for the Accelerator Production of Tritium (APT) program require high-power circulators at 350 MHz and 700 MHz to protect 1 MW Continuous Wave (CW) klystrons from reflected power. The 350 MHz circulator is based on the CERN, EXF, and APS designs and has performed very well. The 700 MHz circulator is a new design. Prototype 700 MHz circulators have been high-power tested at Los Alamos National Laboratory (LANL). The first of these circulators has satisfied performance requirements. The circulator requirements, results from the testing, and lessons learned from this development are presented and discussed.

  8. 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

  9. 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.

  10. N-Terminal Prolactin-Derived Fragments, Vasoinhibins, Are Proapoptoptic and Antiproliferative in the Anterior Pituitary

    PubMed Central

    Ferraris, Jimena; Radl, Daniela Betiana; Zárate, Sandra; Jaita, Gabriela; Eijo, Guadalupe; Zaldivar, Verónica; Clapp, Carmen; Seilicovich, Adriana; Pisera, Daniel

    2011-01-01

    The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this

  11. 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.

  12. The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening.

    PubMed

    Schwarze, Simone; Zwettler, Fabian U; Johnson, Christopher M; Neuweiler, Hannes

    2013-01-01

    Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s(-1). The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.

  13. 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

  14. 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.

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

    PubMed Central

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

    2015-01-01

    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

  16. Circulation control technology applied to propulsive high lift systems

    NASA Technical Reports Server (NTRS)

    Englar, R. J.; Nichols, J. H., Jr.; Harris, M. J.; Eppel, J. C.; Shovlin, M. D.

    1984-01-01

    Technology developed for the Circulation Control Wing high-lift system has been extended to augment lift by entraining and redirecting engine thrust. Ejecting a thin jet sheet tangentially over a small curved deflecting surface adjacent to the slipstream of a turbofan engine causes the slipstream to flow around that deflecting surface. The angle of deflection is controlled pneumatically by varying the momentum of the thin jet sheet. The downward momentum of the slipstream enhances wing lift. This concept of pneumatically deflecting the slipstream has been applied to an upper surface blowing high-lift system and to a thrust deflecting system. The capability of the pneumatic upper surface blowing system was demonstrated in a series of investigations using a wind tunnel model and the NASA Quiet Short-haul Research Aircraft (QSRA). Full-scale thrust deflections greater than 90 deg were achieved. This mechanically simple system can provide increased maneuverability, heavy lift or overload capability, or short takeoff and landing performance.

  17. The pulmonary circulation of some domestic animals at high altitude

    NASA Astrophysics Data System (ADS)

    Anand, I.; Heath, D.; Williams, D.; Deen, M.; Ferrari, R.; Bergel, D.; Harris, P.

    1988-03-01

    Pulmonary haemodynamics and the histology of the pulmonary vasculature have been studied at high altitude in the yak, in interbreeds between yaks and cattle, and in domestic goats and sheep indigenous to high altitudes together with crosses between them and low-altitude strains. Cattle at high altitude had a higher pulmonary arterial pressure than cattle at low altitude. The yak and two interbreeds with cattle (dzos and stols) had a low pulmonary arterial pressure compared with cattle, while the medial thickness of the small pulmonary arteries was less than would be expected in cattle, suggesting that the yak has a low capacity for hypoxic pulmonary vasoconstriction and that this characteristic is transmitted genetically. Goats and sheep showed haemodynamic evidence of a limited response of the pulmonary circulation to high altitude, but no evidence that the high altitude breeds had lost this response. There were no measurable differences in the thickness of the media of the small pulmonary arteries between high- and low-altitude breeds of goats and sheep. All these species showed prominent intimal protrusions of muscle into the pulmonary veins but no specific effect of high altitude in this respect.

  18. Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

    PubMed Central

    Brandt, A; Glanville, R W; Hörlein, D; Bruckner, P; Timpl, R; Fietzek, P P; Kühn, K

    1984-01-01

    The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain. PMID:6331392

  19. N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity

    PubMed Central

    Wang, Shengjun; Xin, Fengxue; Liu, Xiaoyue; Wang, Yuxiao; An, Zhenyi; Qi, Qingsheng; Wang, Peng George

    2009-01-01

    Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-ΔH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application. PMID:20016784

  20. Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3.

    PubMed

    Wu, Ying; Wei, Shuo; Van Doren, Steven R; Brew, Keith

    2011-05-13

    The avid binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is crucial for the regulation of pericellular and extracellular proteolysis. The interactions of the catalytic domain (cd) of MMP-1 with the inhibitory domains of TIMP-1 and TIMP-2 (N-TIMPs) and MMP-3cd with N-TIMP-2 have been characterized by isothermal titration calorimetry and compared with published data for the N-TIMP-1/MMP-3cd interaction. All interactions are largely driven by increases in entropy but there are significant differences in the profiles for the interactions of both N-TIMPs with MMP-1cd as compared with MMP-3cd; the enthalpy change ranges from small for MMP-1cd to highly unfavorable for MMP-3cd (-0.1 ± 0.7 versus 6.0 ± 0.5 kcal mol(-1)). The heat capacity change (ΔC(p)) of binding to MMP-1cd (temperature dependence of ΔH) is large and negative (-210 ± 20 cal K(-1) mol(-1)), indicating a large hydrophobic contribution, whereas the ΔC(p) values for the binding to MMP-3cd are much smaller (-53 ± 3 cal K(-1) mol(-1)), and some of the entropy increase may arise from increased conformational entropy. Apart from differences in ionization effects, it appears that the properties of the MMP may have a predominant influence in the thermodynamic profiles for these N-TIMP/MMP interactions.

  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.

  2. The N-terminal tails of the H2A-H2B histones affect dimer structure and stability.

    PubMed

    Placek, Brandon J; Gloss, Lisa M

    2002-12-17

    The histone proteins of the core nucleosome are highly basic and form heterodimers in a "handshake motif." The N-terminal tails of the histones extend beyond the canonical histone fold of the hand-shake motif and are the sites of posttranslational modifications, including lysine acetylations and serine phosphorylations, which influence chromatin structure and activity as well as alter the charge state of the tails. However, it is not well understood if these modifications are signals for recruitment of other cellular factors or if the removal of net positive charge from the N-terminal tail plays a role in the overall structure of chromatin. To elucidate the effects of the N-terminal tails on the structure and stability of histones, the highly charged N-terminal tails were truncated from the H2A and H2B histones. Three mutant dimers were studied: DeltaN-H2A/WT H2B; WT H2A/DeltaN-H2B, and DeltaN-H2A/DeltaN-H2B. The CD spectra, stabilities to urea-denaturation, and the salt-dependent stabilization of the three truncated dimers were compared with those of the wild-type dimer. The data support four conclusions regarding the effects of the N-terminal tails of H2A and H2B: (1) Removal of the N-terminal tails of H2A and H2B enhance the helical structure of the mutant heterodimers. (2) Relative to the full-length WT heterodimer, the DeltaN-H2A/WT H2B dimer is destabilized, while the WT H2A/DeltaN-H2B and DeltaN-H2A/DeltaN-H2B dimers are slightly stabilized. (3) The truncated dimers exhibit decreased m values, relative to the WT dimer, supporting the hypothesis that the N-terminal tails in the isolated dimer adopt a collapsed structure. (4) Electrostatic repulsion in the N-terminal tails decreases the stability of the H2A-H2B dimer.

  3. N-terminal sequence analysis of proteins and peptides.

    PubMed

    Reim, D F; Speicher, D W

    2001-05-01

    Amino-terminal (N-terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N-terminal end. This unit describes the sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Perkin-Elmer Procise Sequencer. Sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Hewlett-Packard Model G1005A sequencer is also described. Methods are provided for optimizing separation of PTH amino acid derivatives on Perkin-Elmer instruments and for increasing the proportion of sample injected onto the PTH analyzer on older Perkin-Elmer instruments by installing a modified sample loop. The amount of data obtained from a single sequencer run is substantial, and careful interpretation of this data by an experienced scientist familiar with the current operation performance of the instrument used for this analysis is critically important. A discussion of data interpretation is therefore provided. Finally, discussion of optimization of sequencer performance as well as possible solutions to frequently encountered problems is included.

  4. Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation.

    PubMed

    Helsens, Kenny; Van Damme, Petra; Degroeve, Sven; Martens, Lennart; Arnesen, Thomas; Vandekerckhove, Joël; Gevaert, Kris

    2011-08-05

    Initiation of protein translation is a well-studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function, and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a Saccharomyces cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique N(α)-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of N(α)-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5' UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum matches were only accepted at a 99% probability threshold and were subsequently analyzed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based N(α)-acetylated peptides that point to N(α)-acetylation as a post-translational modification since these peptides did not start nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and

  5. 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.

  6. A helical bundle in the N-terminal domain of the BLM helicase mediates dimer and potentially hexamer formation.

    PubMed

    Shi, Jing; Chen, Wei-Fei; Zhang, Bo; Fan, San-Hong; Ai, Xia; Liu, Na-Nv; Rety, Stephane; Xi, Xu-Guang

    2017-04-07

    Helicases play a critical role in processes such as replication or recombination by unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In humans, mutations in genes of three members of the RecQ family helicases (blm, wrn, and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking. Because the helicase core domains are highly conserved, it has been postulated that functional differences among family members might be explained by significant differences in the N-terminal domains, but these domains are poorly characterized. To help fill this gap, we now describe bioinformatics, biochemical, and structural data for three vertebrate BLM proteins. We pair high resolution crystal structures with SAXS analysis to describe an internal, highly conserved sequence we term the dimerization helical bundle in N-terminal domain (DHBN). We show that, despite the N-terminal domain being loosely structured and potentially lacking a defined three-dimensional structure in general, the DHBN exists as a dimeric structure required for higher order oligomer assembly. Interestingly, the unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer, and also, the stable DHBN dimer can be dissociated upon ATP hydrolysis. Thus, the structural and biochemical characterizations of N-terminal domains will provide new insights into how the N-terminal domain affects the structural and functional organization of the full BLM molecule.

  7. 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

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

    PubMed

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

    2014-10-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (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 an 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 within the non

  9. N-terminal chemical protein labeling using the naturally split GOS-TerL intein.

    PubMed

    Bachmann, Anne-Lena; Mootz, Henning D

    2017-03-23

    Chemoselective and regioselective chemical protein labeling is of great importance, yet no current technique is sufficiently general and simple to perform. Protein trans-splicing by split inteins can be used to ligate short tags with chemical labels to either the N or the C terminus of a protein. The CysTag approach exploits split intein fragments without a cysteine fused with such a short tag containing a single cysteine that is easily amenable to selective modification using classical cysteine bioconjugation. Labeling of the protein of interest is achieved through transfer of the pre-labeled tag by protein trans-splicing. This protocol keeps other cysteines unmodified. While split inteins for C-terminal CysTag labeling were previously reported, no high-yielding and naturally split intein for N-terminal labeling has been available. In this work, the recently discovered GOS-TerL intein was explored as the only known naturally split intein that both lacks a cysteine in its N-terminal fragment and is active under ambient conditions. Thioredoxin as a model protein and a camelid nanobody were labeled with a synthetic fluorophore by transferring the pre-labeling CysTag in the protein trans-splicing reaction with yields of about 50 to 90%. The short N-terminal intein fragment was also chemically synthesized with a tag to enable protein labeling by semi-synthetic protein trans-splicing. Our results expand the scope of the CysTag labeling strategy, which achieves selective chemical modification without the requirement for sophisticated biorthogonal functional groups and rather builds on the plethora of commercially available reagents directed at the thiol side chain of cysteine. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  10. Light-regulated nuclear import and degradation of Arabidopsis phytochrome-A N-terminal fragments.

    PubMed

    Wolf, Iris; Kircher, Stefan; Fejes, Erzsébet; Kozma-Bognár, László; Schäfer, Eberhard; Nagy, Ferenc; Adám, Eva

    2011-02-01

    The photoreceptor phytochrome-A (phyA) regulates germination and seedling establishment by mediating very low fluence (VLFR) and far-red high irradiance (FR-HIR) responses in Arabidopsis thaliana. In darkness, phyA homodimers exist in the biologically inactive Pr form and are localized in the cytoplasm. Light induces formation of the biologically active Pfr form and subsequent rapid nuclear import. PhyA Pfr, in contrast to the Pr form, is labile and has a half-life of ∼30 min. We produced transgenic plants in a phyA-201 null background that express the PHYA-yellow fluorescent protein (YFP) or the PHYA686-YFP-dimerization domain (DD) and PHYA686-YFP-DD-nuclear localization signal (NLS) or PHYA686-YFP-DD-nuclear exclusion signal (NES) fusion proteins. The PHYA686-YFP fusion proteins contained the N-terminal domain of phyA (686 amino acid residues), a short DD and the YFP. Here we report that (i) PHYA686-YFP-DD fusion protein is imported into the nucleus in a light-dependent fashion; (ii) neither of the PHYA686 fusion proteins is functional in FR-HIR and nuclear VLFR; and (iii) the phyA-dependent, blue light-induced inhibition of hypocotyl growth is mediated by the PHYA686-YFP-DD-NES but not by the PHYA686-YFP-DD-NLS and PHYA686-YFP-DD fusion proteins. We demonstrate that (i) light induces degradation of all PHYA N-terminal-containing fusion proteins and (ii) these N-terminal domain-containing fusion proteins including the constitutively nuclear PHYA686-YFP-DD-NLS and predominantly cytoplasmic PHYA686-YFP-DD-NES degrade at comparable rates but markedly more slowly than PHYA-YFP, whereas (iii) light-induced degradation of the native phyA is faster compared with PHYA-YFP.

  11. N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

    PubMed Central

    Van Damme, Petra; Lasa, Marta; Polevoda, Bogdan; Gazquez, Cristina; Elosegui-Artola, Alberto; Kim, Duk Soo; De Juan-Pardo, Elena; Demeyer, Kimberly; Hole, Kristine; Larrea, Esther; Timmerman, Evy; Prieto, Jesus; Arnesen, Thomas; Sherman, Fred; Gevaert, Kris; Aldabe, Rafael

    2012-01-01

    Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human. PMID:22814378

  12. 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.

  13. c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

    DTIC Science & Technology

    2015-03-01

    1 AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N- Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis” PRINCIPAL...TITLE AND SUBTITLE “c-jun-N- Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER...ABSTRACT Abstract: 250 aminopyrazoles, a new class of c-jun-N- terminal kinase (JNK) inhibitors, have been synthesized and the biochemical IC50 has

  14. Novel Insights into Structure-Activity Relationships of N-Terminally Modified PACE4 Inhibitors.

    PubMed

    Kwiatkowska, Anna; Couture, Frédéric; Levesque, Christine; Ly, Kévin; Beauchemin, Sophie; Desjardins, Roxane; Neugebauer, Witold; Dory, Yves L; Day, Robert

    2016-02-04

    PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile.

  15. Preparation of protein samples for mass spectrometry and N-terminal sequencing.

    PubMed

    Glenn, Gary

    2014-01-01

    The preparation of protein samples for mass spectrometry and N-terminal sequencing is a key step in successfully identifying proteins. Mass spectrometry is a very sensitive technique, and as such, samples must be prepared carefully since they can be subject to contamination of the sample (e.g., due to incomplete subcellular fractionation or purification of a multiprotein complex), overwhelming of the sample by highly abundant proteins, and contamination from skin or hair (keratin can be a very common hit). One goal of sample preparation for mass spec is to reduce the complexity of the sample - in the example presented here, mitochondria are purified, solubilized, and fractionated by sucrose density gradient sedimentation prior to preparative 1D SDS-PAGE. It is important to verify the purity and integrity of the sample so that you can have confidence in the hits obtained. More protein is needed for N-terminal sequencing and ideally it should be purified to a single band when run on an SDS-polyacrylamide gel. The example presented here involves stably expressing a tagged protein in HEK293 cells and then isolating the protein by affinity purification and SDS-PAGE.

  16. Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis

    NASA Astrophysics Data System (ADS)

    Collin-Hansen, C.; Andersen, R. A.; Steinnes, E.

    2003-05-01

    A Cd-binding protein was isolated from the popular edible mushroom Boletus edulis, which is a hyperaccumulator of both Cd and Hg. Wild-growing samples of B. edulis were collected from soils rich in Cd. Cd radiotracer was added to the crude protein preparation obtained from ethanol precipitation of heat-treated cytosol. Proteins were then further separated in two consecutive steps; gel filtration and anion exchange chromatography. In both steps the Cd radiotracer profile showed only one distinct peak, which corresponded well with the profiles of endogenous Cd obtained by atomic absorption spectrophotometry (AAS). Concentrations of the essential elements Cu and Zn were low in the protein fractions high in Cd. N-terminal sequencing performed on the Cd-binding protein fractions revealed a protein with a novel amino acid sequence, which contained aromatic amino acids as well as proline. Both the N-terminal sequencing and spectrofluorimetric analysis with EDTA and ABD-F (4-aminosulfonyl-7-fluoro-2, 1, 3-benzoxadiazole) failed to detect cysteine in the Cd-binding fractions. These findings conclude that the novel protein does not belong to the metallothionein family. The results suggest a role for the protein in Cd transport and storage, and they are of importance in view of toxicology and food chemistry, but also for environmental protection.

  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. Jun N-terminal kinase signaling makes a face

    PubMed Central

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

    2016-01-01

    ABSTRACT decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions. PMID:27384866

  19. Destabilization of Internal Kink Modes at High Frequency by Energetic Circulating Ions

    SciTech Connect

    Wang, Shaojie

    2001-06-04

    A theoretical model is proposed to interpret the high-frequency fishbone instability observed in tangential neutral-beam-injection discharges in a tokamak. It is shown that, when the beam ion beta exceeds a critical value, energetic circulating ions can indeed destabilize the internal kink mode through circulation resonance at a high frequency comparable to the circulation frequency of the energetic ions. The critical beta value of the energetic ions, the real frequency, and the growth rate of the mode are in general agreement with the high-frequency fishbone instability observed in experiments.

  20. 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.

  1. 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

  2. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    PubMed Central

    Lei, Yan-Chang; Tian, Yong-Jun; Ding, Hong-Hui; Wang, Bao-Ju; Yang, Yan; Hao, You-Hua; Zhao, Xi-Ping; Lu, Meng-Ji; Gong, Fei-Li; Yang, Dong-Liang

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the

  3. N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

    SciTech Connect

    Scott, Daniel C.; Monda, Julie K.; Bennett, Eric J.; Harper, J. Wade; Schulman, Brenda A.

    2012-10-25

    Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

  4. Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Ksiazek, Dorota; Brandstetter, Hans; Israel, Lars; Bourenkov, Gleb P; Katchalova, Galina; Janssen, Klaus-Peter; Bartunik, Hans D; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2003-09-01

    Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

  5. Naa50/San-dependent N-terminal acetylation of Scc1 is potentially important for sister chromatid cohesion

    PubMed Central

    Ribeiro, Ana Luisa; Silva, Rui D.; Foyn, Håvard; Tiago, Margarida N.; Rathore, Om Singh; Arnesen, Thomas; Martinho, Rui Gonçalo

    2016-01-01

    The gene separation anxiety (san) encodes Naa50/San, a N-terminal acetyltransferase required for chromosome segregation during mitosis. Although highly conserved among higher eukaryotes, the mitotic function of this enzyme is still poorly understood. Naa50/San was originally proposed to be required for centromeric sister chromatid cohesion in Drosophila and human cells, yet, more recently, it was also suggested to be a negative regulator of microtubule polymerization through internal acetylation of beta Tubulin. We used genetic and biochemical approaches to clarify the function of Naa50/San during development. Our work suggests that Naa50/San is required during tissue proliferation for the correct interaction between the cohesin subunits Scc1 and Smc3. Our results also suggest a working model where Naa50/San N-terminally acetylates the nascent Scc1 polypeptide, and that this co-translational modification is subsequently required for the establishment and/or maintenance of sister chromatid cohesion. PMID:27996020

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

    SciTech Connect

    Meagher, Martin; Enemark, Eric J.

    2016-06-22

    The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome 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.

  7. New roles for old modifications: emerging roles of N-terminal post-translational modifications in development and disease.

    PubMed

    Tooley, John G; Schaner Tooley, Christine E

    2014-12-01

    The importance of internal post-translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N-terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N-terminal PTMs are now beginning to be elucidated. New findings show that N-terminal methylation, along with N-terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N-terminal ubiquitylation and N-terminal propionylation. Here, will discuss the recent advances in the functional studies of N-terminal PTMs, recount the new N-terminal PTMs being identified, and briefly examine the possibility of dynamic N-terminal PTM exchange.

  8. New roles for old modifications: Emerging roles of N-terminal post-translational modifications in development and disease

    PubMed Central

    Tooley, John G; Schaner Tooley, Christine E

    2014-01-01

    The importance of internal post-translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N-terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N-terminal PTMs are now beginning to be elucidated. New findings show that N-terminal methylation, along with N-terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N-terminal ubiquitylation and N-terminal propionylation. Here, will discuss the recent advances in the functional studies of N-terminal PTMs, recount the new N-terminal PTMs being identified, and briefly examine the possibility of dynamic N-terminal PTM exchange. PMID:25209108

  9. Impact of N-Terminal Acetylation of α-Synuclein on Its Random Coil and Lipid Binding Properties

    PubMed Central

    2012-01-01

    N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson’s disease, is common in mammals. The impact of this modification on the protein’s structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone 3JHH couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and 3JHH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an “initiation–elongation” process of binding to the membrane. PMID:22694188

  10. SSDP1 gene encodes a protein with a conserved N-terminal FORWARD domain.

    PubMed

    Bayarsaihan, Dashzeveg

    2002-09-23

    I describe the characterization of mouse, human and chicken SSDP1 orthologs that encode a highly conserved protein with over 90% identity at the amino acid level. Structurally, the protein consists of a well-preserved FWD (FORWARD)-domain at the N-terminal end and a proline-, glycine-, methionine- and serine-rich sequence in the central and C-terminal regions. The FORWARD domain, comprised of three alpha-helices, is characterized by the presence of a FWD-box of unknown function conserved not only in vertebrates, but also in nematode, plants, fly and yeast. Human SSDP1 spans about 200 kb on the chromosome 1p31-p32 region and consists of 17 exons. The SSDP1 mRNA transcripts are distributed ubiquitously in adult human and mouse tissues.

  11. 157 nm photodissociation of dipeptide ions containing N-terminal arginine.

    PubMed

    Webber, Nathaniel; He, Yi; Reilly, James P

    2014-02-01

    Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

  12. Synaptic transmission and plasticity require AMPA receptor anchoring via its N-terminal domain.

    PubMed

    Watson, Jake F; Ho, Hinze; Greger, Ingo H

    2017-03-14

    AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and are selectively recruited during activity-dependent plasticity to increase synaptic strength. A prerequisite for faithful signal transmission is the positioning and clustering of AMPARs at postsynaptic sites. The mechanisms underlying this positioning have largely been ascribed to the receptor cytoplasmic C-termini and to AMPAR-associated auxiliary subunits, both interacting with the postsynaptic scaffold. Here, using mouse organotypic hippocampal slices, we show that the extracellular AMPAR N-terminal domain (NTD), which projects midway into the synaptic cleft, plays a fundamental role in this process. This highly sequence-diverse domain mediates synaptic anchoring in a subunit-selective manner. Receptors lacking the NTD exhibit increased mobility in synapses, depress synaptic transmission and are unable to sustain long-term potentiation (LTP). Thus, synaptic transmission and the expression of LTP are dependent upon an AMPAR anchoring mechanism that is driven by the NTD.

  13. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains.

    PubMed

    Gibson, S J; Bond, N J; Milne, S; Lewis, A; Sheriff, A; Pettman, G; Pradhan, R; Higazi, D R; Hatton, D

    2017-03-28

    Monoclonal antibodies (mAbs) contain short N-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis-cleavage of the signal peptide can occur, resulting in truncation or elongation at the N-terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N-terminus. This truncation was not caused by mis-processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed-batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N-terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc-containing mAbs. This article is protected by copyright. All rights reserved.

  14. The Solution Structure of the N-Terminal Domain of Human Tubulin Binding Cofactor C Reveals a Platform for Tubulin Interaction

    PubMed Central

    Garcia-Mayoral, Mª Flor; Castaño, Raquel; Fanarraga, Monica L.; Zabala, Juan Carlos; Rico, Manuel; Bruix, Marta

    2011-01-01

    Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers. PMID:22028797

  15. Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

    PubMed

    Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

    2017-04-01

    Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.

  16. Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus.

    PubMed

    Song, Letian; Dumon, Claire; Siguier, Béatrice; André, Isabelle; Eneyskaya, Elena; Kulminskaya, Anna; Bozonnet, Sophie; O'Donohue, Michael Joseph

    2014-03-20

    To understand structure-function relationships in the N-terminal region of GH11 xylanases, the 17 N-terminal amino acids of the GH11 xylanase from Neocallimastix patriciarum (Np-Xyn) have been grafted onto the N-terminal extremity of the untypically short GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn), creating a hybrid enzyme denoted NTfus. The hybrid xylanase displayed properties (pH and temperature optima) similar to those of the parental enzyme, although thermostability was lowered, with the Tm value, being reduced by 5°C. Kinetic assays using oNP-Xylo-oligosaccharides (DP2 and 3) indicated that the N-extension did not procure more extensive substrate binding, even when further mutagenesis was performed to promote this. However, these experiments confirmed weak subsite -3 for both NTfus and the parental enzyme. The catalytic efficiency of NTfus was shown to be 17% higher than that of the parental enzyme on low viscosity wheat arabinoxylan and trials using milled wheat straw as the substrate revealed that NTfus released more substituted oligosaccharide products (Xyl/Ara=8.97±0.13 compared to Xyl/Ara=9.70±0.21 for the parental enzyme), suggesting that the hybrid enzyme possesses wider substrate selectivity. Combining either the parental enzyme or NTfus with the cellulolytic cocktail Accellerase 1500 boosted the impact of the latter on wheat straw, procuring yields of solubilized xylose and glucose of 23 and 24% of theoretical yield, respectively, thus underlining the benefits of added xylanase activity when using this cellulase cocktail. Overall, in view of the results obtained for NTfus, we propose that the N-terminal extension leads to the modification of a putative secondary substrate binding site, a hypothesis that is highly consistent with previous data.

  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. Substrate specificity of mammalian N-terminal α-amino methyltransferase

    PubMed Central

    Petkowski, Janusz J.; Schaner Tooley, Christine E.; Anderson, Lissa C.; Shumilin, Igor A.; Balsbaugh, Jeremy L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Minor, Wladek; Macara, Ian G.

    2012-01-01

    N-terminal methylation of free α-amino-groups is a post-translational modification of proteins that has been known for 30 years but has been very little studied. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono- di- or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-aminomethylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification, and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products. PMID:22769851

  19. 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

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

    PubMed Central

    Song, Albert S.; Poor, Taylor A.; Abriata, Luciano A.; Jardetzky, Theodore S.; Dal Peraro, Matteo; Lamb, Robert A.

    2016-01-01

    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

  1. Functional differences between HOX proteins conferred by two residues in the homeodomain N-terminal arm.

    PubMed Central

    Phelan, M L; Sadoul, R; Featherstone, M S

    1994-01-01

    Hox genes encode homeodomain-containing transcriptional regulators that function during development to specify positional identity along embryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core motif. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregulatory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1.6) is unable to activate transcription through the Hoxd-4 autoregulatory element. Here we show that this is due to an inability of the HOXA-1 homeodomain to bind a HOXD-4 recognition site effectively. We have produced chimeras between HOXD-4 and HOXA-1 to map specific residues responsible for this functional difference. When positions 2 and 3 in the N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both strong DNA binding and transcriptional activation were rescued. This substitution appears to confer an increased DNA-binding ability on the HOXA-1 homeodomain, since we were unable to detect a high-affinity recognition sequence for HOXA-1 in a randomized pool of DNA probes. The contribution of position 3 to DNA binding has been implicated by structural studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific homeodomain residues that confer major differences in DNA binding and transcriptional activation between Hox gene products have not been previously determined. Identity at these two positions is generally conserved among paralogs but varies between Hox gene subfamilies. As a result, these residues may be important for the regulation of target gene expression by specific Hox products. Images PMID:7913516

  2. Structural basis of damage recognition by thymine DNA glycosylase: Key roles for N-terminal residues

    PubMed Central

    Coey, Christopher T.; Malik, Shuja S.; Pidugu, Lakshmi S.; Varney, Kristen M.; Pozharski, Edwin; Drohat, Alexander C.

    2016-01-01

    Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5-formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG82-308, a new construct containing 29 more N-terminal residues than TDG111-308, the construct used for previous structures of DNA-bound TDG. Crystal structures and NMR experiments demonstrate that most of these N-terminal residues are disordered, for substrate- or product-bound TDG82-308. Nevertheless, G·T substrate affinity and glycosylase activity of TDG82-308 greatly exceeds that of TDG111-308 and is equivalent to full-length TDG. We report the first high-resolution structures of TDG in an enzyme-substrate complex, for G·U bound to TDG82-308 (1.54 Å) and TDG111-308 (1.71 Å), revealing new enzyme-substrate contacts, direct and water-mediated. We also report a structure of the TDG82-308 product complex (1.70 Å). TDG82-308 forms unique enzyme–DNA interactions, supporting its value for structure-function studies. The results advance understanding of how TDG recognizes and removes modified bases from DNA, particularly those resulting from deamination. PMID:27580719

  3. Structure of a tropomyosin N-terminal fragment at 0.98 Å resolution

    SciTech Connect

    Meshcheryakov, Vladimir A.; Krieger, Inna; Kostyukova, Alla S.; Samatey, Fadel A.

    2011-09-01

    The crystal structure of the N-terminal fragment of the short nonmuscle α-tropomyosin has been determined at a resolution of 0.98 Å. Tropomyosin (TM) is an elongated two-chain protein that binds along actin filaments. Important binding sites are localized in the N-terminus of tropomyosin. The structure of the N-terminus of the long muscle α-TM has been solved by both NMR and X-ray crystallography. Only the NMR structure of the N-terminus of the short nonmuscle α-TM is available. Here, the crystal structure of the N-terminus of the short nonmuscle α-TM (αTm1bZip) at a resolution of 0.98 Å is reported, which was solved from crystals belonging to space group P3{sub 1} with unit-cell parameters a = b = 33.00, c = 52.03 Å, α = β = 90, γ = 120°. The first five N-terminal residues are flexible and residues 6–35 form an α-helical coiled coil. The overall fold and the secondary structure of the crystal structure of αTM1bZip are highly similar to the NMR structure and the atomic coordinates of the corresponding C{sup α} atoms between the two structures superimpose with a root-mean-square deviation of 0.60 Å. The crystal structure validates the NMR structure, with the positions of the side chains being determined precisely in our structure.

  4. 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.

  5. Preparation of Arabidopsis thaliana seedling proteomes for identifying metacaspase substrates by N-terminal COFRADIC.

    PubMed

    Tsiatsiani, Liana; Stael, Simon; Van Damme, Petra; Van Breusegem, Frank; Gevaert, Kris

    2014-01-01

    Proteome-wide discovery of in vivo metacaspase substrates can be obtained by positional proteomics approaches such as N-terminal COFRADIC, for example by comparing the N-terminal proteomes (or N-terminomes) of wild-type plants to transgenic plants not expressing a given metacaspase. In this chapter we describe a protocol for the preparation of plant tissue proteomes, including differential isotopic labelling allowing for a comparison of in vivo N-terminomes that serves as the starting point for N-terminal COFRADIC studies.

  6. Stability of the heme Fe-N-terminal amino group coordination bond in denatured cytochrome c.

    PubMed

    Tai, Hulin; Munegumi, Toratane; Yamamoto, Yasuhiko

    2009-01-05

    In the denatured states of Hydrogenobacter thermophilus cytochrome c(552) (HT) and Pseudomonas aeruginosa cytochrome c(551) (PA), and their mutants, the N-terminal amino group of the polypeptide chain is coordinated to heme Fe in place of the axial Met, the His-N(term) form being formed. The coordination of the N-terminal amino group to heme Fe leads to loop formation by the N-terminal stretch preceding the first Cys residue bound to the heme, and the N-terminal stretches of HT and PA are different from each other in terms of both the sequence and the number of constituent amino acid residues. The His-N(term) form was shown to be rather stable, and hence it can influence the stability of the denatured state. We have investigated the heme Fe coordination structures and stabilities of the His-N(term) forms emerging upon guanidine hydrochloric acid-induced unfolding of the oxidized forms of the proteins. The Fe-N(term) coordination bond in the His-N(term) form with a 9-residue N-terminal stretch of HT proteins was found to be tilted to some extent away from the heme normal, as reflected by the great heme methyl proton shift spread. On the other hand, the small heme methyl proton shift spread of the His-N(term) form with an 11-residue stretch of PA proteins indicated that its Fe-N(term) bond is nearly parallel with the heme normal. The stability of the His-N(term) form was found to be affected by the structural properties of the N-terminal stretch, such as its length and the N-terminal residue. With a given N-terminal residue, the stability of the His-N(term) form is higher for a 9-residue N-terminal stretch than an 11-residue one. In addition, with a given length of the N-terminal stretch, the His-N(term) form with an N-terminal Glu is stabilized by a few kJ mol(-1) relative to that with an N-terminal Asn. These results provide a novel insight into the stabilizing interactions in the denatured cyts c that will facilitate elucidation of the folding/unfolding mechanisms

  7. High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System

    PubMed Central

    Regmi, Sagar; Fu, Afu; Luo, Kathy Qian

    2017-01-01

    Circulating tumor cells (CTCs) are the primary targets of cancer treatment as they cause distal metastasis. However, how CTCs response to exercise-induced high shear stress is largely unknown. To study the effects of hemodynamic microenvironment on CTCs, we designed a microfluidic circulatory system that produces exercise relevant shear stresses. We explore the effects of shear stresses on breast cancer cells with different metastatic abilities, cancer cells of ovarian, lung and leukemic origin. Three major findings were obtained. 1) High shear stress of 60 dynes/cm2 achievable during intensive exercise killed more CTCs than low shear stress of 15 dynes/cm2 present in human arteries at the resting state. 2) High shear stress caused necrosis in over 90% of CTCs within the first 4 h of circulation. More importantly, the CTCs that survived the first 4 h-circulation, underwent apoptosis during 16–24 h of post-circulation incubation. 3) Prolonged high shear stress treatment effectively reduced the viability of highly metastatic and drug resistant breast cancer cells. As high shear stress had much less damaging effects on leukemic cells mimicking the white blood cells, we propose that intensive exercise may be a good strategy for generating high shear stress that can destroy CTCs and prevent cancer metastasis. PMID:28054593

  8. Investigating the circulation patterns of the northern Adriatic Sea with a very high resolution model.

    NASA Astrophysics Data System (ADS)

    Zavatarelli, M.; Mattia, G.; Lovato, T.

    2015-12-01

    The NEMO model was implemented on the northern Adriatic sea with an high resolution (horizontal resolution of 800 m and vertical resolution of 2 m). The model is off-line nested with a general circulation model of Mediterranean sea providing open boundary data. Hindcast simulations with high frequency atmospheric forcing and daily river runoff, were performed and analyzed in order to study the interannual variability of the circulation pattern and of the shelf dense water (Northern Adriatic dense Water, NADW) production in relation to the freshwater input and heat and wind forcing. Whenever possible, the results were validated against available observations. The simulations highlighted the large interannual variability of the circulation patterns. This is a contribution of the EU-FP7 Project "PERSEUS" (Policy oriented environmental research in the Southern European Seas)

  9. 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.

  10. Experimental and Quantum Chemical Approaches to Develop Highly Selective Nanocatalysts for CO2 -free Power Circulation.

    PubMed

    Yamauchi, Miho; Ozawa, Nobuki; Kubo, Momoji

    2016-10-01

    Renewable electricity must be utilized to usefully suppress the atmospheric CO2 concentration and slow the progression of global warming. We have thus proposed a new concept involving CO2 -free electric power circulation systems via highly selective electrochemical reactions of alcohol/carboxylic acid redox couples. Design concepts for nanocatalysts able to catalyze highly selective electrochemical reactions are provided from both experimental and quantum mechanical perspectives.

  11. High power circulator for the 433.3 MHz proton linac

    NASA Astrophysics Data System (ADS)

    Shirai, T.; Inoue, M.; Iwashita, Y.; Noda, A.; Takekoshi, H.; Tsuruoka, S.

    1992-10-01

    A 433.3 MHz T-junction waveguide circulator with a dummy-load was constructed to isolate the klystron and a linac cavity at the high rf power level of 1 MW. The isolation is -34.9 dB at a low duty factor and it decreases to -23.8 dB at a high duty factor (1%), which shows enough performance for our linac operation.

  12. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    PubMed

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  13. Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.

    PubMed

    Minteer, Christopher J; Siegart, Nicolle M; Colelli, Kathryn M; Liu, Xinyue; Linhardt, Robert J; Wang, Chunyu; Gomez, Alvin V; Reitter, Julie N; Mills, Kenneth V

    2017-02-28

    Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

  14. Dissecting the Functional Role of the N-Terminal Domain of the Human Small Heat Shock Protein HSPB6

    PubMed Central

    Heirbaut, Michelle; Beelen, Steven; Strelkov, Sergei V.; Weeks, Stephen D.

    2014-01-01

    HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function. PMID:25157403

  15. 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.

  16. 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

    2016-04-13

    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.

  17. Peptide scrambling during collision-induced dissociation is influenced by N-terminal residue basicity.

    PubMed

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

    2014-11-01

    '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)+H(2)O 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.

  18. SHP-1 inhibition by 4-hydroxynonenal activates Jun N-terminal kinase and glutamate cysteine ligase.

    PubMed

    Rinna, Alessandra; Forman, Henry Jay

    2008-07-01

    4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, is toxic at high concentrations, but at near-physiological concentrations it induces detoxifying enzymes. Previous data established that in human bronchial epithelial (HBE1) cells, both genes for glutamate cysteine ligase (GCL) are induced by HNE through the c-Jun N-terminal kinase (JNK) pathway. The protein-tyrosine phosphatase SH2 domain containing phosphatase-1 (SHP-1) is thought to play a role as a negative regulator of cell signaling, and has been implicated as such in the JNK pathway. In the present study, SHP-1 was demonstrated to contribute to HNE-induced-gclc expression via regulation of the JNK pathway in HBE1 cells. Treatment of HBE1 cells with HNE induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4), JNK, and c-Jun. HNE was able to inhibit protein tyrosine phosphatase activity of SHP-1 through increased degradation of the protein. Furthermore, transfection with small interference RNA SHP-1 showed an enhancement of JNK and c-Jun phosphorylation, but not of MKK4, leading to increased gclc expression. These results demonstrate that SHP-1 plays a role as a negative regulator of the JNK pathway and that HNE activated the JNK pathway by inhibiting SHP-1. Thus, SHP-1 acts as a sensor for HNE and is responsible for an important adaptive response to oxidative stress.

  19. 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.

  20. N-terminal truncated carboxypeptidase E expression is associated with poor prognosis of lung adenocarcinoma

    PubMed Central

    Sun, Jing; Meng, Dawei; Li, Li; Tian, Xin; Jia, Yunji; Wang, Hongyue; Yu, Huihui; Sun, Tiemin; Qu, Aibing; Shen, Hui; Bao, Jimin; Zhang, Guirong

    2016-01-01

    Lung cancer is a malignant tumor with high morbidity and mortality rates. To date, no suitable molecular diagnostic tool to predict disease recurrence and metastasis has been identified. The current study aimed to evaluate the potential of N-terminal truncated carboxypeptidase E (CPEΔN) to predict the recurrence and metastasis of lung adenocarcinoma. Western blotting revealed the co-expression of CPE and CPEΔN in the surgically collected pathological and pericarcinoma tissues tissues of 62.1% (59/95) lung adenocarcinoma patients. The full length CPE protein was predominantly expressed in pericarcinoma tissues and CPEΔN expression was identified in the pericarcinoma normal tissues of only 5.26% (5/95) patients. The 3-year postoperative recurrence and metastasis rates were significantly higher in patients with positive CPEΔN expression than in patients with negative CPEΔN expression (P=0.009). Furthermore, the overall survival rate of patients with predominant nuclear CPE expression was lower than that of patients with predominant cytoplasmic CPE expression (46.3 vs. 64.7%); however, no statistically significant difference was identified (P=0.125). Thus, the results of the current study indicated that CPEΔN may present a novel molecular biomarker for predicting recurrence and metastasis of lung adenocarcinoma, which may aid with stratifying patients by risk and thus, may facilitate individualized therapy. PMID:28101219

  1. c-Jun N-terminal kinase activity is required for efficient respiratory syncytial virus production.

    PubMed

    Caly, Leon; Li, Hong-Mei; Bogoyevitch, Marie A; Jans, David A

    2017-01-29

    Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year worldwide. With no RSV antiviral or efficacious vaccine currently available, improved understanding of the host-RSV interaction is urgently required. Here we examine the contribution to RSV infection of the host stress-regulated c-Jun N-terminal kinase (JNK), for the first time. Peak JNK1/2 phosphoactivation is observed at ∼24 h post-infection, correlating with the time of virus assembly. The release of infectious RSV virions from infected cells was significantly reduced by either JNK1/2 siRNA knockdown or treatment with the JNK-specific inhibitor, JNK-IN-VIII. High resolution microscopy confirmed RSV accumulation in the host cell cytoplasm. The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics.

  2. 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.

  3. Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration.

    PubMed

    Tasaki, Junichi; Shibata, Norito; Sakurai, Toshihide; Agata, Kiyokazu; Umesono, Yoshihiko

    2011-04-01

    The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.

  4. Synaptic transmission and plasticity require AMPA receptor anchoring via its N-terminal domain

    PubMed Central

    Watson, Jake F; Ho, Hinze; Greger, Ingo H

    2017-01-01

    AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and are selectively recruited during activity-dependent plasticity to increase synaptic strength. A prerequisite for faithful signal transmission is the positioning and clustering of AMPARs at postsynaptic sites. The mechanisms underlying this positioning have largely been ascribed to the receptor cytoplasmic C-termini and to AMPAR-associated auxiliary subunits, both interacting with the postsynaptic scaffold. Here, using mouse organotypic hippocampal slices, we show that the extracellular AMPAR N-terminal domain (NTD), which projects midway into the synaptic cleft, plays a fundamental role in this process. This highly sequence-diverse domain mediates synaptic anchoring in a subunit-selective manner. Receptors lacking the NTD exhibit increased mobility in synapses, depress synaptic transmission and are unable to sustain long-term potentiation (LTP). Thus, synaptic transmission and the expression of LTP are dependent upon an AMPAR anchoring mechanism that is driven by the NTD. DOI: http://dx.doi.org/10.7554/eLife.23024.001 PMID:28290985

  5. Inhibitors of c-Jun N-terminal kinases: JuNK no more?

    PubMed

    Bogoyevitch, Marie A; Arthur, Peter G

    2008-01-01

    The c-Jun N-terminal kinases (JNKs) have been the subject of intense interest since their discovery in the early 1990s. Major research programs have been directed to the screening and/or design of JNK-selective inhibitors and testing their potential as drugs. We begin this review by considering the first commercially-available JNK ATP-competitive inhibitor, SP600125. We focus on recent studies that have evaluated the actions of SP600125 in lung, brain, kidney and liver following exposure to a range of stress insults including ischemia/reperfusion. In many but not all cases, SP600125 administration has proved beneficial. JNK activation can also follow infection, and we next consider recent examples that demonstrate the benefits of SP600125 administration in viral infection. Additional ATP-competitive JNK inhibitors have now been described following high throughput screening of small molecule libraries, but information on their use in biological systems remains limited and thus these inhibitors will require further evaluation. Peptide substrate-competitive ATP-non-competitive inhibitors of JNK have also now been described, and we discuss the recent advances in the use of JNK inhibitory peptides in the treatment of neuronal death, diabetes and viral infection. We conclude by raising a number of questions that should be considered in the quest for JNK-specific inhibitors.

  6. High-Reynolds Number Circulation Control Testing in the National Transonic Facility

    NASA Technical Reports Server (NTRS)

    Milholen, William E., II; Jones, Gregory S.; Chan, David T.; Goodliff, Scott L.

    2012-01-01

    A new capability to test active flow control concepts and propulsion simulations at high Reynolds numbers in the National Transonic Facility at the NASA Langley Research Center is being developed. The first active flow control experiment was completed using the new FAST-MAC semi-span model to study Reynolds number scaling effects for several circulation control concepts. Testing was conducted over a wide range of Mach numbers, up to chord Reynolds numbers of 30 million. The model was equipped with four onboard flow control valves allowing independent control of the circulation control plenums, which were directed over a 15% chord simple-hinged flap. Preliminary analysis of the uncorrected lift data showed that the circulation control increased the low-speed maximum lift coefficient by 33%. At transonic speeds, the circulation control was capable of positively altering the shockwave pattern on the upper wing surface and reducing flow separation. Furthermore, application of the technique to only the outboard portion of the wing demonstrated the feasibility of a pneumatic based roll control capability.

  7. Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori.

    PubMed

    Hu, Heidi Q; Johnson, Ryan C; Merrell, D Scott; Maroney, Michael J

    2017-02-28

    The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

  8. Amount of newspaper coverage of high school athletics for boys and girls on sports page and newspaper circulation.

    PubMed

    Pedersen, Paul M; Whisenant, Warren A

    2002-02-01

    This study analyzed the amount of coverage for high school athletics in 43 newspapers with small circulation by devoting 40% of their interscholastic athletics coverage to girls in athletics, printed significantly more articles about girls' athletics than did the newspapers with medium (33%) or large (32%) circulation. Therefore, the smaller the newspaper circulation, the more equitable the coverage of athletics for girls and boys. This finding was consistent with some prior work but not all.

  9. Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion.

    PubMed

    Netter, Catharina; Weber, Gert; Benecke, Heike; Wahl, Markus C

    2009-07-01

    RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B'' proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the beta-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B'' proteins. However, unlike U1A and U2B'', some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an alpha-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.

  10. 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.

  11. C-Jun N-terminal Kinase and Apoptotic Signaling in Prostate Cancer

    DTIC Science & Technology

    2002-01-01

    hydrogen peroxide (H20 2) to induce JNK activation varied in different cell types. Pyrrolidine dithiocarbamate (PDTC), a presumed antioxidant (13,14...Down-regulation of the c-Jun N-terminal kinase (JNK) phosphatase M3/6 and activation of JNK by hydrogen peroxide and pyrrolidine dithiocarbamate...and Tan, T.-H. (2001) Down-regulation of the c-Jun N-terminal kinase (JNK) phosphatase M3/6 and activation of JNK by hydrogen peroxide and pyrrolidine

  12. Yeast strains with N-terminally truncated ribosomal protein S5: implications for the evolution, structure and function of the Rps5/Rps7 proteins.

    PubMed

    Lumsden, Thomas; Bentley, Amber A; Beutler, William; Ghosh, Arnab; Galkin, Oleksandr; Komar, Anton A

    2010-03-01

    Ribosomal protein (rp)S5 belongs to the family of the highly conserved rp's that contains rpS7 from prokaryotes and rpS5 from eukaryotes. Alignment of rpS5/rpS7 from metazoans (Homo sapiens), fungi (Saccharomyces cerevisiae) and bacteria (Escherichia coli) shows that the proteins contain a conserved central/C-terminal core region and possess variable N-terminal regions. Yeast rpS5 is 69 amino acids (aa) longer than the E. coli rpS7 protein; and human rpS5 is 48 aa longer than the rpS7, respectively. To investigate the function of the yeast rpS5 and in particular the role of its N-terminal region, we obtained and characterized yeast strains in which the wild-type yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. All mutant yeast strains were viable and displayed only moderately reduced growth rates, with the exception of the strain lacking 46 N-terminal amino acids, which had a doubling time of about 3 h. Biochemical analysis of the mutant yeast strains suggests that the N-terminal part of the eukaryotic and, in particular, yeast rpS5 may impact the ability of 40S subunits to function properly in translation and affect the efficiency of initiation, specifically the recruitment of initiation factors eIF3 and eIF2.

  13. Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein 1

    PubMed Central

    Laín, Ana; Elguezabal, Natalia; Brena, Sonia; García-Ruiz, Juan Carlos; del Palacio, Amalia; Moragues, María D; Pontón, José

    2007-01-01

    Background The diagnosis of invasive candidiasis is difficult because there are no specific clinical manifestations of the disease and colonization and infection are difficult to distinguish. In the last decade, much effort has been made to develop reliable tests for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use. Results Antibodies against a recombinant N-terminal fragment of the Candida albicans germ tube-specific antigen hyphal wall protein 1 (Hwp1) generated in Escherichia coli were detected by both immunoblotting and ELISA tests in a group of 36 hematological or Intensive Care Unit patients with invasive candidiasis and in a group of 45 control patients at high risk for the mycosis who did not have clinical or microbiological data to document invasive candidiasis. Results were compared with an immunofluorescence test to detect antibodies to C. albicans germ tubes (CAGT). The sensitivity, specificity, positive and negative predictive values of a diagnostic test based on the detection of antibodies against the N-terminal fragment of Hwp1 by immunoblotting were 27.8 %, 95.6 %, 83.3 % and 62.3 %, respectively. Detection of antibodies to the N-terminal fragment of Hwp1 by ELISA increased the sensitivity (88.9 %) and the negative predictive value (90.2 %) but slightly decreased the specificity (82.6 %) and positive predictive values (80 %). The kinetics of antibody response to the N-terminal fragment of Hwp1 by ELISA was very similar to that observed by detecting antibodies to CAGT. Conclusion An ELISA test to detect antibodies against a recombinant N-terminal fragment of the C. albicans germ tube cell wall antigen Hwp1 allows the diagnosis of invasive candidiasis with similar results to those obtained by detecting antibodies to CAGT but without the need of treating the sera to adsorb the antibodies against the cell wall surface of the blastospore. PMID:17448251

  14. N-Terminal Truncation of an Isolated Human IgG1 CH2 Domain Significantly Increases its Stability and Aggregation Resistance

    PubMed Central

    Gong, Rui; Wang, Yanping; Ying, Tianlei; Feng, Yang; Streaker, Emily; Prabakaran, Ponraj; Dimitrov, Dimiter S.

    2013-01-01

    Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5°C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak shFcRn binding to CH2 as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins. PMID:23641816

  15. Effects of prior acute exercise on circulating cytokine concentration responses to a high-fat meal.

    PubMed

    Brandauer, Josef; Landers-Ramos, Rian Q; Jenkins, Nathan T; Spangenburg, Espen E; Hagberg, James M; Prior, Steven J

    2013-08-01

    High-fat meal consumption alters the circulating cytokine profile and contributes to cardiometabolic diseases. A prior bout of exercise can ameliorate the triglyceride response to a high-fat meal, but the interactive effects of exercise and high-fat meals on cytokines that mediate cardiometabolic risk are not fully understood. We investigated the effects of prior exercise on the responses of circulating tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8, leptin, retinol-binding protein 4 (RBP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1) to a high-fat meal. Ten healthy men were studied before and 4 h after ingestion of a high-fat meal either with or without ∼50 min of endurance exercise at 70% of VO2 max on the preceding day. In response to the high-fat meal, lower leptin and higher VEGF, bFGF, IL-6, and IL-8 concentrations were evident (P < 0.05 for all). There was no effect of the high-fat meal on PlGF, TNF-α, or RBP4 concentrations. We found lower leptin concentrations with prior exercise (P < 0.05) and interactive effects of prior exercise and the high-fat meal on sFlt-1 (P < 0.05). The high-fat meal increased IL-6 by 59% without prior exercise and 218% with prior exercise (P < 0.05). In conclusion, a prior bout of endurance exercise does not affect all high-fat meal-induced changes in circulating cytokines, but does affect fasting or postprandial concentrations of IL-6, leptin, and sFlt-1. These data may reflect a salutary effect of prior exercise on metabolic responses to a high-fat meal.

  16. High-Lift Capability of Low Aspect Ratio Wings Utilizing Circulation Control and Upper Surface Blowing

    DTIC Science & Technology

    1980-07-01

    the Upper Surface Blowing (USB) and the Circulation Control Wing (CCW). Both concepts use the Coanda effect as a means of augmenting aerodynamic lift...USB), and a unique combination of the two (CCW/USB). Wing tip sails were used as a means of increasing th(, effective aspect ratio of these wings...wing tip sails are effective in reducing the induced drag of these powered- lift low aspect ratio wings under high-lift conditions. The induced drag

  17. Ocean circulation model predicts high genetic structure observed in a long-lived pelagic developer.

    PubMed

    Sunday, J M; Popovic, I; Palen, W J; Foreman, M G G; Hart, M W

    2014-10-01

    Understanding the movement of genes and individuals across marine seascapes is a long-standing challenge in marine ecology and can inform our understanding of local adaptation, the persistence and movement of populations, and the spatial scale of effective management. Patterns of gene flow in the ocean are often inferred based on population genetic analyses coupled with knowledge of species' dispersive life histories. However, genetic structure is the result of time-integrated processes and may not capture present-day connectivity between populations. Here, we use a high-resolution oceanographic circulation model to predict larval dispersal along the complex coastline of western Canada that includes the transition between two well-studied zoogeographic provinces. We simulate dispersal in a benthic sea star with a 6-10 week pelagic larval phase and test predictions of this model against previously observed genetic structure including a strong phylogeographic break within the zoogeographical transition zone. We also test predictions with new genetic sampling in a site within the phylogeographic break. We find that the coupled genetic and circulation model predicts the high degree of genetic structure observed in this species, despite its long pelagic duration. High genetic structure on this complex coastline can thus be explained through ocean circulation patterns, which tend to retain passive larvae within 20-50 km of their parents, suggesting a necessity for close-knit design of Marine Protected Area networks.

  18. Hydrogen ion titration of 12 S rape seed protein and partial N-terminal sequence of one of it's subunits.

    PubMed

    Bhushan, R; Mahesh, V K; Mallikharjun, P V

    1989-10-01

    The high molecular weight 12 S protein from rape seed was isolated in a homogeneous form and characterized. Six subunits were isolated by PAGE in the presence of SDS and 0.2 M 2-mercaptoethanol. These subunits (s1 to s6) were found in the protein in the weight ratio of 1.32:1.2:1.15:1.0:1.21:1.11. The molecular weights and first two N-terminal amino acids of the isolated subunits were 64,800 and phenylalanine, alanine (s1), 50,650 and valine, tyrosine (s2), 42,500 and phenylalanine, leucine (s3), 28,800 and threonine, glutamic acid (s4), 19,100 and cystine, isoleucine (s5) and 15,600 and alanine, phenylalanine (s6). The number of side chain carboxyl, imidazole and epsilon-amino groups were calculated from the hydrogen ion titrations, which were in agreement with the amino acid assay. Besides, the N-terminal amino acid sequence upto 43 residues for one subunit (s6) is reported using Edman degradation.

  19. Structural evidence for variable oligomerization of the N-terminal domain of cyclase-associated protein (CAP).

    PubMed

    Yusof, Adlina Mohd; Hu, Nien-Jen; Wlodawer, Alexander; Hofmann, Andreas

    2005-02-01

    Cyclase-associated protein (CAP) is a highly conserved and widely distributed protein that links the nutritional response signaling to cytoskeleton remodeling. In yeast, CAP is a component of the adenylyl cyclase complex and helps to activate the Ras-mediated catalytic cycle of the cyclase. While the N-terminal domain of CAP (N-CAP) provides a binding site for adenylyl cyclase, the C-terminal domain (C-CAP) possesses actin binding activity. Our attempts to crystallize full-length recombinant CAP from Dictyostelium discoideum resulted in growth of orthorhombic crystals containing only the N-terminal domain (residues 42-227) due to auto-proteolytic cleavage. The structure was solved by molecular replacement with data at 2.2 A resolution. The present crystal structure allows the characterization of a head-to-tail N-CAP dimer in the asymmetric unit and a crystallographic side-to-side dimer. Comparison with previously published structures of N-CAP reveals variable modes of dimerization of this domain, but the presence of a common interface for the side-to-side dimer.

  20. 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.

  1. Heterologous expression and N-terminal His-tagging processes affect the catalytic properties of staphylococcal lipases: a monolayer study.

    PubMed

    Horchani, Habib; Sabrina, Lignon; Régine, Lebrun; Sayari, Adel; Gargouri, Youssef; Verger, Robert

    2010-10-15

    The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant forms of three staphylococcal lipases (SSL, SXL and SAL3) were compared using the monomolecular film technique. A kinetic study on the dependence of the stereoselectivity of these nine lipase forms on the surface pressure was performed using the three dicaprin isomers spread in the form of monomolecular films at the air-water interface. New parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC), were introduced. The findings obtained showed that with all the lipases tested, the recombinant expression process and the N-terminal His-tag slightly affect the sn-1 preference for dicaprin enantiomers as well as the penetration capacity into monomolecular films of phosphatidylcholine but significantly decrease the catalytic rate of hydrolysis of three dicaprin isomers. This rate reduction is more pronounced at high surface pressures, i.e. at low interfacial energies. In conclusion, the effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension. In the case of the situation most commonly encountered in the literature, i.e. the heterologous expression of a tagged lipase, the rate of catalysis can be decreased by these processes by 42-83% on average in comparison with the values measured with the corresponding wild type form.

  2. Enhancing the antimicrobial activity of Sus scrofa lysozyme by N-terminal fusion of a sextuple unique homologous peptide.

    PubMed

    Zhu, Dewei; Cai, Guolin; Li, Xiaomin; Lu, Jian; Zhang, Liang

    2017-02-10

    Sus scrofa lysozyme (SSL), an important component of the pig immune system, is a potential candidate to replace antibiotics in feed. However, there is little antimicrobial activity of natural SSL against gram-negative bacteria, which limits its application. In this study, a unique peptide (A-W-V-A-W-K) with antimicrobial activity against gram-negative bacteria was discovered and purified from trypsin hydrolysate of natural SSL. This unique peptide was fused to natural SSL and the recombinant fused SSL exhibited improved activity against gram-negative bacteria. The N-terminal fusion likely increased the membrane penetrability and induced programmed bacterial cell death. The recombinant fused SSL also showed higher activity against some gram-positive bacteria with O-acetylation. By N-terminal fusion of the sextuple peptide, the anti-microbial activity, either to gram-positive or negative bacteria, of the recombinant SSL was higher than the fusion of only one copy of the peptide. This study provides a general, feasible, and highly useful strategy to enhance the antimicrobial activity of lysozyme.

  3. Interaction of thioflavin T with amyloid fibrils of apolipoprotein A-I N-terminal fragment: resonance energy transfer study.

    PubMed

    Girych, Mykhailo; Gorbenko, Galyna; Trusova, Valeriya; Adachi, Emi; Mizuguchi, Chiharu; Nagao, Kohjiro; Kawashima, Hiroyuki; Akaji, Kenichi; Lund-Katz, Sissel; Phillips, Michael C; Saito, Hiroyuki

    2014-01-01

    Apolipoprotein A-I is amenable to a number of specific mutations associated with hereditary systemic amyloidoses. Amyloidogenic properties of apoA-I are determined mainly by its N-terminal fragment. In the present study Förster resonance energy transfer between tryptophan as a donor and Thioflavin T as an acceptor was employed to obtain structural information on the amyloid fibrils formed by apoA-I variant 1-83/G26R/W@8. Analysis of the dye-fibril binding data provided evidence for the presence of two types of ThT binding sites with similar stoichiometries (bound dye to monomeric protein molar ratio ∼10), but different association constants (∼6 and 0.1μM(-1)) and ThT quantum yields in fibril-associated state (0.08 and 0.05, respectively). A β-strand-loop-β-strand structural model of 1-83/G26R/W@8 apoA-I fibrils has been proposed, with potential ThT binding sites located in the solvent-exposed grooves of the N-terminal β-sheet layer. Reasoning from the expanded FRET analysis allowing for heterogeneity of ThT binding centers and fibril polymorphism, the most probable locations of high- and low-affinity ThT binding sites were attributed to the grooves T16_Y18 and D20_L22, respectively.

  4. 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.

  5. The high latitude circulation and temperature structure of the thermosphere near solstice

    NASA Technical Reports Server (NTRS)

    Roble, R. G.; Dickinson, R. E.; Ridley, E. C.; Emery, B. A.; Hays, P. B.; Killeen, T. L.; Spencer, N. W.

    1983-01-01

    NCAR thermospheric-general-circulation-model (TGCM) computations of solar-maximum thermospheric neutral-gas temperature and circulation around the December solstice are presented and discussed. The TGCM uses a 5 x 5-deg grid and 24 constant-pressure layers, corresponding to altitudes of about 97-500 km. The results are mapped as electron-density contours, polar plots, cylindrical equidistant projections, meridional cross sections, and F-region polar plots comparing the TGCM predictions with DE-2 satellite observations. The significant differences between summer and winter high-latitude F-region winds are attributed to the ion drag momentum associated with magnetospheric convection. The TGCM wind predictions follow the same pattern as the satellite measurements but are too small; possible model corrections are considered.

  6. Helium circulator design considerations for modular high temperature gas-cooled reactor plant

    SciTech Connect

    McDonald, C.F.; Nichols, M.K.

    1986-12-01

    Efforts are in progress to develop a standard modular high temperature gas-cooled reactor (MHTGR) plant that is amenable to design certification and serial production. The MHTGR reference design, based on a steam cycle power conversion system, utilizes a 350 MW(t) annular reactor core with prismatic fuel elements. Flexibility in power rating is afforded by utilizing a multiplicity of the standard module. The circulator, which is an electric motor-driven helium compressor, is a key component in the primary system of the nuclear plant, since it facilitates thermal energy transfer from the reactor core to the steam generator; and, hence, to the external turbo-generator set. This paper highlights the helium circulator design considerations for the reference MHTGR plant and includes a discussion on the major features of the turbomachine concept, operational characteristics, and the technology base that exists in the US.

  7. Extensive Mycobacterium tuberculosis circulation in a highly endemic prison and the need for urgent environmental interventions.

    PubMed

    Sánchez, A; Huber, F D; Massari, V; Barreto, A; Camacho, L A B; Cesconi, V; Saad, M H; Larouze, B

    2012-10-01

    Aimed at assessing the circulation of Mycobacterium tuberculosis in a highly endemic prison, this 13-month prospective study was performed on strains isolated from tuberculosis (TB) cases detected passively and actively. We used X-ray screening of newly admitted inmates and mass screening at the beginning of the study and again 1 year later. Of the 94 strains genotyped by restriction fragment-length polymorphism, 79 (84·0%) belonged to one of the 12 identified clusters (2-21 strains each), including two main clusters (18 and 21 cases, respectively). A history of TB treatment was reported in 22/79 (27·8%) clustered cases. Time-space distribution of clustered cases was predominantly consistent with transmission, in micro-epidemics. Given the dominant pattern of exogenous infection and the extensive strain circulation, effective TB control should emphasize reduction of overcrowding and improvement of environmental measures as a complement to detection and treatment of cases.

  8. NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

    PubMed

    Petkowski, Janusz J; Bonsignore, Lindsay A; Tooley, John G; Wilkey, Daniel W; Merchant, Michael L; Macara, Ian G; Schaner Tooley, Christine E

    2013-12-15

    NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

  9. NRMT2 is an N-terminal monomethylase that primes for its homolog NRMT1

    PubMed Central

    Petkowski, Janusz J.; Bonsignore, Lindsay A.; Tooley, John G.; Wilkey, Daniel W.; Merchant, Michael L.; Macara, Ian G.; Schaner Tooley, Christine E.

    2014-01-01

    N-terminal RCC1 methyltransferase (NRMT) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homolog of NRMT, Methyltransferase-like protein 11B (METTL11B, now renamed NRMT2). We show here for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and mass spectrometry experiments indicate they differ in their specific catalytic functions. While NRMT1 is a distributive methyltransferase that can mono-, di-, and trimethylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation. PMID:24090352

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

    PubMed

    Edamatsu, M

    2016-02-11

    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.

  11. Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane

    PubMed Central

    Lee, Chien-Hsien; Chou, Chia-Cheng; Hsu, Min-Feng; Wang, Andrew H.-J.

    2015-01-01

    In silico algorithms have been the common approach for transmembrane (TM) protein topology prediction. However, computational tools may produce questionable results and experimental validation has proven difficult. Although biochemical strategies are available to determine the C-terminal orientation of TM proteins, experimental strategies to determine the N-terminal orientation are still limited but needed because the N-terminal end is essential for membrane targeting. Here, we describe a new and easy method to effectively determine the N-terminal orientation of the target TM proteins in Escherichia coli plasma membrane environment. D94N, the mutant of bacteriorhodopsin from Haloarcula marismortui, can be a fusion partner to increase the production of the target TM proteins if their N-termini are in cytoplasm (Nin orientation). To create a suitable linker for orientating the target TM proteins with the periplasmic N-termini (Nout orientation) correctly, we designed a three-TM-helix linker fused at the C-terminus of D94N fusion partner (termed D94N-3TM) and found that D94N-3TM can specifically improve the production of the Nout target TM proteins. In conclusion, D94N and D94N-3TM fusion partners can be applied to determine the N-terminal end of the target TM proteins oriented either Nin or Nout by evaluating the net expression of the fusion proteins. PMID:26462555

  12. Cytoplasmic c-Jun N-terminal immunoreactivity: a hallmark of retinal apoptosis.

    PubMed

    Chiarini, Luciana B; de Freitas, Fabíola G; Leal-Ferreira, Mona Lisa; Tolkovsky, Aviva; Linden, Rafael

    2002-12-01

    1. We investigated the association of c-Jun with apoptosis within retinal tissue. Explants of the retina of neonatal rats were subject to a variety of procedures that cause apoptosis of specific classes of retinal cells at distinct stages of differentiation. The expression of c-Jun was detected by Western Blot, and immunohistochemistry was done with antibodies made for either N-terminal or C-terminal domains of c-Jun, and correlated with apoptosis detected either by chromatin condensation or by in situ nick end labeling of fragmented DNA. 2. c-Jun protein content was increased in retinal tissue subject to induction of both photoreceptor and ganglion cell death. 3. c-Jun N-terminal immunoreactivity was found mainly in the cytoplasm of apoptotic cells regardless of cell type, of the stage of differentiation, including proliferating cells, or of the means of induction of apoptosis. 4. The data are consistent with the hypothesis that c-Jun is involved in the control of cell death in retinal tissue, but other proteins that cross-react with c-Jun N-terminal antibodies may also be major markers of retinal apoptosis. 5. Antibodies directed to c-Jun N-terminal (aa 91-105) are useful tools to follow apoptotic changes in retinal tissue.

  13. Ascorbate as a pro-oxidant: mild N-terminal modification with vinylboronic acids.

    PubMed

    Ohata, Jun; Ball, Zachary T

    2017-02-04

    We describe divergent reactivity of vinylboronic acids for protein modification. In addition to previously reported copper-catalyzed backbone N-H modification, ascorbate in air mediates N-terminal functionalization with the same vinylboronate reagents. This mild and selective aqueous reactivity enables selective single-modification of the B chain of human insulin.

  14. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    PubMed

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

  15. Lymph node and circulating T cell characteristics are strongly correlated in end-stage renal disease patients, but highly differentiated T cells reside within the circulation.

    PubMed

    Dedeoglu, B; de Weerd, A E; Huang, L; Langerak, A W; Dor, F J; Klepper, M; Verschoor, W; Reijerkerk, D; Baan, C C; Litjens, N H R; Betjes, M G H

    2017-05-01

    Ageing is associated with changes in the peripheral T cell immune system, which can be influenced significantly by latent cytomegalovirus (CMV) infection. To what extent changes in circulating T cell populations correlate with T cell composition of the lymph node (LN) is unclear, but is crucial for a comprehensive understanding of the T cell system. T cells from peripheral blood (PB) and LN of end-stage renal disease patients were analysed for frequency of recent thymic emigrants using CD31 expression and T cell receptor excision circle content, relative telomere length and expression of differentiation markers. Compared with PB, LN contained relatively more CD4(+) than CD8(+) T cells (P < 0·001). The percentage of naive and central memory CD4(+) and CD8(+) T cells and thymic output parameters showed a strong linear correlation between PB and LN. Highly differentiated CD28(null) T cells, being CD27(-) , CD57(+) or programmed death 1 (PD-1(+) ), were found almost exclusively in the circulation but not in LN. An age-related decline in naive CD4(+) and CD8(+) T cell frequency was observed (P = 0·035 and P = 0·002, respectively) within LN, concomitant with an increase in central memory CD8(+) T cells (P = 0·033). Latent CMV infection increased dramatically the frequency of circulating terminally differentiated T cells, but did not alter T cell composition and ageing parameters of LN significantly. Overall T cell composition and measures of thymic function in PB and LN are correlated strongly. However, highly differentiated CD28(null) T cells, which may comprise a large part of circulating T cells in CMV-seropositive individuals, are found almost exclusively within the circulation.

  16. Gulf of Mexico circulation within a high-resolution numerical simulation of the North Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Romanou, Anastasia; Chassignet, Eric P.; Sturges, Wilton

    2004-01-01

    The Gulf of Mexico circulation is examined from the results of a high-resolution (1/12°) North Atlantic simulation using the Miami Isopycnic Coordinate Ocean Model. The motivation for this paper is twofold: first, we validate the model's performance in the Gulf of Mexico by comparing the model fields to past and recent observations, and second, given the good agreement with the observed Gulf of Mexico surface circulation and Loop Current variability, we expand the discussion and analysis of the model circulation to areas that have not been extensively observed/analyzed, such as the vertical structure of the Loop Current and associated eddies, especially the deep circulation below 1500 m. The interval between successive model eddy sheddings is 3 to 15 months, the eddy diameters range between 140 and 500 km, the life span is about 1 year, and the translational speeds are 2-3 km d-1, in good agreement with observations. Areas of high cyclonic eddy occurrence in the model are southwest of Florida, the Loop Current boundary, and the western Campeche Bay area. The cyclonic eddy diameters range between 50 and 375 km, the orbital speeds range between 1 and 55 cm s-1, the translational speeds range between 0.5 and 14 km d-1, and the eddy life spans range between 1 and 3 months. The vertical structure of the temperature and salinity of each modeled eddy, from the moment it is shed until it disintegrates in the western Gulf of Mexico, is in agreement with the few available observations. Below 1500 m, deep cyclonic eddies are associated with the surface Loop Current anticyclones. The eddy variability is consistent with Rossby waves propagating westward, and there is bottom intensification of the flow close to steep topography. Overall, we show that this very high horizontal resolution isopycnic coordinate ocean model, which is able to produce a quite realistic surface circulation for the North and equatorial Atlantic, is also able to reproduce well the smaller-scale, basin

  17. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

    PubMed Central

    Liu, Xiang; Zaid, Ali; Goh, Lucas Y. H.; Hobson-Peters, Jody; Hall, Roy A.; Merits, Andres

    2017-01-01

    ABSTRACT Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. PMID:28223458

  18. N-terminal amino acid sequences of D-serine deaminases of wild-type and operator-constitutive strains of Escherichia coli K-12.

    PubMed Central

    Heincz, M C; McFall, E

    1975-01-01

    The N-terminal amino acid sequences of the D-serine deaminases from strains of Escherichia coli K-12 that harbor wild-type and high-level constitutive catabolite-insensitive operator-initiator regions are identical: Met-Ser-GluNH2-Ser-Gly-Arg-His-Cys. This result indicates that the operator-initiator region is probably distinct from the D-serine deaminase structural gene. Images PMID:1099073

  19. NASA High-Reynolds Number Circulation Control Research - Overview of CFD and Planned Experiments

    NASA Technical Reports Server (NTRS)

    Milholen, W. E., II; Jones, Greg S.; Cagle, Christopher M.

    2010-01-01

    A new capability to test active flow control concepts and propulsion simulations at high Reynolds numbers in the National Transonic Facility at the NASA Langley Research Center is being developed. This technique is focused on the use of semi-span models due to their increased model size and relative ease of routing high-pressure air to the model. A new dual flow-path high-pressure air delivery station has been designed, along with a new high performance transonic sem -si pan wing model. The modular wind tunnel model is designed for testing circulation control concepts at both transonic cruise and low-speed high-lift conditions. The ability of the model to test other active flow control techniques will be highlighted. In addition, a new higher capacity semi-span force and moment wind tunnel balance has been completed and calibrated to enable testing at transonic conditions.

  20. TOPK is highly expressed in circulating tumor cells, enabling metastasis of prostate cancer

    PubMed Central

    Shi, Changhong; Hu, Peizhen; Yan, Wei; Wang, Zhe; Duan, Qiuhong; Lu, Fan; Qin, Lipeng; Lu, Tao; Xiao, Juanjuan; Wang, Yingmei; Zhu, Feng; Shao, Chen

    2015-01-01

    Circulating tumor cells (CTCs) are important for metastasis in prostate cancer. T-LAK cell-originated protein kinase (TOPK) is highly expressed in cancer cells. Herein, we established a xenograft animal model, isolated and cultured the CTCs, and found CTCs have significantly greater migratory capacity than parental cells. TOPK is more highly expressed in the CTCs than in parental cells and is also highly expressed in the metastatic nodules caused by CTCs in mice. Knocking down TOPK decreased the migration of CTCs both in vitro and in vivo. TOPK was modulated by the PI3K/PTEN and ERK pathways during the metastasis of prostate cancer. High levels of TOPK in the tumors of patients were correlated with advanced stages of prostate cancer, especially for high-risk patients of Gleason score≥8, PSA>20ng/ml. In summary, TOPK was speculated to be one of a potential marker and therapeutic target in advanced prostate cancer. PMID:25881543

  1. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Mavoungou, Chrystelle; Israel, Lars; Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state (1)H-(15)N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an alpha-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by beta-strands.

  2. Amenorrhoea, Galactorrhoea, and Primary Hypothyroidism with High Circulating Levels of Prolactin

    PubMed Central

    Edwards, C. R. W.; Forsyth, Isabel A.; Besser, G. M.

    1971-01-01

    A 22-year-old woman with primary hypothyroidism developed amenorrhoea and galactorrhoea during oral contraceptive therapy. Investigation showed high levels of circulating prolactin which rose in response to insulin-induced hypoglycaemia and were suppressed by an oral glucose load. After treatment with thyroxine normal periods returned, the galactorrhoea improved, and the prolactin levels fell to undetectable levels. The results of the prolactin and human growth hormone assays confirm that it is possible to distinguish between human growth hormone and human prolactin. PMID:5105903

  3. Multiscale dynamical analysis of a high-resolution numerical model simulation of the Solomon Sea circulation

    NASA Astrophysics Data System (ADS)

    Djath, Bughsin'; Verron, Jacques; Melet, Angelique; Gourdeau, Lionel; Barnier, Bernard; Molines, Jean-Marc

    2014-09-01

    A high 1/36° resolution numerical model is used to study the ocean circulation in the Solomon Sea. An evaluation of the model with (the few) available observation shows that the 1/36° resolution model realistically simulates the Solomon Sea circulations. The model notably reproduces the high levels of mesoscale eddy activity observed in the Solomon Sea. With regard to previous simulations at 1/12° resolution, the average eddy kinetic energy levels are increased by up to ˜30-40% in the present 1/36° simulation, and the enhancement extends at depth. At the surface, the eddy kinetic energy level is maximum in March-April-May and is minimum in December-January-February. The high subsurface variability is related to the variability of the western boundary current (New Guinea Coastal Undercurrent). Moreover, the emergence of submesoscales is clearly apparent in the present simulations. A spectral analysis is conducted in order to evidence and characterize the modeled submesoscale dynamics and to provide a spectral view of scales interactions. The corresponding spectral slopes show a strong consistency with the Surface Quasi-Geostrophic turbulence theory.

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

    PubMed

    Viriyakosol, S; Kirkland, T N

    1996-02-01

    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.

  5. 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.

  6. Bordetella dermonecrotic toxin binds to target cells via the N-terminal 30 amino acids.

    PubMed

    Fukui-Miyazaki, Aya; Ohnishi, Shinya; Kamitani, Shigeki; Abe, Hiroyuki; Horiguchi, Yasuhiko

    2011-03-01

    Bordetella dermonecrotic toxin (DNT) affects the biological function of host cells by activating intracellular Rho GTPases. The toxin binds to unidentified receptor(s) via 54 N-terminal amino acids, undergoes intramolecular cleavage on the C-terminal side of Arg(44) by furin or furin-like protease, and eventually enters the cytoplasm where the Rho GTPases reside. The binding to the receptor(s) and intramolecular cleavage are essential for DNT to intoxicate cells, and the 54 amino-acid binding domain encompasses the cleavage site, however, it is unclear whether these two events are related. In this study, we could narrow down the cell-binding domain to the N-terminal amino acids 2-30. The region does not contain the furin-recognition site, indicating that the cell binding and the intramolecular cleavage are independent events.

  7. 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.

  8. 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.

  9. NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1.

    PubMed

    Parnham, Stuart; Gaines, William A; Duggan, Brendan M; Marcotte, William R; Hennig, Mirko

    2011-10-01

    The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35-40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all (1)H, (13)C, and (15)N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.

  10. Thermostabilization of glutamate decarboxylase B from Escherichia coli by structure-guided design of its pH-responsive N-terminal interdomain.

    PubMed

    Jun, Chanha; Joo, Jeong Chan; Lee, Jung Heon; Kim, Yong Hwan

    2014-03-20

    Glutamate decarboxylase B (GadB) from Escherichia coli is a highly active biocatalyst that can convert l-glutamate to γ-aminobutyrate (GABA), a precursor of 2-pyrrolidone (a monomer of Nylon 4). In contrast to vigorous studies of pH shifting of GadB, mesophilic GadB has not been stabilized by protein engineering. In this study, we improved the thermostability of GadB through structural optimization of its N-terminal interdomain. According to structural analysis, the N-terminal fourteen residues (1-14) of homo-hexameric GadB formed a triple-helix bundle interdomain at acidic pH and contributed to the thermostability of GadB in preliminary tests as the pH shifted from 7.6 to 4.6. GadB thermostabilization was achieved by optimization of hydrophobic and electrostatic interactions at the N-terminal interdomain. A triple mutant (GadB-TM: Gln5Asp/Val6Ile/Thr7Glu) showed higher thermostability than the wild-type (GadB-WT), i.e., 7.9 and 7.7°C increases in the melting temperature (Tm) and the temperature at which 50% of the initial activity remained after 10min incubation (T50(10)), respectively. The triple mutant showed no reduction of catalytic activity in enzyme kinetics. Molecular dynamics (MD) simulation at high temperature showed that reinforced interactions of the triple mutant rigidified the N-terminal interdomain compared to the wild-type, leading to GadB thermostabilization.

  11. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    PubMed

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

  12. 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

    2013-06-07

    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.

  13. Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

    SciTech Connect

    Krejciríková, Veronika; Pachl, Petr; Fábry, Milan; Malý, Petr; Rezácová, Pavlína; Brynda, Jirí

    2011-11-18

    Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{sub d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.

  14. 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

  15. c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

    DTIC Science & Technology

    2013-10-01

    Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR: Dr. Philip LoGrasso CONTRACTING ORGANIZATION: The Scripps Research... Lateral Sclerosis ” 5a. CONTRACT NUMBER W81XWH-12-1-0431 5b. GRANT NUMBER W81XWH-12-1-0431 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Philip...Annual 3. DATES COVERED 30September2012-29September2013 4. TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic

  16. Variational data assimilation system with nesting model for high resolution ocean circulation

    NASA Astrophysics Data System (ADS)

    Ishikawa, Yoichi; In, Teiji; Nakada, Satoshi; Nishina, Kei; Igarashi, Hiromichi; Hiyoshi, Yoshimasa; Sasaki, Yuji; Wakamatsu, Tsuyoshi; Awaji, Toshiyuki

    2015-10-01

    To obtain the high-resolution analysis fields for ocean circulation, a new incremental approach is developed using a four-dimensional variational data assimilation system with nesting models. The results show that there are substantial biases when using a classical method combined with data assimilation and downscaling, caused by different dynamics resulting from the different resolutions of the models used within the nesting models. However, a remarkable reduction in biases of the low-resolution model relative to the high-resolution model was observed using our new approach in narrow strait regions, such as the Tsushima and Tsugaru straits, where the difference in the dynamics represented by the high- and low-resolution models is substantial. In addition, error reductions are demonstrated in the downstream region of these narrow channels associated with the propagation of information through the model dynamics.

  17. 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.

  18. Critical roles of CTP synthase N-terminal in cytoophidium assembly.

    PubMed

    Huang, Yong; Wang, Jin-Jun; Ghosh, Sanjay; Liu, Ji-Long

    2017-03-22

    Several metabolic enzymes assemble into distinct intracellular structures in prokaryotes and eukaryotes suggesting an important functional role in cell physiology. The CTP-generating enzyme CTP synthase forms long filamentous structures termed cytoophidia in bacteria, yeast, fruit flies and human cells independent of its catalytic activity. However, the amino acid determinants for protein-protein interaction necessary for polymerisation remained unknown. In this study, we systematically analysed the role of the conserved N-terminal of Drosophila CTP synthase in cytoophidium assembly. Our mutational analyses identified three key amino acid residues within this region that play an instructive role in organisation of CTP synthase into a filamentous structure. Co-transfection assays demonstrated formation of heteromeric CTP synthase filaments which is disrupted by protein carrying a mutated N-terminal alanine residue thus revealing a dominant-negative activity. Interestingly, the dominant-negative activity is supressed by the CTP synthase inhibitor DON. Furthermore, we found that the amino acids at the corresponding position in the human protein exhibit similar properties suggesting conservation of their function through evolution. Our data suggest that cytoophidium assembly is a multi-step process involving N-terminal-dependent sequential interactions between correctly folded structural units and provide insights into the assembly of these enigmatic structures.

  19. Molecular Basis of Substrate Specific Acetylation by N-Terminal Acetyltransferase NatB.

    PubMed

    Hong, Haiyan; Cai, Yongfei; Zhang, Shijun; Ding, Hongyan; Wang, Haitao; Han, Aidong

    2017-04-04

    The NatB N-terminal acetyltransferase specifically acetylates the N-terminal group of substrate protein peptides starting with Met-Asp/Glu/Asn/Gln. How NatB recognizes and acetylates these substrates remains unknown. Here, we report crystal structures of a NatB holoenzyme from Candida albicans in the presence of its co-factor CoA and substrate peptides. The auxiliary subunit Naa25 of NatB forms a horseshoe-like deck to hold specifically its catalytic subunit Naa20. The first two amino acids Met and Asp of a substrate peptide mediate the major interactions with the active site in the Naa20 subunit. The hydrogen bonds between the substrate Asp and pocket residues of Naa20 are essential to determine the NatB substrate specificity. Moreover, a hydrogen bond between the amino group of the substrate Met and a carbonyl group in the Naa20 active site directly anchors the substrate toward acetyl-CoA. Together, these structures define a unique molecular mechanism of specific N-terminal acetylation acted by NatB.

  20. Mechanism of N-terminal modulation of activity at the melanocortin-4 receptor GPCR

    PubMed Central

    Ersoy, Baran A; Pardo, Leonardo; Zhang, Sumei; Thompson, Darren A; Millhauser, Glenn; Govaerts, Cedric; Vaisse, Christian

    2013-01-01

    Most of our understanding of G protein–coupled receptor (GPCR) activation has been focused on the direct interaction between diffusible ligands and their seven-transmembrane domains. However, a number of these receptors depend on their extracellular N-terminal domain for ligand recognition and activation. To dissect the molecular interactions underlying both modes of activation at a single receptor, we used the unique properties of the melanocortin-4 receptor (MC4R), a GPCR that shows constitutive activity maintained by its N-terminal domain and is physiologically activated by the peptide α-melanocyte stimulating hormone (αMSH). We find that activation by the N-terminal domain and αMSH relies on different key residues in the transmembrane region. We also demonstrate that agouti-related protein, a physiological antagonist of MC4R, acts as an inverse agonist by inhibiting N terminus–mediated activation, leading to the speculation that a number of constitutively active orphan GPCRs could have physiological inverse agonists as sole regulators. PMID:22729149

  1. 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

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

    SciTech Connect

    Marcianò, G.; Huang, D. T.

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. 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.

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

    PubMed

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

    2013-01-01

    Ryanodine receptors are large channels that release Ca(2+) 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.

  4. Bepridil opens the regulatory N-terminal lobe of cardiac troponin C

    PubMed Central

    Li, Yu; Love, Michael L.; Putkey, John A.; Cohen, Carolyn

    2000-01-01

    Cardiac troponin C (cTnC) is the calcium-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of congestive heart failure. This calmodulin-like protein consists of two lobes connected by a central linker; each lobe contains two EF-hand domains. The regulatory N-terminal lobe of cTnC, unlike that of skeletal troponin C (sTnC), contains only one functional EF-hand and does not open fully upon the binding of Ca2+. We have determined the crystal structure of cTnC, with three bound Ca2+ ions, complexed with the calcium-sensitizer bepridil, to 2.15-Å resolution. In contrast to apo- and 3Ca2+-cTnC, the drug-bound complex displays a fully open N-terminal lobe similar to the N-terminal lobes of 4Ca2+-sTnC and cTnC bound to a C-terminal fragment of cardiac troponin I (residues 147–163). The closing of the lobe is sterically hindered by one of the three bound bepridils. Our results provide a structural basis for the Ca2+-sensitizing effect of bepridil and reveal the details of a distinctive two-stage mechanism for Ca2+ regulation by troponin C in cardiac muscle. PMID:10792039

  5. High-Southern Latitudes Sulfur Cycle in an Atmospheric General Circulation Model

    NASA Astrophysics Data System (ADS)

    Cosme, E.; Genthon, C.; Martinerie, P.; Boucher, O.; Pham, M.

    2002-05-01

    This modeling study (Cosme et al., Sulfur cycle in the high southern latitudes in the LMD-ZT General Circulation Model, submitted to JGR) was motivated by the recent publication of annual time-scale records of dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) in Antarctica, completing the available series of sulfate and methanesulfonic acid (MSA). Sulfur chemistry has been incorporated in the Laboratoire de Météorologie Dynamique Atmospheric General Circulation Model (AGCM), LMD-ZT, with high resolution and improved physics in the high-southern latitudes. The model predicts the concentration of 6 major sulfur species through emissions, transport, wet and dry deposition and chemistry in both gaseous and aqueous phases. Model results are broadly realistic when compared with measurements in air and snow or ice, and to results of other modeling studies, at high- and mid- southern latitudes. Although not corrected in this work, defects are identified and discussed: Atmospheric MSA concentrations are underestimated and DMSO concentrations are overestimated in summer, reflecting the lack of a DMSO sink leading to MSA; the deposition scheme used in the model may not be adapted to polar regions; DMS concentrations are underestimated in winter, and the model does not adequately reproduces interannual variability. Oceanic DMS sources appear deciding for the description of the sulfur cycle in these regions. The model suggests that ground atmospheric DMS concentrations are higher in winter than in summer, in a large part of central Antarctica. In the high-southern latitudes, high loads of DMS and DMSO are found and the main chemical sink of sulfur dioxide (SO2) is aqueous oxidation by ozone (O3), whereas oxidation by hydrogen peroxide (H2O2) dominates at the global scale.

  6. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

    PubMed Central

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23–230) as detected by [1H, 15N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif. PMID:27341298

  7. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    PubMed

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-06-24

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

  8. Design of a high-pressure circulating pump for viscous liquids.

    PubMed

    Seifried, Bernhard; Temelli, Feral

    2009-07-01

    The design of a reciprocating dual action piston pump capable of circulating viscous fluids at pressures of up to 34 MPa (5000 psi) and temperatures up to 80 degrees C is described. The piston of this pump is driven by a pair of solenoids energized alternatively by a 12 V direct current power supply controlled by an electronic controller facilitating continuously adjustable flow rates. The body of this seal-less pump is constructed using off-the-shelf parts eliminating the need for custom made parts. Both the electronic controller and the pump can be assembled relatively easily. Pump performance has been evaluated at room temperature (22 degrees C) and atmospheric pressure using liquids with low and moderately high viscosities, such as ethanol and corn oil, respectively. At ambient conditions, the pump delivered continuous flow of ethanol and corn oil at a flow rate of up to 170 and 17 cm3/min, respectively. For pumping viscous fluids comparable to corn oil, an optimum reciprocation frequency was ascertained to maximize flow rate. For low viscosity liquids such as ethanol, a linear relationship between the flow rate and reciprocation frequency was determined up to the maximum reciprocation frequency of the pump. Since its fabrication, the pump has been used in our laboratory for circulating triglycerides in contact with supercritical carbon dioxide at pressures of up to 25 MPa (3600 psi) and temperatures up to 70 degrees C on a daily basis for a total of more than 1500 h of operation functioning trouble free.

  9. NMR structure of the (1-51) N-terminal domain of the HIV-1 regulatory protein Vpr.

    PubMed

    Wecker, K; Roques, B P

    1999-12-01

    The human immunodeficiency virus type 1 (HIV-1) genome encodes a highly conserved 16 kDa regulatory gene product, Vpr (viral protein of regulation, 96 amino acid residues), which is incorporated into virions, in quantities equivalent to those of the viral Gag proteins. In the infected cells, Vpr is believed to function in the early phase of HIV-1 replication, including nuclear migration of preintegration complex, transcription of the provirus genome and viral multiplication by blocking cells in the G2 phase. Vpr has a critical role in long-term AIDS disease by inducing infection in nondividing cells such as monocytes and macrophages. Mutations have suggested that the N-terminal domain of Vpr encompassing the first 40 residues could be required for nuclear localization, packaging into virions and binding of transcription factor (TFIIB, Sp1), viral proteins (p6) and cellular proteins (RIP1, UNG, karyopherins). To gain insight into the structure-function relationship of Vpr, (1-51)Vpr was synthesized and its structure analyzed by circular dichroism and two-dimensional 1H NMR in aqueous trifluoroethanol (30%) solution and refined by restrained molecular dynamics. The structure is characterized by three turns around the first three prolines, Pro5, Pro10, Pro14, followed by a long amphipathic alpha helix-turn-alpha helix (Asp17-Ile46) motif ended by a turn extending from Tyr47 to Thr49. The alpha helix-turn-alpha helix motif and the amphipathic helix are well known for being implicated in protein-protein or protein-nucleic acid interaction. Therefore structural characteristics of the (1-51) N-terminal fragment of Vpr could explain why this region of Vpr plays a role in several biological functions of this protein.

  10. Copper complex species within a fragment of the N-terminal repeat region in opossum PrP protein.

    PubMed

    Vagliasindi, Laura I; Arena, Giuseppe; Bonomo, Raffaele P; Pappalardo, Giuseppe; Tabbì, Giovanni

    2011-03-21

    A spectroscopic (UV-Vis, CD and EPR), thermodynamic and voltammetric study of the copper(ii) complexes with the Ac-PHPGGSNWGQ-NH(2) polypeptide (L), a fragment of the opossum PrP protein N-terminal four-repeat region, was carried out in aqueous solution. It suggests the formation of a highly distorted [Cu(L)H(-2)] complex species in the neutral region, the stereochemistry of which is ascribable to a square base pyramid and a CuN(3)O(2) chromophore, resulting from the coordination of a histidine imidazole and two peptide nitrogen atoms and probably oxygen atoms from water molecules. At basic pH values a [Cu(L)H(-3)](-) species with a pseudo-octahedral geometry was also obtained, with four nitrogen donor atoms in its equatorial plane, coming from the histidine residue and from peptidic nitrogen atoms. Interestingly, at pH values relatively higher than the neutrality, the coordination sphere of the copper complex in the [Cu(L)H(-2)] species changes its stereochemistry towards a pseudo-octahedron, as suggested by the change in the parallel copper hyperfine coupling constant of the EPR spectra at low temperature. A slight difference in the redox potentials between this two-faced [Cu(L)H(-2)] complex species seems to confirm this behaviour. Both potentiometric and spectroscopic data were compared with the analogous species obtained with the Ac-PHGGGWGQ-NH(2) peptide, belonging to the octarepeat domain of the human prion protein (hPrP) N-terminal region. The [Cu(L)H(-2)] species formed by the Ac-PHPGGSNWGQ-NH(2) decapeptide, having a slightly lower stability, turned out to be less abundant and to exist within a narrow pH range.

  11. The cyanobacterial cell division factor Ftn6 contains an N-terminal DnaD-like domain

    PubMed Central

    Marbouty, Martial; Saguez, Cyril; Chauvat, Franck

    2009-01-01

    Background DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation. Results In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain), exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271). We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions. Conclusion Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays. PMID:19698108

  12. Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site*

    PubMed Central

    Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos; Lee, David; Sidhu, Sachdev S.; Li, Sheng; Lai, Jonathan R.; Gavathiotis, Evripidis

    2016-01-01

    The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation. PMID:26565029

  13. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design.

    PubMed

    Taylor, Adam; Liu, Xiang; Zaid, Ali; Goh, Lucas Y H; Hobson-Peters, Jody; Hall, Roy A; Merits, Andres; Mahalingam, Suresh

    2017-02-21

    Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.IMPORTANCE CHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain

  14. Correction of Excessive Precipitation Over Steep and High Mountains in a General Circulation Model

    NASA Technical Reports Server (NTRS)

    Chao, Winston C.

    2012-01-01

    Excessive precipitation over steep and high mountains (EPSM) is a well-known problem in GCMs and meso-scale models. This problem impairs simulation and data assimilation products. Among the possible causes investigated in this study, we found that the most important one, by far, is a missing upward transport of heat out of the boundary layer due to the vertical circulations forced by the daytime upslope winds, which are forced by the heated boundary layer on subgrid-scale slopes. These upslope winds are associated with large subgrid-scale topographic variation, which is found over steep and high mountains. Without such subgridscale heat ventilation, the resolvable-scale upslope flow in the boundary layer generated by surface sensible heat flux along the mountain slopes is excessive. Such an excessive resolvablescale upslope flow combined with the high moisture content in the boundary layer results in excessive moisture transport toward mountaintops, which in turn gives rise to EPSM. Other possible causes of EPSM that we have investigated include 1) a poorly-designed horizontal moisture flux in the terrain-following coordinates, 2) the condition for cumulus convection being too easily satisfied at mountaintops, 3) the presence of conditional instability of the computational kind, and 4) the absence of blocked flow drag. These are all minor or inconsequential. We have parameterized the ventilation effects of the subgrid-scale heated-slope-induced vertical circulation (SHVC) by removing heat from the boundary layer and depositing it in layers higher up when the topographic variance exceeds a critical value. Test results using NASA/Goddard's GEOS-S GCM have shown that this largely solved the EPSM problem.

  15. Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

    PubMed Central

    Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B.; Goldman, Jonathan; Rao, Jianyu; Sledge, George W.; Pegram, Mark D.; Sheth, Shruti; Jeffrey, Stefanie S.; Kulkarni, Rajan P.; Sollier, Elodie; Di Carlo, Dino

    2016-01-01

    Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells. PMID:26863573

  16. Elevation of Circulating miR-210-3p in High-Altitude Hypoxic Environment

    PubMed Central

    Yan, Yan; Wang, Cheng; Zhou, Wanqing; Shi, Yonghui; Guo, Pengtao; Liu, Yuxiu; Wang, Junjun; Zhang, Chen-Yu; Zhang, Chunni

    2016-01-01

    Background: The induction of miR-210-3p, a master hypoxamir, is a consistent feature of the hypoxic response in both normal and malignant cells. However, whether miR-210-3p acts as a circulating factor in response to a hypoxic environment remains unknown. The current study aimed to examine the effect of a high-altitude hypoxic environment on circulating miR-210-3p. Methods: We examined and compared the levels of miR-210-3p using TaqMan-based qRT-PCR in both peripheral blood cells and plasma from 84 ethnic Chinese Tibetans residing at 3560 m, 46 newly arrived migrant Han Chinese (Tibet Han) and 82 Han Chinese residing at 8.9 m (Nanjing Han). Furthermore, we analyzed the correlations of miR-210-3p with hematological indices. Results: The relative concentrations of miR-210-3p to internal reference U6 in blood cells were significantly higher in the Tibet Han group (1.01 ± 0.11, P < 0.001) and in the Tibetan group (1.17 ± 0.09, P < 0.001) than in the Nanjing Han group (0.51 ± 0.04). The absolute concentrations of plasma miR-210-3p were also markedly elevated in the Tibet Han group (503.54 ± 42.95 fmol/L, P = 0.004) and in the Tibetan group (557.78 ± 39.84 fmol/L, P < 0.001) compared to the Nanjing Han group (358.39 ± 16.16 fmol/L). However, in both blood cells and plasma, miR-210-3p levels were not significantly different between the Tibet Han group and the Tibetan group (P = 0.280, P = 0.620, respectively). Plasma miR-210-3p concentrations were positively correlated with miR-210-3p levels in blood cells (r = 0.192, P = 0.005). Furthermore, miR-210-3p levels in both blood cells and plasma showed strong positive correlations with red blood cell counts and hemoglobin and hematocrit values. Conclusion: These data demonstrated, for the first time, that miR-210-3p might act as a circulating factor in response to hypoxic environments and could be associated with human adaptation to life at high altitudes. PMID:27014085

  17. Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

    PubMed Central

    Henderson, L E; Sowder, R; Smythers, G; Benveniste, R E; Oroszlan, S

    1985-01-01

    A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C

  18. Venus atmosphere simulated by a high-resolution general circulation model

    NASA Astrophysics Data System (ADS)

    Sugimoto, Norihiko

    2016-07-01

    An atmospheric general circulation model (AGCM) for Venus on the basis of AFES (AGCM For the Earth Simulator) have been developed (e.g., Sugimoto et al., 2014a) and a very high-resolution simulation is performed. The highest resolution of the model is T319L120; 960 times 480 horizontal grids (grid intervals are about 40 km) with 120 vertical layers (layer intervals are about 1 km). In the model, the atmosphere is dry and forced by the solar heating with the diurnal and semi-diurnal components. The infrared radiative process is simplified by adopting Newtonian cooling approximation. The temperature is relaxed to a prescribed horizontally uniform temperature distribution, in which a layer with almost neutral static stability observed in the Venus atmosphere presents. A fast zonal wind in a solid-body rotation is given as the initial state. Starting from this idealized superrotation, the model atmosphere reaches a quasi-equilibrium state within 1 Earth year and this state is stably maintained for more than 10 Earth years. The zonal-mean zonal flow with weak midlatitude jets has almost constant velocity of 120 m/s in latitudes between 45°S and 45°N at the cloud top levels, which agrees very well with observations. In the cloud layer, baroclinic waves develop continuously at midlatitudes and generate Rossby-type waves at the cloud top (Sugimoto et al., 2014b). At the polar region, warm polar vortex zonally surrounded by a cold latitude band (cold collar) is well reproduced (Ando et al., 2016). As for horizontal kinetic energy spectra, divergent component is broadly (k>10) larger than rotational component compared with that on Earth (Kashimura et al., in preparation). Finally, recent results for thermal tides and small-scale waves will be shown in the presentation. Sugimoto, N. et al. (2014a), Baroclinic modes in the Venus atmosphere simulated by GCM, Journal of Geophysical Research: Planets, Vol. 119, p1950-1968. Sugimoto, N. et al. (2014b), Waves in a Venus general

  19. Molecular Determinants of the N-Terminal Acetyltransferase Naa60 Anchoring to the Golgi Membrane.

    PubMed

    Aksnes, Henriette; Goris, Marianne; Strømland, Øyvind; Drazic, Adrian; Waheed, Qaiser; Reuter, Nathalie; Arnesen, Thomas

    2017-02-14

    Nα-acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) since it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N-termini of transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to study secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C-terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C-terminus is unstructured in solution and only folds into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 towards membranes containing the phosphatidylinositol PI4P, thus possibly explaining the primary residency of Naa60 at the PI4P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.

  20. Miro's N-terminal GTPase domain is required for transport of mitochondria into axons and dendrites.

    PubMed

    Babic, Milos; Russo, Gary J; Wellington, Andrea J; Sangston, Ryan M; Gonzalez, Migdalia; Zinsmaier, Konrad E

    2015-04-08

    Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state.

  1. Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

    PubMed Central

    Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia

    2015-01-01

    Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

  2. Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

    PubMed

    Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

    2011-09-28

    Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

  3. High resolution reconstruction of monthly precipitation of Iberian Peninsula using circulation weather types

    NASA Astrophysics Data System (ADS)

    Cortesi, N.; Trigo, R.; Gonzalez-Hidalgo, J. C.; Ramos, A. M.

    2012-06-01

    Precipitation over the Iberian Peninsula (IP) is highly variable and shows large spatial contrasts between wet mountainous regions, to the north, and dry regions in the inland plains and southern areas. In this work, a high-density monthly precipitation dataset for the IP was coupled with a set of 26 atmospheric circulation weather types (Trigo and DaCamara, 2000) to reconstruct Iberian monthly precipitation from October to May with a very high resolution of 3030 precipitation series (overall mean density one station each 200 km2). A stepwise linear regression model with forward selection was used to develop monthly reconstructed precipitation series calibrated and validated over 1948-2003 period. Validation was conducted by means of a leave-one-out cross-validation over the calibration period. The results show a good model performance for selected months, with a mean coefficient of variation (CV) around 0.6 for validation period, being particularly robust over the western and central sectors of IP, while the predicted values in the Mediterranean and northern coastal areas are less acute. We show for three long stations (Lisbon, Madrid and Valencia) the comparison between model and original data as an example to how these models can be used in order to obtain monthly precipitation fields since the 1850s over most of IP for this very high density network.

  4. Atomic Force Microscopy Imaging of Filamentous Aggregates from an N-Terminal Peptide Fragment of Barnase

    NASA Astrophysics Data System (ADS)

    Shibata-Seki, Teiko; Masai, Junji; Yoshida, Kenji; Sato, Kazuki; Yanagawa, Hiroshi

    1993-06-01

    This paper reports the atomic force microscopy (AFM) imaging of filamentous aggregates derived from an N-terminal peptide fragment of barnase, a ribonuclease from Bacillus amyloliquefaciens. The sample was deposited on a freshly cleaved mica surface and observed in ambient conditions. The overall shapes of the filamentous structures imaged with two different kinds of AFMs were similar to those obtained with a transmission electron microscope (TEM), except that the filaments in AFM images were broader than those in TEM images. This broadening phenomenon characteristic of AFM images was explained in terms of the convolution-type distortion of the specimen diameter by the scanning-tip apex.

  5. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    PubMed

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.

  6. Improved Diffuse Fluorescence Flow Cytometer Prototype for High Sensitivity Detection of Rare Circulating Cells In Vivo

    NASA Astrophysics Data System (ADS)

    Pestana, Noah Benjamin

    Accurate quantification of circulating cell populations is important in many areas of pre-clinical and clinical biomedical research, for example, in the study of cancer metastasis or the immune response following tissue and organ transplants. Normally this is done "ex-vivo" by drawing and purifying a small volume of blood and then analyzing it with flow cytometry, hemocytometry or microfludic devices, but the sensitivity of these techniques are poor and the process of handling samples has been shown to affect cell viability and behavior. More recently "in vivo flow cytometry" (IVFC) techniques have been developed where fluorescently-labeled cells flowing in a small blood vessel in the ear or retina are analyzed, but the sensitivity is generally poor due to the small sampling volume. To address this, our group recently developed a method known as "Diffuse Fluorescence Flow Cytometry" (DFFC) that allows detection and counting of rare circulating cells with diffuse photons, offering extremely high single cell counting sensitivity. In this thesis, an improved DFFC prototype was designed and validated. The chief improvements were three-fold, i) improved optical collection efficiency, ii) improved detection electronics, and iii) development of a method to mitigate motion artifacts during in vivo measurements. In combination, these improvements yielded an overall instrument detection sensitivity better than 1 cell/mL in vivo, which is the most sensitive IVFC system reported to date. Second, development and validation of a low-cost microfluidic device reader for analysis of ocular fluids is described. We demonstrate that this device has equivalent or better sensitivity and accuracy compared a fluorescence microscope, but at an order-of-magnitude reduced cost with simplified operation. Future improvements to both instruments are also discussed.

  7. Influence of high-resolution surface databases on the modeling of local atmospheric circulation systems

    NASA Astrophysics Data System (ADS)

    Paiva, L. M. S.; Bodstein, G. C. R.; Pimentel, L. C. G.

    2014-08-01

    Large-eddy simulations are performed using the Advanced Regional Prediction System (ARPS) code at horizontal grid resolutions as fine as 300 m to assess the influence of detailed and updated surface databases on the modeling of local atmospheric circulation systems of urban areas with complex terrain. Applications to air pollution and wind energy are sought. These databases are comprised of 3 arc-sec topographic data from the Shuttle Radar Topography Mission, 10 arc-sec vegetation-type data from the European Space Agency (ESA) GlobCover project, and 30 arc-sec leaf area index and fraction of absorbed photosynthetically active radiation data from the ESA GlobCarbon project. Simulations are carried out for the metropolitan area of Rio de Janeiro using six one-way nested-grid domains that allow the choice of distinct parametric models and vertical resolutions associated to each grid. ARPS is initialized using the Global Forecasting System with 0.5°-resolution data from the National Center of Environmental Prediction, which is also used every 3 h as lateral boundary condition. Topographic shading is turned on and two soil layers are used to compute the soil temperature and moisture budgets in all runs. Results for two simulated runs covering three periods of time are compared to surface and upper-air observational data to explore the dependence of the simulations on initial and boundary conditions, grid resolution, topographic and land-use databases. Our comparisons show overall good agreement between simulated and observational data, mainly for the potential temperature and the wind speed fields, and clearly indicate that the use of high-resolution databases improves significantly our ability to predict the local atmospheric circulation.

  8. Design of a high-pressure circulating pump for viscous liquids

    NASA Astrophysics Data System (ADS)

    Seifried, Bernhard; Temelli, Feral

    2009-07-01

    The design of a reciprocating dual action piston pump capable of circulating viscous fluids at pressures of up to 34 MPa (5000 psi) and temperatures up to 80 °C is described. The piston of this pump is driven by a pair of solenoids energized alternatively by a 12 V direct current power supply controlled by an electronic controller facilitating continuously adjustable flow rates. The body of this seal-less pump is constructed using off-the-shelf parts eliminating the need for custom made parts. Both the electronic controller and the pump can be assembled relatively easily. Pump performance has been evaluated at room temperature (22 °C) and atmospheric pressure using liquids with low and moderately high viscosities, such as ethanol and corn oil, respectively. At ambient conditions, the pump delivered continuous flow of ethanol and corn oil at a flow rate of up to 170 and 17 cm3/min, respectively. For pumping viscous fluids comparable to corn oil, an optimum reciprocation frequency was ascertained to maximize flow rate. For low viscosity liquids such as ethanol, a linear relationship between the flow rate and reciprocation frequency was determined up to the maximum reciprocation frequency of the pump. Since its fabrication, the pump has been used in our laboratory for circulating triglycerides in contact with supercritical carbon dioxide at pressures of up to 25 MPa (3600 psi) and temperatures up to 70 °C on a daily basis for a total of more than 1500 h of operation functioning trouble free.

  9. Critical particle circulation caused by high-performance steady-state plasma discharge

    NASA Astrophysics Data System (ADS)

    Kasahara, Hiroshi

    2015-11-01

    Steady-state operation focused on the fusion reactor has been investigated in magnetic confined fusion devices, and plasma performance and duration time are steadily extended by the improvement of the quality of plasma heating and sophisticating plasma operation using the understanding of long-pulse plasma experiments. When higher-performance helium steady-state plasma discharges with duration time over 40 min, electron density of 1.2x1019 m-3, ion and electron temperatures over 2 keV and heating power of 1.2MW were repeatedly achieved in LHD, time-evolution of the wall-pumping and increasing frequency of impurity contaminations around the plasma edge clearly occurred. These are strongly related to the increasing mixed-material layer caused by continuous divertor erosion around geometrical dense divertor plates, which consists of carbon (> 90%) and iron (< a few %) with amorphous structure, that can retain the helium particles and affect the particle balance in long-pulse discharges. The mixed-material layer is easily exfoliated by the thermal stress and helium explosion in the layer, and small pieces of exfoliation enter the plasma edge in all toroidal sections. Uncontrolled flake contamination was one of the causes of plasma termination in long-pulse experiments. Increased plasma performance using higher heating power (~ 3.3 MW) with high quality makes robust plasma against impurity contaminations, and then a small amount of contamination of mixed-material does not terminate the helium plasma. Carbon impurity was circulated from the divertor plates and around the plates to the plasma edge in long-pulse plasma discharges, and the circulation was increased by the plasma duration and performance. The eroded material plays an important role in degrading the plasma performance as an impurity source and in the controllability of particle fueling in long-pulse discharges.

  10. Flow Regime Study in a High Density Circulating Fluidized Bed Riser with an Abrupt Exit

    SciTech Connect

    Mei, J.S.; Shadle, L.J.; Yue, P.C.; Monazam, E.R.

    2007-01-01

    Flow regime study was conducted in a 0.3 m diameter, 15.5 m height circulating fluidized bed (CFB) riser with an abrupt exit at the National Energy Technology Laboratory of the U.S. Department of Energy. Local particle velocities were measured at various radial positions and riser heights using an optical fiber probe. On-line measurement of solid circulating rate was continuously recorded by the Spiral. Glass beads of mean diameter 61 μm and particle density of 2,500 kg/m3 were used as bed material. The CFB riser was operated at various superficial gas velocities ranging from 3 to 7.6 m/s and solid mass flux from 20 to 550 kg/m2-s. At a constant riser gas velocity, transition from fast fluidization to dense suspension upflow (DSU) regime started at the bottom of the riser with increasing solid flux. Except at comparatively low riser gas velocity and solid flux, the apparent solid holdup at the top exit region was higher than the middle section of the riser. The solid fraction at this top region could be much higher than 7% under high riser gas velocity and solid mass flux. The local particle velocity showed downward flow near the wall at the top of the riser due to its abrupt exit. This abrupt geometry reflected the solids and, therefore, caused solid particles traveling downward along the wall. However, at location below, but near, the top of the riser the local particle velocities were observed flowing upward at the wall. Therefore, DSU was identified in the upper region of the riser with an abrupt exit while the fully developed region, lower in the riser, was still exhibiting core-annular flow structure. Our data were compared with the flow regime boundaries proposed by Kim et al. [1] for distinguishing the dilute pneumatic transport, fast fluidization, and DSU.

  11. In vivo high spatiotemporal resolution visualization of circulating T lymphocytes in high endothelial venules of lymph nodes

    NASA Astrophysics Data System (ADS)

    Choe, Kibaek; Hwang, Yoonha; Seo, Howon; Kim, Pilhan

    2013-03-01

    Lymph nodes (LN) are major checkpoints for circulating T lymphocytes to recognize foreign antigens collected from peripheral tissue. High endothelial venule (HEV) in LN facilitates the effective transmigration of circulating T lymphocytes from the blood into LN. There have been many efforts to visualize T lymphocytes trafficking across HEV to understand the underlying mechanism. However, due to insufficient spatiotemporal resolution and the lack of an in vivo labeling method, clear visualization of dynamic behaviors of rapidly flowing T lymphocytes in HEV and their transmigration have been difficult. In this work, we adapted a custom-designed video-rate laser scanning confocal microscopy system to track individual flowing T lymphocytes in the HEV in real time in vivo. We demonstrate that the HEVs in LN can be clearly identified in vivo with its distinctive cuboidal morphology of endothelial cells fluorescently labeled by intravenously injected anti-CD31 antibody conjugated with Alexa fluorophore. By visualizing the adaptively transferred T lymphocytes, we successfully analyzed dynamic flowing behaviors of T lymphocytes and their transendothelial migration while interacting with the endothelial cells in HEV.

  12. High-fat meals reduce 24-h circulating leptin concentrations in women.

    PubMed

    Havel, P J; Townsend, R; Chaump, L; Teff, K

    1999-02-01

    Leptin induces weight loss in rodents via its effects on food intake and energy expenditure. High-fat diets induce weight gain, but the mechanism is not well understood. Previous studies have not found an effect of dietary fat content on fasting leptin. There is a nocturnal increase of leptin, however, which is related to insulin responses to meals. We have reported that adipocyte glucose utilization is involved in insulin-induced leptin secretion in vitro. Accordingly, high-fat, low-carbohydrate (HF/LC) meals, which induce smaller insulin and glucose responses, would produce lower leptin concentrations than low-fat, high-carbohydrate (LF/HC) meals. Blood samples were collected every 30-60 min for 24 h from 19 normal-weight (BMI, 24.2 +/- 0.7 kg/m2; percent body fat = 31 +/- 1%) women on 2 days (10 days apart) during which the subjects were randomized to consume three isocaloric 730-kcal meals containing either 60/20 or 20/60% of energy as fat/carbohydrate. Overall insulin and glycemic responses (24-h area under the curve [AUC]) were reduced by 55 and 61%, respectively, on the HF/LC day (P < 0.0001). During LF/HC feeding, there were larger increases of leptin 4-6 h after breakfast (38 +/- 7%, P < 0.001) and lunch (78 +/- 14%, P < 0.001) than after HF/LC meals (both P < 0.02). During LF/HC feeding, leptin increased from a morning baseline of 10.7 +/- 1.6 ng/ml to a nocturnal peak of 21.3 +/- 1.3 ng/ml (change, 10.6 +/- 1.3 ng/ml; percent change, 123 +/- 16%; P < 0.0001). The amplitudes of the nocturnal rise of leptin and the 24-h leptin AUC were 21 +/- 8% (P < 0.005) and 38 +/- 12% (P < 0.0025) larger, respectively, on the LF/HC day. In summary, consumption of HF/LC meals results in lowered 24-h circulating leptin concentrations. This result may be a consequence of decreased adipocyte glucose metabolism. Decreases of 24-h circulating leptin could contribute to the weight gain during consumption of high-fat diets.

  13. Control of breathing and the circulation in high-altitude mammals and birds.

    PubMed

    Ivy, Catherine M; Scott, Graham R

    2015-08-01

    Hypoxia is an unremitting stressor at high altitudes that places a premium on oxygen transport by the respiratory and cardiovascular systems. Phenotypic plasticity and genotypic adaptation at various steps in the O2 cascade could help offset the effects of hypoxia on cellular O2 supply in high-altitude natives. In this review, we will discuss the unique mechanisms by which ventilation, cardiac output, and blood flow are controlled in high-altitude mammals and birds. Acclimatization to high altitudes leads to some changes in respiratory and cardiovascular control that increase O2 transport in hypoxia (e.g., ventilatory acclimatization to hypoxia). However, acclimatization or development in hypoxia can also modify cardiorespiratory control in ways that are maladaptive for O2 transport. Hypoxia responses that arose as short-term solutions to O2 deprivation (e.g., peripheral vasoconstriction) or regional variation in O2 levels in the lungs (i.e., hypoxic pulmonary vasoconstriction) are detrimental at in chronic high-altitude hypoxia. Evolved changes in cardiorespiratory control have arisen in many high-altitude taxa, including increases in effective ventilation, attenuation of hypoxic pulmonary vasoconstriction, and changes in catecholamine sensitivity of the heart and systemic vasculature. Parallel evolution of some of these changes in independent highland lineages supports their adaptive significance. Much less is known about the genomic bases and potential interactive effects of adaptation, acclimatization, developmental plasticity, and trans-generational epigenetic transfer on cardiorespiratory control. Future work to understand these various influences on breathing and circulation in high-altitude natives will help elucidate how complex physiological systems can be pushed to their limits to maintain cellular function in hypoxia.

  14. The Energetic Constraints on the Zonal Mean Atmospheric Circulations in the Tropics, Midlatitudes, and High Latitudes

    NASA Astrophysics Data System (ADS)

    Hwang, Yen-Ting

    In this doctoral thesis, I have studied the processes that affect the atmospheric energy budget and their coupling relationships with atmospheric circulations. The equator-to-pole radiation gradient at the top of the atmosphere is the fundamental driver of atmospheric and oceanic circulations. Any anomaly in the energy budget due to variations in different climate components (such as clouds, aerosols, atmospheric properties, and land surfaces) will have an effect on the atmospheric and oceanic circulations and energy transport. Variations in the energy budget of extratropical regions have a non-local effect on tropical climate and vice versa. We first investigated climate components that affect the atmospheric energy budget and their coupled relationships with the atmospheric energy transport, using CMIP multi-model ensembles. We studied how individual components affect energy transport in three latitude bands: (1) at 70 degrees, where increasing poleward energy transport may cause polar amplification, (2) at 40 degrees, where eddies are the strongest, and (3) in the deep tropics, where global climate models (GCMs) do not agree on the changes in transport in global warming scenarios. In high latitudes, positive radiative effects from melting sea ice decrease the equator-to-pole temperature gradient and prevent poleward fluxes from increasing. Models that have more melting ice tend to predict a smaller increase in the energy transport, which is counterintuitive based on the argument that increasing poleward transport can lead to melting sea ice. The cooling effect of increasing low clouds over newly open ocean along the ice edge sharpens the temperature gradient and increases the energy transport in midlatitudes. Clouds and sea ice in the extratropics can also influence energy transport at the equator. We then shifted our focus to the tropical rain belt, built on the first part that demonstrated a directly linkage from hemispheric asymmetry of the atmospheric energy

  15. An integrated microfluidic device for rapid and high-sensitivity analysis of circulating tumor cells.

    PubMed

    Jiang, Jianing; Zhao, Hui; Shu, Weiliang; Tian, Jing; Huang, Yuqing; Song, Yongxin; Wang, Ruoyu; Li, Encheng; Slamon, Dennis; Hou, Dongmei; Du, Xiaohui; Zhang, Lichuan; Chen, Yan; Wang, Qi

    2017-02-15

    Recently there has been a more focus on the development of an efficient technique for detection of circulating tumor cells (CTCs), due to their significance in prognosis and therapy of metastatic cancer. However, it remains a challenge because of the low count of CTCs in the blood. Herein, a rapid and high-sensitivity approach for CTCs detection using an integrated microfluidic system, consisting of a deterministic lateral displacement (DLD) isolating structure, an automatic purifying device with CD45-labeled immunomagnetic beads and a capturing platform coated with rat-tail collagen was reported. We observed high capture rate of 90%, purity of about 50% and viability of more than 90% at the high throughput of 1 mL/min by capturing green fluorescent protein (GFP)-positive cells from blood. Further capturing of CTCs from metastatic cancers patients revealed a positive capture rate of 83.3%. Furthermore, our device was compared with CellSearch system via parallel analysis of 30 cancer patients, to find no significant difference between the capture efficiency of both methods. However, our device displayed advantage in terms of time, sample volume and cost for analysis. Thus, our integrated device with sterile environment and convenient use will be a promising platform for CTCs detection with potential clinical application.

  16. High rate nitrogen removal by ANAMMOX internal circulation reactor (IC) for old landfill leachate treatment.

    PubMed

    Phan, The Nhat; Van Truong, Thi Thanh; Ha, Nhu Biec; Nguyen, Phuoc Dan; Bui, Xuan Thanh; Dang, Bao Trong; Doan, Van Tuan; Park, Joonhong; Guo, Wenshan; Ngo, Huu Hao

    2017-06-01

    This study aimed to evaluate the performance of a high rate nitrogen removal lab-scale ANAMMOX reactor, namely Internal Circulation (IC) reactor, for old landfill leachate treatment. The reactor was operated with pre-treated leachate from a pilot Partial Nitritation Reactor (PNR) using a high nitrogen loading rate ranging from 2 to 10kgNm(-3)d(-1). High rate removal of nitrogen (9.52±1.11kgNm(-3)d(-1)) was observed at an influent nitrogen concentration of 1500mgNL(-1). The specific ANAMMOX activity was found to be 0.598±0.026gN2-NgVSS(-1)d(-1). Analysis of ANAMMOX granules suggested that 0.5-1.0mm size granular sludge was the dominant group. The results of DNA analysis revealed that Candidatus Kueneniastuttgartiensis was the dominant species (37.45%) in the IC reactor, whereas other species like uncultured Bacteroidetes bacterium only constituted 5.37% in the system, but they were still responsible for removing recalcitrant organic matter.

  17. An integrated microfluidic device for rapid and high-sensitivity analysis of circulating tumor cells

    PubMed Central

    Jiang, Jianing; Zhao, Hui; Shu, Weiliang; Tian, Jing; Huang, Yuqing; Song, Yongxin; Wang, Ruoyu; Li, Encheng; Slamon, Dennis; Hou, Dongmei; Du, Xiaohui; Zhang, Lichuan; Chen, Yan; Wang, Qi

    2017-01-01

    Recently there has been a more focus on the development of an efficient technique for detection of circulating tumor cells (CTCs), due to their significance in prognosis and therapy of metastatic cancer. However, it remains a challenge because of the low count of CTCs in the blood. Herein, a rapid and high-sensitivity approach for CTCs detection using an integrated microfluidic system, consisting of a deterministic lateral displacement (DLD) isolating structure, an automatic purifying device with CD45-labeled immunomagnetic beads and a capturing platform coated with rat-tail collagen was reported. We observed high capture rate of 90%, purity of about 50% and viability of more than 90% at the high throughput of 1 mL/min by capturing green fluorescent protein (GFP)-positive cells from blood. Further capturing of CTCs from metastatic cancers patients revealed a positive capture rate of 83.3%. Furthermore, our device was compared with CellSearch system via parallel analysis of 30 cancer patients, to find no significant difference between the capture efficiency of both methods. However, our device displayed advantage in terms of time, sample volume and cost for analysis. Thus, our integrated device with sterile environment and convenient use will be a promising platform for CTCs detection with potential clinical application. PMID:28198402

  18. High circulating D-dimers are associated with ascites and hepatocellular carcinoma in liver cirrhosis

    PubMed Central

    Spadaro, Aldo; Tortorella, Vincenza; Morace, Carmela; Fortiguerra, Agostino; Composto, Paola; Bonfiglio, Caterina; Alibrandi, Angela; Luigiano, Carmelo; Caro, Giuseppe De; Ajello, Antonino; Ferraù, Oscar; Freni, Maria Antonietta

    2008-01-01

    AIM: To measure plasma D-dimer levels in cirrhotic patients with and without ascites, assessing the effect of ascites resolution in D-dimer concentration. METHODS: Seventy consecutive cirrhotic patients (M = 44, F = 26, mean age 65 years, SD ± 13), observed from October 2005 to March 2006 were enrolled. Circulating D-dimer levels were measured using a latex-enhanced, immunoturbidimetric test. In patients with ascites (n = 42) the test was repeated after ascites resolution. RESULTS: Ascites was present in 42 patients (group A) and absent in 28 (group B). Group A patients had more advanced liver disease. Hepatocellular carcinoma (HCC) was diagnosed in 14 patients and was more frequent in group B. Above normal range D-dimers were found in 45/70 patients. High D-dimers were more frequent in group A than in group B (P = 0.001). High D-dimers were associated with presence of HCC (P = 0.048) only in group B. After ascites resolution, obtained in all patients, mean D-dimer values decreased in those 34 patients with high basal levels (P = 0.007), returning to normal in 17. CONCLUSION: In patients with liver cirrhosis, ascites and HCC are the main factors associated with increased fibrinolytic activity. PMID:18330946

  19. Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites.

    PubMed

    Liew, Oi Wah; Ching Chong, Jenny Pek; Yandle, Tim G; Brennan, Stephen O

    2005-06-01

    C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited

  20. 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.

  1. N-Terminal region is responsible for chemotaxis-inducing activity of flounder IL-8.

    PubMed

    Kurata, Osamu; Wada, Shinpei; Matsuyama, Tomomasa; Sakai, Takamitsu; Takano, Tomokazu

    2014-06-01

    The objective of this study was to locate the functional region responsible for the chemotaxis-inducing activity of flounder interleukin 8 (IL-8), which lacks the glutamic acid-leucine-arginine (ELR) motif essential for the induction of neutrophil migration by mammalian IL-8. Using a human cell line, we produced a secretory recombinant protein of flounder IL-8, and analyzed its chemotaxis-inducing activity on leukocytes collected from the flounder kidney. The recombinant IL-8 induced significant migration in neutrophils, which were morphologically and functionally characterized. Using the Edman degradation method, the N-terminal amino acid sequence of rIL-8 was identified as VSLRSLGV. To examine the significance of the N-terminal region for the bioactivity of flounder IL-8, we prepared several recombinant proteins that containing mutations at the N-terminus. Modification of three residues (residues 9-11: serine-leucine-histidine) corresponding in position to the ELR motif in mammalian IL-8 did not reduce its chemotaxis-inducing activity. However, deletion of the first six or more residues significantly reduced its chemotaxis-inducing activity. We propose that residue 6 (leucine) at the N-terminus is important for the chemotaxis-inducing activity of flounder IL-8.

  2. Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail

    PubMed Central

    Ludwigsen, Johanna; Pfennig, Sabrina; Singh, Ashish K; Schindler, Christina; Harrer, Nadine; Forné, Ignasi; Zacharias, Martin; Mueller-Planitz, Felix

    2017-01-01

    ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1. DOI: http://dx.doi.org/10.7554/eLife.21477.001 PMID:28109157

  3. Specific N-terminal CGRP fragments mitigate chronic hypoxic pulmonary hypertension in rats.

    PubMed

    Qing, Xin; Wimalawansa, Sunil J; Keith, Ingegerd M

    2003-01-31

    Chronic hypoxic pulmonary hypertension (HPH) is characterized by elevated pulmonary arterial pressure (P(PA)), right ventricular hypertrophy (RVH), pulmonary vascular remodeling, pulmonary edema and polycythemia. Currently, there is no safe and effective treatment for HPH. Calcitonin gene-related peptide (CGRP) is the most potent peptide vasodilator discovered thus far. We previously demonstrated that exogenous CGRP reversed HPH in rats. However, the CGRP1 receptor antagonist CGRP(8-37) and smaller inhibitory C-terminal CGRP fragments that can be formed by enzymatic cleavage in vivo may compromise the beneficial effects of endogenous or exogenous CGRP. We here examine the agonistic efficacy of N-terminal rat alpha-CGRP peptides containing the disulfide bridge (Cys(2)-Cys(7)) with amidated C-terminal in prevention of HPH. Chronic infusion of CGRP(1-8), CGRP(1-13), or CGRP(1-14) at 7 nmol/h/rat via the right jugular vein during 14 days of hypobaric hypoxia (10% inspired O(2)) significantly decreased the P(PA), RVH and pulmonary arterial medial thickness in comparison with controls, suggesting that these CGRP sequences can mitigate chronic HPH in rats. Systemic pressure was unchanged by infused peptides indicating no carry-over effect. In conclusion, N-terminal CGRP fragments (CGRP(1-8), CGRP(1-13) and CGRP(1-14)) may have a protective role in hypoxic pulmonary hypertension.

  4. 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

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

    PubMed

    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

    2015-05-11

    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.

  6. Supramolecular properties of the proline-rich gamma-Zein N-terminal domain.

    PubMed Central

    Kogan, Marcelo J; Dalcol, Ionara; Gorostiza, Pau; Lopez-Iglesias, Carmen; Pons, Ramon; Pons, Miquel; Sanz, Fausto; Giralt, Ernest

    2002-01-01

    Zeins are maize storages proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner face of the protein body membrane, and its N-terminal proline-rich repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation. In a preliminary recent work we used atomic force microscopy to study the surface organization of the octamer and transmission electron microscopy to visualize aggregates of the peptide from aqueous solution. We previously envisioned two self-assembly models (i.e., the geometric and the micellar) that take into account the observed features. In the present work we studied in detail the self-assembly of the peptide in solution and found that the peptide is able to form cylindrical micelles. Fibrils formed on graphite are generated by assembly of solution micelles. Based on the results of these studies, we focused exclusively on the micellar model. To our knowledge we have characterized for the first time supramolecular aggregates of polyproline structures other than collagen. The spontaneous arrangement of (VHLPPP)(8) suggests a role for the N-terminal domain of gamma-zein in the process of the whole protein deposition in protein bodies. PMID:12124299

  7. Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster

    PubMed Central

    Villar-Garea, Ana; Forne, Ignasi; Vetter, Irene; Kremmer, Elisabeth; Thomae, Andreas; Imhof, Axel

    2012-01-01

    Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other’s activity. PMID:22053083

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

    PubMed

    Giglione, C; Serero, A; Pierre, M; Boisson, B; Meinnel, T

    2000-11-01

    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.

  9. In silico identification and characterization of N-Terminal acetyltransferase genes of poplar (Populus trichocarpa).

    PubMed

    Zhu, Hang-Yong; Li, Chun-Ming; Wang, Li-Feng; Bai, Hui; Li, Yan-Ping; Yu, Wen-Xi; Xia, De-An; Liu, Chang-Cai

    2014-01-27

    N-terminal acetyltransferase (Nats) complex is responsible for protein N-terminal acetylation (Nα-acetylation), which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS) and auxiliary subunits (AS) have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A-F), being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  10. Reduced circulating oxytocin and High-Molecular-Weight adiponectin are risk factors for metabolic syndrome.

    PubMed

    Yuan, Guoyue; Qian, Weiyun; Pan, Ruirong; Jia, Jue; Jiang, Dan; Yang, Qichao; Wang, Su; Liu, Yuanxin; Yu, Shuqin; Hu, Hao; Sun, Wenjun; Ye, Jingjing; Mao, Chaoming; Zhuang, Ruo; Zhou, Libin

    2016-07-30

    The neurohypophysial hormone, oxytocin, is involved in the regulation of energy metabolism. Adiponectin (APN) is an adipose tissue-specific serum protein that inversely associates with metabolic syndrome (MetS). High-molecular-weight adiponectin (HMW APN) is considered the active form. In the present study, we aimed to determine the relationships of oxytocin and HMW APN to MetS and investigate whether or not the combination of oxytocin and HMW APN is associated with further metabolic abnormalities compared to each of them alone. A total of 170 subjects (75 with MetS and 95 non-MetS) were enrolled. Anthropometric parameters, oral glucose tolerance test (OGTT), blood lipids, hs-CRP, oxytocin and HMW APN levels were measured. Compared with non-MetS subjects, serum oxytocin and HMW APN levels were significantly lower in subjects with MetS (P<0.01). We then classified the subjects into three groups: high oxytocin and high HMW APN levels (high score group), low oxytocin and low HMW APN levels (low score group) and others. Participants in low score group showed the worst metabolic profiles and were more likely to have MetS compared to the other two group. In Spearman rank correlation coefficient, the classification by the combination of oxytocin and HMW APN was significantly correlated with a larger number of metabolic risk factors compared with classification by each of them alone. Individuals with low circulating oxytocin levels coupled with low HMW APN levels were at significantly increased risk of MetS. The combination of both markers would be useful for identifying MetS high risk patients.

  11. The sulfur cycle at high-southern latitudes in the LMD-ZT General Circulation Model

    NASA Astrophysics Data System (ADS)

    Cosme, E.; Genthon, C.; Martinerie, P.; Boucher, O.; Pham, M.

    2002-12-01

    This modeling study was motivated by the recent publication of year-round records of dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) in Antarctica, completing the available series of sulfate and methanesulfonic acid (MSA). Sulfur chemistry has been incorporated in the Laboratoire de Météorologie Dynamique-Zoom Tracers (LMD-ZT) Atmospheric General Circulation Model (AGCM), with high-resolution and improved physics at high-southern latitudes. The model predicts the concentration of six major sulfur species through emissions, transport, wet and dry deposition, and chemistry in both gas and aqueous phases. Model results are broadly realistic when compared with measurements in air and snow or ice, as well as to results of other modeling studies, at high- and middle-southern latitudes. Atmospheric MSA concentrations are underestimated and DMSO concentrations are overestimated in summer, reflecting the lack of a DMSO heterogeneous sink leading to MSA. Experiments with various recently published estimates of the rate of this sink are reported. Although not corrected in this work, other defects are identified and discussed: DMS concentrations are underestimated in winter, MSA and non-sea-salt (nss) sulfate concentrations may be underestimated at the South Pole, the deposition scheme used in the model may not be adapted to polar regions, and the model does not adequately reproduces interannual variability. Oceanic DMS sources have a major contribution to the variability of sulfur in these regions. The model results suggest that in a large part of central Antarctica ground-level atmospheric DMS concentrations are larger in winter than in summer. At high-southern latitudes, high loads of DMS and DMSO are found and the main chemical sink of sulfur dioxide (SO2) is aqueous oxidation by ozone (O3), whereas oxidation by hydrogen peroxide (H2O2) dominates at the global scale. A comprehensive modeled sulfur budget of Antarctica is provided.

  12. Complex mean circulation over the inner shelf south of Martha's Vineyard revealed by observations and a high-resolution model

    USGS Publications Warehouse

    Ganju, Neil K.; Lentz, Steven J.; Kirincich, Anthony R.; Farrar, J. Thomas

    2011-01-01

    Inner-shelf circulation is governed by the interaction between tides, baroclinic forcing, winds, waves, and frictional losses; the mean circulation ultimately governs exchange between the coast and ocean. In some cases, oscillatory tidal currents interact with bathymetric features to generate a tidally rectified flow. Recent observational and modeling efforts in an overlapping domain centered on the Martha's Vineyard Coastal Observatory (MVCO) provided an opportunity to investigate the spatial and temporal complexity of circulation on the inner shelf. ADCP and surface radar observations revealed a mean circulation pattern that was highly variable in the alongshore and cross-shore directions. Nested modeling incrementally improved representation of the mean circulation as grid resolution increased and indicated tidal rectification as the generation mechanism of a counter-clockwise gyre near the MVCO. The loss of model skill with decreasing resolution is attributed to insufficient representation of the bathymetric gradients (Δh/h), which is important for representing nonlinear interactions between currents and bathymetry. The modeled momentum balance was characterized by large spatial variability of the pressure gradient and horizontal advection terms over short distances, suggesting that observed inner-shelf momentum balances may be confounded. Given the available observational and modeling data, this work defines the spatially variable mean circulation and its formation mechanism—tidal rectification—and illustrates the importance of model resolution for resolving circulation and constituent exchange near the coast. The results of this study have implications for future observational and modeling studies near the MVCO and other inner-shelf locations with alongshore bathymetric variability.

  13. a 24/7 High Resolution Storm Surge, Inundation and Circulation Forecasting System for Florida Coast

    NASA Astrophysics Data System (ADS)

    Paramygin, V.; Davis, J. R.; Sheng, Y.

    2012-12-01

    A 24/7 forecasting system for Florida is needed because of the high risk of tropical storm surge-induced coastal inundation and damage, and the need to support operational management of water resources, utility infrastructures, and fishery resources. With the anticipated climate change impacts, including sea level rise, coastal areas are facing the challenges of increasing inundation risk and increasing population. Accurate 24/7 forecasting of water level, inundation, and circulation will significantly enhance the sustainability of coastal communities and environments. Supported by the Southeast Coastal Ocean Observing Regional Association (SECOORA) through NOAA IOOS, a 24/7 high-resolution forecasting system for storm surge, coastal inundation, and baroclinic circulation is being developed for Florida using CH3D Storm Surge Modeling System (CH3D-SSMS). CH3D-SSMS is based on the CH3D hydrodynamic model coupled to a coastal wave model SWAN and basin scale surge and wave models. CH3D-SSMS has been verified with surge, wave, and circulation data from several recent hurricanes in the U.S.: Isabel (2003); Charley, Dennis and Ivan (2004); Katrina and Wilma (2005); Ike and Fay (2008); and Irene (2011), as well as typhoons in the Pacific: Fanapi (2010) and Nanmadol (2011). The effects of tropical cyclones on flow and salinity distribution in estuarine and coastal waters has been simulated for Apalachicola Bay as well as Guana-Tolomato-Matanzas Estuary using CH3D-SSMS. The system successfully reproduced different physical phenomena including large waves during Ivan that damaged I-10 Bridges, a large alongshore wave and coastal flooding during Wilma, salinity drop during Fay, and flooding in Taiwan as a result of combined surge and rain effect during Fanapi. The system uses 4 domains that cover entire Florida coastline: West, which covers the Florida panhandle and Tampa Bay; Southwest spans from Florida Keys to Charlotte Harbor; Southeast, covering Biscayne Bay and Miami and

  14. Hydrodechlorination of TCE in a circulated electrolytic column at high flow rate.

    PubMed

    Fallahpour, Noushin; Yuan, Songhu; Rajic, Ljiljana; Alshawabkeh, Akram N

    2016-02-01

    Palladium-catalytic hydrodechlorination of trichloroethylene (TCE) by cathodic H2 produced from water electrolysis has been tested. For a field in-well application, the flow rate is generally high. In this study, the performance of Pd-catalytic hydrodechlorination of TCE using cathodic H2 is evaluated under high flow rate (1 L min(-1)) in a circulated column system, as expected to occur in practice. An iron anode supports reduction conditions and it is used to enhance TCE hydrodechlorination. However, the precipitation occurs and high flow rate was evaluated to minimize its adverse effects on the process (electrode coverage, clogging, etc.). Under the conditions of 1 L min(-1) flow, 500 mA current, and 5 mg L(-1) initial TCE concentration, removal efficacy using iron anodes (96%) is significantly higher than by mixed metal oxide (MMO) anodes (66%). Two types of cathodes (MMO and copper foam) in the presence of Pd/Al2O3 catalyst under various currents (250, 125, and 62 mA) were used to evaluate the effect of cathode materials on TCE removal efficacy. The similar removal efficiencies were achieved for both cathodes, but more precipitation generated with copper foam cathode (based on the experiments done by authors). In addition to the well-known parameters such as current density, electrode materials, and initial TCE concentration, the high velocities of groundwater flow can have important implications, practically in relation to the flush out of precipitates. For potential field application, a cost-effective and sustainable in situ electrochemical process using a solar panel as power supply is being evaluated.

  15. An RNA-based signature enables high specificity detection of circulating tumor cells in hepatocellular carcinoma

    PubMed Central

    Kalinich, Mark; Bhan, Irun; Kwan, Tanya T.; Miyamoto, David T.; Javaid, Sarah; LiCausi, Joseph A.; Milner, John D.; Hong, Xin; Goyal, Lipika; Sil, Srinjoy; Choz, Melissa; Ho, Uyen; Kapur, Ravi; Muzikansky, Alona; Zhang, Huidan; Weitz, David A.; Sequist, Lecia V.; Ryan, David P.; Chung, Raymond T.; Zhu, Andrew X.; Isselbacher, Kurt J.; Ting, David T.; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A.

    2017-01-01

    Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent. PMID:28096363

  16. Hydrodechlorination of TCE in a circulated electrolytic column at high flow rate

    PubMed Central

    Fallahpour, Noushin; Yuan, Songhu; Rajic, Ljiljana; Alshawabkeh, Akram N.

    2015-01-01

    Palladium-catalytic hydrodechlorination of trichloroethylene (TCE) by cathodic H2 produced from water electrolysis has been tested. For a field in-well application, the flow rate is generally high. In this study, the performance of Pd-catalytic hydrodechlorination of TCE using cathodic H2 is evaluated under high flow rate (1 L min−1) in a circulated column system, as expected to occur in practice. An iron anode supports reduction conditions and it is used to enhance TCE hydrodechlorination. However, the precipitation occurs and high flow rate was evaluated to minimize its advers effects on the process (electrode coverage, clogging, etc.). Under the conditions of 1 L min−1 flow, 500 mA current, and 5 mg L−1 initial TCE concentration, removal efficacy using iron anodes (96%) is significantly higher than by mixed metal oxide (MMO) anodes (66%). Two types of cathodes (MMO and copper foam) in the presence of Pd/Al2O3 catalyst under various currents (250, 125, and 62 mA) were used to evaluate the effect of cathode materials on TCE removal efficacy. The similar removal efficiencies were achieved for both cathodes, but more precipitation generated with copper foam cathode (based on the experiments done by authors). In addition to the well-known parameters such as current density, electrode materials, and initial TCE concentration, the high velocities of groundwater flow can have important implications, practically in relation to the flush out of precipitates. For potential field application, a cost-effective and sustainable in situ electrochemical process using a solar panel as power supply is being evaluated. PMID:26344148

  17. Pharmacological Inhibition of c-Jun N-terminal Kinase Reduces Food Intake and Sensitizes Leptin's Anorectic Signaling Actions.

    PubMed

    Gao, Su; Howard, Shannon; LoGrasso, Philip V

    2017-02-06

    The role for c-Jun N-terminal Kinase (JNK) in the control of feeding and energy balance is not well understood. Here, by use of novel and highly selective JNK inhibitors, we investigated the actions of JNK in the control of feeding and body weight homeostasis. In lean mice, intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of SR-3306, a brain-penetrant and selective pan-JNK (JNK1/2/3) inhibitor, reduced food intake and body weight. Moreover, i.p. and i.c.v. administrations of SR11935, a brain-penetrant and JNK2/3 isoform-selective inhibitor, exerted similar anorectic effects as SR3306, which suggests JNK2 or JNK3 mediates aspect of the anorectic effect by pan-JNK inhibition. Furthermore, daily i.p. injection of SR3306 (7 days) prevented the increases in food intake and weight gain in lean mice upon high-fat diet feeding, and this injection paradigm reduced high-fat intake and obesity in diet-induced obese (DIO) mice. In the DIO mice, JNK inhibition sensitized leptin's anorectic effect, and enhanced leptin-induced STAT3 activation in the hypothalamus. The underlying mechanisms likely involve the downregulation of SOCS3 by JNK inhibition. Collectively, our data suggest that JNK activity promotes positive energy balance, and the therapeutic intervention inhibiting JNK activities represents a promising approach to ameliorate diet-induced obesity and leptin resistance.

  18. High expression of MnSOD promotes survival of circulating breast cancer cells and increases their resistance to doxorubicin

    PubMed Central

    Fu, Afu; Ma, Shijun; Wei, Na; Xuan Tan, Blanche Xiao; Tan, Ern Yu; Luo, Kathy Qian

    2016-01-01

    Understanding the survival mechanism of metastatic cancer cells in circulation will provide new perspectives on metastasis prevention and also shed new light on metastasis-derived drug resistance. In this study, we made it feasible to detect apoptosis of circulating tumor cells (CTCs) in real-time by integrating a fluorescence resonance energy transfer (FRET)-based caspase sensor into one in vitro microfluidic circulatory system, and two in vivo models: zebrafish circulation and mouse lung metastatic model. Our study demonstrated that fluid shear stresses triggered apoptosis of breast cancer cells in circulation by elevating the mitochondrial production of the primary free radical, superoxide anion. Cancer cells with high levels of manganese superoxide dismutase (MnSOD) exhibited stronger resistance to shear force-induced apoptosis and formed more lung metastases in mice. These metastasized cells further displayed higher resistance to chemotherapeutic agent doxorubicin, which also generates superoxide in mitochondria. Specific siRNA-mediated MnSOD knockdown reversed all three phenotypes. Our findings therefore suggest that MnSOD plays an important integrative role in supporting cancer cell survival in circulation, metastasis, and doxorubicin resistance. MnSOD can serve as a new biomarker for identifying metastatic CTCs and a novel therapeutic target for inhibiting metastasis and destroying doxorubicin-resistant breast cancer cells. PMID:27384484

  19. Evaluation of Cloud Parameterizations in a High Resolution Atmospheric General Circulation Model Using ARM Data

    SciTech Connect

    Govindasamy, B; Duffy, P

    2002-04-12

    Typical state of the art atmospheric general circulation models used in climate change studies have horizontal resolution of approximately 300 km. As computing power increases, many climate modeling groups are working toward enhancing the resolution of global models. An important issue that arises when resolution of a model is changed is whether cloud and convective parameterizations, which were developed for use at coarser resolutions, will need to be reformulated or re-tuned. We propose to investigate this issue and specifically cloud statistics using ARM data. The data streams produced by highly instrumented sections of Cloud and Radiation Testbeds (CART) of ARM program will provide a significant aid in the evaluation of cloud and convection parameterization in high-resolution models. Recently, we have performed multiyear global-climate simulations at T170 and T239 resolutions, corresponding to grid cell sizes of 0.7{sup 0} and 0.5{sup 0} respectively, using the NCAR Community Climate Model. We have also a performed climate change simulation at T170. On the scales of a T42 grid cell (300 km) and larger, nearly all quantities we examined in T170 simulation agree better with observations in terms of spatial patterns than do results in a comparable simulation at T42. Increasing the resolution to T239 brings significant further improvement. At T239, the high-resolution model grid cells approach the dimensions of the highly instrumented sections of ARM Cloud and Radiation Testbed (CART) sites. We propose to form a cloud climatology using ARM data for its CART sites and evaluate cloud statistics of the NCAR Community Atmosphere Model (CAM) at higher resolutions over those sites using this ARM cloud climatology. We will then modify the physical parameterizations of CAM for better agreement with ARM data. We will work closely with NCAR in modifying the parameters in cloud and convection parameterizations for the high-resolution model. Our proposal to evaluate the cloud

  20. 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.

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

    PubMed Central

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

    2015-01-01

    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. Increased circulating estradiol in mice fed a high-fat diet does not attenuate ovariectomy-induced bone structural deterioration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ovariectomy-induced estrogen deficiency increases adiposity and induces substantial bone loss by increasing osteoclast activity. This study investigated whether obesity induced by a high-fat diet alter circulating estradiol levels, mitigates or exacerbates bone structure deterioration, and changes m...

  4. Hurricane Forecasting with the High-resolution NASA Finite-volume General Circulation Model

    NASA Technical Reports Server (NTRS)

    Atlas, R.; Reale, O.; Shen, B.-W.; Lin, S.-J.; Chern, J.-D.; Putman, W.; Lee, T.; Yeh, K.-S.; Bosilovich, M.; Radakovich, J.

    2004-01-01

    A high-resolution finite-volume General Circulation Model (fvGCM), resulting from a development effort of more than ten years, is now being run operationally at the NASA Goddard Space Flight Center and Ames Research Center. The model is based on a finite-volume dynamical core with terrain-following Lagrangian control-volume discretization and performs efficiently on massive parallel architectures. The computational efficiency allows simulations at a resolution of a quarter of a degree, which is double the resolution currently adopted by most global models in operational weather centers. Such fine global resolution brings us closer to overcoming a fundamental barrier in global atmospheric modeling for both weather and climate, because tropical cyclones and even tropical convective clusters can be more realistically represented. In this work, preliminary results of the fvGCM are shown. Fifteen simulations of four Atlantic tropical cyclones in 2002 and 2004 are chosen because of strong and varied difficulties presented to numerical weather forecasting. It is shown that the fvGCM, run at the resolution of a quarter of a degree, can produce very good forecasts of these tropical systems, adequately resolving problems like erratic track, abrupt recurvature, intense extratropical transition, multiple landfall and reintensification, and interaction among vortices.

  5. Martian atmospheric gravity waves simulated by a high-resolution general circulation model

    NASA Astrophysics Data System (ADS)

    Kuroda, Takeshi; Yiǧit, Erdal; Medvedev, Alexander S.; Hartogh, Paul

    2016-07-01

    Gravity waves (GWs) significantly affect temperature and wind fields in the Martian middle and upper atmosphere. They are also one of the observational targets of the MAVEN mission. We report on the first simulations with a high-resolution general circulation model (GCM) and present a global distributions of small-scale GWs in the Martian atmosphere. The simulated GW-induced temperature variances are in a good agreement with available radio occultation data in the lower atmosphere between 10 and 30 km. For the northern winter solstice, the model reveals a latitudinal asymmetry with stronger wave generation in the winter hemisphere and two distinctive sources of GWs: mountainous regions and the meandering winter polar jet. Orographic GWs are filtered upon propagating upward, and the mesosphere is primarily dominated by harmonics with faster horizontal phase velocities. Wave fluxes are directed mainly against the local wind. GW dissipation in the upper mesosphere generates a body force per unit mass of tens of m s^{-1} per Martian solar day (sol^{-1}), which tends to close the simulated jets. The results represent a realistic surrogate for missing observations, which can be used for constraining GW parameterizations and validating GCMs.

  6. Elevated Circulating Interleukin 33 Levels in Stable Renal Transplant Recipients at High Risk for Cardiovascular Events

    PubMed Central

    Mansell, Holly; Soliman, Mahmoud; Elmoselhi, Hamdi; Shoker, Ahmed

    2015-01-01

    Background The Major Adverse Cardiovascular Events calculator (CRCRTR-MACE) estimates the burden of cardiovascular risk in renal transplant recipients (RTR). Our recent study of 95 RTR reported the 7-year median risk of cardiovascular events (CVE) to be 9.97%, ranging from 1.93 to 84.27%. Nearly a third (28.4%) of the cohort was above 20% risk for a CVE. Since interleukins (ILs) as part of the inflammatory response may play a role in the pathogenesis of cardiovascular disease (CVD), we extended this study to identify which ILs are associated with high cardiovascular risk in this population. Methods Twenty-two ILs were measured by multiplexed fluorescent bead-based immunoassay in 95 RTR and 56 normal controls. Stepwise analysis after multivariate determination of significant demographic and inflammatory variables was performed between the high and low-CVD risk groups (which were arbitrarily set at scores <10% and ≥20%, respectively). Normalized data was presented as mean ± SD and non-normalized data as median (minimum–maximum). Significance was measured at <0.05. Results 27.5% of the low-risk and 31.3% of the high-risk groups had mean IL levels above the 95 percentile of the normal control levels. In the non-parametric analysis IL-6, 9, 16, 17 and 33 were significantly higher in the high-risk group compared to the control. Univariate analysis (UVA) of the high-risk group identified IL-33 as the only IL that remained significantly higher than the control and low-risk groups (p = 0.000). The percentage of patients with IL-33 levels above the 90 percentile of control value in the low and high-risk groups were 15.6% and 52.0%, respectively (p<0.002). UVA of factors significant to high IL-33 levels included estimated glomerular filtration rate (eGFR), while diabetes mellitus, serum phosphorus, microalbuminuria and age also remained significant in the multivariate analysis. Conclusion Circulating IL-33 level is positively associated with high CRCRTR-MACE score

  7. Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26

    PubMed Central

    Won, Eun-Young; Lee, Sang-Ok; Lee, Dong-Hwa; Lee, Daeyoup; Bae, Kwang-Hee; Lee, Sang Chul; Kim, Seung Jun; Chi, Seung-Wook

    2016-01-01

    Human dual-specificity phosphatase 26 (DUSP26) is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61–211, DUSP26-C), the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39–211, DUSP26-N) with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S) monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop) observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS) measurements showed that DUSP26-N (C152S) exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S) revealed that the N-terminal region of DUSP26-N (C152S) serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics. PMID:27583453

  8. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    SciTech Connect

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  9. Spidroin N-terminal Domain Promotes a pH-dependent Association of Silk Proteins during Self-assembly*

    PubMed Central

    Gaines, William A.; Sehorn, Michael G.; Marcotte, William R.

    2010-01-01

    Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD. PMID:20959449

  10. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    PubMed Central

    Albada, H Bauke; Chiriac, Alina-Iulia; Wenzel, Michaela; Penkova, Maya; Bandow, Julia E; Sahl, Hans-Georg

    2012-01-01

    Summary A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO) and ruthenocene (RcCO) was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW)2 and 1–11 µM for (RW)3 were determined. Interestingly, W(RW)2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW)2- and (RW)3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW)2-peptide versus killing kinetics of the (RW)3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW)2-peptide, although MIC values indicated higher activity for the (RW)3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW)3 and 250 µg/mL for RcCO-W(RW)2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7) showed that the (RW)3-peptide had an IC50 value of ~140 µM and the RcW(RW)2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to

  11. 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.

  12. The Impact of N-terminal Acetylation of α-Synuclein on Phospholipid Membrane Binding and Fibril Structure*

    PubMed Central

    Iyer, Aditya; Roeters, Steven J.; Schilderink, Nathalie; Hommersom, Bob; Heeren, Ron M. A.; Woutersen, Sander; Claessens, Mireille M. A. E.

    2016-01-01

    Human α-synuclein (αS) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of αS into amyloid fibrils. Using bacterially expressed acetylated-αS (NTAc-αS) and endogenous αS (Endo-αS) from human erythrocytes, we show that N-terminal acetylation has little impact on αS binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on αS aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of αS in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-αS in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated αS. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in αS, thus predisposing αS toward specific interactions with other binding partners or alternatively decrease nonspecific interactions. PMID:27531743

  13. Crystallization of Galectin-8 Linker Reveals Intricate Relationship between the N-terminal Tail and the Linker

    PubMed Central

    Si, Yunlong; Wang, Yue; Gao, Jin; Song, Chenyang; Feng, Shiqiong; Zhou, Yifa; Tai, Guihua; Su, Jiyong

    2016-01-01

    Galectin-8 (Gal-8) plays a significant role in normal immunological function as well as in cancer. This lectin contains two carbohydrate recognition domains (CRD) connected by a peptide linker. The N-terminal CRD determines ligand binding specificity, whereas the linker has been proposed to regulate overall Gal-8 function, including multimerization and biological activity. Here, we crystallized the Gal-8 N-terminal CRD with the peptide linker using a crystallization condition that contains Ni2+. The Ni2+ ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni2+ and Asp25 plays an indirect role in determining the structure of β-strand F0 and in influencing the linker conformation which could not be defined due to its dynamic nature. The linker was also shortened in situ and crystallized under a different condition, leading to a higher resolution structure refined to 1.08 Å. This crystal structure allowed definition of a short portion of the linker interacting with the Gal-8 N-terminal tail via ionic interactions and hydrogen bonds. Observation of two Gal-8 N-terminal CRD structures implies that the N-terminal tail and the linker may influence each other’s conformation. In addition, under specific crystallization conditions, glycerol could replace lactose and was observed at the carbohydrate binding site. However, glycerol did not show inhibition activity in hemagglutination assay. PMID:27973456

  14. Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins.

    PubMed

    Tang, Yanan; Li, Liang

    2013-08-20

    The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC-MS) with the use of isotope analog standards.

  15. High resolution modelling of the oceanic circulation and winter vertical mixing in the northwestern Mediterranean

    NASA Astrophysics Data System (ADS)

    Damien, Pierre; Estournel, Claude; Marsaleix, Patrick

    2013-04-01

    The North Western Mediterranean Sea is one of the few regions in the world where open-ocean deep convection occurs. The local cyclonic circulation brings weakly stratified waters close to the surface. In winter, atmospheric conditions (strong cold winds and high heat losses) trigger the deep convection. When the strong forcing stops, restratification of the mixed patch occurs by lateral advection of surrounding lighter water. Mesoscale and submesoscale structures play an important role during these events both in the sinking and spreading of the new dense water formed and in the advection of light surrounding water. The objective is first to check the capabilities of a high resolution model to reproduce the oceanic response to strong wind and, second, to identify processes involved in the water column restratification in terms of spacial and temporal scales. The SYMPHONIE model was implemented at 1 km resolution over the north-western Mediterranean. Simulations were initialized and forced at the open boundaries by the recent MERCATOR release PSY2V4R3. Two atmospheric forcings were use at the surface, ECMWF through bulk formulae and ARPERA. The recent years were simulated and comparisons were performed with the available data set particularly Argo and glider floats and the data of the CASCADE experiment in March 2011. A special attention was paid to the representation of the vertical stratification, of the mixed layer depth and of the properties of the water masses. The characteristics of the deep convection event and of its restratification are examined in terms of water mass formation and budgets. The role played by small scale structures is quantified.

  16. THREE-DIMENSIONAL ATMOSPHERIC CIRCULATION OF HOT JUPITERS ON HIGHLY ECCENTRIC ORBITS

    SciTech Connect

    Kataria, T.; Showman, A. P.; Lewis, N. K.; Fortney, J. J.; Marley, M. S.; Freedman, R. S.

    2013-04-10

    Of the over 800 exoplanets detected to date, over half are on non-circular orbits, with eccentricities as high as 0.93. Such orbits lead to time-variable stellar heating, which has major implications for the planet's atmospheric dynamical regime. However, little is known about the fundamental dynamical regime of such planetary atmospheres, and how it may influence the observations of these planets. Therefore, we present a systematic study of hot Jupiters on highly eccentric orbits using the SPARC/MITgcm, a model which couples a three-dimensional general circulation model (the MITgcm) with a plane-parallel, two-stream, non-gray radiative transfer model. In our study, we vary the eccentricity and orbit-average stellar flux over a wide range. We demonstrate that the eccentric hot Jupiter regime is qualitatively similar to that of planets on circular orbits; the planets possess a superrotating equatorial jet and exhibit large day-night temperature variations. As in Showman and Polvani, we show that the day-night heating variations induce momentum fluxes equatorward to maintain the superrotating jet throughout its orbit. We find that as the eccentricity and/or stellar flux is increased (corresponding to shorter orbital periods), the superrotating jet strengthens and narrows, due to a smaller Rossby deformation radius. For a select number of model integrations, we generate full-orbit light curves and find that the timing of transit and secondary eclipse viewed from Earth with respect to periapse and apoapse can greatly affect what we see in infrared (IR) light curves; the peak in IR flux can lead or lag secondary eclipse depending on the geometry. For those planets that have large temperature differences from dayside to nightside and rapid rotation rates, we find that the light curves can exhibit 'ringing' as the planet's hottest region rotates in and out of view from Earth. These results can be used to explain future observations of eccentric transiting exoplanets.

  17. An ultra-high-throughput spiral microfluidic biochip for the enrichment of circulating tumor cells.

    PubMed

    Warkiani, Majid Ebrahimi; Khoo, Bee Luan; Tan, Daniel Shao-Weng; Bhagat, Ali Asgar S; Lim, Wan-Teck; Yap, Yoon Sim; Lee, Soo Chin; Soo, Ross A; Han, Jongyoon; Lim, Chwee Teck

    2014-07-07

    The detection and characterization of rare circulating tumor cells (CTCs) from the blood of cancer patients can potentially provide critical insights into tumor biology and hold great promise for cancer management. The ability to collect a large number of viable CTCs for various downstream assays such as quantitative measurements of specific biomarkers or targeted somatic mutation analysis is increasingly important in medical oncology. Here, we present a simple yet reliable microfluidic device for the ultra-high-throughput, label-free, size-based isolation of CTCs from clinically relevant blood volumes. The fast processing time of the technique (7.5 mL blood in less than 10 min) and the ability to collect more CTCs from larger blood volumes lends itself to a broad range of potential genomic and transcriptomic applications. A critical advantage of this protocol is the ability to return all fractions of blood (i.e., plasma (centrifugation), CTCs and white blood cells (WBCs) (size-based sorting)) that can be utilized for diverse biomarker studies or time-sensitive molecular assays such as RT-PCR. The clinical use of this biochip was demonstrated by detecting CTCs from 100% (10/10) of blood samples collected from patients with advanced-stage metastatic breast and lung cancers. The CTC recovery rate ranged from 20 to 135 CTCs mL(-1) and obtained under high purity (of 1 CTC out of every 30-100 WBCs which gives ∼4 log depletion of WBCs). They were identified with immunofluorescence assays (pan-cytokeratin+/CD45-) and molecular probes such as HER2/neu.

  18. 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.

  19. 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

  20. Cyclic N-terminal loop of amylin forms non amyloid fibers.

    PubMed

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

    2013-10-01

    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.

  1. 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

  2. N-terminal helix reorients in recombinant C-fragment of Clostridium botulinum type B.

    PubMed

    Jayaraman, Seetharaman; Eswaramoorthy, Subramaniam; Ahmed, S Ashraf; Smith, Leonard A; Swaminathan, Subramanyam

    2005-04-29

    Botulinum neurotoxins comprise seven distinct serotypes (A-G) produced by Clostridium botulinum. The crystal structure of the binding domain of the botulinum neurotoxin type B (BBHc) has been determined to 2A resolution. The overall structure of BBHc is well ordered and similar to that of the binding domain of the holotoxin. However, significant structural changes occur at what would be the interface of translocation and binding domains of the holotoxin. The loop 911-924 shows a maximum displacement of 14.8A at the farthest point. The N-terminal helix reorients and moves by 19.5A from its original position. BBHc is compared with the binding domain of the holotoxin of botulinum type A and B, and the tetanus C-fragment to characterize the heavy chain-carbohydrate interactions. The probable reasons for different binding affinity of botulinum and tetanus toxins are discussed.

  3. 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.

  4. [Proteolysis of semax analogues with different N-terminal amino acids by aminopeptidases].

    PubMed

    Shevchenko, K V; V'iunova, T V; Nagaev, I Iu; Andreeva, L A; Alfeeva, L Iu; Miasoedov, N F

    2011-01-01

    Proteolysis of semax (Met-Glu-His-Phe-Pro-Gly-Pro, Sem) and its analogues ([Ala1]Sem, [Gly1]Sem, [Thr1]Sem, [Trp1]Sem) that are differ from semax in substitution of N-terminal Met residue were studied. It is shown that such replacement changes the rate of peptides degradation by N-aminopeptidases (EC 3.4.11.2, Sigma, Type VI, 9.2 units. Akt. / mg). [Ala1]Sem, [Gly1]Sem and [Thr1]Sem semax analogues proved to be more stable to proteolysis than semax (Sem), and their initial product of proteolysis is His-Phe-Pro-Gly-Pro (Sem-5). For triptophan analogue both Glu-His-Phe-Pro-Gly-Pro (Sem-6) and Sem-5 product are formed in similar quantities. It is found that all investigated analogues can be used as inhibitors in Sem proteolysis.

  5. Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B.

    PubMed

    Mondol, Tanumoy; Åden, Jörgen; Wittung-Stafshede, Pernilla

    2016-02-12

    Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

  6. 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.

  7. Discrete Molecular Dynamics Study of Oligomer Formation by N-Terminally Truncated Amyloid β-Protein

    PubMed Central

    Meral, Derya; Urbanc, Brigita

    2013-01-01

    In Alzheimer’s disease (AD), amyloid β-protein (Aβ) self–assembles into toxic oligomers. Of the two predominant Aβ alloforms, Aβ1–40 and Aβ1–42, the latter is particularly strongly linked to AD. N-terminally truncated and pyroglutamated Aβ peptides were recently shown to seed Aβ aggregation and contribute significantly to Aβ–mediated toxicity, yet their folding and assembly were not explored computationally. Discrete molecular dynamics (DMD) approach previously captured in vitro–derived distinct Aβ1–40 and Aβ1–42 oligomer size distributions and predicted that the more toxic Aβ1–42 oligomers had more flexible and solvent exposed N-termini than Aβ1–40 oligomers. Here, we examined oligomer formation of Aβ3–40, Aβ3–42, Aβ11–40, and Aβ11–42 by the DMD approach. The four N-terminally truncated peptides showed increased oligomerization propensity relative to the full–length peptides, consistent with in vitro findings. Conformations formed by Aβ3–40/42 had significantly more flexible and solvent–exposed N-termini than Aβ1–40/42 conformations. In contrast, in Aβ11–40/42 conformations the N-termini formed more contacts and were less accessible to the solvent. The compactness of the Aβ11–40/42 conformations was in part facilitated by Val12. Two single amino acid substitutions that reduced and abolished hydrophobicity at position 12, respectively, resulted in a proportionally increased structural variability. Our results suggest that Aβ11–40 and Aβ11–42 oligomers might be less toxic than Aβ1–40 and Aβ1–42 oligomers and offer a plausible explanation for the experimentally–observed increased toxicity of Aβ3–40 and Aβ3–42 and their pyroglutamated forms. PMID:23500806

  8. 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.

  9. 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

  10. 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.

  11. Specific cleavage of N-terminal acetyl-methionine from peptides by rabbit muscle fractions

    SciTech Connect

    Krishna, G.R.; Wold, F.

    1986-05-01

    The authors have investigated the hypothesis that the processing of eukaryotic proteins involves acetylation of the N-terminal Met, followed by removal of the resulting Ac-Met to expose the second amino acid in the sequence. An activity was identified in rabbit muscle extracts that effectively removes Ac-Met from a synthetic peptide I (AcMDETGDTALVA) resembling the N-terminus of actin. The activity is associated with microsomes and free ribosomes, but can be extracted from the ribosomes by treatment with 0.5 M NaCl in the presence of 2 mM Mg/sup + +/; a 200 fold purification is achieved by this differential centrifugation procedure. A number of chemically acetylated (/sup 14/C-Ac) peptides have been tested as substrates using an HPLC assay for liberated /sup 14/C-Ac-Met. The results suggest that the partially purified activity is specific for N-terminal Ac-Met in that other Ac-amino acids (Gly, Ala, Ser, Asp) were not released from similar peptides, including a derivative of peptide I with Met missing and with Ac-Asp as n-terminus. The amino acid in the second position also appears to be involved as a specificity determinant; a peptide Ac-Met-Arg-Phe-Ala was inert and the tripeptide Ac-Met/sub 3/ was a poor substrate. The dipeptide Ac-Met-Glu was also a very poor substrate, while several tripeptides Ac-Met-X-Y, were good substrates, although not as good as the actinlike peptide I.

  12. Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity.

    PubMed

    Yuan, Dongfen; Yi, Xiang; Zhao, Yuling; Poon, Chi-Duen; Bullock, Kristin M; Hansen, Kim M; Salameh, Therese S; Farr, Susan A; Banks, William A; Kabanov, Alexander V

    2017-03-24

    Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

  13. Micellar environments induce structuring of the N-terminal tail of the prion protein.

    PubMed

    Renner, Christian; Fiori, Stella; Fiorino, Ferdinando; Landgraf, Dirk; Deluca, Dominga; Mentler, Matthias; Grantner, Klaus; Parak, Fritz G; Kretzschmar, Hans; Moroder, Luis

    2004-03-01

    In the physiological form, the prion protein is a glycoprotein tethered to the cell surface via a C-terminal glycosylphosphatidylinositol anchor, consisting of a largely alpha-helical globular C-terminal domain and an unstructured N-terminal portion. This unstructured part of the protein contains four successive octapeptide repeats, which were shown to bind up to four Cu(2+) ions in a cooperative manner. To mimic the location of the protein on the cell membrane and to analyze possible structuring effects of the lipid/water interface, the conformational preferences of a single octapeptide repeat and its tetrameric form, as well of the fragment 92-113, proposed as an additional copper binding site, were comparatively analyzed in aqueous and dodecylphosphocholine micellar solution as a membrane mimetic. While for the downstream fragment 92-113 no conformational effects were detectable in the presence of DPC micelles by CD and NMR, both the single octapeptide repeat and, in an even more pronounced manner, its tetrameric form are restricted into well-defined conformations. Because of the repetitive character of the rigid structural subdomain in the tetrarepeat molecule, the spatial arrangement of these identical motifs could not be resolved by NMR analysis. However, the polyvalent nature of the repetitive subunits leads to a remarkably enhanced interaction with the micelles, which is not detectably affected by copper complexation. These results strongly suggest interactions of the cellular form of PrP (PrP(c)) N-terminal tail with the cell membrane surface at least in the octapeptide repeat region with preorganization of these sequence portions for copper complexation. There are sufficient experimental facts known that support a physiological role of copper complexation by the octapeptide repeat region of PrP(c) such as a copper-buffering role of the PrP(c) protein on the extracellular surface.

  14. Defining Lipid Interacting Domains in the N-terminal Region of Apolipoprotein B

    PubMed Central

    Jiang, Zhenghui Gordon; Gantz, Donald; Bullitt, Esther; McKnight, C. James

    2008-01-01

    Apolipoprotein B (ApoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the “bad cholesterol” that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted α-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogenous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous to lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins. PMID:17002280

  15. The ABRF Edman Sequencing Research Group 2008 Study: Investigation into Homopolymeric Amino Acid N-Terminal Sequence Tags and Their Effects on Automated Edman Degradation

    PubMed Central

    Thoma, R. S.; Smith, J. S.; Sandoval, W.; Leone, J. W.; Hunziker, P.; Hampton, B.; Linse, K. D.; Denslow, N. D.

    2009-01-01

    were demonstrated to be responsible for the high lag during N-terminal sequence analysis. PMID:19721823

  16. Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins.

    PubMed

    Jbara, Muhammad; Maity, Suman Kumar; Seenaiah, Mallikanti; Brik, Ashraf

    2016-04-20

    Facilitating the process of chemical protein synthesis is an important goal in order to enable the efficient preparation of large and novel protein analogues. Native chemical ligation, which is widely used in the synthesis and semisynthesis of proteins, has been going through several developments to expedite the synthetic process and to obtain the target protein in high yield. A key aspect of this approach is the utilization of protecting groups for the N-terminal Cys in the middle fragments, which bear simultaneously the two reactive groups, i.e., N-terminal Cys and C-terminal thioester. Despite important progress in this area, as has been demonstrated in the use of thiazolidine protecting group in the synthesis of over 100 proteins, finding optimal protecting group(s) remains a challenge. For example, the thiazolidine removal step is very slow (>8 h), and in some cases the applied conditions lead to undesired side reactions. Here we show that water-soluble palladium(II) complexes are excellent reagents for the effective unmasking of thiazolidine, enabling its complete removal within 15 min under native chemical ligation conditions. Moreover, palladium is also able to rapidly remove propargyloxycarbonyl-protecting group from the N-terminal Cys in a similar efficiency. The utility of the new removal conditions for both protecting groups is exemplified in the rapid and efficient synthesis of Lys34-ubiquitinated H2B and for the first time neddlyated peptides derived from cullin1. The current approach expands the use of palladium in protein chemistry and should significantly facilitate the chemical and semisynthesis of synthetically challenging proteins from multiple fragments.

  17. The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

    PubMed Central

    Warnhoff, Kurt; Murphy, John T.; Kumar, Sandeep; Schneider, Daniel L.; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

    2014-01-01

    The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. PMID:25330323

  18. Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

    PubMed

    Songsiriritthigul, Chomphunuch; Suebka, Suwimon; Chen, Chun Jung; Fuengfuloy, Pitchayada; Chuawong, Pitak

    2017-02-01

    The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNA(Asp) and tRNA(Asn). Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Å resolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNA(Asp) and tRNA(Asn). It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNA(Asp) ((34)GUC(36)) and tRNA(Asn) ((34)GUU(36)). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

  19. Biosynthesis, glycosylation, and partial N-terminal amino acid sequence of the T-cell-activating protein TAP.

    PubMed Central

    Reiser, H; Coligan, J; Benacerraf, B; Rock, K L

    1987-01-01

    We have characterized the TAP molecule, an Ly-6 linked T-cell-activating glycoprotein. The three TAP bands that are precipitated from metabolically labeled cells display a common migration pattern in isoelectric focusing/NaDodSO4/PAGE gels and have common N-terminal sequences. This sequence is rich in cysteine and is homologous to that previously reported for the Ly-6.1E antigen. We, therefore, compared TAP and Ly-6.1E biochemically and found them to be structurally distinct. Given the role of TAP in T-cell activation, we further studied whether the molecule was phosphorylated. We have not found evidence for phosphorylation of the TAP protein. The carbohydrates present on the TAP molecule are resistant to peptide N-glycosidase F in vitro and tunicamycin in vivo. The upper band of the TAP triplet is susceptible to treatment with trifluoromethanesulfonic acid and thus seems to be of the O-linked rather than of the N-linked variety. The biosynthetic processing of TAP was studied in pulse-chase experiments. The middle band of the TAP triplet appears to be the earliest detectable species. Its conversion to the O-linked high molecular weight species can be blocked by monensin. Images PMID:3033645

  20. Temperature effects on the hydrodynamic radius of the intrinsically disordered N-terminal region of the p53 protein.

    PubMed

    Langridge, Timothy D; Tarver, Micheal J; Whitten, Steven T

    2014-04-01

    Intrinsically disordered proteins (IDPs) are often characterized in terms of the hydrodynamic radius, Rh . The Rh of IDPs are known to depend on fractional proline content and net charge, where increased numbers of proline residues and increased net charge cause larger Rh . Though sequence and charge effects on the Rh of IDPs have been studied, the temperature sensitivity has been noted only briefly. Reported here are Rh measurements in the temperature range of 5-75°C for the intrinsically disordered N-terminal region of the p53 protein, p53(1-93). Of note, the Rh of this protein fragment was highly sensitive to temperature, decreasing from 35 Å at 5°C to 26 Å at 75°C. Computer generated simulations of conformationally dynamic and disordered polypeptide chains were performed to provide a hypothesis for the heat-induced compaction of p53(1-93) structure, which was opposite to the heat-induced increase in Rh observed for a model folded protein. The simulations demonstrated that heat caused Rh to trend toward statistical coil values for both proteins, indicating that the effects of heat on p53(1-93) structure could be interpreted as thermal denaturation. The simulation data also predicted that proline content contributed minimally to the native Rh of p53(1-93), which was confirmed by measuring Rh for a substitution variant that had all 22 proline residues changed for glycine.

  1. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

    2015-01-01

    Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

  2. Evidence of Molecular Interactions of Aβ1-42 with N-Terminal Truncated Beta Amyloids by NMR.

    PubMed

    Tomaselli, Simona; Pagano, Katiuscia; D'Arrigo, Cristina; Molinari, Henriette; Ragona, Laura

    2017-02-06

    Aβ peptides, the main protein components of Alzheimer's disease (AD) plaques, derive from a proteolytic cleavage of the amyloid precursor protein. Due to heterogeneous cleavage sites, a series of Aβ peptides, including the major and widely studied species Aβ1-40 (Aβ40) and Aβ1-42 (Aβ42), are produced. In addition to the C-terminal heterogeneity of Aβ peptides, significant amounts of N-terminal truncated (Aβ3-42) and pyroglutamate-modified amyloid-β peptides (AβpE3-42) have been identified in AD affected brains and shown to be more cytotoxic than unmodified Aβ peptides. Little is known about the properties of their mixtures with Aβ42. Nuclear Magnetic Resonance spectroscopy is here employed to investigate the interaction of N-truncated peptides with Aβ42 at different molar ratios. We highlight the critical concentration of N-truncated forms influencing the aggregation kinetics of Aβ42. We provide evidence, at residue level, that the C-terminal region of Aβ42 is the locus of transient specific interactions with highly aggregation prone N-truncated alloforms.

  3. Role of N-terminal domain of HMW 1Dx5 in the functional and structural properties of wheat dough.

    PubMed

    Wang, Jing Jing; Liu, Guang; Huang, Yan-Bo; Zeng, Qiao-Hui; Song, Guo-Sheng; Hou, Yi; Li, Lin; Hu, Song-Qing

    2016-12-15

    Effects of N-terminal domain of high molecular weight glutenin subunit (HMW-GS) 1Dx5 (1Dx5-N) on functional and structural properties of wheat dough were determined by farinographic and rheological analysis, size exclusion chromatography, non-reducing/reducing SDS-PAGE, total free sulfhydryl determination, scanning electron microscopy and Fourier transform infrared spectroscopy. Results showed that 1Dx5-N improved the quality of dough with the increased water absorption, dough stability time, elastic and viscous modulus, and the decreased degree of softening, loss tangent. These improvements could be attributed to the formation of the macro-molecular weight aggregates and massive protein networks, which were favored by 1Dx5-N through disulfide bonds and hydrophobic interactions. Additionally, 1Dx5-N drove the transition of α-helix and random coil conformations to β-sheet and β-turn conformations, further demonstrating the formation of HMW-GS polymers and the enhancement of dough strength. Moreover, all the positive effects of 1Dx5-N were reinforced by edible salt NaCl.

  4. Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

    SciTech Connect

    Kwon, Hyock Joo; Abi-Mosleh, Lina; Wang, Michael L.; Deisenhofer, Johann; Goldstein, Joseph L.; Brown, Michael S.; Infante, Rodney E.

    2010-09-21

    LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3{beta}-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3{beta}-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

  5. Biosynthesis, glycosylation, and partial N-terminal amino acid sequence of the T-cell-activating protein TAP

    SciTech Connect

    Reiser, H.; Coligan, J.; Benacerraf, B.; Rock, K.L.

    1987-05-01

    The authors have characterized the TAP molecule, an Ly-6 linked T-cell-activating glycoprotein. The three TAP bands that are precipitated from metabolically labeled cells display a common migration pattern in isoelectric focusing/NaDodSO/sub 4//PAGE gels and have common N-terminal sequences. This sequence is rich in cysteine and is homologous to that previously reported for the Ly-6.1E antigen. They therefore, compared TAP and Ly-6.1E biochemically and found them to be structurally distinct. Given the role of TAP in T-cell activation, they further studied whether the molecule was phosphorylated. We have not found evidence for phosphorylation of the TAP protein. The carbohydrates present on the TAP molecule are resistant to peptide N-glycosidase F in vitro and tunicamycin in vivo. The upper band of the TAP triplet is susceptible to treatment with trifluoromethanesulfonic acid and thus seems to be of the O-linked rather than of the N-linked variety. The biosynthetic processing of TAP was studied in pulse-chase experiments. The middle band of the TAP triplet appears to be the earliest detectable species. Its conversion to the O-linked high molecular weight species can be blocked by monensin.

  6. [Cloning and expression of N-terminal protective domain of spaA gene from Erysipelothrix rhusiopathiae C43311].

    PubMed

    Nazierbieke, Wulumuhan; Liu, Zhuxiang; Li, Ke; Chen, Yiguang; Borrathybay, Entomack

    2008-02-01

    The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the previously reported different serotype strains of E. rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fusion protein of GST-SpaA-N possessed high immunoreactivity.

  7. Crystal Structure of the Lamprey Variable Lymphocyte Receptor C Reveals an Unusual Feature in Its N-Terminal Capping Module

    PubMed Central

    Kanda, Ryo; Sutoh, Yoichi; Kasamatsu, Jun; Maenaka, Katsumi; Kasahara, Masanori; Ose, Toyoyuki

    2014-01-01

    Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner. PMID:24465760

  8. Regulation of the Structurally Dynamic N-terminal Domain of Progesterone Receptor by Protein-induced Folding*

    PubMed Central

    Kumar, Raj; Moure, Carmen M.; Khan, Shagufta H.; Callaway, Celetta; Grimm, Sandra L.; Goswami, Devrishi; Griffin, Patrick R.; Edwards, Dean P.

    2013-01-01

    The N-terminal domain (NTD) of steroid receptors harbors a transcriptional activation function (AF1) that is composed of an intrinsically disordered polypeptide. We examined the interaction of the TATA-binding protein (TBP) with the NTD of the progesterone receptor (PR) and its ability to regulate AF1 activity through coupled folding and binding. As assessed by solution phase biophysical methods, the isolated NTD of PR contains a large content of random coil, and it is capable of adopting secondary α-helical structure and more stable tertiary folding either in the presence of the natural osmolyte trimethylamine-N-oxide or through a direct interaction with TBP. Hydrogen-deuterium exchange coupled with mass spectrometry confirmed the highly dynamic intrinsically disordered property of the NTD within the context of full-length PR. Deletion mapping and point mutagenesis defined a region of the NTD (amino acids 350–428) required for structural folding in response to TBP interaction. Overexpression of TBP in cells enhanced transcriptional activity mediated by the PR NTD, and deletion mutations showed that a region (amino acids 327–428), similar to that required for TBP-induced folding, was required for functional response. TBP also increased steroid receptor co-activator 1 (SRC-1) interaction with the PR NTD and cooperated with SRC-1 to stimulate NTD-dependent transcriptional activity. These data suggest that TBP can mediate structural reorganization of the NTD to facilitate the binding of co-activators required for maximal transcriptional activation. PMID:23995840

  9. Phenyl amide linker improves the pharmacokinetics and pharmacodynamics of N-terminally mono-PEGylated human growth hormone.

    PubMed

    Wu, Ling; Ji, Shaoyang; Shen, Lijuan; Hu, Tao

    2014-09-02

    Human growth hormone (hGH) suffers from a short plasma half-life of ∼15 min, necessitating frequent injections to maintain its physiological effect. PEGylation, conjugation of polyethylene glycol (PEG), is an effective strategy to prolong the plasma half-life of hGH. However, PEGylation can significantly decrease the bioactivity of hGH. Thus, a new PEGylation approach is desired to improve the pharmacokinetics (PK) and pharmacodynamics (PD) of the PEGylated hGH. In the present study, two N-terminally mono-PEGylated hGHs were prepared using aldehyde chemistry. Phenyl amide and ethyl moieties were used as the linkers between PEG and hGH, respectively. The hydrodynamic volume, proteolytic sensitivity, and immunogenicity of the PEGylated hGH with phenyl amide linker (hGH-phenyl-PEG) were lower than those of the one with propyl linker (hGH-prop-PEG). In addition, hGH-phenyl-PEG showed a higher in vitro bioactivity and better PK and PD than hGH-prop-PEG. The better PK of hGH-phenyl-PEG was mainly due to its lower proteolytic sensitivity and low immunogenicity. The better PD of hGH-phenyl-PEG was mainly due to its higher in vitro bioactivity. Thus, the phenyl amide linker can improve the overall pharmacological profiles of the PEGylated hGH. Our study is expected to advance the development of long-acting protein biotherapeutics with high therapeutic efficacy.

  10. Crystal structure of the lamprey variable lymphocyte receptor C reveals an unusual feature in its N-terminal capping module.

    PubMed

    Kanda, Ryo; Sutoh, Yoichi; Kasamatsu, Jun; Maenaka, Katsumi; Kasahara, Masanori; Ose, Toyoyuki

    2014-01-01

    Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.

  11. Inhibition of polyglutamine aggregation by SIMILAR huntingtin N-terminal sequences: Prospective molecules for preclinical evaluation in Huntington's disease.

    PubMed

    Burra, Gunasekhar; Thakur, Ashwani Kumar

    2017-04-12

    The mutant huntingtin protein (mHtt) fragments with expanded polyglutamine sequence forms microscopically visible aggregates in neurons, a hallmark of Huntington's disease (HD). The aggregation process and aggregates are possible targets of therapeutic intervention in HD. Due to lack of treatment and cure, the patients die within 15-20 years after the disease onset. Therefore, discovering therapeutic molecules that may either inhibit the aggregation mechanism or downregulate the toxic effects of mhtt are highly needed. The present study demonstrates the design and use of peptide inhibitors based on the role played by the N-terminal seventeen amino acid sequence (NT17 ) of huntingtin fragment in its aggregation. Fug-NT17 (Fugu), Xen-NT17 (Xenopus), Dro-NT17 (Drosophila), Aib-NT17 , and Pro-NT17 sequences were tested for their ability to inhibit aggregation. Among them, the first three are the sequence variants of human NT17 from evolutionarily distant organisms and the latter two are the analogs of human NT17 containing aminoisobutyric acid (Aib) and proline (Pro). Four out of five inhibited the aggregation of huntingtin fragment, NT17 Q35 P10 K2 polypeptide. Data indicates that the physicochemical properties of the inhibitors play a crucial role in exhibiting the inhibitory effect. These inhibitors can be tested in cell and animal models for the preclinical evaluation in the treating of HD. This article is protected by copyright. All rights reserved.

  12. Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.

    PubMed

    Tanghe, Magali; Danneels, Barbara; Camattari, Andrea; Glieder, Anton; Vandenberghe, Isabel; Devreese, Bart; Stals, Ingeborg; Desmet, Tom

    2015-12-01

    The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.

  13. Involvement of hippocampal jun-N terminal kinase pathway in the enhancement of learning and memory by nicotine.

    PubMed

    Kenney, Justin W; Florian, Cédrick; Portugal, George S; Abel, Ted; Gould, Thomas J

    2010-01-01

    Despite intense scrutiny over the past 20 years, the reasons for the high addictive liability of nicotine and extreme rates of relapse in smokers have remained elusive. One factor that contributes to the development and maintenance of nicotine addiction is the ability of nicotine to produce long-lasting modifications of behavior, yet little is known about the mechanisms by which nicotine alters the underlying synaptic plasticity responsible for behavioral changes. This study is the first to explore how nicotine interacts with learning to alter gene transcription, which is a process necessary for long-term memory consolidation. Transcriptional upregulation of hippocampal jun-N terminal kinase 1 (JNK1) mRNA was found in mice that learned contextual fear conditioning (FC) in the presence of nicotine, whereas neither learning alone nor nicotine administration alone exerted an effect. Furthermore, the upregulation of JNK1 was absent in beta2 nicotinic receptor subunit knockout mice, which are mice that do not show enhanced learning by nicotine. Finally, hippocampal JNK activation was increased in mice that were administered nicotine before conditioning, and the inhibition of JNK during consolidation prevented the nicotine-induced enhancement of contextual FC. These data suggest that nicotine and learning interact to alter hippocampal JNK1 gene expression and related signaling processes, thus resulting in strengthened contextual memories.

  14. A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT

    PubMed Central

    Mao, Peng; Kyriss, McKenna N. M.; Hodges, Amelia J.; Duan, Mingrui; Morris, Robert T.; Lavine, Mark D.; Topping, Traci B.; Gloss, Lisa M.; Wyrick, John J.

    2016-01-01

    Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A–H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A–H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A–H2B deposition by FACT. PMID:27369377

  15. c-Jun N-terminal kinase (JNK) is involved in immune defense against bacterial infection in Crassostrea hongkongensis.

    PubMed

    Qu, Fufa; Xiang, Zhiming; Xiao, Shu; Wang, Fuxuan; Li, Jun; Zhang, Yang; Zhang, Yuehuan; Qin, Yanping; Yu, Ziniu

    2017-02-01

    c-Jun N-terminal kinase (JNK) is a universal and essential subgroup of the mitogen-activated protein kinase (MAPK) superfamily, which is highly conserved from yeast to mammals and functions in a variety of physiological and pathological processes. In this study, we report the first oyster JNK gene homolog (ChJNK) and its biological functions in the Hong Kong oyster Crassostrea hongkongensis. The ChJNK protein consists of 383 amino acids and contains a conserved serine/threonine protein kinase (S_TKc) domain with a typical TPY motif. Phylogenetic analysis revealed that ChJNK shared a close evolutionary relationship with Crassostrea gigas JNK. Quantitative RT-PCR analyses revealed broad expression patterns of ChJNK mRNA in various adult tissues and different embryonic and larval stages of C. hongkongensis. When exposed to Vibrio alginolyticus or Staphylococcus haemolyticus, ChJNK mRNA expression levels were significantly up-regulated in the hemocytes and gills in a time-dependent manner. Additionally, subcellular localization studies that ChJNK is a cytoplasm-localized protein, and that its overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. In summary, this study provided the first experimental demonstration that oysters possess a functional JNK that participates in host defense against bacterial infection in C. hongkongensis.

  16. Ex vivo generation of a highly potent population of circulating angiogenic cells using a collagen matrix.

    PubMed

    Kuraitis, Drew; Hou, Chenchen; Zhang, Yan; Vulesevic, Branka; Sofrenovic, Tanja; McKee, Daniel; Sharif, Zahra; Ruel, Marc; Suuronen, Erik J

    2011-08-01

    Biomaterials that have the ability to augment angiogenesis are highly sought-after for applications in regenerative medicine, particularly for revascularization of ischemic and infarcted tissue. We evaluated the culture of human circulating angiogenic cells (CAC) on collagen type I-based matrices, and compared this to traditional selective-adhesion cultures on fibronectin. Culture on a collagen matrix supported the proliferation of CD133(+) and CD34(+)CD133(+) CACs. When subjected to serum starvation, the matrix conferred a resistance to cell death for CD34(+) and CD133(+) progenitors and increased phosphorylation of Akt. After 4days of culture, phenotypically enriched populations of endothelial cells (CD31(+)CD144(+)) and progenitor cells (CD34(+)CD133(+)) emerged. Culture on matrix upregulated the phosphorylation and activation of ERK1/2 pathway members, and matrix-cultured cells also had an enhanced functional capacity for adhesion and invasion. These functional improvements were abrogated when cultured in the presence of ERK inhibitors. The formation of vessel-like structures in an angiogenesis assay was augmented with matrix-cultured cells, which were also more likely to physically associate with such structures compared to CACs taken from culture on fibronectin. In vivo, treatment with matrix-cultured cells increased the size and density of arterioles, and was superior at restoring perfusion in a mouse model of hindlimb ischemia, compared to fibronectin-cultured cell treatment. This work suggests that a collagen-based matrix, as a novel substrate for CAC culture, possesses the ability to enrich endothelial and angiogenic populations and lead to clinically relevant functional enhancements.

  17. High-Relaxivity Superparamagnetic Iron Oxide Nanoworms with Decreased Immune Recognition and Long-Circulating Properties

    PubMed Central

    Wang, Guankui; Inturi, Swetha; Serkova, Natalie J.; Merkulov, Sergey; McCrae, Keith; Russek, Stephen E.; Banda, Nirmal K.; Simberg, Dmitri

    2015-01-01

    One of the core issues of nanotechnology involves masking the foreignness of nanomaterials to enable in vivo longevity and long-term immune evasion. Dextran-coated superparamagnetic iron oxide nanoparticles are very effective magnetic resonance imaging (MRI) contrast agents, and strategies to prevent immune recognition are critical for their clinical translation. Here we prepared 20 kDa dextran-coated SPIO nanoworms (NWs) of 250 nm diameter and a high molar transverse relaxivity rate R2 (~400 mM−1 s−1) to study the effect of cross-linking-hydrogelation with 1-chloro-2,3-epoxypropane (epichlorohydrin) on the immune evasion both in vitro and in vivo. Cross-linking was performed in the presence of different concentrations of NaOH (0.5 to 10 N) and different temperatures (23 and 37 °C). Increasing NaOH concentration and temperature significantly decrease the binding of anti-dextran antibody and dextran-binding lectin conconavalin A to the NWs. The decrease in dextran immunoreactivity correlated with the decrease in opsonization by complement component 3 (C3) and with the decrease in the binding of the lectin pathway factor MASP-2 in mouse serum, suggesting that cross-linking blocks the lectin pathway of complement. The decrease in C3 opsonization correlated with the decrease in NW uptake by murine peritoneal macrophages. Optimized NWs demonstrated up to 10 h circulation half-life in mice and minimal uptake by the liver, while maintaining the large 250 nm size in the blood. We demonstrate that immune recognition of large iron oxide nanoparticles can be efficiently blocked by chemical cross-linking-hydrogelation, which is a promising strategy to improve safety and bioinertness of MRI contrast agents. PMID:25419856

  18. Improved detection of circulating tumor cells in non-metastatic high-risk prostate cancer patients

    PubMed Central

    Kuske, Andra; Gorges, Tobias M.; Tennstedt, Pierre; Tiebel, Anne-Kathrin; Pompe, Raisa; Preißer, Felix; Prues, Sandra; Mazel, Martine; Markou, Athina; Lianidou, Evi; Peine, Sven; Alix-Panabières, Catherine; Riethdorf, Sabine; Beyer, Burkhard; Schlomm, Thorsten; Pantel, Klaus

    2016-01-01

    The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system, an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points, CTCs were detected in 37%, 54.9% and 58.7% of patients using CellSearch, CellCollector and EPISPOT, respectively. The cumulative positivity rate of the three CTC assays was 81.3% (87/107) with 21.5% (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66% before RP to 34% after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p < 0.0001) and clinical tumor stage (p = 0.04), while the other assays showed no significant correlations. In conclusion, CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer. PMID:28000772

  19. Future climate of the Caribbean from a super-high-resolution atmospheric general circulation model

    NASA Astrophysics Data System (ADS)

    Hall, Trevor C.; Sealy, Andrea M.; Stephenson, Tannecia S.; Kusunoki, Shoji; Taylor, Michael A.; Chen, A. Anthony; Kitoh, Akio

    2013-07-01

    Present-day (1979-2003) and future (2075-2099) simulations of mean and extreme rainfall and temperature are examined using data from the Meteorological Research Institute super-high-resolution atmospheric general circulation model. Analyses are performed over the 20-km model grid for (1) a main Caribbean basin, (2) sub-regional zones, and (3) specific Caribbean islands. Though the model's topography underestimates heights over the eastern Caribbean, it captures well the present-day spatial and temporal variations of seasonal and annual climates. Temperature underestimations range from 0.1 °C to 2 °C with respect to the Japanese Reanalysis and the Climatic Research Unit datasets. The model also captures fairly well sub-regional scale variations in the rainfall climatology. End-of-century projections under the Intergovernmental Panel on Climate Change SRES A1B scenario indicate declines in rainfall amounts by 10-20 % for most of the Caribbean during the early (May-July) and late (August-October) rainy seasons relative to the 1979-2003 baselines. The early dry season (November-January) is also projected to get wetter in the far north and south Caribbean by approximately 10 %. The model also projects a warming of 2-3 °C over the Caribbean region. Analysis of future climate extremes indicate a 5-10 % decrease in the simple daily precipitation intensity but no significant change in the number of consecutive dry days for Cuba, Jamaica, southern Bahamas, and Haiti. There is also indication that the number of hot days and nights will significantly increase over the main Caribbean basin.

  20. 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

    2014-01-03

    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.

  1. Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

    PubMed Central

    Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

    2013-01-01

    This study investigated the functional roles of the N-terminal Ca2+ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca2+-binding site of PAD4 were mutated to disrupt the binding of Ca2+ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the kcat/Km,BAEE values were 0.02, 0.63 and 0.01 s−1mM−1 (20.8 s−1mM−1 for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a kcat value of 0.3 s−1 (13.3 s−1 for wild-type), whereas D176A retained some catalytic power with a kcat of 9.7 s−1. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the kcat/Km,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca2+ indicated that the conformational stability of the enzyme is highly dependent on Ca2+ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca2+ ions in the N-terminal Ca2+-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca2+ ions play critical roles in the full activation of the PAD4 enzyme. PMID:23382808

  2. Functional roles of the non-catalytic calcium-binding sites in the N-terminal domain of human peptidylarginine deiminase 4.

    PubMed

    Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

    2013-01-01

    This study investigated the functional roles of the N-terminal Ca(2+) ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca(2+)-binding site of PAD4 were mutated to disrupt the binding of Ca(2+) ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k(cat)/K(m,BAEE) values were 0.02, 0.63 and 0.01 s(-1)mM(-1) (20.8 s(-1)mM(-1) for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k(cat) value of 0.3 s(-1) (13.3 s(-1) for wild-type), whereas D176A retained some catalytic power with a k(cat) of 9.7 s(-1). Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k(cat)/K(m,BAEE) values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca(2+) indicated that the conformational stability of the enzyme is highly dependent on Ca(2+) ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca(2+) ions in the N-terminal Ca(2+)-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca(2+) ions play critical roles in the full activation of the PAD4 enzyme.

  3. SAXS Structural Studies of Dps from Deinococcus radiodurans Highlights the Conformation of the Mobile N-Terminal Extensions.

    PubMed

    Santos, Sandra P; Cuypers, Maxime G; Round, Adam; Finet, Stephanie; Narayanan, Theyencheri; Mitchell, Edward P; Romão, Célia V

    2017-03-10

    The radiation-resistant bacterium Deinococcus radiodurans contains two DNA-binding proteins from starved cells (Dps): Dps1 (DR2263) and Dps2 (DRB0092). These are suggested to play a role in DNA interaction and manganese and iron storage. The proteins assemble as a conserved dodecameric structure with structurally uncharacterised N-terminal extensions. In the case of DrDps1, these extensions have been proposed to be involved in DNA interactions, while in DrDps2, their function has yet to be established. The reported data reveal the relative position of the N-terminal extensions to the dodecameric sphere in solution for both Dps. The low-resolution small angle X-ray scattering (SAXS) results show that the N-terminal extensions protrude from the spherical shell of both proteins. The SAXS envelope of a truncated form of DrDps1 without the N-terminal extensions appears as a dodecameric sphere, contrasting strongly with the protrusions observed in the full-length models. The effect of iron incorporation into DrDps2 was investigated by static and stopped-flow SAXS measurements, revealing dynamic structural changes upon iron binding and core formation, as reflected by a quick alteration of its radius of gyration. The truncated and full-length versions of DrDps were also compared on the basis of their interaction with DNA to analyse functional roles of the N-terminal extensions. DrDps1 N-terminal protrusions appear to be directly involved with DNA, whilst those from DrDps2 are indirectly associated with DNA binding. Furthermore, detection of DrDps2 in the D. radiodurans membrane fraction suggests that the N-terminus of the protein interacts with the membrane.

  4. Role of Prion Disease-Linked Mutations in the Intrinsically Disordered N-Terminal Domain of the Prion Protein.

    PubMed

    Cong, Xiaojing; Casiraghi, Nicola; Rossetti, Giulia; Mohanty, Sandipan; Giachin, Gabriele; Legname, Giuseppe; Carloni, Paolo

    2013-11-12

    Prion diseases are fatal neurodegenerative disorders in mammals and other animal species. In humans, about 15% of these maladies are caused by pathogenic mutations (PMs) in the gene encoding for the prion protein (PrP(C)). Seven PMs are located in the naturally unfolded PrP(C) N-terminal domain, which constitutes about half of the protein. Intriguingly and in sharp contrast to other PMs clustered in the folded domain, N-terminal PMs barely affect the conversion to the pathogenic (scrapie, or PrP(Sc)) isoform of PrP(C). Here, we hypothesize that the neurotoxicity of these PMs arises from changes in structural determinants of the N-terminal domain, affecting the protein binding with its cellular partners and/or the cotranslational translocation during the PrP(C) biosynthesis. We test this idea by predicting the conformational ensemble of the wild-type (WT) and mutated mouse PrP(C) N-terminal domain, whose sequence is almost identical to that of the human one and for which the largest number of in vivo data is available. The conformational properties of the WT are consistent with those inferred experimentally. Importantly, the PMs turn out to affect in a subtle manner the intramolecular contacts in the putative N-terminal domain binding sites for Cu(2+) ions, sulphated glycosaminoglycans, and other known PrP(C) cellular partners. The PMs also alter the local structural features of the transmembrane domain and adjacent stop transfer effector, which act together to regulate the protein topology. These results corroborate the hypothesis that N-terminal PMs affect the PrP(C) binding to functional interactors and/or the translocation.

  5. Soluble N-terminal fragment of mutant Huntingtin protein impairs mitochondrial axonal transport in cultured hippocampal neurons.

    PubMed

    Tian, Jun; Yan, Ya-Ping; Zhou, Rui; Lou, Hui-Fang; Rong, Ye; Zhang, Bao-Rong

    2014-02-01

    Huntington's disease (HD) is an autosomal dominant, progressive, neurodegenerative disorder caused by an unstable expansion of CAG repeats (>35 repeats) within exon 1 of the interesting transcript 15 (IT15) gene. This gene encodes a protein called Huntingtin (Htt), and mutation of the gene results in a polyglutamine (polyQ) near the N-terminus of Htt. The N-terminal fragments of mutant Htt (mHtt), which tend to aggregate, are sufficient to cause HD. Whether these aggregates are causal or protective for HD remains hotly debated. Dysfunctional mitochondrial axonal transport is associated with HD. It remains unknown whether the soluble or aggregated form of mHtt is the primary cause of the impaired mitochondrial axonal transport in HD pathology. Here, we investigated the impact of soluble and aggregated N-terminal fragments of mHtt on mitochondrial axonal transport in cultured hippocampal neurons. We found that the N-terminal fragment of mHtt formed aggregates in almost half of the transfected neurons. Overexpression of the N-terminal fragment of mHtt decreased the velocity of mitochondrial axonal transport and mitochondrial mobility in neurons regardless of whether aggregates were formed. However, the impairment of mitochondrial axonal transport in neurons expressing the soluble and aggregated N-terminal fragments of mHtt did not differ. Our findings indicate that both the soluble and aggregated N-terminal fragments of mHtt impair mitochondrial axonal transport in cultured hippocampal neurons. We predict that dysfunction of mitochondrial axonal transport is an early-stage event in the progression of HD, even before mHtt aggregates are formed.

  6. Circulation of multiple serotypes of highly divergent enterovirus C in the Xinjiang Uighur Autonomous Region of China

    PubMed Central

    Zhang, Yong; Sun, Qiang; Cui, Hui; Yan, Dongmei; Fan, Qin; Song, Yang; Zhu, Shuangli; Li, Xiaolei; Huang, Guohong; Ji, Tianjiao; Hu, Lan; Wang, Dongyan; Yang, Qian; Xu, Wenbo

    2016-01-01

    Poliomyelitis associated with circulating vaccine-derived polioviruses (cVDPVs) is a serious public health issue in the post-eradication era, and the occurrence of recombinant cVDPVs emphasizes the need to elucidate enterovirus C (EV-C) epidemiology. Stool samples were collected from 826 healthy children in Southern Xinjiang in 2011 to investigate EV-C circulation and epidemiology. Thirty-six EV-Cs were isolated and assigned to eight EV-C serotypes by molecular serotyping, suggesting the circulation of diverse EV-Cs in Xinjiang. Phylogenetic analysis showed that the Xinjiang EV-C strains had larger variation compared to the prototype and other modern strains. Additionally, the results showed unique characteristics of Xinjiang EV-Cs, such as the cytopathicity of CV-A1 strains to RD cells; the high divergence in CV-A11, CV-A13, CV-A17, and CV-A20 strains; the divergence of Xinjiang CV-A24 from AHC-related CV-A24 variant stains distributed worldwide; and the circulation of two novel EV-C serotypes (EV-C96 and EV-C99). Evaluations of this dense and diverse EV-C ecosystem will help elucidate the processes shaping enteroviral biodiversity. This study will improve our understanding of the evolution of enteroviruses and the recombination potential between polioviruses and other EV-Cs. PMID:27642136

  7. Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

    PubMed

    Murison, David A; Ollivierre, Jaylene N; Huang, Qiuying; Budil, David E; Beuning, Penny J

    2017-01-01

    Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD' dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuD', but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

  8. Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine modified proteins by mass spectrometry

    PubMed Central

    Chowdhury, Saiful M.; Munske, Gerhard R.; Yang, Jonathon; Zhukova, Daria; Nguen, Hamilton; Bruce, James E.

    2014-01-01

    Rationale Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies recently published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed. Methods We present here a mass spectrometry-based study to evaluate and optimize the trypsin proteolysis on a guanidinated modified protein. Trypsin proteolysis was studied using different amount of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution. Results We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counter parts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS. Conclusion We found enzyme trypsin is active in guanidinated form of protein depending on enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on

  9. Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions

    PubMed Central

    Huang, Qiuying; Budil, David E.

    2017-01-01

    Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD′. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD′ dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1–7) and UmuD 18 (UmuD Δ1–17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuDʹ, but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD. PMID:28273172

  10. The C-Terminal SynMuv/DdDUF926 Domain Regulates the Function of the N-Terminal Domain of DdNKAP

    PubMed Central

    Burgute, Bhagyashri D.; Peche, Vivek S.; Müller, Rolf; Matthias, Jan; Gaßen, Berthold; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A.

    2016-01-01

    NKAP (NF-κB activating protein) is a highly conserved SR (serine/arginine-rich) protein involved in transcriptional control and splicing in mammals. We identified DdNKAP, the Dictyostelium discoideum ortholog of mammalian NKAP, as interacting partner of the nuclear envelope protein SUN-1. DdNKAP harbors a number of basic RDR/RDRS repeats in its N-terminal domain and the SynMuv/DUF926 domain at its C-terminus. We describe a novel and direct interaction between DdNKAP and Prp19 (Pre mRNA processing factor 19) which might be relevant for the observed DdNKAP ubiquitination. Genome wide analysis using cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq) revealed DdNKAP association with intergenic regions, exons, introns and non-coding RNAs. Ectopic expression of DdNKAP and its domains affects several developmental aspects like stream formation, aggregation, and chemotaxis. We conclude that DdNKAP is a multifunctional protein, which might influence Dictyostelium development through its interaction with RNA and RNA binding proteins. Mutants overexpressing full length DdNKAP and the N-terminal domain alone (DdN-NKAP) showed opposite phenotypes in development and opposite expression profiles of several genes and rRNAs. The observed interaction between DdN-NKAP and the DdDUF926 domain indicates that the DdDUF926 domain acts as negative regulator of the N-terminus. PMID:27997579

  11. DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the Saccharomyces cervisiae TATA Binding Protein

    SciTech Connect

    Gupta,S.; Cheng, H.; Mollah, A.; Jamison, E.; Morris, S.; Chance, M.; Khrapunov, S.; Brenowitz, M.

    2007-01-01

    Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by 'protein footprinting' with hydroxyl radical ({center_dot}OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

  12. 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

  13. Higher Frequency of Circulating PD-1(high) CXCR5(+)CD4(+) Tfh Cells in Patients with Chronic Schistosomiasis.

    PubMed

    Zhang, Yumei; Jiang, Yanyan; Wang, Yanjuan; Liu, Hua; Shen, Yujuan; Yuan, Zhongying; Hu, Yuan; Xu, Yuxin; Cao, Jianping

    2015-01-01

    The current knowledge of immunological responses to schistosomiasis is insufficient for the development of vaccine and therapies. The role of T follicular helper (Tfh) cells in schistosome infections is not fully defined. The frequency of circulating Tfh cells and serum cytokine levels were analyzed in 11 patients with chronic schistosomiasis and 10 healthy controls (HC), who reside in an endemic area for Schistosomiasis japonicum. Significantly higher frequencies of circulating CXCR5(+) CD4(+) Tfh cells and higher expression levels of ICOS and PD-1 in CXCR5(+) CD4(+) Tfh cells were observed in patients with chronic schistosomiasis compared to HC. The levels of IL-21 in serum and the expression of IL-21 mRNA were higher in chronic schistosomiasis patients than in HC. Moreover, the frequency of circulating PD-1(high) CXCR5(+) CD4(+) Tfh cells positively correlated with the levels of IL-21 in serum from patients with chronic schistosomiasis. A positive correlation was also found between the frequency of PD-1(high) CXCR5(+) CD4(+) Tfh cells and the levels of soluble egg antigen (SEA)-specific antibodies in serum samples from the patient group. Our study is the first regarding Tfh cells in chronic human schistosomiasis and the finding indicate that PD-1(high) CXCR5(+) CD4(+)Tfh cells might play an important role in the production of specific antibodies in schistosomiasis. This study contributes to the understanding of immune response to schistosomiasis and may provide helpful support in vaccine development.

  14. Structure analysis of the membrane-bound PhoD signal peptide of the Tat translocase shows an N-terminal amphiphilic helix.

    PubMed

    Klein, Marco J; Grage, Stephan L; Muhle-Goll, Claudia; Bürck, Jochen; Afonin, Sergii; Ulrich, Anne S

    2012-12-01

    Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution ¹³C/¹⁵N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD.

  15. Characterization of an invertase with pH tolerance and truncation of its N-terminal to shift optimum activity toward neutral pH.

    PubMed

    Du, Liqin; Pang, Hao; Wang, Zilong; Lu, Jian; Wei, Yutuo; Huang, Ribo

    2013-01-01

    Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.

  16. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    SciTech Connect

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan

    2013-08-15

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

  17. Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

    PubMed Central

    Batt, Sarah M.; Bingle, Lewis E.H.; Dafforn, Tim R.; Thomas, Christopher M.

    2009-01-01

    ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning. PMID:19109978

  18. Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.

    PubMed

    Pavlou, Demetria; Kirmizis, Antonis

    2016-03-01

    Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.

  19. Application of a Short, Disordered N-Terminal Flagellin Segment, a Fully Functional Flagellar Type III Export Signal, to Expression of Secreted Proteins ▿

    PubMed Central

    Dobó, József; Varga, János; Sajó, Ráchel; Végh, Barbara M.; Gál, Péter; Závodszky, Péter; Vonderviszt, Ferenc

    2010-01-01

    Recently, we have demonstrated that the 26-47 segment of Salmonella enterica serovar Typhimurium flagellin is capable of mediating flagellar export. In order to reveal whether other parts of the N-terminal region have any significant influence on secretion, a series of plasmids were constructed containing the lac promoter followed by the 26-47, 2-65, or 2-192 portion of Salmonella flagellin, to which various heterologous proteins of different size were fused (18 constructs overall). Essentially, all three segments could drive protein export; however, the nature of the attached polypeptide also had a significant effect on secretion efficiency. When low export efficiency was observed, it was mainly caused by inclusion body formation. Our data provide strong support for the idea that a short segment within the disordered N-terminal region of axial proteins is recognized by the flagellar type III export machinery. The 26-47 segment of flagellin contains all of the necessary information to direct translocation of attached polypeptide chains. This short (positions 26 to 47) flagellin segment attached to recombinant proteins can be used for secreted protein expression. Certain fusion proteins that are easily degraded within the cells were found to be intact in the medium, implying a potential application of this expression system for proteins with high proteolytic susceptibility. PMID:20008166

  20. SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation.

    PubMed

    Bienvenut, Willy V; Giglione, Carmela; Meinnel, Thierry

    2017-01-01

    Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.

  1. Modeling Black Sea circulation with high resolution in the coastal zone

    NASA Astrophysics Data System (ADS)

    Zalesnyi, V. B.; Gusev, A. V.; Agoshkov, V. I.

    2016-05-01

    We present a numerical model of Black Sea circulation based on primitive equations with improved spatial resolution in the coastal zone. The model equations are formulated in a two-pole orthogonal coordinate system with arbitrary locations of the poles and a vertical σ coordinate. Increased horizontal resolution is gained by displacing the pole into the vicinity of the separated subdomain. The problem is solved over a grid with a variable step. The northern coordinate pole is displaced to the vicinity of Gelendzhik; the grid step varies from 150 m in the coastal zone to 4.6 km in the main basin. We simulated the fields of currents, sea level, temperature, and salinity under the given atmospheric forcing in 2007. The model is capable of reproducing the large-scale Black Sea circulation and submesoscale variations in the coastal currents.

  2. CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells.

    PubMed

    Filippo, Thais R M; Galindo, Layla T; Barnabe, Gabriela F; Ariza, Carolina B; Mello, Luiz E; Juliano, Maria A; Juliano, Luiz; Porcionatto, Marimélia A

    2013-09-01

    Neural stem/progenitor cells (NSC) respond to injury after brain injuries secreting IL-1, IL-6, TNF-α, IL-4 and IL-10, as well as chemokine members of the CC and CXC ligand families. CXCL12 is one of the chemokines secreted at an injury site and is known to attract NSC-derived neuroblasts, cells that express CXCL12 receptor, CXCR4. Activation of CXCR4 by CXCL12 depends on two domains located at the N-terminal of the chemokine. In the present work we aimed to investigate if the N-terminal end of CXCL12, where CXCR4 binding and activation domains are located, was sufficient to induce NSC-derived neuroblast chemotaxis. Our data show that a synthetic peptide analogous to the first 21 amino acids of the N-terminal end of CXCL12, named PepC-C (KPVSLSYRCPCRFFESHIARA), is able to promote chemotaxis of neuroblasts in vivo, and stimulate chemotaxis and proliferation of CXCR4+ cells in vitro, without affecting NSC fate. We also show that PepC-C upregulates CXCL12 expression in vivo and in vitro. We suggest the N-terminal end of CXCL12 is responsible for a positive feedback loop to maintain a gradient of CXCL12 that attracts neuroblasts from the subventricular zone into an injury site.

  3. Molecular evolution of troponin I and a role of its N-terminal extension in nematode locomotion

    PubMed Central

    Barnes, Dawn E.; Hwang, Hyundoo; Ono, Kanako; Lu, Hang; Ono, Shoichiro

    2016-01-01

    Summary 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 C. 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. PMID:26849746

  4. Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity.

    PubMed

    Minagawa, Shun; Kondoh, Yasumitsu; Sueoka, Keigo; Osada, Hiroyuki; Nakamoto, Hitoshi

    2011-04-01

    Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.

  5. N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Bonneil, Eric; Thibault, Pierre; Meloche, Sylvain

    2004-01-01

    Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized. PMID:15226418

  6. High Altitude Dust Layers on Mars: Observations and Impact on the General Circulation

    NASA Astrophysics Data System (ADS)

    Guzewich, S. D.; Talaat, E. R.; Toigo, A. D.; Waugh, D. W.; McConnochie, T. H.

    2012-12-01

    Limb-scanning observations of dust mixing ratio with the Thermal Emission Spectrometer (TES) onboard Mars Global Surveyor spanning three Mars years (MY 24-27) show two discrete layers of enhanced dust mixing ratios at 20-30 km and 45-65 km. This represents a significant departure from the canonical Conrath-ν (Conrath, 1975) vertical dust profile used in most Mars general circulation models (GCMs). The lower layer was previously identified with the Mars Climate Sounder (Heavens et al., 2011) and is primarily a tropical phenomenon. It is persistent year-round, save a brief period each year from Ls = 20°-50°, reaches highest altitudes and amplitudes during the dust storm season, and exhibits significant diurnal variability associated with water ice clouds. The 45-65 km layer is nearly absent in nighttime observations, reaches highest amplitudes during northern summer, and is not observed during the dust storm season. The TES dust climatology was adapted to radiatively force the MarsWRF GCM and simulations were performed to investigate the effect of the new dust profiles. Changes in the vertical profile of dust result in significant changes to the general circulation of the atmosphere including an enhanced polar warming, decoupled middle and lower atmosphere meridional circulations, and an improved match to TES temperature observations.

  7. 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.

  8. Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences

    PubMed Central

    Ivanov, Ivaylo P.; Firth, Andrew E.; Michel, Audrey M.; Atkins, John F.; Baranov, Pavel V.

    2011-01-01

    In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5′ cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized—both for increased coding capacity and potentially also for novel regulatory mechanisms—remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5′ untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data. PMID:21266472

  9. 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.

  10. Trehalose induces functionally active conformation in the intrinsically disordered N-terminal domain of glucocorticoid receptor.

    PubMed

    Khan, Shagufta H; Jasuja, Ravi; Kumar, Raj

    2016-08-05

    Glucocorticoid receptor (GR) is a classic member of the nuclear receptor superfamily and plays pivotal roles in human physiology at the level of gene regulation. Various constellations of cellular cofactors are required to associate with GR to activate/repress genes. The effects of specific ligands on the AF2 structure and consequent preferential binding of co-activators or co-repressors have helped our understanding of the mechanisms involved. But the data so far fall short of fully explaining GR actions. We believe that this is because work so far has largely avoided detailed examination of the contributions of AF1 to overall GR actions. It has been shown that the GR containing only the N-terminal domain (NTD) and the DNA-binding domain (GR500) is constitutively quite active in stimulating transcription from simple promoters. However, we are only beginning to understand structure and functions of GR500 in spite of the fact that AF1 located within the NTD serves as major transactivation domain for GR. Lack of this information has hampered our complete understanding of how GR regulates its target gene(s). The major obstacle in determining GR500 structure has been due to its intrinsically disordered NTD conformation, frequently found in transcription factors. In this study, we tested whether a naturally occurring osmolyte, trehalose, can promote functionally ordered conformation in GR500. Our data show that in the presence of trehalose, GR500 is capable of formation of a native-like functionally folded conformation.

  11. An N-terminal region of mot-2 binds to p53 in vitro.

    PubMed

    Kaul, S C; Reddel, R R; Mitsui, Y; Wadhwa, R

    2001-01-01

    The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312-352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its N-terminal amino acid residues 253-282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.

  12. c-Jun N-Terminal Kinase Phosphorylation Is a Biomarker of Plitidepsin Activity

    PubMed Central

    Muñoz-Alonso, María J.; Álvarez, Enrique; Guillén-Navarro, María José; Pollán, Marina; Avilés, Pablo; Galmarini, Carlos M.; Muñoz, Alberto

    2013-01-01

    Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin. PMID:23697951

  13. 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.

  14. Calcium-controlled conformational choreography in the N-terminal half of adseverin.

    PubMed

    Chumnarnsilpa, Sakesit; Robinson, Robert C; Grimes, Jonathan M; Leyrat, Cedric

    2015-09-14

    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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.

  15. 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

  16. 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.

  17. 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

  18. Functional regions of the N-terminal domain of the antiterminator RfaH

    PubMed Central

    Belogurov, Georgiy A; Sevostyanova, Anastasia; Svetlov, Vladimir; Artsimovitch, Irina

    2010-01-01

    RfaH is a bacterial elongation factor that increases expression of distal genes in several long, horizontally acquired operons. RfaH is recruited to the transcription complex during RNA chain elongation through specific interactions with a DNA element called ops. Following recruitment, RfaH remains bound to RNA polymerase (RNAP) and acts as an antiterminator by reducing RNAP pausing and termination at some factor-independent and Rho-dependent signals. RfaH consists of two domains connected by a flexible linker. The N-terminal RfaH domain (RfaHN) recognizes the ops element, binds to the RNAP and reduces pausing and termination in vitro. Functional analysis of single substitutions in this domain reported here suggests that three separate RfaHN regions mediate these functions. We propose that a polar patch on one side of RfaHN interacts with the non-template DNA strand during recruitment, whereas a hydrophobic surface on the opposite side of RfaHN remains bound to the β′ subunit clamp helices domain throughout transcription of the entire operon. The third region is apparently dispensable for RfaH binding to the transcription complex but is required for the antitermination modification of RNAP. PMID:20132437

  19. 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.

  20. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab.

    PubMed

    Tan, Shuguang; Zhang, Hao; Chai, Yan; Song, Hao; Tong, Zhou; Wang, Qihui; Qi, Jianxun; Wong, Gary; Zhu, Xiaodong; Liu, William J; Gao, Shan; Wang, Zhongfu; Shi, Yi; Yang, Fuquan; Gao, George F; Yan, Jinghua

    2017-02-06

    Cancer immunotherapy by targeting of immune checkpoint molecules has been a research 'hot-spot' in recent years. Nivolumab, a human monoclonal antibody targeting PD-1, has been widely used clinically since 2014. However, the binding mechanism of nivolumab to PD-1 has not yet been shown, despite a recent report describing the complex structure of pembrolizumab/PD-1. It has previously been speculated that PD-1 glycosylation is involved in nivolumab recognition. Here we report the complex structure of nivolumab with PD-1 and evaluate the effects of PD-1 N-glycosylation on the interactions with nivolumab. Structural and functional analyses unexpectedly reveal an N-terminal loop outside the IgV domain of PD-1. This loop is not involved in recognition of PD-L1 but dominates binding to nivolumab, whereas N-glycosylation is not involved in binding at all. Nivolumab binds to a completely different area than pembrolizumab. These results provide the basis for the design of future inhibitory molecules targeting PD-1.

  1. N-Terminal Peptide Detection with Optimized Peptide-Spectrum Matching and Streamlined Sequence Libraries.

    PubMed

    Lycette, Brynne E; Glickman, Jacob W; Roth, Samuel J; Cram, Abigail E; Kim, Tae Hee; Krizanc, Danny; Weir, Michael P

    2016-09-02

    We identified tryptic peptides in yeast cell lysates that map to translation initiation sites downstream of the annotated start sites using the peptide-spectrum matching algorithms OMSSA and Mascot. To increase the accuracy of peptide-spectrum matching, both algorithms were run using several standardized parameter sets, and Mascot was run utilizing a, b, and y ions from collision-induced dissociation. A large fraction (22%) of the detected N-terminal peptides mapped to translation initiation downstream of the annotated initiation sites. Expression of several truncated proteins from downstream initiation in the same reading frame as the full-length protein (frame 1) was verified by western analysis. To facilitate analysis of the larger proteome of Drosophila, we created a streamlined sequence library from which all duplicated trypsin fragments had been removed. OMSSA assessment using this "stripped" library revealed 171 peptides that map to downstream translation initiation sites, 76% of which are in the same reading frame as the full-length annotated proteins, although some are in different reading frames creating new protein sequences not in the annotated proteome. Sequences surrounding implicated downstream AUG start codons are associated with nucleotide preferences with a pronounced three-base periodicity N1^G2^A3.

  2. 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

    2010-10-22

    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 {angstrom} 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 {alpha}-helix. The structure of the first subdomain (residues 1-117), which consists mostly of {beta}-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.

  3. Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis.

    PubMed

    Cunha, A F; Sousa, M V; Silva, S P; Jesuíno, R S; Soares, C M; Felipe, M S

    1999-04-01

    Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.

  4. PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

    PubMed

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Kim, Bo Seong; Bak, Hye Jin; Lee, Jin Young; Park, Myoung-Ae; Park, Ju Hyeon; Lee, Hyung Ho; Chung, Joon Ki

    2013-10-10

    Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

  5. 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

  6. Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

    PubMed

    Ishmukhametov, Robert R; DeLeon-Rangel, Jessica; Zhu, Shaotong; Vik, Steven B

    2017-04-01

    Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

  7. N-Terminal Truncated UCH-L1 Prevents Parkinson's Disease Associated Damage

    PubMed Central

    Kim, Hee-Jung; Kim, Hyun Jung; Jeong, Jae-Eun; Baek, Jeong Yeob; Jeong, Jaeho; Kim, Sun; Kim, Young-Mee; Kim, Youhwa; Nam, Jin Han; Huh, Sue Hee; Seo, Jawon; Jin, Byung Kwan; Lee, Kong-Joo

    2014-01-01

    Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. In vivo experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H2O2, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD. PMID:24959670

  8. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.

    PubMed

    Emanuelsson, O; Nielsen, H; Brunak, S; von Heijne, G

    2000-07-21

    A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.

  9. Ion Channels of Alamethicin Dimer N-Terminally Linked by Disulfide Bond

    PubMed Central

    Okazaki, Takashi; Sakoh, Machiko; Nagaoka, Yasuo; Asami, Koji

    2003-01-01

    A covalent dimer of alamethicin Rf30 was synthesized by linking the N-termini by a disulfide bond. When the dimer peptides were added to the cis-side of a diphytanoyl PC membrane, macroscopic channel current was induced only at cis positive voltages. The single-channel recordings showed several conductance levels that were alternately stabilized. These results indicate that the dimer peptides form stable channels by N-terminal insertion like alamethicin and that most of the pores are assembled from even numbers of helices. Taking advantages of the long open duration of the dimer peptide channels, the current-voltage (I-V) relations of the single-channels were obtained by applying fast voltage ramps during the open states. The I-V relations showed rectification, such that current from the cis-side toward the trans-side is larger than that in the opposite direction. The intrinsic rectification is mainly attributed to the macro dipoles of parallel peptide helices surrounding a central pore. PMID:12829482

  10. Ion channels of alamethicin dimer N-terminally linked by disulfide bond.

    PubMed

    Okazaki, Takashi; Sakoh, Machiko; Nagaoka, Yasuo; Asami, Koji

    2003-07-01

    A covalent dimer of alamethicin Rf30 was synthesized by linking the N-termini by a disulfide bond. When the dimer peptides were added to the cis-side of a diphytanoyl PC membrane, macroscopic channel current was induced only at cis positive voltages. The single-channel recordings showed several conductance levels that were alternately stabilized. These results indicate that the dimer peptides form stable channels by N-terminal insertion like alamethicin and that most of the pores are assembled from even numbers of helices. Taking advantages of the long open duration of the dimer peptide channels, the current-voltage (I-V) relations of the single-channels were obtained by applying fast voltage ramps during the open states. The I-V relations showed rectification, such that current from the cis-side toward the trans-side is larger than that in the opposite direction. The intrinsic rectification is mainly attributed to the macro dipoles of parallel peptide helices surrounding a central pore.

  11. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab

    PubMed Central

    Tan, Shuguang; Zhang, Hao; Chai, Yan; Song, Hao; Tong, Zhou; Wang, Qihui; Qi, Jianxun; Wong, Gary; Zhu, Xiaodong; Liu, William J.; Gao, Shan; Wang, Zhongfu; Shi, Yi; Yang, Fuquan; Gao, George F.; Yan, Jinghua

    2017-01-01

    Cancer immunotherapy by targeting of immune checkpoint molecules has been a research ‘hot-spot' in recent years. Nivolumab, a human monoclonal antibody targeting PD-1, has been widely used clinically since 2014. However, the binding mechanism of nivolumab to PD-1 has not yet been shown, despite a recent report describing the complex structure of pembrolizumab/PD-1. It has previously been speculated that PD-1 glycosylation is involved in nivolumab recognition. Here we report the complex structure of nivolumab with PD-1 and evaluate the effects of PD-1 N-glycosylation on the interactions with nivolumab. Structural and functional analyses unexpectedly reveal an N-terminal loop outside the IgV domain of PD-1. This loop is not involved in recognition of PD-L1 but dominates binding to nivolumab, whereas N-glycosylation is not involved in binding at all. Nivolumab binds to a completely different area than pembrolizumab. These results provide the basis for the design of future inhibitory molecules targeting PD-1. PMID:28165004

  12. Evidence of Presynaptic Localization and Function of the c-Jun N-Terminal Kinase

    PubMed Central

    Biggi, Silvia; Buccarello, Lucia; Sclip, Alessandra; Lippiello, Pellegrino; Rumio, Cristiano; Di Marino, Daniele

    2017-01-01

    The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity. PMID:28367336

  13. c-Jun N-terminal kinase - c-Jun pathway transactivates Bim to promote osteoarthritis.

    PubMed

    Ye, Zhiqiang; Chen, Yuxian; Zhang, Rongkai; Dai, Haitao; Zeng, Chun; Zeng, Hua; Feng, Hui; Du, Gengheng; Fang, Hang; Cai, Daozhang

    2014-02-01

    Osteoarthritis (OA) is a chronic degenerative joint disorder. Previous studies have shown abnormally increased apoptosis of chondrocytes in patients and animal models of OA. TNF-α and nitric oxide have been reported to induce chondrocyte ageing; however, the mechanism of chondrocyte apoptosis induced by IL-1β has remained unclear. The aim of this study is to identify the role of the c-Jun N-terminal kinase (JNK) - c-Jun pathway in regulating induction of Bim, and its implication in chondrocyte apoptosis. This study showed that Bim is upregulated in chondrocytes obtained from the articular cartilage of OA patients and in cultured mouse chondrocytes treated with IL-1β. Upregulation of Bim was found to be critical for chondrocyte apoptosis induced by IL-1β, as revealed by the genetic knockdown of Bim, wherein apoptosis was greatly reduced in the chondrocytes. Moreover, activation of the JNK-c-Jun pathway was observed under IL-1β treatment, as indicated by the increased expression levels of c-Jun protein. Suppression of the JNK-c-Jun pathway, using chemical inhibitors and RNA interference, inhibited the Bim upregulation induced by IL-1β. These findings suggest that the JNK-c-Jun pathway is involved in the upregulation of Bim during OA and that the JNK-c-Jun-Bim pathway is vital for chondrocyte apoptosis.

  14. Control of protein life-span by N-terminal methionine excision

    PubMed Central

    Giglione, Carmela; Vallon, Olivier; Meinnel, Thierry

    2003-01-01

    Peptide deformylases (PDFs) have been discovered recently in eukaryotic genomes, and it appears that N-terminal methionine excision (NME) is a conserved pathway in all compartments where protein synthesis occurs. This work aimed at uncovering the function(s) of NME in a whole proteome, using the chloroplast-encoded proteins of both Arabidopsis thaliana and Chlamydomonas reinhardtii as model systems. Dis ruption of PDF1B in A.thaliana led to an albino phenotype, and an extreme sensitivity to the PDF- specific inhibitor actinonin. In contrast, a knockout line for PDF1A exhibited no apparent phenotype. Photosystem II activity in C.reinhardtii cells was substantially reduced by the presence of actinonin. Pulse–chase experiments revealed that PDF inhibi tion leads to destabilization of a crucial subset of chloroplast-encoded photosystem II components in C.reinhardtii. The same proteins were destabilized in pdf1b. Site-directed substitutions altering NME of the most sensitive target, subunit D2, resulted in similar effects. Thus, plastid NME is a critical mechanism specifically influencing the life-span of photosystem II polypeptides. A general role of NME in modulating the half-life of key subsets of proteins is suggested. PMID:12505980

  15. Evolutionary Conserved Role of c-Jun-N-Terminal Kinase in CO2-Induced Epithelial Dysfunction

    PubMed Central

    Vadász, István; Dada, Laura A.; Briva, Arturo; Helenius, Iiro Taneli; Sharabi, Kfir; Welch, Lynn C.; Kelly, Aileen M.; Grzesik, Benno A.; Budinger, G. R. Scott; Liu, Jing; Seeger, Werner; Beitel, Greg J.; Gruenbaum, Yosef; Sznajder, Jacob I.

    2012-01-01

    Elevated CO2 levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes. PMID:23056407

  16. 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.

  17. Hemispheric asymmetries in the quasibiennial oscillation signature on the mid- to high-latitude circulation of the stratosphere.

    PubMed

    Peña-Ortiz, C; García-Herrera, R; Ribera, P; Calvo, N

    2008-12-01

    The quasibiennial oscillation (QBO) dominates the variability of the equatorial stratosphere and also affects the circulation and temperature of the extratropical region. In this paper we review previous work showing that the mid- to high-latitude circulation is weaker (stronger) when QBO easterlies (westerlies) dominate in the low equatorial stratosphere. The accepted explanation for the extratropical QBO signature is based on the QBO modulation of upward propagating planetary Rossby waves. This mechanism is consistent with the strong seasonality observed in the extratropical QBO. The largest QBO signature in the northern extratropical stratosphere occurs during winter when the dominating westerly wind allows the penetration of planetary waves in the northern stratosphere. However, during the southern winter, planetary waves do not disrupt the southern stratospheric vortex and the largest QBO signature is found during the late spring (November). To further illustrate these mechanisms, we analyze the QBO signature on the mid- to high-latitude circulation of the stratosphere using data from the ERA-40 reanalysis. The extratropical signature in both hemispheres is evaluated as a function of the latitude-height structure of the zonal wind in the tropical region in order to determine how the extratropical response depends on the vertical phase structure of the tropical QBO. We also analyze the QBO impact on planetary wave activity in order to determine how this modulation can explain the observed extratropical QBO signal.

  18. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

    SciTech Connect

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  19. Calcium occupancy of N-terminal sites within calmodulin induces inhibition of the ryanodine receptor calcium release channel.

    PubMed

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-09-18

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614-N3643) located within the central portion of the primary sequence. However, it is presently unclear whether the identified CaM-binding sequence in association with CaM (a) senses calcium over the physiological range of calcium concentrations associated with RyR1 regulation or alternatively, (b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene)maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association of both apo-CaM and calcium-activated CaM with RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-dependent activation of these individual domains. Fluorescence changes upon calcium activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM at resting calcium levels; at calcium levels associated with muscle contraction, activation of the N-terminal domain occurs with concomitant increases in the fluorescence intensity of PyC-CaM that is associated with structural changes within the CaM-binding sequence of RyR1. Occupancy of calcium-binding sites in the N

  20. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect.

  1. Hydroxyl Radical-Mediated Novel Modification of Peptides: N-Terminal Cyclization through the Formation of α-Ketoamide.

    PubMed

    Lee, Seon Hwa; Kyung, Hyunsook; Yokota, Ryo; Goto, Takaaki; Oe, Tomoyuki

    2015-01-20

    The hydroxyl radical-mediated oxidation of peptides and proteins constitutes a large group of post-translational modifications that can result in structural and functional changes. These oxidations can lead to hydroxylation, sulfoxidation, or carbonylation of certain amino acid residues and cleavage of peptide bonds. In addition, hydroxyl radicals can convert the N-terminus of peptides to an α-ketoamide via abstraction of the N-terminal α-hydrogen and hydrolysis of the ketimine intermediate. In the present study, we identified N-terminal cyclization as a novel modification mediated by a hydroxyl radical. The reaction of angiotensin (Ang) II (DRVYIHPF) and the hydroxyl radical generated by the Cu(II)/ascorbic acid (AA) system or UV/hydrogen peroxide system produced N-terminal cyclized-Ang II (Ang C) and pyruvamide-Ang II (Ang P, CH3COCONH-RVYIHPF). The structure of Ang C was confirmed by mass spectrometry and comparison to an authentic standard. The subsequent incubation of isolated Ang P in the presence of Cu(II)/AA revealed that Ang P was the direct precursor of Ang C. The proposed mechanism involves the formation of a nitrogen-centered (aminyl) radical, which cyclizes to form a five-membered ring containing the alkoxy radical. The subsequent β-scission reaction of the alkoxyl radical results in the cleavage of the terminal CH3CO group. The initial aminyl radical can be stabilized by chelation to the Cu(II) ions. The affinity of Ang C toward the Ang II type 1 receptor was significantly lower than that of Ang II or Ang P. Ang C was not further metabolized by aminopeptidase A, which converts Ang II to Ang III. Hydroxyl radical-mediated N-terminal cyclization was also observed in other Ang peptides containing N-terminal alanine, arginine, valine, and amyloid β 1-11 (DAEFRHDSGYE).

  2. 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.

  3. A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization.

    PubMed

    Haslbeck, Martin; Ignatiou, Athanasios; Saibil, Helen; Helmich, Sonja; Frenzl, Elke; Stromer, Thusnelda; Buchner, Johannes

    2004-10-15

    Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.

  4. 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.

  5. N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

    PubMed

    Girgenrath, Tanya; Mahalingam, Mohana; Svensson, Bengt; Nitu, Florentin R; Cornea, Razvan L; Fessenden, James D

    2013-05-31

    We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

  6. Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury

    PubMed Central

    Jang, Sehwan; Yu, Li-Rong; Abdelmegeed, Mohamed A.; Gao, Yuan; Banerjee, Atrayee; Song, Byoung-Joon

    2015-01-01

    The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl4 (50 mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl4-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1 h while liver damage was maximal at 24 h post-CCl4 injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8 h following CCl4 exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins. PMID:26491845

  7. c-JUN N-terminal kinase (JNK) is activated and contributes to tumor cell proliferation in classical Hodgkin lymphoma.

    PubMed

    Leventaki, Vasiliki; Drakos, Elias; Karanikou, Maria; Psatha, Konstantina; Lin, Pei; Schlette, Ellen; Eliopoulos, Aris; Vassilakopoulos, Theodoros P; Papadaki, Helen; Patsouris, Efstratios; Medeiros, L Jeffrey; Rassidakis, George Z

    2014-03-01

    c-JUN N-terminal Kinase (JNK) is activated/phosphorylated by upstream MAPK kinases (MKK), and, in turn, phosphorylates and activates its major substrate c-JUN, a member of the activator protein-1 (AP-1) transcription factors. c-JUN is overexpressed and activated in Hodgkin and Reed Sternberg cells (HRS) of classical Hodgkin lymphoma (cHL), however, the mechanism of its activation remains unknown. JNK activation was immunohistochemically assessed in 60 cases of HL and in a control group of 151 B-cell non-Hodgkin lymphomas. The biologic effects of JNK activation in cultured HRS cells were investigated using colony formation, cell growth and viability assays and cell cycle analysis by flow cytometry. Western blotting was used to assess protein levels. p-JNK was expressed in 90% of HL, 83% of Burkitt lymphomas, 28% of mantle cell lymphomas, 23% of diffuse large B-cell lymphomas, 19% of follicular lymphomas, and 18% of extranodal marginal zone lymphomas of MALT type. None of the 48 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma and 18 cases of plasma cell myeloma showed JNK phosphorylation (P < 001, Kruskall-Wallis test). Pharmacological inhibition of JNK activity in cultured HRS cells resulted in a significant decrease of cell growth, which was associated with cell cycle arrest at the G2/M phase. The cell cycle effects were linked to deactivation of c-JUN and upregulation of its known target, the cyclin-dependent kinase inhibitor p21. JNK is highly activated in HRS cells, and may contribute to uncontrolled cell cycle progression and proliferation of tumor cells in cHL.

  8. Induction of experimental autoimmune encephalomyelitis in the absence of c-Jun N-terminal kinase 2.

    PubMed

    Nicolson, Kirsty; Freland, Sofia; Weir, Catherine; Delahunt, Brett; Flavell, Richard A; Bäckström, B Thomas

    2002-08-01

    Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-dependent, organ-specific autoimmune model commonly used to investigate mechanisms involved in the activation of autoreactive T(h)1 cells. Mitogen-activated protein kinases such as c-Jun N-terminal kinase (Jnk) 1 and 2 play an important role in the differentiation of naive precursors into T(h)1 or T(h)2 effector cells. To investigate the role of Jnk2 on autoimmunity, Jnk2(-/-) and wild-type mice were immunized with the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide and the onset of EAE studied. Surprisingly, Jnk2(-/-) mice were as susceptible to EAE as wild-type mice, regardless of whether low or high antigen doses were used to induce disease. In vitro stimulation of lymph node cells from Jnk2(-/-) and wild-type mice resulted in comparable proliferation in response to MOG35-55, Mycobacterium tuberculosis and concanavalin A. MOG35-55-specific T cells lacking Jnk2 showed a T(h)1 cytokine profile with IFN-gamma, but no IL-4 or IL-5 production. No differences in the types of infiltrating cells or myelin destruction in the central nervous system were found between Jnk2(-/-) and wild-type mice, indicating that lack of Jnk2 does not alter the effector phase of EAE. Our results suggest that, despite involvement in T(h)1/T(h)2 differentiation in vitro, Jnk2 is necessary neither for the induction nor effector phase of MOG35-55-induced EAE and nor is it required for antigen-specific IFN-gamma production.

  9. c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    PubMed Central

    Suzuki, Akiko; Kakisaka, Keisuke; Suzuki, Yuji; Wang, Ting; Takikawa, Yasuhiro

    2016-01-01

    AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed “lipoapoptosis,” in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12 (AML12) cells treated with palmitate (PA) were used as an in vitro model. RESULTS: LC3-II, p62, and Run domain Beclin-1 interacting and cysteine-rich containing (Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase (JNK) inhibition at both the mRNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-II and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown. CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide anti-fibrosis in the NASH liver. PMID:27605885

  10. The dynamics of interconverting D- and E-forms of the HIV-1 integrase N-terminal domain.

    PubMed

    Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

    2014-11-01

    The N-terminal domain (NTD) of HIV-1 integrase adopts two inter-converting forms (D- and E-) due to their specific coordination of a Zn(2+) ion by an HHCC motif. Mutational studies on NTD have suggested the importance of conformational transition in regulating the functions of tetramers and dimers of HIV-1 integrase. This study explores the stability and dynamics of native NTD forms and the conformational transition between D- and E-forms using molecular dynamics simulations elucidating their role in regulation of viral and host DNA integration. Simulation of native forms of NTD revealed stable dynamics. Transition studies between D- and E-forms using conventional molecular dynamics simulations for 50 ns partially revealed conformational change towards the target during D- to -E simulation (the extension of α1-helix), which failed in the E- to -D simulation. This could be attributed to the existence of the D-form (-1,945.907 kCal/mol) in higher energy than the E-form (-2,002.383 kCal/mol). The conformational transition pathway between these two states was explored using targeted molecular dynamics simulations. Analysis of the targeted molecular dynamics trajectories revealed conformations closer to the experimentally-reported intermediate form of an NTD during the transition phase. The role of Met22 in stabilizing the E-form was studied by simulating the E-form with Met22Ala mutation, revealing a highly dynamic α1-helix as compared to the native form. The present study reveals the significant role of the Zn(2+) ion-coordinated HHCC motif and its interaction with Met22 as the basis for understanding the biological implications of D- and E-forms of the NTD in regulating integration reaction.

  11. N-terminal prohormone brain natriuretic peptide (NT-proBNP) as a noninvasive marker for restrictive syndromes.

    PubMed

    Mady, C; Fernandes, F; Ramires, F J A; Nastari, L; Buck, P C; Arteaga, E; Ianni, B M; Salemi, V M C

    2008-08-01

    Constrictive pericarditis (CP) and restrictive cardiomyopathy share many similarities in both their clinical and hemodynamic characteristics and N-terminal prohormone brain natriuretic peptide (NT-proBNP) is a sensitive marker of cardiac diastolic dysfunction. The objectives of the present study were to determine whether serum NT-proBNP was high in patients with endomyocardial fibrosis (EMF) and CP, and to investigate how this relates to diastolic dysfunction. Thirty-three patients were divided into two groups: CP (16 patients) and EMF (17 patients). The control group consisted of 30 healthy individuals. Patients were evaluated by bidimensional echocardiography, with restriction syndrome evaluated by pulsed Doppler of the mitral flow and serum NT-proBNP measured by immunoassay and detected by electrochemiluminescence. Spearman correlation coefficient was used to analyze the association between log NT-proBNP and echocardiographic parameters. Log NT-proBNP was significantly higher (P < 0.05) in CP patients (log mean: 2.67 pg/mL; 95%CI: 2.43-2.92 log pg/mL) and in EMF patients (log mean: 2.91 pg/mL; 95%CI: 2.70-3.12 log pg/mL) compared with the control group (log mean: 1.45; 95%CI: 1.32-1.60 log pg/mL). There were no statistical differences between EMF and CP patients (P = 0.689) in terms of NT-proBNP. The NT-proBNP log tended to correlate with peak velocity of the E wave (r = 0.439; P = 0.060, but not with A wave (r = -0.399; P = 0.112). Serum NT-proBNP concentration can be used as a marker to detect the presence of diastolic dysfunction in patients with restrictive syndrome; however, serum NT-proBNP levels cannot be used to differentiate restrictive cardiomyopathy from CP.

  12. Association of N-terminal domain polymorphisms of the porcine glucocorticoid receptor with carcass composition and meat quality traits.

    PubMed

    Reyer, Henry; Ponsuksili, Siriluck; Wimmers, Klaus; Murani, Eduard

    2014-02-01

    The glucocorticoid receptor (GR) is a ubiquitously acting transcription factor that is responsible for mediating the physiological response to stress and adaptation to environmental conditions. Genetic variation of a GR gene (NR3C1) may therefore contribute to multiple phenotypic alterations and influence relevant traits of animal production. Here, we examined effects of two non-synonymous mutations of the porcine NR3C1, leading to amino acid exchanges p.Glu13Asp (c.39A>C) and p.Val19Leu (c.55G>C) in the N-terminal domain of the GR, on meat quality and carcass composition. In addition, we explored their influence on transcriptional activity of GR in vitro. A commercial crossbreed Pietrain × (German Large White × German Landrace) herd (n = 545) in which genotypes and relevant traits had been collected was used to perform the association analysis. The single nucleotide polymorphism (SNP) c.55G>C was significantly associated with conductivity and meat color scores. These effects were highly consistent considering the physiological relationship between these traits. Association analysis of SNP c.39A>C also revealed significant effects on closely connected meat quality traits. In addition, SNP c.55G>C showed association with carcass traits, mainly those related to muscle deposition. The molecular mechanism of action of both amino acid substitutions remains obscure because neither showed significant influence on transcriptional activity of GR. Our study emphasizes NR3C1 as an important candidate gene for muscle-related traits in pigs, but further work is necessary to clarify the molecular background of the identified associations.

  13. Antigenic analysis of highly pathogenic avian influenza virus H5N1 sublineages co-circulating in Egypt.

    PubMed

    Watanabe, Yohei; Ibrahim, Madiha S; Ellakany, Hany F; Kawashita, Norihito; Daidoji, Tomo; Takagi, Tatsuya; Yasunaga, Teruo; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2012-10-01

    Highly pathogenic avian influenza virus H5N1 has spread across Eurasia and Africa, and outbreaks are now endemic in several countries, including Indonesia, Vietnam and Egypt. Continuous circulation of H5N1 virus in Egypt, from a single infected source, has led to significant genetic diversification with phylogenetically separable sublineages, providing an opportunity to study the impact of genetic evolution on viral phenotypic variation. In this study, we analysed the phylogeny of H5 haemagglutinin (HA) genes in influenza viruses isolated in Egypt from 2006 to 2011 and investigated the effect of conserved amino acid mutations in the HA genes in each of the sublineages on their antigenicity. The analysis showed that viruses in at least four sublineages still persisted in poultry in Egypt as of 2011. Using reverse genetics to generate HA-reassortment viruses with specific HA mutations, we found antigenic drift in the HA in two influenza virus sublineages, compared with the other currently co-circulating influenza virus sublineages in Egypt. Moreover, the two sublineages with significant antigenic drift were antigenically distinguishable. Our findings suggested that phylogenetically divergent H5N1 viruses, which were not antigenically cross-reactive, were co-circulating in Egypt, indicating that there was a problem in using a single influenza virus strain as seed virus to produce influenza virus vaccine in Egypt and providing data for designing more efficacious control strategies in H5N1-endemic areas.

  14. Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood.

    PubMed

    Li, Junjie; Zhang, Weixia; Chung, Ting-Fung; Slipchenko, Mikhail N; Chen, Yong P; Cheng, Ji-Xin; Yang, Chen

    2015-07-23

    We report a transient absorption (TA) imaging method for fast visualization and quantitative layer analysis of graphene and GO. Forward and backward imaging of graphene on various substrates under ambient condition was imaged with a speed of 2 μs per pixel. The TA intensity linearly increased with the layer number of graphene. Real-time TA imaging of GO in vitro with capability of quantitative analysis of intracellular concentration and ex vivo in circulating blood were demonstrated. These results suggest that TA microscopy is a valid tool for the study of graphene based materials.

  15. Simulation of the mantle and crustal Helium isotope signature in the Mediterranean Sea using a high resolution regional circulation model

    NASA Astrophysics Data System (ADS)

    Ayache, Mohamed; Dutay, Jean-claude; Jean-baptiste, Philippe

    2015-04-01

    Helium isotopes (3He, 4He) are useful tracers for investigating the deep ocean circulation and for evaluating ocean general circulation models, because helium is a stable and conservative nuclide that does not take part in any chemical or biological processes. Helium in the ocean originates from three different sources: namely, (i) gas dissolution in equilibrium with atmospheric helium, (ii) helium-3 addition by radioactive decay of tritium (called tritiugenic helium), and (iii) injection of helium-3 and helium-4 by the submarine volcanic activity which occurs mainly at plate boundaries, and also addition of helium-4 from the crust and sedimentary cover by α-decay of uranium and thorium contained in various minerals (called terrigenic helium). Here we present the first simulation of the helium isotope distribution in the whole Mediterranean Sea, using a high resolution model (NEMO-MED12). The simulation was produced by building a simple source function for helium produced by submarine volcanic degassing in the main active areas of the Mediterranean, and by crustal degassing at sea bottom, based on previous estimates of the total flux of helium into the oceans. In addition to providing constraints on the degassing flux, our work provides information on the variability of the thermohaline circulation and the ventilation of the deep waters to constrain the degree to which the NEMO-MED12 can reproduce correctly the main hydrographic features of the Mediterranean Sea circulation. This study is part of the work carried out to assess the robustness of the NEMO-MED12 model, which will be used to study the evolution of the climate and its effect on the biogeochemical cycles in the Mediterranean Sea, and to improve our ability to predict the future evolution of the Mediterranean Sea under the increasing anthropogenic pressure.

  16. Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

    PubMed Central

    Díaz Lozano, Isabel María; De Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

    2017-01-01

    The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease. PMID:28294160

  17. Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

    NASA Astrophysics Data System (ADS)

    Díaz Lozano, Isabel María; de Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

    2017-03-01

    The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.

  18. Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region.

    PubMed

    Díaz Lozano, Isabel María; De Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

    2017-03-15

    The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.

  19. Chemically Modified Plastic Tube for High Volume Removal and Collection of Circulating Tumor Cells

    PubMed Central

    Gaitas, Angelo; Kim, Gwangseong

    2015-01-01

    In this preliminary effort, we use a commercially available and chemically modified tube to selectively capture circulating tumor cells (CTCs) from the blood stream by immobilizing human anti-EpCAM antibodies on the tube's interior surface. We describe the requisite and critical steps required to modify a tube into a cancer cell-capturing device. Using these simple modifications, we were able to capture or entrap about 85% of cancer cells from suspension and 44% of cancer cells from spiked whole blood. We also found that the percentage of cells captured was dependent on the tube's length and also the number of cancer cells present. It is our strong belief that with the utilization of appropriate tube lengths and procedures, we can ensure capture and removal of nearly the entire CTC population in whole blood. Importantly after a patient’s entire blood volume has circulated through the tube, the tube can then be trypsinized to release the captured live CTCs for further analysis and testing. PMID:26176235

  20. Copper and zinc binding properties of the N-terminal histidine-rich sequence of Haemophilus ducreyi Cu,Zn superoxide dismutase.

    PubMed

    Paksi, Zoltán; Jancsó, Attila; Pacello, Francesca; Nagy, Nóra; Battistoni, Andrea; Gajda, Tamás

    2008-09-01

    The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H(2)N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine epsilon-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K(D,Zn)=1.6x10(-9)M and K(D,Cu)=5.0x10(-12)M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K(D) values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.

  1. 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

  2. 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.

  3. 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.

  4. Folding and stability studies on C-PE and its natural N-terminal truncant.

    PubMed

    Anwer, Khalid; Parmar, Asha; Rahman, Safikur; Kaushal, Avani; Madamwar, Datta; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2014-03-01

    The conformational and functional state of biliproteins can be determined by optical properties of the covalently linked chromophores. α-Subunit of most of the phycoerythrin contains 164 residues. Recently determined crystal structure of the naturally truncated form of α-subunit of cyanobacterial phycoerythrin (Tr-αC-PE) lacks 31 N-terminal residues present in its full length form (FL-αC-PE). This provides an opportunity to investigate the structure-function relationship between these two natural forms. We measured guanidinium chloride (GdmCl)-induced denaturation curves of FL-αC-PE and Tr-αC-PE proteins, followed by observing changes in absorbance at 565nm, fluorescence at 350 and 573nm, and circular dichroism at 222nm. The denaturation curve of each protein was analyzed for ΔGD(∘), the value of Gibbs free energy change on denaturation (ΔGD) in the absence of GdmCl. The main conclusions of the this study are: (i) GdmCl-induced denaturation (native state↔denatured state) of FL-αC-PE and Tr-αC-PE is reversible and follows a two-state mechanism, (ii) FL-αC-PE is 1.4kcalmol(-1) more stable than Tr-αC-PE, (iii) truncation of 31-residue long fragment that contains two α-helices, does not alter the 3-D structure of the remaining protein polypeptide chain, protein-chromophore interaction, and (iv) amino acid sequence of Tr-αC-PE determines the functional structure of the phycoerythrin.

  5. N-terminal Half of Transportin SR2 Interacts with HIV Integrase.

    PubMed

    Tsirkone, Vicky G; Blokken, Jolien; De Wit, Flore; Breemans, Jolien; De Houwer, Stéphanie; Debyser, Zeger; Christ, Frauke; Strelkov, Sergei V

    2017-03-29

    The karyopherin transportin SR2 (TRN-SR2, TNPO3) is responsible for shuttling specific cargoes such as serine/arginine-rich splicing factors from the cytoplasm to the nucleus. This protein plays a key role in HIV infection by facilitating the nuclear import of the pre-integration complex (PIC) which contains the viral DNA as well as several cellular and HIV proteins including the integrase. The process of nuclear import is considered to be the bottleneck of the viral replication cycle and therefore represents a promising target for anti-HIV drug design. Previous studies have demonstrated that the direct interaction between TRN-SR2 and HIV integrase predominantly involves the catalytic core domain (CCD) and the C-terminal domain (CTD) of the latter. We aimed at providing a detailed molecular view of this interaction through a biochemical characterization of the respective protein complex. Size-exclusion chromatography was used to characterize the interaction of TRN-SR2 with a truncated variant of HIV-1 integrase including both the CCD and CTD. These experiments indicate that one TRN-SR2 molecule can specifically bind one CCD-CTD dimer. Next, the regions of the solenoid-like TRN-SR2 molecule that are involved in the interaction with integrase were identified using AlphaScreen binding assays, revealing that the integrase interacts with the N-terminal half of TRN-SR2 principally through the HEAT repeats 4, 10 and 11. Combining these results with small-angle X-ray scattering data for the complex of TRN-SR2 with truncated integrase we propose a molecular model of the complex. We speculate that nuclear import of the PIC may proceed concurrently with the normal nuclear transport.

  6. Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

    PubMed Central

    Lassy, Rachel A. L.; Miller, Charles G.

    2000-01-01

    Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available. PMID:10762256

  7. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

    PubMed

    Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter; Münk, Carsten

    2016-12-01

    Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain.

  8. Structural and functional relationships of the steroid hormone receptors’ N-terminal transactivation domain

    PubMed Central

    Kumar, Raj; Litwack, Gerald

    2009-01-01

    Steroid hormone receptors are members of a family of ligand inducible transcription factors, and regulate the transcriptional activation of target genes by recruiting coregulatory proteins to the pre-initiation machinery. The binding of these coregulatory proteins to the steroid hormone receptors is often mediated through their two activation functional domains, AF1, which resides in the N-terminal domain, and the ligand-dependent AF2, which is localized in the C-terminal ligand binding domain. Compared to other important functional domains of the steroid hormone receptors, our understanding of the mechanisms of action of the AF1 are incomplete, in part, due to the fact that, in solution, AF1 is intrinsically disordered (ID). However, recent studies have shown that AF1 must adopt a functionally active and folded conformation for its optimal activity under physiological conditions. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF1-mediated gene activation, focusing on AF1 conformation and coactivator binding. We further propose models for the binding/folding of the AF1 domains of the steroid hormone receptors and their protein-protein interactions. The population of ID AF1 can be visualized as a collection of many different conformations, some of which may be assuming the proper functional folding for other critical target binding partners that result in ultimate assembly of AF1:coactivator complexes and subsequent gene regulation. Knowledge of the mechanisms involved therein will significantly help in understanding how signals from a steroid to a specific target gene are conveyed. PMID:19666041

  9. 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

  10. Stable proline box motif at the N-terminal end of alpha-helices.

    PubMed Central

    Viguera, A. R.; Serrano, L.

    1999-01-01

    We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis shows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an alpha-helix, but not when Pro is at position N1. N-cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix formers results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This suggests that the Pro-box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position. PMID:10493574

  11. Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region.

    PubMed Central

    Markoff, L; Lin, B C; Sveda, M M; Lai, C J

    1984-01-01

    A full-length double-stranded DNA copy of an influenza A virus N2 neuraminidase (NA) gene was cloned into the late region of pSV2330, a hybrid expression vector that includes pBR322 plasmid DNA sequences and the simian virus 40 early region and simian virus 40 late region promoters, splice sequences, and transcription termination sites. The protein encoded by the cloned wild-type NA gene was shown to be present in the cytoplasm of fixed cells and at the surface of "live" or unfixed cells by indirect immunofluorescence with N2 monoclonal antibodies. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [35S]methionine-labeled proteins from wild-type vector-infected cells with heterospecific N2 antibody showed that the product of the cloned NA DNA comigrated with glycosylated NA from influenza virus-infected cells, remained associated with internal membranes of cells fractionated into membrane and cytoplasmic fractions, and could form an immunoprecipitable dimer. NA enzymatic activity was detectable after simian virus 40 lysis of vector-infected cells. These properties of the product of the cloned wild-type gene were compared with those of the polypeptides produced by three deletion mutant NA DNAs that were also cloned into the late region of the pSV2330 vector. These mutants lacked 7 (dlk), 21 (dlI), or all 23 amino acids (dlZ) of the amino (N)-terminal variable hydrophobic region that anchors the mature wild-type NA tetrameric structure in the infected cell or influenza viral membrane. Comparison of the phenotypes of these mutants showed that this region in the NA molecule also includes sequences that control translocation of the nascent polypeptide into membrane organelles for glycosylation. Images PMID:6700587

  12. 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

  13. Involvement of c-Jun N-Terminal Kinase in TNF-α-Driven Remodeling.

    PubMed

    Eurlings, Irene M J; Reynaert, Niki L; van de Wetering, Cheryl; Aesif, Scott W; Mercken, Evi M; de Cabo, Rafael; van der Velden, Jos L; Janssen-Heininger, Yvonne M; Wouters, Emiel F M; Dentener, Mieke A

    2017-03-01

    Lung tissue remodeling in chronic obstructive pulmonary disease (COPD) is characterized by airway wall thickening and/or emphysema. Although the bronchial and alveolar compartments are functionally independent entities, we recently showed comparable alterations in matrix composition comprised of decreased elastin content and increased collagen and hyaluronan contents of alveolar and small airway walls. Out of several animal models tested, surfactant protein C (SPC)-TNF-α mice showed remodeling in alveolar and airway walls similar to what we observed in patients with COPD. Epithelial cells are able to undergo a phenotypic shift, gaining mesenchymal properties, a process in which c-Jun N-terminal kinase (JNK) signaling is involved. Therefore, we hypothesized that TNF-α induces JNK-dependent epithelial plasticity, which contributes to lung matrix remodeling. To this end, the ability of TNF-α to induce a phenotypic shift was assessed in A549, BEAS2B, and primary bronchial epithelial cells, and phenotypic markers were studied in SPC-TNF-α mice. Phenotypic markers of mesenchymal cells were elevated both in vitro and in vivo, as shown by the expression of vimentin, plasminogen activator inhibitor-1, collagen, and matrix metalloproteinases. Concurrently, the expression of the epithelial markers, E-cadherin and keratin 7 and 18, was attenuated. A pharmacological inhibitor of JNK attenuated this phenotypic shift in vitro, demonstrating involvement of JNK signaling in this process. Interestingly, activation of JNK signaling was also clearly present in lungs of SPC-TNF-α mice and patients with COPD. Together, these data show a role for TNF-α in the induction of a phenotypic shift in vitro, resulting in increased collagen production and the expression of elastin-degrading matrix metalloproteinases, and provide evidence for involvement of the TNF-α-JNK axis in extracellular matrix remodeling.

  14. GPR37 Surface Expression Enhancement via N-Terminal Truncation or Protein-Protein Interactions1

    PubMed Central

    Dunham, Jill H.; Meyer, Rebecca C.; Garcia, Erin L.; Hall, Randy A.

    2009-01-01

    GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that exhibits poor plasma membrane expression when expressed in most cell types. We sought to find ways to enhance GPR37 trafficking to the cell surface in order to facilitate studies of GPR37 functional activity in heterologous cells. In truncation studies, we found that removing the GPR37 N-terminus (NT) dramatically enhanced the receptor’s plasma membrane insertion. Further studies on sequential NT truncations revealed that removal of the first 210 amino acids increased surface expression nearly as much as removal of the entire NT. In studies examining the effects of co-expression of GPR37 with a variety of other GPCRs, we observed significant increases in GPR37 surface expression when the receptor was co-expressed with the adenosine receptor A2AR or the dopamine receptor D2R. Co-immunoprecipitation experiments revealed that full-length GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly associating with D2R, resulting in modestly-altered D2R affinity for both agonists and antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds, we observed a specific interaction between GPR37 and syntenin-1, which resulted in a dramatic increase in GPR37 surface expression in HEK-293 cells. These findings reveal three independent approaches – N-terminal truncation, co-expression with other receptors and co-expression with syntenin-1 – by which GPR37 surface trafficking in heterologous cells can be greatly enhanced to facilitate functional studies on this orphan receptor. PMID:19799451

  15. The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-terminal sequence extension.

    PubMed

    Martin-Eauclaire, Marie-France; Salvatierra, Juan; Bosmans, Frank; Bougis, Pierre E

    2016-09-01

    We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.

  16. Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

    PubMed

    Vickaryous, Nicola K; Teh, Evelyn M; Stewart, Bruce; Dolphin, Peter J; Too, Catherine K L; McLeod, Roger S

    2003-03-21

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the

  17. De novo assembly and identification of unique contigs in the bovine oviduct from animals with high and low circulating estradiol concentrations during timed artificial insemination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reproductive efficiency is a large concern for many cattle producers and understanding the mechanisms responsible for biological variation in reproduction is key to improving reproductive efficiency. Timed artificial insemination of beef cows with high circulating estradiol concentrations at time o...

  18. 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

  19. 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

    2016-04-08

    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.

  20. [Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue].

    PubMed

    Lasta, S; Fajloun, Z; Mansuelle, P; Sabatier, J M; Boudabous, A; Sampieri, F

    2008-01-01

    The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

  1. 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.

  2. Release of an HtrA-Like Protease from the Cell Surface of Thermophilic Brevibacillus sp. WF146 via Substrate-Induced Autoprocessing of the N-terminal Membrane Anchor.

    PubMed

    Zhu, Fengtao; Yang, Xing; Wu, Yan; Wang, Yasi; Tang, Xiao-Feng; Tang, Bing

    2017-01-01

    High-temperature requirement A (HtrA)-like proteases participate in protein quality control in prokaryotes and eukaryotes by degrading damaged proteins; however, little is known about HtrAs produced by thermophiles. HtrAw is an HtrA-like protease of thermophilic Brevibacillus sp. WF146. The intact form of HtrAw (iHtrAw) consisting of a transmembrane segment-containing N-terminal domain, a trypsin-like protease domain, and a C-terminal PDZ domain was produced in Escherichia coli. Purified iHtrAw itself is unable to cleave the N-terminal domain, but requires protein substrates to autoprocess the N-terminal domain intermolecularly, yielding a short form (sHtrAw). Mutation at the substrate-binding site in the PDZ domain affects the conversion of iHtrAw to sHtrAw. Deletion analysis revealed that the N-terminal domain is not necessary for enzyme folding, activity, and thermostability. Compared with other known HtrAs, HtrAw contains an additional Ca(2+)-binding Dx[DN]xDG motif important for enzyme stability and/or activity. When produced in an htrA/htrB double deletion mutant of Bacillus subtilis, iHtrAw localized predominantly to the cell pellet, and the amount of sHtrAw in the culture supernatant increased at elevated temperatures. Moreover, HtrAw increased the heat resistance of the B. subtilis mutant. In strain WF146, HtrAw exists in both a cell-associated intact form and a cell-free short form; an increase in growth temperature enhanced HtrAw production and the amount of cell-free short form. Release of the short form of HtrAw from the membrane may have the advantage of allowing the enzyme to freely access and degrade damaged proteins surrounding the bacterium living at high temperatures.

  3. Release of an HtrA-Like Protease from the Cell Surface of Thermophilic Brevibacillus sp. WF146 via Substrate-Induced Autoprocessing of the N-terminal Membrane Anchor

    PubMed Central

    Zhu, Fengtao; Yang, Xing; Wu, Yan; Wang, Yasi; Tang, Xiao-Feng; Tang, Bing

    2017-01-01

    High-temperature requirement A (HtrA)-like proteases participate in protein quality control in prokaryotes and eukaryotes by degrading damaged proteins; however, little is known about HtrAs produced by thermophiles. HtrAw is an HtrA-like protease of thermophilic Brevibacillus sp. WF146. The intact form of HtrAw (iHtrAw) consisting of a transmembrane segment-containing N-terminal domain, a trypsin-like protease domain, and a C-terminal PDZ domain was produced in Escherichia coli. Purified iHtrAw itself is unable to cleave the N-terminal domain, but requires protein substrates to autoprocess the N-terminal domain intermolecularly, yielding a short form (sHtrAw). Mutation at the substrate-binding site in the PDZ domain affects the conversion of iHtrAw to sHtrAw. Deletion analysis revealed that the N-terminal domain is not necessary for enzyme folding, activity, and thermostability. Compared with other known HtrAs, HtrAw contains an additional Ca2+-binding Dx[DN]xDG motif important for enzyme stability and/or activity. When produced in an htrA/htrB double deletion mutant of Bacillus subtilis, iHtrAw localized predominantly to the cell pellet, and the amount of sHtrAw in the culture supernatant increased at elevated temperatures. Moreover, HtrAw increased the heat resistance of the B. subtilis mutant. In strain WF146, HtrAw exists in both a cell-associated intact form and a cell-free short form; an increase in growth temperature enhanced HtrAw production and the amount of cell-free short form. Release of the short form of HtrAw from the membrane may have the advantage of allowing the enzyme to freely access and degrade damaged proteins surrounding the bacterium living at high temperatures. PMID:28377763

  4. Serum-circulating miRNAs predict neuroblastoma progression in mouse model of high-risk metastatic disease

    PubMed Central

    Ramraj, Satish Kumar; Aravindan, Sheeja; Somasundaram, Dinesh Babu; Herman, Terence S.; Natarajan, Mohan; Aravindan, Natarajan

    2016-01-01

    Background Circulating miRNAs have momentous clinical relevance as prognostic biomarkers and in the progression of solid tumors. Recognizing novel candidates of neuroblastoma-specific circulating miRNAs would allow us to identify potential prognostic biomarkers that could predict the switch from favorable to high-risk metastatic neuroblastoma (HR-NB). Results Utilizing mouse models of favorable and HR-NB and whole miRnome profiling, we identified high serum levels of 34 and low levels of 46 miRNAs in animals with HR-NB. Preferential sequence homology exclusion of mouse miRNAs identified 25 (11 increased; 14 decreased) human-specific prognostic marker candidates, of which, 21 were unique to HR-NB. miRNA QPCR validated miRnome profile. Target analysis defined the candidate miRNAs' signal transduction flow-through and demonstrated their converged roles in tumor progression. miRNA silencing studies verified the function of select miRNAs on the translation of at least 14 target proteins. Expressions of critical targets that correlate tumor progression in tissue of multifarious organs identify the orchestration of HR-NB. Significant (>10 fold) increase in serum levels of miR-381, miR-548h, and miR-580 identify them as potential prognostic markers for neuroblastoma progression. Conclusion For the first time, we identified serum-circulating miRNAs that predict the switch from favorable to HR-NB and, further imply that these miRNAs could play a functional role in tumor progression. PMID:26921195

  5. 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

  6. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains

    PubMed Central

    Lucena, Pedro I.; Faget, Douglas V.; Pachulec, Emilia; Robaina, Marcela C.; Klumb, Claudete E.

    2015-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4+ T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions. PMID:26483414

  7. 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.

  8. NFAT2 Isoforms Differentially Regulate Gene Expression, Cell Death, and Transformation through Alternative N-Terminal Domains.

    PubMed

    Lucena, Pedro I; Faget, Douglas V; Pachulec, Emilia; Robaina, Marcela C; Klumb, Claudete E; Robbs, Bruno K; Viola, João P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4(+) T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions.

  9. 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.

  10. A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

    1998-01-01

    To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

  11. 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.

  12. Functional Interrogation of the N-Terminal Lid of MDMX in p53 Binding via Native Chemical Ligation.

    PubMed

    Chen, Xishan; Lu, Weiyue

    2016-01-01

    The homologous proteins MDM2 and MDMX negatively regulate the tumor suppressor protein p53 by antagonizing p53 transactivation activity and targeting p53 for degradation. MDM2 and MDMX bind to p53 via N-terminal p53-binding domains to control the level of p53. The N-terminal regions of MDM2 and MDMX are modified in vivo under stressed conditions, suggesting that modifications to MDM2/MDMX also may affect the p53-MDM2/MDMX interaction. Ample evidence suggests that the MDM2 lid (residues 1-24) is partially structured and significantly reduces its binding affinity with p53 several fold. Since MDM2 and MDMX possess very similar p53-binding domains but different lids, however, the function of the N-terminal lid of MDMX still remains poorly understood. Using a native chemical ligation technique, the p53-binding domain of MDMX, (1-108)MDMX, and its N-terminal lid (residues 1-23) truncated analogue (24-108)MDMX were chemically synthesized. We comparatively characterized their structures by circular dichroism (CD) spectra, and measured their binding affinities with a panel of p53-derived peptide ligands by fluorescence polarization and surface plasmon resonance assays. Our results indicate that, as opposed to the lid of MDM2, the lid of MDMX has little effect on p53-binding, adopts no structural conformation, and has rare auto-inhibitory function. Different lid modifications of MDM2 and MDMX are functionally different with respect to p53 binding, which should be considered when designing dual specific inhibitors of MDM2 and MDMX.

  13. Conformational unfolding in the N-terminal region of ribonuclease A detected by nonradiative energy transfer.

    PubMed

    McWherter, C A; Haas, E; Leed, A R; Scheraga, H A

    1986-04-22

    Unfolding in the N-terminal region of RNase A was studied by the nonradiative energy-transfer technique. RNase A was labeled with a nonfluorescent acceptor (2,4-dinitrophenyl) on the alpha-amino group and a fluorescent donor (ethylenediamine monoamide of 2-naphthoxyacetic acid) on a carboxyl group in the vicinity of residue 50 (75% at Glu-49 and 25% at Asp-53). The distribution of donor labeling sites does not affect the results of this study since they are close in both the sequence and the three-dimensional structure. The sites of labeling were determined by peptide mapping. The derivatives possessed full enzymatic activity and underwent reversible thermal transitions. However, there were some quantitative differences in the thermodynamic parameters. When the carboxyl groups were masked, there was a 5 degrees C lowering of the melting temperature at pH 2 and 4, and no significant change in delta H(Tm). Labeling of the alpha-amino group had no effect on the melting temperature or delta H(Tm) at pH 2 but did result in a dramatic decrease in delta H(Tm) of the unfolding reaction at pH 4. The melting temperature did not change appreciably at pH 4, indicating that an enthalpy/entropy compensation had occurred. The efficiencies of energy transfer determined with both fluorescence intensity and lifetime measurements were in reasonably good agreement. The transfer efficiency dropped from about 60% under folding conditions to roughly 20% when the derivatives were unfolded with disulfide bonds intact and was further reduced to 5% when the disulfide bonds were reduced. The interprobe separation distance was estimated to be 35 +/- 2 A under folding conditions. The contribution to the interprobe distance resulting from the finite size of the probes was treated by using simple geometric considerations and a rotational isomeric state model of the donor probe linkage. With this model, the estimated average interprobe distance of 36 A is in excellent agreement with the

  14. Determination of the pKa of the N-terminal amino group of ubiquitin by NMR

    PubMed Central

    Oregioni, Alain; Stieglitz, Benjamin; Kelly, Geoffrey; Rittinger, Katrin; Frenkiel, Tom

    2017-01-01

    Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains. PMID:28252051

  15. 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.

  16. Loss of the N-terminal methyltransferase NRMT1 increases sensitivity to DNA damage and promotes mammary oncogenesis

    PubMed Central

    Bonsignore, Lindsay A.; Butler, Jill Sergesketter; Klinge, Carolyn M.; Tooley, Christine E. Schaner

    2015-01-01

    Though discovered over four decades ago, the function of N-terminal methylation has mostly remained a mystery. Our discovery of the first mammalian N-terminal methyltransferase, NRMT1, has led to the discovery of many new functions for N-terminal methylation, including regulation of DNA/protein interactions, accurate mitotic division, and nucleotide excision repair (NER). Here we test whether NRMT1 is also important for DNA double-strand break (DSB) repair, and given its previously known roles in cell cycle regulation and the DNA damage response, assay if NRMT1 is acting as a tumor suppressor. We find that NRMT1 knockdown significantly enhances the sensitivity of breast cancer cell lines to both etoposide treatment and γ-irradiation, as well as, increases proliferation rate, invasive potential, anchorage-independent growth, xenograft tumor size, and tamoxifen sensitivity. Interestingly, this positions NRMT1 as a tumor suppressor protein involved in multiple DNA repair pathways, and indicates, similar to BRCA1 and BRCA2, its loss may result in tumors with enhanced sensitivity to diverse DNA damaging chemotherapeutics. PMID:25909287

  17. The SAS-5 N-terminal domain is a tetramer, with implications for centriole assembly in C. elegans.

    PubMed

    Shimanovskaya, Ekaterina; Qiao, Renping; Lesigang, Johannes; Dong, Gang

    2013-07-01

    The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. It has a unique 9-fold symmetry and its assembly is governed by at least five component proteins (SPD-2, ZYG-1, SAS-5, SAS-6 and SAS-4), which are recruited in a hierarchical order. Recently published structural studies of the SAS-6 N-terminal domain have greatly advanced our understanding of the mechanisms of centriole assembly. However, it remains unclear how the weak interaction between the SAS-6 N-terminal head groups could drive the assembly of a closed ring-like structure, and what determines the stacking of multiple rings on top one another in centriole duplication. We recently reported that SAS-5 binds specifically to a very narrow region of the SAS-6 central coiled coil through its C-terminal domain (CTD, residues 391-404). Here, we further demonstrate by both static light scattering and small angle X-ray scattering that the SAS-5 N-terminal domain (NTD, residues 1-260) forms a tetramer. Specifically, we found that the tetramer is formed by SAS-5 residues 82-260, whereas residues 1-81 are intrinsically disordered. Taking these results together, we propose a working model for SAS-5-mediated assembly of the multi-layered central tube structure.

  18. Determination of the pKa of the N-terminal amino group of ubiquitin by NMR.

    PubMed

    Oregioni, Alain; Stieglitz, Benjamin; Kelly, Geoffrey; Rittinger, Katrin; Frenkiel, Tom

    2017-03-02

    Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains.

  19. 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

  20. Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge.

    PubMed

    Wang, Yawei; Fu, Zheng; Huang, Huoqing; Zhang, Huashan; Yao, Bin; Xiong, Hairong; Turunen, Ossi

    2012-05-01

    In order to increase the stability of thermophilic Thermomyces lanuginosus GH11 xylanase, TLX, a disulfide bridge Q1C-Q24C was introduced into the N-terminal region of the enzyme. The apparent temperature optimum shifted upwards at pH 6.5 by about 10°C to 75°C. The resistance to thermal inactivation also increased by about 10°C. The melting temperature measured by CD spectroscopy increased from 66 to 74°C. Therefore the N-terminal disulfide bridge increased both kinetic and thermodynamic stability almost equally. At pH 8 and 70°C, the disulfide bridge increased the enzyme half-life 20-fold in the presence of substrate. In contrast to the situation in acidic-neutral pH, the substrate decreased the thermostability of xylanases in alkaline pH. The upper limit for the performance of the disulfide bridge mutant at pH 9 was 75°C. This study showed that N-terminal disulfide bridges can stabilize even thermostable family GH11 xylanases.

  1. Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

    SciTech Connect

    H Wang; H Robinson; H Ke

    2011-12-31

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.

  2. Conformation changes, N-terminal involvement and cGMP signal relay in phosphodiesterase-5 GAF domain

    SciTech Connect

    Wang, H.; Robinson, H.; Ke, H.

    2010-12-03

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.

  3. Mean circulation and high-frequency flow amplification in the Sable Gully

    NASA Astrophysics Data System (ADS)

    Greenan, Blair J. W.; Petrie, Brian D.; Cardoso, Diana A.

    2014-06-01

    The Sable Gully, a broad, shelf break submarine canyon approximately 40 km east of Sable Island on the eastern Scotian Shelf, separates Banquereau and Sable Island Banks. Unique among canyons on the eastern Canadian continental shelf because of its depth, steep slopes and extension far onto the shelf, its ecological significance and increasing human pressures led to its designation in 2004 under Canada's Oceans Act as the first Marine Protected Area (MPA) in the Atlantic Region. To improve the state of knowledge of the Gully MPA, a multi-disciplinary field program was carried out in 2006-07; the physical oceanographic component consisted of the deployment (April 2006) and recovery (August 2007) of four current meter moorings and CTD surveys. Analysis of this 16-month mooring deployment demonstrates that the mean circulation above the canyon rim (~200 m) is characterized by a southwestward flow that appears unaffected by the canyon topography. There is also some indication of the existence of an eddy at rim depth. Below 500 m, the circulation is dominated by an upcanyon flow (of order 0.02 m s-1) at the mooring array (halfway between the canyon head and mouth). The mean, 200 m-bottom transport towards the head of the Gully was estimated as 35,500 m3 s-1, implying an upwelling velocity of 1.7×10-4 m s-1 (14 m d-1) over the area. Results also show bottom-intensified tidal flows and non-linear constituents due to the interaction of K1, O1, M2 and S2 components along the thalweg of the canyon; the strong overtides and compound tides observed in the Gully make it unique among canyons. Further analyses provide evidence of enhanced mixing in the Gully (Kv~180×10-4 m2 s-1), which is approximately 20 times that observed on the adjoining Scotian Shelf. Total variance of the currents in the Gully is about 2.5 times greater than that observed on the nearby continental slope with an equivalent water depth.

  4. In Developping a Bench-Scale Circulating Fluidized Bed Combustor to Burn High Ash Brazilian Coal-Dolomites Mixtures

    NASA Astrophysics Data System (ADS)

    Ramírez Behainne, Jhon Jairo; Hory, Rogério Ishikawa; Goldstein, Leonardo; Bernárdez Pécora, Araí Augusta

    This work considers some of the questions in burning high ash Brazilian coal-dolomite mixtures in a bench-scale circulating fluidized bed combustor (CFBC). Experimental tests were performed with the CE4500 coal from Santa Catarina State, in southern Brazil, with a Sauter mean diameter d p =43 μm. The coal particles were mixed with dolomite particles of d p = 111 μm and this fuel mixture was fed into the circulating fluidized reactor, previously loaded with quartz sand particles of d p =353 μm. This inert material was previously heated by the combustion of liquefied petroleum gas up to the ignition temperature of the fuel mixture. The CFBC unit has a 100mm internal diameter riser, 4.0m high, as well as a 62.8mm internal diameter downcomer. The loop has a cyclone, a sampling valve to collect particles and a 62.8mm internal diameter L-valve to recirculate the particles in the loop. A screw feeder with a rotation control system was used to feed the fuel mixture to the reactor. The operational conditions were monitored by pressure taps and thermocouples installed along the loop. A data acquisition system showed the main operational conditions to control. Experimental tests performed put in evidence the problems found during bed operation, with special attention to the solids feed device, to the L-valve operation, to particle size, solids inventory, fluidized gas velocity, fuel mixture and recirculated solids feeding positions.

  5. Seasonal circulation on the western shelf of the Gulf of Mexico using a high-resolution numerical model

    NASA Astrophysics Data System (ADS)

    Zavala-Hidalgo, Jorge; Morey, Steven L.; O'Brien, James J.

    2003-12-01

    The seasonal circulation on the western shelf of the Gulf of Mexico is studied using a high-resolution numerical simulation, historical hydrographic data, sea level data, and satellite images. Three regions are distinguished, the Tamaulipas-Veracruz (TAVE) shelf, the Louisiana-Texas (LATEX) shelf, and the western Campeche Bank. On the TAVE shelf there is a swift reversal of the along-shelf current, downcoast from September to March and upcoast from May to August when there is upwelling due to offshore Ekman transport. Circulation on the western Campeche Bank is upcoast throughout the year. The LATEX shelf has a cyclonic circulation, except during summer months when the flow is eastward. During spring-summer the upcoast current on the TAVE shelf reaches the southern Texas shelf where it encounters a downcoast coastal current favoring offshore transports. In the fall-winter, the downcoast current reaches the southern Bay of Campeche where it meets an opposing along-shelf current, generating seasonal offshore transports. During fall and winter, cool low-salinity water from the Mississippi and Atchafalaya Rivers is advected westward along the LATEX shelf onto the TAVE shelf, developing along-shelf fronts and temperature inversions commonly observed over the outer shelf and shelf break. The main forcing over the western shelf of the gulf is the along-coast wind stress component. The existence of the cross-shelf transports in the confluence regions is supported by chlorophyll a data. Up to 80% of the seasonal sea level variability is explained by the along shelf currents and the low-frequency variability of the atmospheric sea level pressure.

  6. A Study of The Eastern Mediterranean Hydrology and Circulation By Comparing Observation and High Resolution Numerical Model Results.

    NASA Astrophysics Data System (ADS)

    Alhammoud, B.; Béranger, K.; Mortier, L.; Crépon, M.

    The Eastern Mediterranean hydrology and circulation are studied by comparing the results of a high resolution primitive equation model (described in dedicated session: Béranger et al.) with observations. The model has a horizontal grid mesh of 1/16o and 43 z-levels in the vertical. The model was initialized with the MODB5 climatology and has been forced during 11 years by the daily sea surface fluxes provided by the European Centre for Medium-range Weather Forecasts analysis in a perpetual year mode corresponding to the year March 1998-February 1999. At the end of the run, the numerical model is able to accurately reproduce the major water masses of the Eastern Mediterranean Basin (Levantine Surface Water, modi- fied Atlantic Water, Levantine Intermediate Water, and Eastern Mediterranean Deep Water). Comparisons with the POEM observations reveal good agreement. While the initial conditions of the model are somewhat different from POEM observations, dur- ing the last year of the simulation, we found that the water mass stratification matches that of the observations quite well in the seasonal mean. During the 11 years of simulation, the model drifts slightly in the layers below the thermocline. Nevertheless, many important physical processes were reproduced. One example is that the dispersal of Adriatic Deep Water into the Levantine Basin is rep- resented. In addition, convective activity located in the northern part of the Levantine Basin occurs in Spring as expected. The surface circulation is in agreement with in-situ and satellite observations. Some well known mesoscale features of the upper thermocline circulation are shown. Sea- sonal variability of transports through Sicily, Otranto and Cretan straits are inves- tigated as well. This work was supported by the french MERCATOR project and SHOM.

  7. Surface circulation in the Strait of Gibraltar: a comparison study between HF radar and high resolution model data.

    NASA Astrophysics Data System (ADS)

    Soto-Navarro, Javier; Lorente, Pablo; Álvarez-Fanjul, Enrique; Ruiz-Gil de la Serna, M. Isabel

    2015-04-01

    Surface currents from the HF radar system deployed by Puertos del Estado (PdE) at the Strait of Gibraltar and an operational high resolution configuration of the MIT global circulation model, implemented in the strait area in the frame of the SAMPA project, have been analyzed and compared in the period February 2013 - September 2014. The comparison have been carried out in the time and frequency domains, by statistical a geophysical (tide ellipses, wind forcing, EOF) methods. Results show good agreement between both current fields in the strait axis, with correlation around 0.6 (reaching 0.9 in the low frequency band). Higher discrepancies are found in the boundaries of the domain, due to the differences in the meridional components, likely related to the sparser and less accurate radar measurements in these areas. Rotary spectral analysis show a very good agreement between both systems, which is reflected in a very similar and realistic representation of the main tide constituents (M2, S2 and K1). The wind forced circulation pattern, of special interest in the mouth of the Bay of Algeciras, is also precisely represented by radar and model. Finally, the spatial patterns of the first four EOF modes of both fields are rather close, reinforcing the previous results. As conclusion, the analysis points out the proper representation of the surface circulation of the area performed by the PdE HF radar system and the SAMPA model. However, weak and strong points are found in both, stressing the importance of having two complementary tools in the area.

  8. Characteristics of high temperature cementitious lost-circulation control materials for geothermal wells

    SciTech Connect

    Sugama, T.; Kukacka, L.E.; Galen, B.G.; Milestone, N.B.

    1986-01-01

    Materials systems have been formulated for the in situ conversion of water-based bentonite drilling fluids into cementitious lost-circulation control materials (CLCM) for use in geothermal wells at temperatures up to 300/sup 0/C. The formulations consist of a cement hardener, a borax admixture, and a fiber glass bridging material which are added to the bentonite fluids. Evaluations of the properties of the slurry and the cured CLCMS revealed that the ions supplied by dissociation of the borax in the CLCM slurry acted to suppress the bentonite hydration and retarded the hardening rate of the cement at elevated temperatures. The CaO-SiO/sub 2/-H/sub 2/O (C-S-H) phases formed during curing of the CLCM play essential roles in improving the quality of the hardened CLCMs. It was observed that xonotlite-truscottite transformation resulted in strength reductions and increased water permeability. The plugging ability of fiber glass depends on the conentration and fiber size. The silicate ions dissolved by hot alkaline disintegration of the fiber glass were chemisorbed with Ca/sup 2 +/ ions from the cement and led to the precipitation of C-S-H compounds on the fiber surfaces, which improved bond strength at the matrix-fiber interfaces.

  9. 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.

  10. N-Terminal Lipid Modification Is Required for the Stable Accumulation of CyanoQ in Synechocystis sp. PCC 6803

    PubMed Central

    Juneau, Andrea D.; Frankel, Laurie K.; Bricker, Terry M.; Roose, Johnna L.

    2016-01-01

    The CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex. PMID:27656895

  11. The effects of phosphomimetic lid mutation on the thermostability of the N-terminal domain of MDM2.

    PubMed

    Worrall, Erin G; Worrall, Liam; Blackburn, Elizabeth; Walkinshaw, Malcolm; Hupp, Ted R

    2010-05-07

    The multidomain E3 ubiquitin ligase MDM2 catalyzes p53 ubiquitination by a "dual-site" docking mechanism whereby MDM2 binding to at least two distinct peptide motifs on p53 promotes ubiquitination. One protein-protein interaction occurs between the N-terminal hydrophobic pocket of MDM2 and the transactivation motif of p53, and the second interaction occurs between the acidic domain of MDM2 and a motif in the DNA-binding domain of p53. A flexible N-terminal pseudo-substrate or "lid" adjacent to the N-terminal hydrophobic pocket of MDM2 has a phosphorylation site, and there are distinct models proposed on how the phosphorylated lid could affect MDM2 function. Biochemical studies have predicted that phosphomimetic mutation will stabilize the lid on the surface of MDM2 and will "open" the hydrophobic pocket and stabilize the MDM2-p53 complex, while NMR studies proposed that phosphomimetic mutation "closes" the lid over the MDM2 pocket and inhibits MDM2-p53 complex formation. To resolve these discrepancies, we utilized a quantitative fluorescence-based dye binding assay to measure the thermal unfolding of wild-type (wt), DeltaLid, and S17D N-terminal domains of MDM2 as a function of increasing ligand concentration. Our data reveal that S17D lid mutation increases, rather than decreases, the thermostability of the N-terminal domain of MDM2 in the absence or in the presence of ligand. DeltaLid mutation, by contrast, increases MDM2 thermoinstability. This is consistent with biochemical data, using full-length MDM2, showing that the S17D mutation stabilizes the MDM2-p53 complex and increases the specific activity of the E3 ubiquitin ligase function of MDM2. These data indicate that phosphomimetic lid mutation results in an "opening," rather than a "closing," of the pocket of MDM2 and highlight the ability of small intrinsically disordered or unstructured peptide motifs to regulate the specific activity of a protein.

  12. The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

    PubMed Central

    Varela Chavez, Carolina; Haustant, Georges Michel; Baron, Bruno; England, Patrick; Chenal, Alexandre; Pauillac, Serge; Blondel, Arnaud; Popoff, Michel-Robert

    2016-01-01

    Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. PMID:27023605

  13. Abnormal splicing in the N-terminal variable region of cardiac troponin T impairs systolic function of the heart with preserved Frank-Starling compensation.

    PubMed

    Feng, Han-Zhong; Chen, Guozhen; Nan, Changlong; Huang, Xupei; Jin, Jian-Ping

    2014-09-01

    Abnormal splice-out of the exon 7-encoded segment in the N-terminal variable region of cardiac troponin T (cTnT-ΔE7) was found in turkeys and, together with the inclusion of embryonic exon (eTnT), in adult dogs with a correlation with dilated cardiomyopathy. Overexpression of these cTnT variants in transgenic mouse hearts significantly decreased cardiac function. To further investigate the functional effect of cTnT-ΔE7 or ΔE7+eTnT in vivo under systemic regulation, echocardiography was carried out in single and double-transgenic mice. No atrial enlargement, ventricular hypertrophy or dilation was detected in the hearts of 2-month-old cTnT-ΔE7 and ΔE7+eTnT mice in comparison to wild-type controls, indicating a compensated state. However, left ventricular fractional shortening and ejection fraction were decreased in ΔE7 and ΔE7+eTnT mice, and the response to isoproterenol was lower in ΔE7+eTnT mice. Left ventricular outflow tract velocity and gradient were decreased in the transgenic mouse hearts, indicating decreased systolic function. Ex vivo working heart function showed that high afterload or low preload resulted in more severe decreases in the systolic function and energetic efficiency of cTnT-ΔE7 and ΔE7+eTnT hearts. On the other hand, increases in preload demonstrated preserved Frank-Starling responses and minimized the loss of cardiac function and efficiency. The data demonstrate that the N-terminal variable region of cardiac TnT regulates systolic function of the heart.

  14. Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth.

    PubMed Central

    Wang, H G; Rikitake, Y; Carter, M C; Yaciuk, P; Abraham, S E; Zerler, B; Moran, E

    1993-01-01

    Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function. Images PMID:8416379

  15. Direct interaction between Tks proteins and the N-terminal proline-rich region (PRR) of NoxA1 mediates Nox1-dependent ROS generation.

    PubMed

    Gianni, Davide; DerMardirossian, Céline; Bokoch, Gary M

    2011-01-01

    NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been implicated in several physiological and pathophysiological processes. To date seven members of this family have been reported, including Nox1-5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, the organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate proteins Tks4 and Tks5 as functional members of a p47(phox)-related organizer superfamily. As a functional consequence of this interaction, Nox1 localizes to invadopodia, actin-rich membrane protrusions of cancer cells which facilitate pericellular proteolysis and invasive behavior. Here, we report that Tks4 and Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of NoxA1 is essential for this direct interaction with the Tks proteins. When the PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5 are unable to act as organizers for Nox2 because of their inability to interact with p67(phox), which lacks the N-terminal PRR, thus conferring a unique specificity to Tks4 and 5. Taken together, these results clarify the molecular basis for the interaction between NoxA1 and the Tks proteins and may provide new insights into the pharmacological design of a more effective anti-metastatic strategy.

  16. Characterization of mutant xylanases using fourier transform ion cyclotron resonance mass spectrometry: stabilizing contributions of disulfide bridges and N-terminal extensions.

    PubMed

    Jänis, Janne; Turunen, Ossi; Leisola, Matti; Derrick, Peter J; Rouvinen, Juha; Vainiotalo, Pirjo

    2004-07-27

    Structural properties and thermal stability of Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) and its three recombinant mutants were characterized using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry and hydrogen/deuterium (H/D) exchange reactions. TRX II has been previously stabilized by a disulfide bridge C110-C154 and other site-directed mutations (TRX II mutants DS2 and DS5). Very recently, a highly thermostable mutant was introduced by combining mutations of DS5 with an N-terminal disulfide bridge C2-C28 (mutant DB1). Accurate mass measurements of TRX II, DS2, DS5, and DB1 verified the expected DNA-encoded protein sequences (average mass error 1.3 ppm) and allowed unequivocal assignment of the disulfides without chemical reduction and subsequent alkylation of the expected cross-links. Moreover, H/D exchange reactions provided means for the detection of a major heat-induced conformational change comprising two interconverting conformers of very different H/D exchange rates as well as allowed the apparent melting temperatures (T(m)) to be determined (62.6, 65.1, 68.0, and 82.2 degrees C for TRX II, DS2, DS5, and DB1, respectively). Residual activity measurements verified that the enzymes inactivated at significantly lower temperatures than expected on the basis of the apparent T(m) values, strongly suggesting that the inactivation takes place through minor conformational change other than observed by H/D exchange. ESI FT-ICR analyses also revealed molecular heterogeneity in DS5 and DB1 due to the propeptide incorporation. Resulting unintentional N-terminal extensions were observed to further improve the stability of the DB1 mutant. The extension of six amino acid residues upstream from the protein N-terminus increased stability by approximately 5 degrees C.

  17. The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

    PubMed

    Varela Chavez, Carolina; Haustant, Georges Michel; Baron, Bruno; England, Patrick; Chenal, Alexandre; Pauillac, Serge; Blondel, Arnaud; Popoff, Michel-Robert

    2016-03-25

    Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1-93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

  18. Association between N-terminal proB-type Natriuretic Peptide and Depressive Symptoms in Patients with Acute Myocardial Infarction

    PubMed Central

    Ren, Yan; Jia, Jiao; Sa, Jian; Qiu, Li-Xia; Cui, Yue-Hua; Zhang, Yue-An; Yang, Hong; Liu, Gui-Fen

    2017-01-01

    Background: While depression and certain cardiac biomarkers are associated with acute myocardial infarction (AMI), the relationship between them remains largely unexplored. We examined the association between depressive symptoms and biomarkers in patients with AMI. Methods: We performed a cross-sectional study using data from 103 patients with AMI between March 2013 and September 2014. The levels of depression, N-terminal proB-type natriuretic peptide (NT-proBNP), and troponin I (TnI) were measured at baseline. The patients were divided into two groups: those with depressive symptoms and those without depressive symptoms according to Zung Self-rating Depression Scale (SDS) score. Baseline comparisons between two groups were made using Student's t-test for continuous variables, Chi-square or Fisher's exact test for categorical variables, and Wilcoxon test for variables in skewed distribution. Binomial logistic regression and multivariate linear regression were performed to assess the association between depressive symptoms and biomarkers while adjusting for demographic and clinical variables. Results: Patients with depressive symptoms had significantly higher NT-proBNP levels as compared to patients without depressive symptoms (1135.0 [131.5, 2474.0] vs. 384.0 [133.0, 990.0], Z = −2.470, P = 0.013). Depressive symptoms were associated with higher NT-proBNP levels (odds ratio [OR] = 2.348, 95% CI: 1.344 to 4.103, P = 0.003) and higher body mass index (OR = 1.169, 95% confidence interval [CI]: 1.016 to 1.345, P = 0.029). The total SDS score was associated with the NT-proBNP level (β = 0.327, 95% CI: 1.674 to 6.119, P = 0.001) after multivariable adjustment. In particular, NT-proBNP was associated with three of the depressive dimensions, including core depression (β = 0.299, 95% CI: 0.551 to 2.428, P = 0.002), cognitive depression (β = 0.320, 95% CI: 0.476 to 1.811, P = 0.001), and somatic depression (β = 0.333, 95% CI: 0.240 to 0.847, P = 0.001). Neither the

  19. N-Terminal Prosomatostatin as a Risk Marker for Cardiovascular Disease and Diabetes in a General Population

    PubMed Central

    Almgren, Peter; Nilsson, Peter M.; Melander, Olle

    2016-01-01

    Context: Somatostatin inhibits a range of hormones, including GH, insulin, and glucagon, but little is known about its role in the development of cardiometabolic disease. Objective: The objective of the study was to investigate whether fasting plasma concentration of N-terminal prosomatostatin (NT-proSST) is associated with the development of diabetes, coronary artery disease (CAD), and mortality. Design, Setting, and Participants: NT-proSST was measured in plasma from 5389 fasting participants of the population-based study Malmö Preventive Project, with a mean baseline age of 69.4 ± 6.2 years. Cox proportional hazards models adjusted for traditional cardiovascular risk factors were used to investigate the relationships between baseline NT-proSST and end points, with a mean follow-up of 5.6 ± 1.4 years. Main Outcome Measures: CAD, diabetes, and mortality were measured. Results: Overall, NT-proSST (hazard ratio [HR] per SD increment of log transformed NT-proSST) was unrelated to the risk of incident diabetes (220 events; HR 1.05; 95% confidence interval [CI] 0.91–1.20; P = .531) but was related to the risk of incident CAD (370 events; HR 1.17; 95% CI 1.06–1.30; P = .003), all-cause mortality (756 events; HR 1.24; 95% CI 1.15–1.33; P < .001), and cardiovascular mortality (283 events; HR 1.33; 95% CI 1.19–1.43; P < .001). The relationships were not linear, with most of the excess risk observed in subjects with high values of NT-proSST. Subjects in the top vs bottom decile had a severely increased risk of incident CAD (HR 2.41; 95% CI 1.45–4.01; P < .001), all-cause mortality (HR 1.84; 95% CI 1.33–2.53; P < .001), and cardiovascular mortality (HR 2.44; 95% CI 1.39–4.27; P < .001). Conclusion: NT-proSST was significantly and independently associated with the development of CAD, all-cause mortality, and cardiovascular mortality. PMID:27399347

  20. Melanin or a Melanin-Like Substance Interacts with the N-Terminal Portion of Prion Protein and Inhibits Abnormal Prion Protein Formation in Prion-Infected Cells.

    PubMed

    Hamanaka, Taichi; Nishizawa, Keiko; Sakasegawa, Yuji; Oguma, Ayumi; Teruya, Kenta; Kurahashi, Hiroshi; Hara, Hideyuki; Sakaguchi, Suehiro; Doh-Ura, Katsumi

    2017-03-15

    Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP region of amino acids 23 to 50 including a positively charged amino acid cluster and weakly interacted with the PrP octarepeat peptide region of residues 51 to 90. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that of control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis, even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N terminus of PrP, but the pathophysiological roles of the PrP-melanin interaction remain unclear.IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors, such as metal ions, lipids, nucleic acids, antiprion compounds, and several

  1. Characteristics of self-alkalization in high-rate denitrifying automatic circulation (DAC) reactor fed with methanol and sodium acetate.

    PubMed

    Li, Wei; Zheng, Ping; Guo, Jun; Ji, Junyuan; Zhang, Meng; Zhang, Zonghe; Zhan, Enchao; Abbas, Ghulam

    2014-02-01

    Denitrification is a self-alkalization process. In this experiment, the characteristics of self-alkalization in high-rate heterotrophic denitrifying automatic circulation (DAC) reactor fed with methanol and sodium acetate were investigated, respectively. The results showed that, (1) The self-alkalization of high-rate denitrifying reactors was remarkably strong both with methanol and sodium acetate as carbon sources, while the effluent pH was much lower than the stoichiometric values and the malfunction from self-alkalization of denitrification was far less serious than expected. (2) The self-adaptation of the reactors was attributed to the neutralization of carbon dioxide (oxidization product of organic matter) and the self-adaptation of denitrifying sludge. The formation and discharge of calcium carbonate precipitates gave rise to lower effluent pH and alkalinity than the stoichiometric values.

  2. Inter-comparison of the mean circulation in the Coral and Solomon Sea simulated by high resolution ocean models

    NASA Astrophysics Data System (ADS)

    Maes, C.; Durand, F.; Gasparin, F.; Melet, A.; Ganachaud, A.

    2010-12-01

    Of primary importance to the properties of water masses transported by the northern limb of the South Pacific subtropical gyre toward the equatorial band, the transfer within the Coral and, ultimately, the Solomon Sea is perturbed by the labyrinthine topography of the region. It results in highly energetic currents and complex pathways through the Vanuatu Archipelago and New Caledonia, at the entrance of the Coral Sea, and through the Solomon Sea once the flow has bifurcated northward along the coasts of Australia and of the Louisiade Archipelago of Papua New Guinea. In the Coral Sea, the existence of the North Vanuatu Jet and North Caledonian Jet is now well established but their variations as well as their detailed characteristics, including for instance their vertical extension, remain largely unknown. In this study, recourse to ocean simulations is made in order to highlight the representation of such complex circulation of the south western Pacific Ocean and to analyze the long term variability and physical mechanism implied in the jet dynamics. A brief overview of recent observations collected through the comprehensive observational SPICE program (CLIVAR/WCRP) will first be presented in order to set the context. Then, 6 different state-of-the-art numerical experiments with high horizontal resolution, ranging from 1/10 to 1/12 degree, and realistic topography regionally focused on the Coral and Solomon Sea or extracted from global experiments, are analyzed. Here, we will consider OGCMs forced by realistic and observed atmospheric fields but each model has its own strategy in terms of diffusion, topography representation and boundaries condition when appropriate. The focus is set primarily on the annual mean circulation of the upper ocean layers (above the 1000-m depth) and on the water mass transports simulated in the vicinity of the various topographic obstacles. The results will underline that most of high resolution numerical models have reached a high

  3. [Designing of hybrid human interferon alfa-2 strain-producers and the use of enteropeptidase for obtaining N-terminal methionine-free interferons].

    PubMed

    Shirokov, D A; Riabichenko, V V; Akishina, R I; Ospel'nikova, T P; Glazunov, A V; Chestukhina, G G; Veĭko, V P

    2011-01-01

    A system for production of human interferon-alpha2a (IFN-alpha2a) and IFN-alpha2b lacking N-terminal methionine has been developed. Plasmids containing genes of hybrid IFN-alpha2 under the control of different promoters were constructed; a sequence encoding the enteropeptidase hydrolysis site being introduced in proximal part of the genes. As the result, 4 strains of Escherichia coli producing hybrid IFN-alpha2 have been obtained. The methodology for IFN-alpha2 renaturation, hydrolysis of its N-terminal part, chromatographic purification of N-terminal methionine-free IFN-alpha2 has been developed.

  4. High-circulating leptin levels are associated with increased blood pressure in uncontrolled resistant hypertension.

    PubMed

    de Haro Moraes, C; Figueiredo, V N; de Faria, A P C; Barbaro, N R; Sabbatini, A R; Quinaglia, T; Ferreira-Melo, S E; Martins, L C; Demacq, C; Júnior, H M

    2013-04-01

    Leptin and aldosterone have been associated with the pathophysiological mechanisms of hypertension. However, despite studies showing the association of leptin with intima-media thickness, arterial distensibility and sympathetic nerve activation, the relationship between leptin and blood pressure (BP) in resistant hypertension (RHTN) is unknown. We aimed to assess the correlation of plasma leptin and aldosterone levels with BP in uncontrolled controlled RHTN (UCRHTN) and CRHTN patients. Plasma leptin and aldosterone levels, office BP, ambulatory BP monitoring and heart rate were measured in 41 UCRHTN, 39 CRHTN and 31 well-controlled HTN patients. No differences were observed between the three groups regarding gender, body mass index and age. The UCRHTN group had increased leptin when compared with CRHTN and well-controlled HTN patients (38.2±21.4, 19.6±8.7 and 20.94±13.9 ng ml(-1), respectively; P<0.05). Aldosterone levels values were also statistically different when comparing RHTN, CRHTN and well-controlled HTN patients (9.6±3.8, 8.1±5.0 and 8.0±4.7 ng dl(-1), respectively; P<0.05). As expected, UCRHTN patients had higher heart rate values compared with CRHTN and well-controlled HTN patients (86.2±7.2, 83.5±6.7 and 83.4±8.5, respectively; P<0.05). Plasma leptin positively correlated with systolic (SBP) and diastolic BP (DBP), and aldosterone (r=0.43, 0.35 and 0.47, respectively; all P<0.05) in UCRHTN, but neither in the CRHTN nor in the HTN group. Simple linear regression showed that SBP, DBP and aldosterone may be predicted by leptin (r(2)=0.16, 0.15 and 0.19, respectively; all P<0.05) only in the UCRHTN subgroup. In conclusion, UCRHTN patients have higher circulating leptin levels associated with increased plasma aldosterone and BP levels when compared with CRHTN and HTN subjects.

  5. High-frequency daily temperature variability in China and its relationship to large-scale circulation

    SciTech Connect

    Wu, Fu-Ting; Fu, Congbin; Qian, Yun; Gao, Yang; Wang, Shu-Yu

    2016-04-18

    Two measures of intra-seasonal variability, indicated respectively by standard deviations (SD) and day-to-day (DTD) fluctuations denoted by absolute differences between adjacent 2-day periods, as well as their relationships with large-scale circulation patterns were investigated in China during 1962–2008 on the basis of homogenized daily temperature records from 549 local stations and reanalysis data. Our results show that both the SD and DTD of daily minimum temperatures (Tmin) in summer as well as the minimum and maximum temperatures in winter have been decreasing, while the daily maximum temperature (Tmax) variability in summer is fluctuating more, especially over southern China. In summer, an attribution analysis indicates that the intensity of the Western Pacific Subtropical High (WPSH) and high-level East Asian Subtropical Jet stream (EASJ) are positively correlated with both SD and DTD, but the correlation coefficients are generally greater with the SD than with the DTD of the daily maximum temperature, Tmax. In contrast, the location of the EASJ shows the opposite correlation pattern, with intensity regarding the correlation with both SD and DTD. In winter, the Arctic Oscillation (AO) is negatively correlated with both the SD and DTD of the daily minimum temperature, but its intra-seasonal variability exhibits good agreement with the SD of the Tmin. The Siberian High acts differently with respect to the SD and DTD of the Tmin, demonstrating a regionally consistent positive correlation with the SD. Overall, the large-scale circulation can well explain the intra-seasonal SD, but DTD fluctuations may be more local and impacted by local conditions, such as changes in the temperature itself, the land surface, and so on.

  6. High-resolution numerical simulation of Venus atmosphere by AFES (Atmospheric general circulation model For the Earth Simulator)

    NASA Astrophysics Data System (ADS)

    Sugimoto, Norihiko; AFES project Team

    2016-10-01

    We have developed an atmospheric general circulation model (AGCM) for Venus on the basis of AFES (AGCM For the Earth Simulator) and performed a high-resolution simulation (e.g., Sugimoto et al., 2014a). The highest resolution is T639L120; 1920 times 960 horizontal grids (grid intervals are about 20 km) with 120 vertical layers (layer intervals are about 1 km). In the model, the atmosphere is dry and forced by the solar heating with the diurnal and semi-diurnal components. The infrared radiative process is simplified by adopting Newtonian cooling approximation. The temperature is relaxed to a prescribed horizontally uniform temperature distribution, in which a layer with almost neutral static stability observed in the Venus atmosphere presents. A fast zonal wind in a solid-body rotation is given as the initial state.Starting from this idealized superrotation, the model atmosphere reaches a quasi-equilibrium state within 1 Earth year and this state is stably maintained for more than 10 Earth years. The zonal-mean zonal flow with weak midlatitude jets has almost constant velocity of 120 m/s in latitudes between 45°S and 45°N at the cloud top levels, which agrees very well with observations. In the cloud layer, baroclinic waves develop continuously at midlatitudes and generate Rossby-type waves at the cloud top (Sugimoto et al., 2014b). At the polar region, warm polar vortex surrounded by a cold latitude band (cold collar) is well reproduced (Ando et al., 2016). As for horizontal kinetic energy spectra, divergent component is broadly (k > 10) larger than rotational component compared with that on Earth (Kashimura et al., in preparation). We will show recent results of the high-resolution run, e.g., small-scale gravity waves attributed to large-scale thermal tides. Sugimoto, N. et al. (2014a), Baroclinic modes in the Venus atmosphere simulated by GCM, Journal of Geophysical Research: Planets, Vol. 119, p1950-1968.Sugimoto, N. et al. (2014b), Waves in a Venus general

  7. Antigenic analysis of H5N1 highly pathogenic avian influenza viruses circulating in Egypt (2006-2012).

    PubMed

    Ibrahim, Mahmoud; Eladl, Abdel-Fattah; Sultan, Hesham A; Arafa, Abdel Satar; Abdel Razik, Alaa G; Abd El Rahman, Sahar; El-Azm, Kamel I Abou; Saif, Yehia M; Lee, Chang-Won

    2013-12-27

    The highly pathogenic avian influenza (HPAI) H5N1 in Egypt circulated continuously after its introduction in February 2006 with substantial economic losses and frequent human infections. Phylogenetic analysis of the available HA sequences revealed the presence of two main sublineages; the classic 2.2.1 and the variant 2.2.1.1. The classic 2.2.1 had subdivided into two clusters of viruses; cluster C1 contained the originally introduced virus and isolates from 2006 to 2009 and cluster C2 emerged in 2007 and continues to circulate. The variant 2.2.1.1 represents the isolates mainly from chickens and subdivided into two clusters; cluster V1 contains isolates from 2007 to 2009 and cluster V2 contains isolates from 2008 to 2011. Sequence analysis revealed 28 amino acid mutations in the previously reported antigenic sites and high evolution rate which may be due to selective pressure from vaccination and/or natural infection. Antigenic analysis of 18 H5N1 isolates from 2006 to 2012 that represent different clusters was conducted using hemagglutination inhibition (HI) and virus neutralization (VN) assays using hyperimmune sera produced by immunizing SPF chickens with inactivated whole-virus. Antigenic relatedness of ancestral Egyptian H5N1 isolate (459-3/06) with other isolates ranged from 30.7% to 79.1% indicating significant antigenic drift of the H5N1 viruses from the ancestral strains. The antigenic relatedness between C2 and V2 clusters ranged from 28.9% to 68% supporting the need for vaccine seed strains from both clusters. Interestingly, A/CK/EG/1709-6/2008 H5N1 strain showed a broad cross reactivity against viruses in different H5N1 clusters (antigenic relatedness ranged from 63.9% to 85.8%) demonstrating a potential candidate as a vaccine strain. Antigenic cartography which facilitates a quantitative interpretation and easy visualization of serological data was constructed based on HI results and further demonstrated the several antigenic groups among Egyptian H5N

  8. [PATTERNS IN CIRCULATION AND TRANSMISSION OF MARINE BIRD PARASITES IN HIGH ARCTIC: A CASE OF ACANTHOCEPHALAN POLYMORPHUS PHIPPSI (PALAEACANTHOCEPHALA, POLYMORPHIDAE)].

    PubMed

    Galaktionov, K V; Atrashkevich, G I

    2015-01-01

    This study, based on the materials on parasitic infection of marine birds and invertebrates in Frantz Josef Land (FJL) collected in 1991-1993, focussed on the acanthocephalan Polymorphus phippsi. We identified this parasite, confirmed its species status and analysed its circulation and transmission patterns in high Arctic. The causes of its erroneous identification as P. minutus in several studies were also examined. In contrast to P. minutus, the transmission of P. phippsi is realized in marine coastal ecosystems. Its' main intermediate host in the Arctic is the amphipod Gammarus (Lagunogammarus) setosus, commonin coastal. areas of the shelf zone throughout the Arctic basin. P. phippsi population in FJL and the entire European Arctic is on the whole maintained by a single obligate final host, the common eider Somateria mollissima. Prevalence (P) of P. phippsi in this bird reached 100 %, with the maximal infection intensity (IImax) of 1188 and the mean abundance (MA) of 492.1. Other species of birds found to be infected with P. phippsi (Arctic turn, black guillemot, purple sandpiper and several gulls) are facultative and/or eliminative hosts. The most heavily infected birds were Arctic terns (P = 72.7%, IImax = 227, MA = = 47.1), which contained single mature acanthocephalans. For one of the FJL regions, infections flows of P. phippsi through various host categories were calculated. Involvement of birds unrelated to the common eider into the circulation of P. phippsi is facilitated by their feeding character in the Arctic. While coastal crustaceans are abundant, fish food is relatively scarce (polar cod, snailfishes), and so amphipods make up a considerable part of the diet of marine birds in FJL, if not most of it, as for instance in case of Arctic tern. This promotes an easy entry of the larvae of crustaceans-parasitizing helminthes (cestodes and acanthocephalans, including cystacanths P. phippsi) into non-specific hosts and opens broad colonization possibilities

  9. Oil spill model coupled to an ultra-high-resolution circulation model: implementation for the Adriatic Sea

    NASA Astrophysics Data System (ADS)

    Korotenko, K.

    2003-04-01

    An ultra-high-resolution version of DieCAST was adjusted for the Adriatic Sea and coupled with an oil spill model. Hydrodynamic module was developed on base of th low dissipative, four-order-accuracy version DieCAST with the resolution of ~2km. The oil spill model was developed on base of particle tracking technique The effect of evaporation is modeled with an original method developed on the base of the pseudo-component approach. A special dialog interface of this hybrid system allowing direct coupling to meteorlogical data collection systems or/and meteorological models. Experiments with hypothetic oil spill are analyzed for the Northern Adriatic Sea. Results (animations) of mesoscale circulation and oil slick modeling are presented at wabsite http://thayer.dartmouth.edu/~cushman/adriatic/movies/

  10. A high resolution Adriatic-Ionian Sea circulation model for operational forecasting

    NASA Astrophysics Data System (ADS)

    Ciliberti, Stefania Angela; Pinardi, Nadia; Coppini, Giovanni; Oddo, Paolo; Vukicevic, Tomislava; Lecci, Rita; Verri, Giorgia; Kumkar, Yogesh; Creti', Sergio

    2015-04-01

    System (MFS, http://medforecast.bo.ingv.it/) using daily means fields computed from daily outputs of the 1/16° general circulation model. One-way nesting is done by a novel pre-processing tool for an on-the-fly computation of boundary datasets compatible with BDY module provided by NEMO. It imposes the interpolation constraint and correction as in Pinardi et al. (2003) on the total velocity, ensuring that the total volume transport across boundaries is preserved after the interpolation procedures. In order to compute the lateral open boundary conditions, the model applies the Flow Relaxation Scheme (Engerdhal, 1995) for temperature, salinity and velocities and the Flather's radiation condition (Flather, 1976) for the depth-mean transport. Concerning the forecasting production cycle, AIFS produces 9-days forecast every day, producing hourly and daily means of temperature, salinity, surface currents, heat flux, water flux and shortwave radiation fields. AIREG model performances have been verified by using statistics (root mean square errors and BIAS) with respect to observed data (ARGO and CDT datasets)

  11. Surface circulation in the Iroise Sea (western Brittany) derived from high resolution current mapping by HF radars

    NASA Astrophysics Data System (ADS)

    Sentchev, Alexei; Forget, Philippe; Barbin, Yves; Marié, Louis; Ardhuin, Fabrice

    2010-05-01

    The use of high frequency radar (HFR) systems for near-real-time coastal ocean monitoring necessities that short time scale motions of the radar-derived velocities are better understood. While the ocean radar systems are able to describe coastal flow patterns with unprecedented details, the data they produce are often too sparse or gappy for applications such as the identification of coherent structures and fronts or understanding transport and mixing processes. In this study, we address two challenges. First, we report results from the HF radar system (WERA) which is routinely operating since 2006 on the western Brittany coast to monitor surface circulation in the Iroise Sea, over an area extending up to 100 km offshore. To obtain more reliable records of vector current fields at high space and time resolution, the Multiple Signal Classification (MUSIC) direction finding algorithm is employed in conjunction with the variational interpolation (2dVar) of radar-derived velocities. This provides surface current maps at 1 km spacing and time resolution of 20 min. Removing the influence of the sea state on radar-derived current measurements is discussed and performed on some data sequences. Second, we examine in deep continuous 2d velocity records for a number of periods, exploring the different modes of variability of surface currents in the region. Given the extent, duration, and resolution of surface current velocity measurements, new quantitative insights from various time series and spatial analysis on higher frequency kinematics will be discussed. By better characterizing the full spectrum of flow regimes that contribute to the surface currents and their shears, a more complete picture of the circulation in the Iroise Sea can be obtained.

  12. High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers for Measuring Puberty Onset in Chicken (Gallus gallus)

    PubMed Central

    Su, Yijun; Li, Guohui; Qu, Liang; Zhang, Huiyong; Wang, Kehua; Zou, Jianmin; Liu, Honglin

    2016-01-01

    There are still no highly sensitive and unique biomarkers for measurement of puberty onset. Circulating miRNAs have been shown to be promising biomarkers for diagnosis of various diseases. To identify circulating miRNAs that could be served as biomarkers for measuring chicken (Gallus gallus) puberty onset, the Solexa deep sequencing was performed to analyze the miRNA expression profiles in serum and plasma of hens from two different pubertal stages, before puberty onset (BO) and after puberty onset (AO). 197 conserved and 19 novel miRNAs (reads > 10) were identified as serum/plasma-expressed miRNAs in the chicken. The common miRNA amounts and their expression changes from BO to AO between serum and plasma were very similar, indicating the different treatments to generate serum and plasma had quite small influence on the miRNAs. 130 conserved serum-miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) from BO to AO, with 68 up-regulated and 62 down-regulated. 4829 putative genes were predicted as the targets of the 40 most differentially expressed miRNAs (|log2(fold-change)|>1.0, P < 0.01). Functional analysis revealed several pathways that were associated with puberty onset. Further quantitative real-time PCR (RT-qPCR) test found that a seven-miRNA panel, including miR-29c, miR-375, miR-215, miR-217, miR-19b, miR-133a and let-7a, had great potentials to serve as novel biomarkers for measuring puberty onset in chicken. Due to highly conserved nature of miRNAs, the findings could provide cues for measurement of puberty onset in other animals as well as humans. PMID:27149515

  13. Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

    PubMed Central

    Schaal, H; Klein, M; Gehrmann, P; Adams, O; Scheid, A

    1995-01-01

    An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion

  14. 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

    2007-01-09

    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.

  15. An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities.

    PubMed

    Wemhöner, Konstantin; Kanyshkova, Tatyana; Silbernagel, Nicole; Fernandez-Orth, Juncal; Bittner, Stefan; Kiper, Aytug K; Rinné, Susanne; Netter, Michael F; Meuth, Sven G; Budde, Thomas; Decher, Niels

    2015-01-01

    Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in I h activation curve, and an altered responsiveness of I h to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats.

  16. An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities

    PubMed Central

    Wemhöner, Konstantin; Kanyshkova, Tatyana; Silbernagel, Nicole; Fernandez-Orth, Juncal; Bittner, Stefan; Kiper, Aytug K.; Rinné, Susanne; Netter, Michael F.; Meuth, Sven G.; Budde, Thomas; Decher, Niels

    2015-01-01

    Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in Ih activation curve, and an altered responsiveness of Ih to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats. PMID:26578877

  17. Complete mapping of substrate translocation highlights the role of LeuT N-terminal segment in regulating transport cycle.

    PubMed

    Cheng, Mary Hongying; Bahar, Ivet

    2014-10-01

    Neurotransmitter: sodium symporters (NSSs) regulate neuronal signal transmission by clearing excess neurotransmitters from the synapse, assisted by the co-transport of sodium ions. Extensive structural data have been collected in recent years for several members of the NSS family, which opened the way to structure-based studies for a mechanistic understanding of substrate transport. Leucine transporter (LeuT), a bacterial orthologue, has been broadly adopted as a prototype in these studies. This goal has been elusive, however, due to the complex interplay of global and local events as well as missing structural data on LeuT N-terminal segment. We provide here for the first time a comprehensive description of the molecular events leading to substrate/Na+ release to the postsynaptic cell, including the structure and dynamics of the N-terminal segment using a combination of molecular simulations. Substrate and Na+-release follows an influx of water molecules into the substrate/Na+-binding pocket accompanied by concerted rearrangements of transmembrane helices. A redistribution of salt bridges and cation-π interactions at the N-terminal segment prompts substrate release. Significantly, substrate release is followed by the closure of the intracellular gate and a global reconfiguration back to outward-facing state to resume the transport cycle. Two minimally hydrated intermediates, not structurally resolved to date, are identified: one, substrate-bound, stabilized during the passage from outward- to inward-facing state (holo-occluded), and another, substrate-free, along the reverse transition (apo-occluded).