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Sample records for high circulating n-terminal

  1. High-dose aprotinin does not affect troponin I, N-Terminal pro-B-type natriuretic peptid and renal function in children submitted to surgical correction with extracorporeal circulation.

    PubMed

    Ferreira, Cesar Augusto; Vicente, Walter Villela de Andrade; Evora, Paulo Roberto Barbosa; Rodrigues, Alfredo José; Klamt, Jyrson Guilherme; Carlotti, Ana Paula de Carvalho Panzeri; Carmona, Fábio; Manso, Paulo Henrique

    2009-01-01

    To evaluate if the use of hemostatic high-dose aprotinin seems influence to myocardial, renal and metabolic functions in children submitted to surgical correction with extracorporeal circulation (ECC). Material and Methods A prospective randomized study was conducted on children aged 30 days to 4 years submitted to correction of acyanogenic congenital heart disease with ECC and divided into two groups: Control (n=9) and Aprotinin (n=10). In the Aprotinin Group the drug was administered before and during ECC and the myocardial and multiorgan dysfunctions were analyzed on the basis of clinical and biochemical markers. Differences were considered to be significant when P<0.05. The groups were similar regarding demographic and intraoperative variables, except for a greater hemodilution in the Aprotinin Group. The drug had no benefit regarding time of mechanical pulmonary ventilation, permanence in the pediatric postoperative intensive care unit (ICU) and length of hospitalization, or regarding the use of inotropic drugs and renal function. The partial arterial oxygen pressure/inspired oxygen fraction ratio (PaO2/FiO2) was significantly reduced 24h after surgery in the Control Group. Blood loss was similar for both groups. Cardiac troponin I (cTnI), creatine kinase MB fraction (CKMB), serum glutamic-oxaloacetic transaminase (SGOT) and the aminoterminal fraction of natriuretic peptide type B (NT-proBNP) did not differ significantly between groups. Post-ECC blood lactate concentration and metabolic acidosis was more intense in the Aprotinin Group. There were no complications with the use of aprotinin. High-dose aprotinin did not significant influence in serum markers troponin I, NT-proBNP and renal function, but did associated with hemodilution, blood lactate concentration and metabolic acidosis more intense.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. High-performance liquid chromatography of N-terminal tyrosine-containing oligopeptides by pre-column fluorescence derivatization with hydroxylamine, cobalt (II) and borate reagents.

    PubMed

    Nakano, M; Kai, M; Ohno, M; Ohkura, Y

    1987-12-18

    A pre-column fluorescence derivatization method is described for the high-performance liquid chromatographic determination of N-terminal tyrosine-containing oligopeptides involving methionine-enkephalin and leucine-enkephalin. The peptides are converted into fluorescent derivatives by heating in a weakly alkaline medium (pH 8.5) containing hydroxylamine, cobalt(II) ion and borate. The derivatives are separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in a mobile phase containing borate buffer (pH 8.5) and tetra-n-butylammonium chloride, and then determined by fluorimetry. The derivatization provides a single fluorescent product for each N-terminal tyrosine-containing oligopeptide, but does not allow the production of fluorescent derivatives for peptides having no tyrosyl residue at the N-terminal. The method is selective and sensitive; the lower limits of detection for the N-terminal tyrosine-containing oligopeptides tested were 140-310 fmol per 100 microliters injected.

  9. 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

  10. 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.

  11. N-terminal pro-brain natriuretic peptide and high-sensitivity troponin in the evaluation of acute chest pain of uncertain etiology. A PITAGORAS substudy.

    PubMed

    Sanchis, Juan; Bardají, Alfredo; Bosch, Xavier; Loma-Osorio, Pablo; Marín, Francisco; Sánchez, Pedro L; Calvo, Francisco; Avanzas, Pablo; Hernández, Carolina; Serrano, Silvia; Carratalá, Arturo; Barrabés, José A

    2013-07-01

    High-sensitivity troponin assays have improved the diagnosis of acute coronary syndrome in patients presenting with chest pain and normal troponin levels as measured by conventional assays. Our aim was to investigate whether N-terminal pro-brain natriuretic peptide provides additional information to troponin determination in these patients. A total of 398 patients, included in the PITAGORAS study, presenting to the emergency department with chest pain and normal troponin levels as measured by conventional assay in 2 serial samples (on arrival and 6 h to 8h later) were studied. The samples were also analyzed in a central laboratory for high-sensitivity troponin T (both samples) and for N-terminal pro-brain natriuretic peptide (second sample). The endpoints were diagnosis of acute coronary syndrome and the composite endpoint of in-hospital revascularization or a 30-day cardiac event. Acute coronary syndrome was adjudicated to 79 patients (20%) and the composite endpoint to 59 (15%). When the N-terminal pro-brain natriuretic peptide quartile increased, the diagnosis of acute coronary syndrome also increased (12%, 16%, 23% and 29%; P=.01), as did the risk of the composite endpoint (6%, 13%, 16% and 24%; P=.004). N-terminal pro-brain natriuretic peptide elevation (>125ng/L) was associated with both endpoints (relative risk= 2.0; 95% confidence interval, 1.2-3.3; P=.02; relative risk=2.4; 95% confidence interval, 1.4-4.2; P=.004). However, in the multivariable models adjusted by clinical and electrocardiographic data, a predictive value was found for high-sensitivity T troponin but not for N-terminal pro-brain natriuretic peptide. In low-risk patients with chest pain of uncertain etiology evaluated using high-sensitivity T troponin, N-terminal pro-brain natriuretic peptide does not contribute additional predictive value to diagnosis or the prediction of short-term outcomes. Copyright © 2012 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. "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.

  18. 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.

  19. 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.

  20. The invertase inhibitor Nt-CIF from tobacco: a highly thermostable four-helix bundle with an unusual N-terminal extension.

    PubMed

    Hothorn, Michael; D'Angelo, Igor; Márquez, José Antonio; Greiner, Steffen; Scheffzek, Klaus

    2004-01-23

    Plant invertases are sucrolytic enzymes essential for plant metabolism and development. Enzyme activity is regulated on a posttranslational level via inhibitory proteins, referred to as invertase inhibitors. Ectopic expression of invertase inhibitors in crop plants has high biotechnological potential. However, little biochemical and up to now no detailed structural information is available about this class of plant regulatory proteins. Here, we present the crystal structure of the cell wall-associated invertase inhibitor Nt-CIF from tobacco at a resolution of 1.87A. The structural model reveals an asymmetric four-helix bundle with an uncommon N-terminal extension that appears to be critical for the structural integrity of the protein. Structure analysis of a second crystal form grown in the presence of CdCl(2) reveals two metal binding sites. Nt-CIF is highly thermostable and retains full inhibitory activity after cooling to ambient temperatures. The structure of Nt-CIF provides the first three-dimensional information source for the posttranslational regulation of plant invertases. Based on the recently discovered sequence homology between inhibitors of invertases and pectin methylesterases, our structural model is likely to represent a scaffold also used for the regulation of the latter enzymes, which do not share sequence similarity with invertases. Thus, our structural model sets the 3D-stage for the investigation of posttranslational regulation of invertases as well as pectin methylesterases.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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 Central

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

    2012-01-01

    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. PMID:22837419

  8. 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.

  9. Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo*

    PubMed Central

    Foyn, Håvard; Van Damme, Petra; Støve, Svein I.; Glomnes, Nina; Evjenth, Rune; Gevaert, Kris; Arnesen, Thomas

    2013-01-01

    N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators. PMID:23043182

  10. 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

  11. Structural basis for substrate recognition by the human N-terminal methyltransferase 1

    SciTech Connect

    Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; Qin, Su; Li, Li; Loppnau, Peter; Huang, Rong; Min, Jinrong

    2015-11-05

    α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides, respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.

  12. Oxidative stress-mediated N-terminal protein modifications and MS-based approaches for N-terminal proteomics.

    PubMed

    Lee, Seon Hwa; Oe, Tomoyuki

    2016-02-01

    The N-termini of peptides and proteins can be subjected to highly diverse modifications, including acetylation, myristoylation, pyroglutamylation, and epimerization. These modifications affect protein stability, localization, and activity as well as alter the chemical properties of the N-terminus. Oxidative stress is known to induce the direct oxidation of amino acid side chains and peptide backbones in proteins. Alternatively, polyunsaturated fatty acids can be oxidized to lipid hydroperoxides, which further decompose to form highly reactive aldehydes such as 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE). ONE and HNE modify various amino acid residues and induce protein cross-linking. However, there have been few studies on oxidative stress-mediated N-terminal modifications and the resulting functional changes. Our recent studies have reported several novel N-terminal modifications that result in the formation of α-ketoamide, transamination, cyclization, and epimerization. These novel N-terminal modifications are the focus of this review. We also outline recent advances in approaches for N-terminal analysis, which have been developed over the last several decades. Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Structural basis for substrate recognition by the human N-terminal methyltransferase 1

    DOE PAGES

    Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

    2015-11-05

    α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

  18. Relation of adiponectin and high-sensitivity C-reactive protein to pulse-wave velocity and N-terminal pro-B-type natriuretic peptide in the general population.

    PubMed

    Sung, Shih-Hsien; Chuang, Shao-Yuan; Sheu, Wayne Huey-Herng; Lee, Wen-Jane; Chou, Pesus; Chen, Chen-Huan

    2009-05-15

    The roles of metabolic syndrome and chronic subclinical inflammation in arterial stiffening and the development of heart failure remain to be elucidated. Whether adiponectin and high-sensitivity C-reactive protein (hs-CRP) were independently related to brachial-ankle pulse-wave velocity (ba-PWV) and N-terminal pro-brain natriuretic peptide (NT-pro-BNP) in the general population were investigated. Eligible study subjects were 445 Chinese residents aged > or =40 years who participated in a community-based survey, underwent examination of ba-PWV, and had complete data of serum adiponectin, hs-CRP (<10 mg/L), and NT-pro-BNP. Adiponectin, but not hs-CRP, was independently related to ba-PWV (standardized regression parameter -0.107, p <0.05) when age, gender, body mass index, and number of metabolic syndrome components were accounted for. On the other hand, ba-PWV, adiponectin, and hs-CRP were independently related to NT-pro-BNP (standardized regression parameters 0.116, 0.188, and 0.094, respectively; all p <0.05) when age, gender, body mass index, number of metabolic syndrome components, and renal function were accounted for. In conclusion, adiponectin, but not hs-CRP, is independently associated with both ba-PWV and NT-pro-BNP in the general population. Because adiponectin, hs-CRP, ba-PWV, and NT-pro-BNP may represent markers for metabolic syndrome, chronic subclinical inflammation, arterial stiffness, and ventricular dysfunction, respectively, our results suggest that adiponectin may directly modulate both arterial stiffening and ventricular dysfunction. In contrast, hs-CRP may independently contribute to ventricular dysfunction, but not arterial stiffening.

  19. 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.

  20. Liver failure induces a systemic inflammatory response. Prevention by recombinant N-terminal bactericidal/permeability-increasing protein.

    PubMed Central

    Boermeester, M. A.; Houdijk, A. P.; Meyer, S.; Cuesta, M. A.; Appelmelk, B. J.; Wesdorp, R. I.; Hack, C. E.; Van Leeuwen, P. A.

    1995-01-01

    The observed increased susceptibility of patients with fulminant hepatic failure for local and systemic infections has been hypothesized to be due to a failure for the hepatic clearance function and subsequent leaking of endogenous endotoxins into the systemic circulation. However, experimental evidence for such a systemic inflammation during liver failure due to endogenous endotoxemia is lacking. Therefore, we designed a study to clarify whether circulating endotoxins due to liver failure could lead to the development of systemic inflammations. In a rat model for liver failure induced by a two-thirds partial hepatectomy, we evaluated the course of circulating tumor necrosis factor and interleukin-6, changes in blood chemistry and hemodynamics, and histopathological changes in the lungs. Partially hepatectomized animals, but not sham-operated animals, demonstrated cardiac failure, increased levels of creatinin and urea, metabolic acidosis, high plasma levels of tumor necrosis factor and interleukin-6, and an influx of PMNs in the lungs-together indicating the development of a systemic inflammatory response. Continuous infusion of recombinant N-terminal bactericidal/permeability-increasing protein (rBPI23), a well described endotoxin-neutralizing protein, prevented these inflammatory reactions. Ex vivo experiments with rat plasma samples confirmed the presence of circulating endotoxins in partially hepatectomized rats as opposed to those treated with rBPI23. Thus, our results indicate that the early phase of liver failure induces a systemic inflammatory response triggered by circulating endotoxins, which can be prevented by perioperative infusion of rBPI23. Images Figure 2 PMID:7485405

  1. 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.

  2. 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.

  3. N-terminal propeptide of type III procollagen as a biomarker of anabolic response to recombinant human GH and testosterone

    USDA-ARS?s Scientific Manuscript database

    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...

  4. Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

    PubMed

    Scott, Daniel C; Hammill, Jared T; Min, Jaeki; Rhee, David Y; Connelly, Michele; Sviderskiy, Vladislav O; Bhasin, Deepak; Chen, Yizhe; Ong, Su-Sien; Chai, Sergio C; Goktug, Asli N; Huang, Guochang; Monda, Julie K; Low, Jonathan; Kim, Ho Shin; Paulo, Joao A; Cannon, Joe R; Shelat, Anang A; Chen, Taosheng; Kelsall, Ian R; Alpi, Arno F; Pagala, Vishwajeeth; Wang, Xusheng; Peng, Junmin; Singh, Bhuvanesh; Harper, J Wade; Schulman, Brenda A; Guy, R Kip

    2017-08-01

    N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Self-biased circulators for high power applications

    NASA Astrophysics Data System (ADS)

    Sokolov, Alexander S.

    Self-biased circulators exploit the properties of high anisotropy magnetic field in hexagonal ferrites, thus allowing operation without biasing magnets and a significant size and weight reduction. Although first self-biased circulators were demonstrated more than 20 years ago, all the prototypes constructed so far are unsuitable for practical applications. An attempt to design a self-biased circulator from scratch was made. Novel exceptionally low dielectric loss and high heat conductivity ceramic materials were developed and innovative substrate synthesis techniques were employed. Low temperature cofiring of green body ferrite compacts and dielectric ceramic slurries were mastered, resulting in solid composite substrates. Original device design was developed. Key features (including wide coupling angles, wide microstriplines, thick substrate, and absence of impedance transformers) enable low insertion loss, broadband operation, high power handling, and compact size. Fabrication and testing of Ka band Y-junction self-biased circulator are reported herein. Furthermore, design approach and fabrication techniques developed here can be readily applied for the construction of X-band self-biased circulators, provided that suitable ferrite materials are available. Low temperature cofiring of ferrite and dielectric materials is especially beneficial for various RF and high-frequency applications. Multiple devices can be readily fabricated on a single wafer using conventional lithographic techniques, resulting in true microwave monolithic integrated circuit.

  10. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco

    PubMed Central

    Sayanova, Olga; Smith, Mark A.; Lapinskas, Peter; Stobart, A. Keith; Dobson, Gary; Christie, William W.; Shewry, Peter R.; Napier, Johnathan A.

    1997-01-01

    γ-Linolenic acid (GLA; C18:3 Δ6,9,12) 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 Δ6-fatty acid desaturase using linoleic acid (C18:2 Δ9,12) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the Δ6-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 Δ6,9,12,15) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage Δ6-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. PMID:9108131

  11. 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.

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

    PubMed

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

    2016-05-01

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

  13. Vasoinhibins: a family of N-terminal prolactin fragments that inhibit angiogenesis and vascular function.

    PubMed

    Clapp, Carmen; González, Carmen; Macotela, Yazmín; Aranda, Jorge; Rivera, José C; García, Celina; Guzmán, Jessica; Zamorano, Miriam; Vega, Claudia; Martín, Cecilia; Jeziorski, Michael C; de la Escalera, Gonzalo Martínez

    2006-01-01

    Antiangiogenic molecules derived from prolactin (PRL) are not a single entity, but rather a family of peptides with different molecular masses, all containing the N-terminal region of PRL. Cleavage of PRL by cathepsin-D or by matrix metalloproteases generates N-terminal fragments that act on endothelial cells to suppress vasodilation and angiogenesis and promote vascular regression. N-terminal PRL fragments have been identified in cartilage and retina, where angiogenesis is highly restricted. In vivo experiments demonstrate that these PRL fragments exert a tonic and essential suppression of retinal blood vessel growth and dilation. Similar PRL fragments have been detected in the pituitary gland, a highly vascularized organ where the control of vascular growth may differ from that in tissues where angiogenesis is highly restricted. We have previously proposed the name vasoinhibins to describe the collection of N-terminal PRL fragments having blood vessel-blocking activity, and here we discuss their promise as factors to control vascular function in health and disease.

  14. 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.

  15. 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.

  16. Functional characterization of an N-terminally-truncated mitochondrial porin expressed in Neurospora crassa.

    PubMed

    Shuvo, Sabbir R; Kovaltchouk, Uliana; Zubaer, Abdullah; Kumar, Ayush; Summers, William A T; Donald, Lynda J; Hausner, Georg; Court, Deborah A

    2017-08-01

    Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-β-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

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

    PubMed

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

    2016-02-10

    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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  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. 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.

  12. 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

  13. 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.

  14. HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor

    PubMed Central

    Chain, Benjamin M.; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward

    2008-01-01

    The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes. PMID:18765264

  15. 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

  16. 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

  17. High efficiency vortex trapping of circulating tumor cells

    PubMed Central

    Wong, Jessica; Karimi, Armin; Che, James; Renier, Corinne; Matsumoto, Melissa; Triboulet, Melanie; Garon, Edward B.; Goldman, Jonathan W.; Rettig, Matthew B.; Jeffrey, Stefanie S.; Kulkarni, Rajan P.; Sollier, Elodie; Di Carlo, Dino

    2015-01-01

    Circulating tumor cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. Several technologies have been demonstrated to isolate CTCs with high efficiency but achieve a low purity from a large background of blood cells. We have previously shown the ability to enrich CTCs with high purity from large volumes of blood through selective capture in microvortices using the Vortex Chip. The device consists of a narrow channel followed by a series of expansion regions called reservoirs. Fast flow in the narrow entry channel gives rise to inertial forces, which direct larger cells into trapping vortices in the reservoirs where they remain circulating in orbits. By studying the entry and stability of particles following entry into reservoirs, we discover that channel cross sectional area plays an important role in controlling the size of trapped particles, not just the orbital trajectories. Using these design modifications, we demonstrate a new device that is able to capture a wider size range of CTCs from clinical samples, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream interventions, including genetic analysis, cell culture, and ultimately personalized cancer therapy. PMID:26697126

  18. 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.

  19. 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.

  20. The effects of super-flux (high performance) dialyzer on plasma glycosylated pro-B-type natriuretic peptide (proBNP) and glycosylated N-Terminal proBNP in end-stage renal disease patients on dialysis.

    PubMed

    Nakagawa, Yasuaki; Nishikimi, Toshio; Kuwahara, Koichiro; Yasuno, Shinji; Kinoshita, Hideyuki; Kuwabara, Yoshihiro; Nakao, Kazuhiro; Minami, Takeya; Yamada, Chinatsu; Ueshima, Kenji; Ikeda, Yoshihiro; Okamoto, Hiroyuki; Horii, Kazukiyo; Nagata, Kiyoshi; Kangawa, Kenji; Minamino, Naoto; Nakao, Kazuwa

    2014-01-01

    Plasma BNP levels are predictive of prognosis in hemodialysis patients. However, recent studies showed that the current BNP immunoassay cross-reacts with glycosylated proBNP, and the NT-proBNP assay underestimates glycosylated NT-proBNP. In addition, the recently developed high performance dialyzer removes medium-sized molecular solutes such as β2-microgloburin. We therefore investigated the effects of high performance dialysis on measured levels of glycosylated proBNP, glycosylated NT-proBNP and other BNP-related peptides in end-stage renal disease (ESRD) patients on hemodialysis. The relationships between clinical parameters and BNP-related molecule were also investigated. We used our newly developed immunoassay to measure plasma total BNP and proBNP in 105 normal subjects and 36 ESRD patients before and after hemodialysis. Plasma NT-proBNP was measured using Elecsys II after treatment with or without deglycosylating enzymes. We also measured plasma ANP and cGMP using radioimmunoassays. All the measured BNP-related peptides were significantly higher in ESRD patients than healthy subjects. Total BNP (-38.9%), proBNP (-29.7%), glycoNT-proBNP (-45.5%), nonglycoNT-proBNP (-53.4%), ANP (-50.4%) and cGMP (-72.1%) were all significantly reduced after hemodialysis, and the magnitude of the reduction appeared molecular weight- dependent. Both the proBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios were increased after hemodialysis. The former correlated positively with hemodialysis vintage and negatively with systolic blood pressure, while the latter correlated positively with parathyroid hormone levels. These results suggest that hemodialysis using super-flux dialyzer removes BNP-related peptides in a nearly molecular weight-dependent manner. The ProBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios appear to be influenced by hemodialysis-related parameters in ESRD patients on hemodialysis.

  1. 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.

  2. 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.

  3. Transglutaminase 5 is acetylated at the N-terminal end.

    PubMed

    Rufini, A; Vilbois, F; Paradisi, A; Oddi, S; Tartaglione, R; Leta, A; Bagetta, G; Guerrieri, P; Finazzi-Agro', A; Melino, G; Candi, E

    2004-07-01

    Transglutaminases (TGases) are calcium-dependent enzymes that catalyse cross-linking between proteins by acyl transfer reaction; they are involved in many biological processes including coagulation, differentiation, and tissue repair. Transglutaminase 5 was originally cloned from keratinocytes, and a partial biochemical characterisation showed its involvement in skin differentiation, in parallel to TGase 1 and TGase 3. Here, we demonstrate, by electrospray tandem mass spectrometry that TGase 5 is acetylated at the N-terminal end. Moreover, in situ measurement of TGase activity shows that endogenous TGase 5 is active upon treatment with phorbol acetate, and the enzyme co-localises with vimentin intermediate filaments.

  4. High-temperature self-circulating thermoacoustic heat exchanger

    NASA Astrophysics Data System (ADS)

    Backhaus, S.; Swift, G. W.; Reid, R. S.

    2005-07-01

    Thermoacoustic and Stirling engines and refrigerators use heat exchangers to transfer heat between the oscillating flow of their thermodynamic working fluids and external heat sources and sinks. An acoustically driven heat-exchange loop uses an engine's own pressure oscillations to steadily circulate its own thermodynamic working fluid through a physically remote high-temperature heat source without using moving parts, allowing for a significant reduction in the cost and complexity of thermoacoustic and Stirling heat exchangers. The simplicity and flexibility of such heat-exchanger loops will allow thermoacoustic and Stirling machines to access diverse heat sources and sinks. Measurements of the temperatures at the interface between such a heat-exchange loop and the hot end of a thermoacoustic-Stirling engine are presented. When the steady flow is too small to flush out the mixing chamber in one acoustic cycle, the heat transfer to the regenerator is excellent, with important implications for practical use.

  5. The N-terminal extension of the P. falciparum GBP130 signal peptide is irrelevant for signal sequence function.

    PubMed

    Meyer, Corinna; Barniol, Luis; Hiss, Jan A; Przyborski, Jude M

    2017-07-17

    The malaria parasite P. falciparum exports a large number of proteins to its host cell, the mature human erythrocyte. Although the function of the majority of these proteins is not well understood, many exported proteins appear to play a role in modification of the erythrocyte following invasion. Protein export to the erythrocyte is a secretory process that begins with entry to the endoplasmic reticulum. For most exported proteins, this step is mediated by hydrophobic signal peptides found towards the N-terminal end of proteins. The signal peptides present on P. falciparum exported proteins often differ in length from those found in other systems, and generally contain a highly extended N-terminal region. Here we have investigated the function of these extended N-terminal regions, using the exported parasite protein GBP130 as a model. Surprisingly, several deletions of the extended N-terminal regions of the GBP130 signal peptide have no effect on the ability of the signal peptide to direct a fluorescent reporter to the secretory pathway. Addition of the same N-terminal extension to a canonical signal peptide does not affect transport of either soluble or membrane proteins to their correct respective subcellular localisations. Finally, we show that extended signal peptides are able to complement canonical signal peptides in driving protein traffic to the apicoplast of the parasite, and are also functional in a mammalian cell system. Our study is the first detailed analysis of an extended P. falciparum signal peptide and suggests that N-terminal extensions of exported Plasmodium falciparum proteins are not required for entry to the secretory system, and are likely to be involved in other, so far unknown, processes. Copyright © 2017. Published by Elsevier GmbH.

  6. 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.

  7. 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.

  8. N-terminal pro-brain natriuretic peptide in acute Kawasaki disease correlates with coronary artery involvement.

    PubMed

    Adjagba, Philippe M; Desjardins, Laurent; Fournier, Anne; Spigelblatt, Linda; Montigny, Martine; Dahdah, Nagib

    2015-10-01

    We have lately documented the importance of N-terminal pro-brain natriuretic peptide in aiding the diagnosis of Kawasaki disease. We sought to investigate the potential value of N-terminal pro-brain natriuretic peptide pertaining to the prediction of coronary artery dilatation (Z-score>2.5) and/or of resistance to intravenous immunoglobulin therapy. We hypothesised that increased serum N-terminal pro-brain natriuretic peptide level correlates with increased coronary artery dilatation and/or resistance to intravenous immunoglobulin. We carried out a prospective study involving newly diagnosed patients treated with 2 g/kg intravenous immunoglobulin within 5-10 days of onset of fever. Echocardiography was performed in all patients at onset, then weekly for 3 weeks, then at month 2, and month 3. Coronary arteries were measured at each visit, and coronary artery Z-score was calculated. All the patients had N-terminal pro-brain natriuretic peptide serum level measured at onset, and the Z-score calculated. There were 109 patients enrolled at 6.58±2.82 days of fever, age 3.79±2.92 years. High N-terminal pro-brain natriuretic peptide level was associated with coronary artery dilatation at onset in 22.2 versus 5.6% for normal N-terminal pro-brain natriuretic peptide levels (odds ratio 4.8 [95% confidence interval 1.05-22.4]; p=0.031). This was predictive of cumulative coronary artery dilatation for the first 3 months (p=0.04-0.02), but not during convalescence at 2-3 months (odds ratio 1.28 [95% confidence interval 0.23-7.3]; p=non-significant). Elevated N-terminal pro-brain natriuretic peptide levels did not predict intravenous immunoglobulin resistance, 15.3 versus 13.5% (p=1). Elevated N-terminal pro-brain natriuretic peptide level correlates with acute coronary artery dilatation in treated Kawasaki disease, but not with intravenous immunoglobulin resistance.

  9. High-stability algorithm for the three-pattern decomposition of global atmospheric circulation

    NASA Astrophysics Data System (ADS)

    Cheng, Jianbo; Gao, Chenbin; Hu, Shujuan; Feng, Guolin

    2017-07-01

    In order to study the atmospheric circulation from a global-wide perspective, the three-pattern decomposition of global atmospheric circulation (TPDGAC) has been proposed in our previous studies. In this work, to easily and accurately apply the TPDGAC in the diagnostic analysis of atmospheric circulation, a high-stability algorithm of the TPDGAC has been presented. By using the TPDGAC, the global atmospheric circulation is decomposed into the three-dimensional (3D) horizontal, meridional, and zonal circulations (three-pattern circulations). In particular, the global zonal mean meridional circulation is essentially the three-cell meridional circulation. To demonstrate the rationality and correctness of the proposed numerical algorithm, the climatology of the three-pattern circulations and the evolution characteristics of the strength and meridional width of the Hadley circulation during 1979-2015 have been investigated using five reanalysis datasets. Our findings reveal that the three-pattern circulations capture the main features of the Rossby, Hadley, and Walker circulations. The Hadley circulation shows a significant intensification during boreal winter in the Northern Hemisphere and shifts significantly poleward during boreal (austral) summer and autumn in the Northern (Southern) Hemisphere.

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

    PubMed Central

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

    2009-01-01

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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. Molecular cloning and biologically active production of IpaD N-terminal region.

    PubMed

    Hesaraki, Mahdi; Saadati, Mojtaba; Honari, Hossein; Olad, Gholamreza; Heiat, Mohammad; Malaei, Fatemeh; Ranjbar, Reza

    2013-07-01

    Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD(72-162) could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni-NTA column. Western blot analysis using, His-tag and IpaD(72-162) polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD(72-162) provides a protective system against Shigella flexneri 5a and S. dysenteriae type I. Copyright © 2013. Published by Elsevier Ltd.

  19. 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.

  20. Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation.

    PubMed

    Soldano, Karen L; Jivan, Arif; Nicchitta, Christopher V; Gewirth, Daniel T

    2003-11-28

    GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 A of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.

  1. Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major beta-lactamases.

    PubMed Central

    Franceschini, N; Amicosante, G; Perilli, M; Maccarrone, M; Oratore, A; van Beeumen, J; Frère, J M

    1991-01-01

    The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A. Images Fig. 2. PMID:2039443

  2. 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.

  3. 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.

  4. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    SciTech Connect

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

    2013-01-01

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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Crystals of the hydrogenase maturation factor HypF N-terminal domain grown in microgravity, display improved internal order

    NASA Astrophysics Data System (ADS)

    Ponassi, Marco; Felli, Lamberto; Parodi, Stefania; Valbusa, Ugo; Rosano, Camillo

    2011-01-01

    Synthesis of the active [Ni-Fe]-hydrogenase in prokaryotes requires a series of ancillary maturation factors. Among them, the HypF maturation factor is a multidomain 82 kDa protein, whose N-terminal domain displays sequence and structural similarities to acylphosphatases. Acylphosphatases are small enzymes that are able to catalyze carboxyl-phosphate bond hydrolysis in acylphosphates, as well as in nucleoside di- and tri-phosphates and in arylphosphates. Here, we present a crystallographic comparison between microgravity and earth-grown crystals of the HypF N-terminal domain. Both crystals were of excellent quality, thereby allowing us to collect very high resolution datasets. A detailed analysis of data collection and refinement statistics, together with an analysis of the diffraction pattern showed that microgravity would appear to further improve the internal order of crystals.

  16. 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

  17. 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.

  18. 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

  19. Heparin promotes fibril formation by the N-terminal fragment of amyloidogenic apolipoprotein A-I.

    PubMed

    Mikawa, Shiho; Mizuguchi, Chiharu; Nishitsuji, Kazuchika; Baba, Teruhiko; Shigenaga, Akira; Shimanouchi, Toshinori; Sakashita, Naomi; Otaka, Akira; Akaji, Kenichi; Saito, Hiroyuki

    2016-10-01

    Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β-transition and fibril formation of the highly amyloidogenic region spanning residues 44-65 and colocalizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA-I variants. © 2016 Federation of European Biochemical Societies.

  20. 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.

  1. 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.

  2. An N-terminal Domain of Adenovirus Protein VI Fragments Membranes By Inducing Positive Membrane Curvature

    PubMed Central

    Maier, Oana; Galan, Debra L.; Wodrich, Harald; Wiethoff, Christopher M.

    2010-01-01

    Adenovirus (Ad) membrane penetration during cell entry is poorly understood. Here we show that antibodies which neutralize the membrane lytic activity of the Ad capsid protein VI interfere with Ad endosomal membrane penetration. In vitro studies using a peptide corresponding to an N-terminal amphipathic α-helix of protein VI (VI-Φ), as well as other truncated forms of protein VI suggest that VI-Φ is largely responsible for protein VI binding to and lysing of membranes. Additional studies suggest that VI-Φ lies nearly parallel to the membrane surface. Protein VI fragments membranes and induces highly curved structures. Further studies suggest that Protein VI induces positive membrane curvature. These data support a model in which protein VI binds membranes, inducing positive curvature strain which ultimately leads to membrane fragmentation. These results agree with previous observations of Ad membrane permeabilization during cell entry and provide an initial mechanistic description of a nonenveloped virus membrane lytic protein. PMID:20409568

  3. 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.

  4. 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

  5. Effect of sodium chloride on the structure and stability of spider silk's N-terminal protein domain.

    PubMed

    Gronau, Greta; Qin, Zhao; Buehler, Markus J

    2013-03-01

    A spider's ability to store silk protein solutions at high concentration is believed to be related to the protein's terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage.

  6. 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.

  7. Plasmatic levels of N-terminal pro-atrial natriuretic peptide in preeclamptic patients and healthy normotensive pregnant women.

    PubMed

    Reyna-Villasmil, Eduardo; Mejia-Montilla, Jorly; Reyna-Villasmil, Nadia; Mayner-Tresol, Gabriel; Herrera-Moya, Pedro; Fernández-Ramírez, Andreina; Rondón-Tapía, Marta

    2017-08-31

    To compare plasma N-terminal pro-atrial natriuretic peptide concentrations in preeclamptic patients and healthy normotensive pregnant women. A cases-controls study was done with 180 patients at Hospital Central Dr. Urquinaona, Maracaibo, Venezuela, that included 90 preeclamptic patients (group A; cases) and 90 healthy normotensive pregnant women selected with the same age and body mass index similar to group A (group B; controls). Blood samples were collected one hour after admission and prior to administration of any medication in group A to determine plasma N-terminal pro-atrial natriuretic peptide and other laboratory parameters. Plasma N-terminal pro-atrial natriuretic peptide concentrations in group A (mean 1.01 [0.26] pg/mL) showed a significant difference when compared with patients in group B (mean 0.55 [0.07] pg/mL; P<.001]. There was no significant correlation with systolic and diastolic blood pressure values in preeclamptic patients (P=ns). A cut-off value of 0.66ng/mL had an area under the curve of 0.93, sensitivity of 87.8%, specificity of 83.3%, a positive predictive value of 84.0% and a negative predictive value of 87.2%, with a diagnostic accuracy of 85.6%. Preeclamptic patients have significantly higher concentrations of plasma N-terminal pro-atrial natriuretic peptide compared with healthy normotensive pregnant women, with high predictive values for diagnosis. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  8. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. 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

  10. 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.

  11. The histone H3 N-terminal tail: a computational analysis of the free energy landscape and kinetics.

    PubMed

    Zheng, Yuqing; Cui, Qiang

    2015-05-28

    Histone tails are the short peptide protrusions outside of the nucleosome core particle and they play a critical role in regulating chromatin dynamics and gene activity. A histone H3 N-terminal tail, like other histone tails, can be covalently modified on different residues to activate or repress gene expression. Previous studies have indicated that, despite its intrinsically disordered nature, the histone H3 N-terminal tail has regions of notable secondary structural propensities. To further understand the structure-dynamics-function relationship in this system, we have carried out 75.6 μs long implicit solvent simulations and 29.3 μs long explicit solvent simulations. The extensive samplings allow us to better characterize not only the underlying free energy landscape but also kinetic properties through Markov state models (MSM). Dihedral principal component analysis (dPCA) and locally scaled diffusion map (LSDMap) analysis yield consistent results that indicate an overall flat free energy surface with several shallow basins that correspond to conformations with a high α-helical propensity in two regions of the peptide. Kinetic information extracted from Markov state models reveals rapid transitions between different metastable states with mean first passage times spanning from several hundreds of nanoseconds to hundreds of microseconds. These findings shed light on how the dynamical nature of the histone H3 N-terminal tail is related to its function. The complementary nature of dPCA, LSDMap and MSM for the analysis of biomolecules is also discussed.

  12. 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

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

    PubMed Central

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

    2016-01-01

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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. Molecular analysis of laminin N-terminal domains mediating self-interactions.

    PubMed

    Odenthal, Uwe; Haehn, Sebastian; Tunggal, Patrick; Merkl, Barbara; Schomburg, Dietmar; Frie, Christian; Paulsson, Mats; Smyth, Neil

    2004-10-22

    The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from alpha, beta, and gamma chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five alpha, three beta, and three gamma chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all alpha chains and from the beta2 and beta3 subunits. The gamma chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the gamma3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Functional role of the flexible N-terminal extension of FKBP38 in catalysis.

    PubMed

    Kang, Congbao; Ye, Hong; Chia, Joel; Choi, Bo-Hwa; Dhe-Paganon, Sirano; Simon, Bernd; Schütz, Ulrike; Sattler, Michael; Yoon, Ho Sup

    2013-10-22

    FKBP38 regulates apoptosis through unique interactions with multiple regulators including Bcl-2. Interestingly, the peptidylprolyl isomerase activity of FKBP38 is only detectable when it binds to calcium-saturated calmodulin (CaM/Ca(2+)). This, in turn, permits the formation of a complex with Bcl-2. FKBP38 thereby provides an important link between isomerase activity and apoptotic pathways. Here, we show that the N-terminal extension (residues 1-32) preceding the catalytic domain of FKBP38 has an autoinhibitory activity. The core isomerase activity of FKBP38 is inhibited by transient interactions involving the flexible N-terminal extension that precedes the catalytic domain. Notably, CaM/Ca(2+) binds to this N-terminal extension and thereby releases the autoinhibitory contacts between the N-terminal extension and the catalytic domain, thus potentiating the isomerase activity of FKBP38. Our data demonstrate how CaM/Ca(2+) modulates the catalytic activity of FKBP38.

  4. Functional role of the flexible N-terminal extension of FKBP38 in catalysis

    NASA Astrophysics Data System (ADS)

    Kang, Congbao; Ye, Hong; Chia, Joel; Choi, Bo-Hwa; Dhe-Paganon, Sirano; Simon, Bernd; Schütz, Ulrike; Sattler, Michael; Yoon, Ho Sup

    2013-10-01

    FKBP38 regulates apoptosis through unique interactions with multiple regulators including Bcl-2. Interestingly, the peptidylprolyl isomerase activity of FKBP38 is only detectable when it binds to calcium-saturated calmodulin (CaM/Ca2+). This, in turn, permits the formation of a complex with Bcl-2. FKBP38 thereby provides an important link between isomerase activity and apoptotic pathways. Here, we show that the N-terminal extension (residues 1-32) preceding the catalytic domain of FKBP38 has an autoinhibitory activity. The core isomerase activity of FKBP38 is inhibited by transient interactions involving the flexible N-terminal extension that precedes the catalytic domain. Notably, CaM/Ca2+ binds to this N-terminal extension and thereby releases the autoinhibitory contacts between the N-terminal extension and the catalytic domain, thus potentiating the isomerase activity of FKBP38. Our data demonstrate how CaM/Ca2+ modulates the catalytic activity of FKBP38.

  5. Characterization of the circulating serum amyloid A in bottlenose dolphins.

    PubMed

    Segawa, Takao; Otsuka, Toru; Itou, Takuya; Suzuki, Miwa; Karatani, Nana; Sakai, Takeo

    2013-04-15

    Several isoforms of serum amyloid A (SAA) have been identified so far and because the plasma concentration of it increases dramatically, it is used as an indicator of inflammation in animals. In many terrestrial mammals, the circulating isoforms are SAA1 and SAA2, which are synthesized in the liver. Extra-hepatically synthesized SAA3, however, is a predominantly local SAA isoform with a characteristic N-terminal TFLK motif and a highly alkaline isoelectric point (pI). The aim of this study was to characterize the circulating SAA isoforms in bottlenose dolphins (dSAA) by determining the deduced amino acid sequence isolated from liver and the pI of plasma from healthy dolphins and those with inflammation. The deduced amino acid sequences of dSAA showed characteristics of SAA3 with an N-terminal TFLK motif, a predicted alkaline pI and were phylogenetically clustered with the SAA3 group rather than the SAA1 and SAA2 groups. Various tissues contained dSAA mRNA with the highest levels being detected in the liver. Isoelectric focusing and western blot analysis showed that one highly alkaline SAA was markedly detected in plasma obtained from dolphins affected by inflammation. These results suggest that, unlike other mammals, the circulating SAA in dolphins exhibits SAA3 properties, as is the case in pigs. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Identification of the sequence determinants of protein N-terminal acetylation through a decision tree approach.

    PubMed

    Yamada, Kazunori D; Omori, Satoshi; Nishi, Hafumi; Miyagi, Masaru

    2017-06-02

    N-terminal acetylation is one of the most common protein modifications in eukaryotes and occurs co-translationally when the N-terminus of the nascent polypeptide is still attached to the ribosome. This modification has been shown to be involved in a wide range of biological phenomena such as protein half-life regulation, protein-protein and protein-membrane interactions, and protein subcellular localization. Thus, accurately predicting which proteins receive an acetyl group based on their protein sequence is expected to facilitate the functional study of this modification. As the occurrence of N-terminal acetylation strongly depends on the context of protein sequences, attempts to understand the sequence determinants of N-terminal acetylation were conducted initially by simply examining the N-terminal sequences of many acetylated and unacetylated proteins and more recently by machine learning approaches. However, a complete understanding of the sequence determinants of this modification remains to be elucidated. We obtained curated N-terminally acetylated and unacetylated sequences from the UniProt database and employed a decision tree algorithm to identify the sequence determinants of N-terminal acetylation for proteins whose initiator methionine ((i)Met) residues have been removed. The results suggested that the main determinants of N-terminal acetylation are contained within the first five residues following (i)Met and that the first and second positions are the most important discriminator for the occurrence of this phenomenon. The results also indicated the existence of position-specific preferred and inhibitory residues that determine the occurrence of N-terminal acetylation. The developed predictor software, termed NT-AcPredictor, accurately predicted the N-terminal acetylation, with an overall performance comparable or superior to those of preceding predictors incorporating machine learning algorithms. Our machine learning approach based on a decision tree

  7. The N-Terminal Methionine of Cellular Proteins as a Degradation Signal

    PubMed Central

    Kim, Heon-Ki; Kim, Ryu-Ryun; Oh, Jang-Hyun; Cho, Hanna; Varshavsky, Alexander; Hwang, Cheol-Sang

    2014-01-01

    SUMMARY The Arg/N-end rule pathway targets for degradation proteins that bear specific unacetylated N-terminal residues while the Ac/N-end rule pathway targets proteins through their Nα-terminally acetylated (Nt-acetylated) residues. Here we show that Ubr1, the ubiquitin ligase of the Arg/N-end rule pathway, recognizes unacetylated N-terminal methionine if it is followed by a hydrophobic residue. This capability of Ubr1 expands the range of substrates that can be targeted for degradation by the Arg/N-end rule pathway, because virtually all nascent cellular proteins bear N-terminal methionine. We identified Msn4, Sry1, Arl3, and Pre5 as examples of normal or misfolded proteins that can be destroyed through the recognition of their unacetylated N-terminal methionine. Inasmuch as proteins bearing the Nt-acetylated N-terminal methionine residue are substrates of the Ac/N-end rule pathway, the resulting complementarity of the Arg/N-end rule and Ac/N-end rule pathways enables the elimination of protein substrates regardless of acetylation state of N-terminal methionine in these substrates. PMID:24361105

  8. Antagonistic roles of the N-terminal domain of prion protein to doppel.

    PubMed

    Sakaguchi, Suehiro

    2008-01-01

    Prion protein (PrP)-like molecule, doppel (Dpl), is neurotoxic in mice, causing Purkinje cell degeneration. In contrast, PrP antagonizes Dpl in trans, rescuing mice from Purkinje cell death. We have previously shown that PrP with deletion of the N-terminal residues 23-88 failed to neutralize Dpl in mice, indicating that the N-terminal region, particularly that including residues 23-88, may have trans-protective activity against Dpl. Interestingly, PrP with deletion elongated to residues 121 or 134 in the N-terminal region was shown to be similarly neurotoxic to Dpl, indicating that the PrP C-terminal region may have toxicity which is normally prevented by the N-terminal domain in cis. We recently investigated further roles for the N-terminal region of PrP in antagonistic interactions with Dpl by producing three different types of transgenic mice. These mice expressed PrP with deletion of residues 25-50 or 51-90, or a fusion protein of the N-terminal region of PrP with Dpl. Here, we discuss a possible model for the antagonistic interaction between PrP and Dpl.

  9. N-terminal fragment of probrain natriuretic peptide is associated with diabetes microvascular complications in type 2 diabetes.

    PubMed

    Hamano, Kumiko; Nakadaira, Ikue; Suzuki, Jun; Gonai, Megumi

    2014-01-01

    Circulating levels of N-terminal fragment of probrain natriuretic peptide (NT-proBNP) are established as a risk factor for cardiovascular disease and mortality in patients with diabetes, as well as in the general population. We sought to examine the possibility of NT-proBNP as a biomarker of microvascular complications in patients with type 2 diabetes. In total, 277 outpatients with type 2 diabetes were consecutively enrolled as a hospital cohort. Two hundred and seventeen of these patients (132 males; mean age, 63.4 years) were designated as cases with any of the diabetic complications (retinopathy, neuropathy, nephropathy, ischemic heart disease, strokes, peripheral artery disease), and 60 (42 males; mean age, 54.1 years) were set as controls without clinical evidence of diabetic complications. Diabetic complications were evaluated by medical record and routine laboratory examinations. NT-proBNP was measured and investigated with regard to the associations with diabetic complications. Mean NT-proBNP levels were significantly higher in patients with any of the diabetic complications (59 versus 33 pg/mL; P<0.0001). In logistic regression analysis, NT-proBNP levels >79 pg/mL, which was the highest tertile, were independently associated with a 5.04 fold increased risk of all complications (P<0.0051) compared to the lowest tertile (NT-proBNP levels <31 pg/mL). Odd ratios of cardiovascular disease and nephropathy, neuropathy, and retinopathy were 9.33, 6.23, 6.6 and 13.78 respectively, in patients with NT-proBNP values in the highest tertile (>79 pg/mL), independently of age, sex, duration of diabetes or other risk factors, such as body mass index or hemoglobin A1c. In addition, NT-proBNP levels were associated with surrogate markers of atherosclerosis, such as brachial-ankle pulse wave velocity (r=0.449, P<0.0001) and left ventricular hypertrophy (r=0.212, P<0.001). In this hospital-based cohort of type 2 diabetes, the NT-proBNP levels were associated with systemic

  10. THEORY OF SOLAR MERIDIONAL CIRCULATION AT HIGH LATITUDES

    SciTech Connect

    Dikpati, Mausumi; Gilman, Peter A. E-mail: gilman@ucar.edu

    2012-02-10

    We build a hydrodynamic model for computing and understanding the Sun's large-scale high-latitude flows, including Coriolis forces, turbulent diffusion of momentum, and gyroscopic pumping. Side boundaries of the spherical 'polar cap', our computational domain, are located at latitudes {>=} 60 Degree-Sign . Implementing observed low-latitude flows as side boundary conditions, we solve the flow equations for a Cartesian analog of the polar cap. The key parameter that determines whether there are nodes in the high-latitude meridional flow is {epsilon} = 2{Omega}n{pi}H{sup 2}/{nu}, where {Omega} is the interior rotation rate, n is the radial wavenumber of the meridional flow, H is the depth of the convection zone, and {nu} is the turbulent viscosity. The smaller the {epsilon} (larger turbulent viscosity), the fewer the number of nodes in high latitudes. For all latitudes within the polar cap, we find three nodes for {nu} = 10{sup 12} cm{sup 2} s{sup -1}, two for 10{sup 13}, and one or none for 10{sup 15} or higher. For {nu} near 10{sup 14} our model exhibits 'node merging': as the meridional flow speed is increased, two nodes cancel each other, leaving no nodes. On the other hand, for fixed flow speed at the boundary, as {nu} is increased the poleward-most node migrates to the pole and disappears, ultimately for high enough {nu} leaving no nodes. These results suggest that primary poleward surface meridional flow can extend from 60 Degree-Sign to the pole either by node merging or by node migration and disappearance.

  11. 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

  12. Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

    PubMed Central

    Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

    2009-01-01

    Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

  13. 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)

  14. Controlling Cement Circulation Loss to Both High-Permeability and Fractured Formations

    SciTech Connect

    Nayberg, T.M.; Linafelter, R.L.

    1984-05-01

    A polymeric material is described which has been developed for controlling lost circulation during cementing. The morphology of the material permits sufficient particle deformation to optimize plugging of high permeability matrices, while the particle size of the material is precisely sized so as to optimize sealing of fractures. This optimization of morphology and particle size distribution enables the material to effectively control cement circulation loss to both high permeability and fractured formations. Laboratory data are presented comparing the relative effectiveness of the new material, gilsonite, gilsonite plus cellophane flakes, crushed coal and a graded plastic material in controlling cement slurry loss to simulated high permeability and fractured formations. In simulated high permeability formations the new material outperforms gilsonite and gilsonite plus cellophane flakes, and is slightly better than crushed coal. In simulated fractured formations this material outperforms a graded plastic material. It is shown that the new material does not appreciably affect thickening time or compressive strength of the cement slurries. Free water data for the cement systems tested with this material are satisfactory, but gel should be added to some systems to optimize this slurry property. Wyoming Overthrust, Williston Basin and Powder River Basin case histories are presented which demonstrate the effectiveness of this new material in areas where severe lost circulation problems have been encountered. It has been used to control cement circulation loss in wells with both high permeability and fractured formations, in wells with high bottomhole static temperatures and in applications requiring flow through downhole tools.

  15. Affects of N-terminal variation in the SeM protein of Streptococcus equi on antibody and fibrinogen binding.

    PubMed

    Timoney, John F; DeNegri, Rafaela; Sheoran, Abhineet; Forster, Nathalie

    2010-02-10

    The clonal Streptococcus equi causes equine strangles, a highly contagious suppurative lymphadenopathy and rhinopharyngitis. An important virulence factor and vaccine component, the antiphagocytic fibrinogen binding SeM of S. equi is a surface anchored fibrillar protein. Two recent studies of N. American, Japanese and European isolates have revealed a high frequency of N-terminal amino acid variation in SeM of S. equi CF32 that suggests this region of the protein is subject to immunologic selection pressure. The aims of the present study were firstly to map regions of SeM reactive with convalescent equine IgG and IgA and stimulatory for lymph node cells and secondly to determine effects of N-terminal variation on the functionality of SeM. Variation did not significantly affect fibrinogen binding or susceptibility of S. equi to an opsonic equine serum. Linear epitopes reactive with convalescent IgG and mucosal IgA were concentrated toward the conserved center of SeM. However, IgA but not IgG from every horse reacted with at least one peptide that contained variable sequence. Lymph node cells (CD4+) from horses immunized with SeM were strongly responsive to a peptide (alphaalpha36-138) encoding the entire variable region. SeM (CF32) specific mouse Mab 04D11 which reacted strongly with this larger peptide but not with shorter peptides within that sequence reacted strongly with whole cells of S. equi CF32 but only weakly with cells of any of 14 isolates of S. equi expressing different variants of SeM. These results in combination suggest that N-terminal variation alters a conformational epitope of significance in mucosal IgA and systemic T cell responses but does not affect antibody mediated phagocytosis and killing. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

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

  17. The concept of a plasma centrifuge with a high frequency rotating magnetic field and axial circulation

    NASA Astrophysics Data System (ADS)

    Borisevich, V. D.; Potanin, E. P.

    2017-07-01

    The possibility of using a rotating magnetic field (RMF) in a plasma centrifuge (PC), with axial circulation to multiply the radial separation effect in an axial direction, is considered. For the first time, a traveling magnetic field (TMF) is proposed to drive an axial circulation flow in a PC. The longitudinal separation effect is calculated for a notional model, using specified operational parameters and the properties of a plasma, comprising an isotopic mixture of 20Ne-22Ne and generated by a high frequency discharge. The optimal intensity of a circulation flow, in which the longitudinal separation effect reaches its maximum value, is studied. The optimal parameters of the RMF and TMF for effective separation, as well as the centrifuge performance, are calculated.

  18. 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.

  19. 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.

  20. 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

  1. Tor forms a dimer through an N-terminal helical solenoid with a complex topology.

    PubMed

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M; Williams, Roger L

    2016-04-13

    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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. 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

  8. 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.

  9. 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.

  10. Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae.

    PubMed

    Edwin, Aaron; Persson, Cecilia; Mayzel, Maxim; Wai, Sun Nyunt; Öhman, Anders; Karlsson, B Göran; Sauer-Eriksson, A Elisabeth

    2015-12-01

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important. © 2015 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  11. 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.

  12. Proteolytic cleavage of ostrich and turkey pancreatic lipases: production of an active N-terminal domain.

    PubMed

    Ben Bacha, Abir; Fendri, Ahmed; Gargouri, Youssef; Mejdoub, Hafedh; Miled, Nabil

    2007-10-01

    The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

  13. 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

  14. A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues.

    PubMed

    Güttler, Bert H-O; Cynis, Holger; Seifert, Franziska; Ludwig, Hans-Henning; Porzel, Andrea; Schilling, Stephan

    2013-04-01

    The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid.

    PubMed

    Zheng, Zhen; Chen, Peiyao; Li, Gongyu; Zhu, Yunxia; Shi, Zhonghua; Luo, Yufeng; Zhao, Chun; Fu, Ziyi; Cui, Xianwei; Ji, Chenbo; Wang, Fuqiang; Huang, Guangming; Liang, Gaolin

    2017-01-01

    CBT-Cys click condensation reaction has a high second-order reaction rate constant and has found wide applicability in recent years. However, its reaction mechanism has not been experimentally validated and its application for identifying bioactive N-terminal Cys peptides in real clinical samples has not been reported. Herein, firstly, by employing induced nanoelectrospray ionization-mass spectrometry (InESI-MS) and a home-built micro-reactor, we successfully intercepted and structurally characterized the crucial intermediate in this click reaction for the first time. With the intermediate, the proposed mechanism of this reaction was corroborated. Moreover, we also applied this MS setup to monitor the reaction in real time and obtained the second-order reaction rate constants of this reaction at different pH values. After mechanistic study, we applied this click reaction for identifying bioactive N-terminal cysteine peptides in amniotic fluid (AF). Eight unique N-terminal Cys peptides in AF, three of which are located in the functional domain regions of their corresponding proteins, were identified with a false positive rate less than 1%. One of the three peptides was found able to inhibit the growth of uterine endometrial cancer HEC-1-B cells but not the endometrial normal cells via a typical apoptotic pathway. With its mechanism satisfactorily elucidated, the kinetic parameters obtained, as well as its application for fishing bioactive N-terminal Cys peptides from vast complex clinical samples, we anticipate that this CBT-Cys click reaction could be applied more widely for the facile isolation, site-specific identification, and quantification of N-terminal Cys-containing peptides in complex biological samples.

  3. 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.

  4. 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.

  5. 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.

  6. Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation.

    PubMed

    Kronqvist, Nina; Otikovs, Martins; Chmyrov, Volodymyr; Chen, Gefei; Andersson, Marlene; Nordling, Kerstin; Landreh, Michael; Sarr, Médoune; Jörnvall, Hans; Wennmalm, Stefan; Widengren, Jerker; Meng, Qing; Rising, Anna; Otzen, Daniel; Knight, Stefan D; Jaudzems, Kristaps; Johansson, Jan

    2014-01-01

    The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

  7. 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.

  8. 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.

  9. Impact of the N-terminal amino acid on the formation of pyrazines from peptides in Maillard model systems.

    PubMed

    Van Lancker, Fien; Adams, An; De Kimpe, Norbert

    2012-05-09

    Only a minor part of Maillard reaction studies in the literature focused on the reaction between carbohydrates and peptides. Therefore, in continuation of a previous study in which the influence of the peptide C-terminal amino acid was investigated, this study focused on the influence of the peptide N-terminal amino acid on the production of pyrazines in model reactions of glucose, methylglyoxal, or glyoxal. Nine different dipeptides and three tripeptides were selected. It was shown that the structure of the N-terminal amino acid is determinative for the overall pyrazine production. Especially, the production of 2,5(6)-dimethylpyrazine and trimethylpyrazine was low in the case of proline, valine, or leucine at the N-terminus, whereas it was very high for glycine, alanine, or serine. In contrast to the alkyl-substituted pyrazines, unsubstituted pyrazine was always produced more in the case of experiments with free amino acids. It is clear that different mechanisms must be responsible for this observation. This study clearly illustrates the capability of peptides to produce flavor compounds such as pyrazines.

  10. Site-Specific Protein Bioconjugation via a Pyridoxal 5'-Phosphate-Mediated N-Terminal Transamination Reaction.

    PubMed

    Witus, Leah S; Francis, Matthew

    2010-06-01

    The covalent attachment of chemical groups to proteins is a critically important tool for the study of protein function and the creation of protein-based materials. Methods of site-specific protein modification are necessary for the generation of well defined bioconjugates possessing a new functional group in a single position in the amino acid sequence. This article describes a pyridoxal 5'-phosphate (PLP)-mediated transamination reaction that is specific for the N-terminus of a protein. The reaction oxidizes the N-terminal amine to a ketone or an aldehyde, which can form a stable oxime linkage with an alkoxyamine reagent of choice. Screening studies have identified the most reactive N-terminal residues, facilitating the use of site-directed mutagenesis to achieve high levels of conversion. Additionally, this reaction has been shown to be effective for a number of targets that are not easily accessed through heterologous expression, such as monoclonal antibodies. Curr. Protoc. Chem. Biol. 2:125-134 © 2010 by John Wiley & Sons, Inc.

  11. Direct Interaction of the N-Terminal Domain of Ribosomal Protein S1 with Protein S2 in Escherichia coli

    PubMed Central

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C.; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1–106; S1106) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1106 affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α2) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in Gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions. PMID:22412910

  12. 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.

  13. The two PAN ATPases from Halobacterium display N-terminal heterogeneity and form labile complexes with the 20S proteasome.

    PubMed

    Chamieh, Hala; Guetta, Dorian; Franzetti, Bruno

    2008-04-15

    The PAN (proteasome-activating nucleotidase) proteins from archaea represent homologues of the eukaryotic 26S proteasome regulatory ATPases. In vitro the PAN complex has been previously shown to have a stimulatory effect on the peptidase activities of the 20S core. By using gradient ultracentrifugation we found that, in cellular extracts, the two PAN proteins from Halobacterium do not form stable high-molecular-mass complexes. Only PAN B was found to associate transiently with the 20S proteasome, thus suggesting that the two PAN proteins are not functionally redundant. The PAN B-20S proteasome complexes associate in an ATP-dependent manner and are stabilized upon nucleotide binding. The two PAN proteins were immunodetected in cellular extracts as N-terminal-truncated polypeptides. RNA-mapping experiments and sequence analysis indicated that this process involved transcript heterogeneities and dual translational initiation mechanisms. Taken together, our results suggest that PAN N-terminal modifications and their intracellular dynamics of assembly/association may constitute important determinants of proteolysis regulation.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. New OprM structure highlighting the nature of the N-terminal anchor.

    PubMed

    Monlezun, Laura; Phan, Gilles; Benabdelhak, Houssain; Lascombe, Marie-Bernard; Enguéné, Véronique Y N; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In Gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved outer membrane factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications (PTMs), such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional PTM could open new research lines in the field of antibiotics resistance. In Escherichia coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any modification. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X-ray structure at 3.8 Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitoylated cysteine.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice*

    PubMed Central

    Biesiadecki, Brandon J.; Tachampa, Kittipong; Yuan, Chao; Jin, Jian-Ping; de Tombe, Pieter P.; Solaro, R. John

    2010-01-01

    The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a β-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca2+ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased β myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function. PMID:20410305

  6. Clinical correlation between N-terminal pro-B-type natriuretic peptide and angiographic coronary atherosclerosis.

    PubMed

    Ribeiro, Demóstenes G L; Silva, Ricardo P; Barboza, Daniella R M M; Lima-Júnior, Roberto C P; Ribeiro, Ronaldo A

    2014-06-01

    This study aimed to investigate the clinical correlation between angiographic coronary atherosclerosis and N-terminal pro-B-type natriuretic peptide along with other known correlated factors. In total, 153 patients with a diagnostic hypothesis of stable angina, unstable angina or acute myocardial infarction were classified as group A (patients with angiographically normal coronary arteries) or group B (patients with angiographic coronary atherosclerosis). The two groups were analyzed with respect to the following factors: gender, age, body mass index, abdominal circumference, smoking, diabetes mellitus, arterial hypertension, early family history of atherosclerosis, statin use, the presence of metabolic syndrome, clinical presentation and biochemical factors, including cholesterol, creatinine and fibrinogen plasma concentrations, monocyte counts and N-terminal pro-B-type natriuretic peptide. Univariate analyses comparing the two groups revealed that group B patients more frequently had diabetes, used statins and had systolic dysfunction, N-terminal pro-B-type natriuretic peptide levels ≥ 250 pg/mL, fibrinogen levels >500 mg/dL and ≥ 501 monocytes/mm3 compared with group A patients (p<0.05). Nevertheless, multivariate logistic regression analysis demonstrated that the independent predictors of angiographic coronary atherosclerosis were an N-terminal pro-B-type natriuretic peptide level ≥ 250 pg/mL, diabetes mellitus and increased monocyte numbers and fibrinogen plasma concentration, regardless of the creatinine level or the presence of systolic dysfunction. An N-terminal pro-B-type natriuretic peptide plasma concentration of ≥ 250 pg/mL is an independent predictor of angiographic coronary atherosclerosis.

  7. An optimization model of EMU circulation problem for high-speed railway

    NASA Astrophysics Data System (ADS)

    Li, Wenjun; Nie, Lei

    2017-05-01

    The electronic multiple unit (EMU) circulation plan schedule EMUs to perform train tasks based on a timetable while satisfying a number of constraints. This paper propose a model considering several constraints such as maintenance capability, accommodation capacity and maintenance management modes. Then, the real-world case of Beijing-Shanghai high-speed railway is studied to analyze the impacts of maintenance-mileage limits and maintenance management modes.

  8. Surface Circulation in the Iroise Sea (W. Brittany) from High Resolution HF Radar Mapping

    DTIC Science & Technology

    2013-01-01

    2011 Keywords: HF radar Tidal current Residual flow Eddy field Iroise SeaThe data from two high-frequency radars (HFR) operating in the Iroise Sea are...previously available. Refined resolution enabled to iden- tify fine-scale structures of surface circulation, to quantify the variability of tidal currents...and the residual (time averaged) velocity field, and to explain spatial intermittence in polarization of the tidal current ellipses. The analyzed data

  9. 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

  10. 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.

  11. 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

  12. Biologic variability of N-terminal pro-brain natriuretic peptide in adult healthy cats.

    PubMed

    Harris, Autumn N; Estrada, Amara H; Gallagher, Alexander E; Winter, Brandy; Lamb, Kenneth E; Bohannon, Mary; Hanscom, Jancy; Mainville, Celine A

    2017-02-01

    Objectives The biologic variability of N-terminal pro-brain natriuretic peptide (NT-proBNP) and its impact on diagnostic utility is unknown in healthy cats and those with cardiac disease. The purpose of this study was to determine the biologic variation of NT-proBNP within-day and week-to-week in healthy adult cats. Methods Adult cats were prospectively evaluated by complete blood count (CBC), biochemistry, total thyroxine, echocardiography, electrocardiography and blood pressure, to exclude underlying systemic or cardiac disease. Adult healthy cats were enrolled and blood samples were obtained at 11 time points over a 6 week period (0, 2 h, 4 h, 6 h, 8 h, 10 h and at weeks 2, 3, 4, 5 and 6). The intra-individual (coefficient of variation [CVI]) biologic variation along with index of individuality and reference change values (RCVs) were calculated. Univariate models were analyzed and included comparison of the six different time points for both daily and weekly samples. This was followed by a Tukey's post-hoc adjustment, with a P value of <0.05 being significant. Results The median daily and weekly CVI for the population were 13.1% (range 0-28.7%) and 21.2% (range 3.9-68.1%), respectively. The index of individuality was 0.99 and 1 for daily and weekly samples, respectively. The median daily and weekly RCVs for the population were 39.8% (range 17.0-80.5%) and 60.5% (range 20.1-187.8%), respectively. Conclusions and relevance This study demonstrates high individual variability for NT-proBNP concentrations in a population of adult healthy cats. Further research is warranted to evaluate NT-proBNP variability, particularly how serial measurements of NT-proBNP may be used in the diagnosis and management of cats with cardiac disease.

  13. The effects of two weeks of recombinant growth hormone administration on the response of IGF-I and N-terminal pro-peptide of collagen type III (P-III-NP) during a single bout of high resistance exercise in resistance trained young men.

    PubMed

    Velloso, C P; Aperghis, M; Godfrey, R; Blazevich, A J; Bartlett, C; Cowan, D; Holt, R I G; Bouloux, P; Harridge, S D R; Goldspink, G

    2013-06-01

    Recombinant human growth hormone (rhGH) is used by some athletes and body builders with the aim of enhancing performance, building muscle and improving physique. Detection of the misuse of rhGH has proved difficult for a number of reasons. One of these is the effect of preceding exercise. In this randomised, double blind placebo-controlled study, we determined the effects of rhGH administration in male amateur athletes on two candidate markers of rhGH abuse, IGF-I and N-terminal pro-peptide of collagen type III (P-III-NP), following a bout of weightlifting exercise. Sixteen men entered a four-week general weight training programme to homogenise their activity profile. They then undertook repeated bouts of standardised leg press weightlifting exercise (AHRET-acute heavy resistance exercise test). Blood samples were taken before and up to one hour after the AHRET. After the first laboratory visit (Test 1), the subjects were randomly assigned to receive daily injections of either rhGH (0.1 IU kg(-1) day(-1)) or placebo for two weeks. The AHRET was repeated after the two-week dosing period (Test 2) and a further test was undertaken following a one-week washout (Test 3). There was no effect of exercise on either IGF-I or P-III-NP in any test. Both markers were markedly elevated at Test 2 (p<0.001), with P-III-NP remaining elevated at Test 3 in the GH administration group (p<0.05). Application of the GH-2000 discriminant function positively identified GH administration in 17 of 40 blood samples taken at Test 2 from the rhGH group and none from the placebo group. The data show that rhGH results in elevated levels of IGF-I and P-III-NP in well-trained individuals and that leg press weightlifting exercise does not affect these markers. The GH-2000 discriminant function identified four of eight subjects taking rhGH with no false positive results. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Secondary gravity waves in the winter mesosphere: Results from a high-resolution global circulation model

    NASA Astrophysics Data System (ADS)

    Becker, Erich; Vadas, Sharon

    2017-04-01

    General circulation models that simulate gravity waves (GWs) explicitly are expected to capture the spatial and temporal variability of the GW sources. Moreover, secondary GWs that result from the nonlinearity and intermittency of the primary GWs in the middle atmosphere can be studied. In this study we describe a new high-resolution version of the Kuehlungsborn Mechanistic general Circulation Model (KMCM). The model includes no GW parameterization, and unresolved dynamical scales are parameterized by a macro-turbulent diffusion scheme that accounts for damping of resolved waves in a self-consistent fashion. We analyze the model with regard to secondary GWs in the mesosphere during Austral winter. The westward GW drag in the lower winter mesosphere agrees well with that from conventional models with parameterized GWs, and it is strongly due to orographic GWs generated by the Andes and the Antartic Pensiula. Due to the high temporal and spatial intermittency of the primary GWs in the southern winter stratopause region, secondary GWs are generated and propagate to higher altitudes. These secondary GWs impose a substantial eastward drag in the mesopause region where they dissipate. The eastward GW drag results in an additional eastward maximum of the mean zonal wind around 90-100 km. We propose that when the polar night jet is strong, secondary GWs may play a significant role in the general circulation of the winter mesopause region. Radar measurements in the northern hemisphere during January 2016 are consistent with this finding.

  15. 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.

  16. 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.

  17. 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.

  18. Using high-resolution global mantle circulation models to understand fast seismic velocity anomalies

    NASA Astrophysics Data System (ADS)

    Webb, P. J.; Davies, J.; Davies, R.

    2011-12-01

    Mantle circulation models (MCMs) incorporate plate motion history into a standard mantle convection model. They are an important tool for studying the link between plate tectonics and Earth's interior. Such models are used to predict temperature fields throughout the mantle, which can be compared to seismic tomography, a present day snapshot of Earth's mantle. Assuming a mantle primarily driven by thermal convection we relate regions of colder than average mantle to regions of faster than average seismic velocity. Traditionally, mantle circulation models have used 120 million years of plate motion history as their surface velocity boundary condition. Such models can reconcile observed large-scale fast seismic velocity anomalies in regions of major plate convergence, to depths of around 1300 km. Recent advances allow for high-resolution mantle circulation models with up to 300 million years of plate motion history. When combined with higher resolution tomographic studies we are now able to study the influence of subduction on mantle heterogeneity structure, in greater detail. In this study we present results from a suite of mantle circulation models across a wide parameter space. By comparing the predicted temperature fields to seismic tomography we gain an understanding of which parameters affect the location and magnitude of subduction related anomalies. We also quantify the effect of using mineral physics data to convert modelled temperature fields to seismic velocity. In the simplest cases, we can accurately predict the larger regional scale, mid-mantle fast seismic velocity anomalies, including remnants of the Farallon slab beneath North America and the Tethys oceans below South East Asia. However, the more realistic high-resolution studies allow us to investigate other areas of interest, including more localised aspects of Tethyan subduction associated with the closure of smaller ocean basins. We also examine the model predictions for subducted material in

  19. 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.

  20. 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

  1. 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.

  2. Performance, kinetics behaviors and microbial community of internal circulation anaerobic reactor treating wastewater with high organic loading rate: Role of external hydraulic circulation.

    PubMed

    Luo, Gan; Li, Jun; Li, Yan; Wang, Zhu; Li, Wen-Tao; Li, Ai-Min

    2016-12-01

    Performance of internal circulation anaerobic reactor (IC) treating wastewater at high organic loading rate (OLR) and role of external hydraulic circulation were evaluated. When the OLR was increased from 2.50 to 18.94kgCOD/m(3)/d, COD removal decreased to 85% slightly and methane production increased to 4.49L/L/d with the upflow velocity of 1.0m/h resulted from the additional hydraulic circulation. Withdrawal of external hydraulic circulation led to decrease of COD removal to lower than 60% drastically and methane production by 81%. Accumulation of volatile fatty acids caused decline of pH to below 6.0 and the shift of substrate metabolic pathway to the hybrid fermentation. In addition, both maximum methane production rate and maximum substrate degradation rate obtained from mathematical models decreased significantly. Hydrogenotrophic methanogens including Methanobacterium and Methanocorpusculum predominated in the anaerobic sludge and the shift of microbial community was also observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. 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. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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

  5. Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry.

    PubMed

    Doucet, Alain; Overall, Christopher M

    2011-01-01

    Edman degradation is a long-established technique for N-terminal sequencing of proteins and cleavage fragments. However, for accurate data analysis and amino acid assignments, Edman sequencing proceeds on samples of single proteins only and so lacks high-throughput capabilities. We describe a new method for the high-throughput determination of N-terminal sequences of multiple protein fragments in solution. Proteolytic processing can change the activity of bioactive proteins and also reveal cryptic binding sites and generate proteins with new functions (neoproteins) not found in the parent molecule. For example, extracellular matrix (ECM) protein processing often produces multiple proteolytic fragments with the generation of cryptic binding sites and neoproteins by ECM protein processing being well documented. The exact proteolytic cleavage sites need to be identified to fully understand the functions of the cleavage fragments and biological roles of proteases in vivo. However, the identification of cleavage sites in complex high molecular proteins such as those composing the ECM is not trivial. N-terminal microsequencing of proteolytic fragments is the usual method employed, but it suffers from poor resolution of sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and is inefficient at identifying multiple cleavages, requiring preparation of numerous gels or membrane slices for analysis. We recently developed Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS) to overcome these limitations as a complement for N-terminal sequencing. ATOMS employs isotopic labeling and quantitative tandem mass spectrometry to identify cleavage sites in a fast and accurate manner. We successfully used ATOMS to identify nearly 100 cleavage sites in the ECM proteins laminin and fibronectin. Presented herein is the detailed step-by-step protocol for ATOMS. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. A unique glycine-rich motif at the N-terminal region of Bamboo mosaic virus coat protein is required for symptom expression.

    PubMed

    Lan, Ping; Yeh, Wen-Bin; Tsai, Chih-Wei; Lin, Na-Sheng

    2010-07-01

    The coat proteins (CP) of many plant viruses are multifunctional proteins. We used N-terminal sequencing and mass spectrometry/mass spectrometry analysis to identify a truncated form of the Bamboo mosaic virus (BaMV) CP missing the N-terminal 35 amino acids (N35). The N35 region is unique in the potexviruses by its containing a glycine-rich motif (GRM) not present in databases but highly conserved among BaMV isolates. Results from site-directed mutagenesis and deletion mutational analysis showed that loss of this region converted necrotic local lesions to chlorotic local lesions on Chenopodium quinoa leaves. Furthermore, this region is required for successful development of mosaic symptoms on Nicotiana benthamiana leaves but is dispensable for BaMV replication and cell-to-cell and long-distance movement as well as virion assembly. This unique GRM-containing region of BaMV CP may be a symptom determinant in specific hosts.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.

    PubMed

    Höög, J O; Weis, M; Zeppezauer, M; Jörnvall, H; von Bahr-Lindström, H

    1987-12-01

    Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.

  12. 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.

  13. 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.

  14. N-terminal-prolonged vinyl ester-based peptides as selective proteasome beta1 subunit inhibitors.

    PubMed

    Baldisserotto, Anna; Destro, Federica; Vertuani, Gianni; Marastoni, Mauro; Gavioli, Riccardo; Tomatis, Roberto

    2009-08-01

    The synthesis and biological properties of vinyl ester peptide-based molecules bearing linear N-terminal amino acids are reported. Compounds were tested in vitro for their capacity to inhibit the chymotryptic-, tryptic-like, and post-acidic activities of the proteasome. Some analogues showed selective inhibition of post-acidic (PGPH) activity, which is attributed to the beta1 subunit. Interestingly, active compounds demonstrated higher inhibitory activity toward 'standard' proteasomes than toward immunoproteasomes. The inhibitory potency was found to be related to the amino acidic sequence and to the length of the N-terminal residues. The new inhibitors demonstrated resistance to plasmatic proteases and a good capacity to permeate the cell membrane.

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

    DTIC Science & Technology

    2014-10-01

    ABSTRACT Abstract: XXX aminopyrazoles, a new class of c-jun-N-terminal kinase (JNK) inhibitors, have been synthesized and the biochemical IC50 has been...have also been generated as backups. XXX compounds from the pyridopyrimidinone class have been synthesized and tested in biochemical and cell based...assays, and XXX compounds from the amino acid transporter analog class have been made and tested in biochemical assays. The goal of this work is to

  16. New OprM structure highlighting the nature of the N-terminal anchor

    PubMed Central

    Monlezun, Laura; Phan, Gilles; Benabdelhak, Houssain; Lascombe, Marie-Bernard; Enguéné, Véronique Y. N.; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In Gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved outer membrane factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications (PTMs), such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional PTM could open new research lines in the field of antibiotics resistance. In Escherichia coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any modification. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X-ray structure at 3.8 Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitoylated cysteine. PMID:26191054

  17. 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

  18. 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

  19. Multicanonical Molecular Dynamics Simulations of the N-terminal Domain of Protein L9

    PubMed Central

    Yaşar, Fatih; Jiang, Ping; Hansmann, Ulrich H. E.

    2014-01-01

    We describe multicanonical molecular dynamic simulations of the N-terminal domain of the protein L9. Analyzing free energy landscapes and thermal ordering, we propose a possible folding mechanism for the protein. By comparing our results with that of molecular dynamics runs of the protein at constant temperature, we find that multicanonical molecular dynamics leads to orders of magnitude higher sampling of folding transitions. PMID:25253918

  20. Numerical study on the Welander oscillatory natural circulation problem using high-order numerical methods

    DOE PAGES

    Zou, Ling; Zhao, Haihua; Kim, Seung Jun

    2016-11-16

    In this study, the classical Welander’s oscillatory natural circulation problem is investigated using high-order numerical methods. As originally studied by Welander, the fluid motion in a differentially heated fluid loop can exhibit stable, weakly instable, and strongly instable modes. A theoretical stability map has also been originally derived from the stability analysis. Numerical results obtained in this paper show very good agreement with Welander’s theoretical derivations. For stable cases, numerical results from both the high-order and low-order numerical methods agree well with the non-dimensional flow rate analytically derived. The high-order numerical methods give much less numerical errors compared to themore » low-order methods. For stability analysis, the high-order numerical methods could perfectly predict the stability map, while the low-order numerical methods failed to do so. For all theoretically unstable cases, the low-order methods predicted them to be stable. The result obtained in this paper is a strong evidence to show the benefits of using high-order numerical methods over the low-order ones, when they are applied to simulate natural circulation phenomenon that has already gain increasing interests in many future nuclear reactor designs.« less

  1. Numerical study on the Welander oscillatory natural circulation problem using high-order numerical methods

    SciTech Connect

    Zou, Ling; Zhao, Haihua; Kim, Seung Jun

    2016-11-16

    In this study, the classical Welander’s oscillatory natural circulation problem is investigated using high-order numerical methods. As originally studied by Welander, the fluid motion in a differentially heated fluid loop can exhibit stable, weakly instable, and strongly instable modes. A theoretical stability map has also been originally derived from the stability analysis. Numerical results obtained in this paper show very good agreement with Welander’s theoretical derivations. For stable cases, numerical results from both the high-order and low-order numerical methods agree well with the non-dimensional flow rate analytically derived. The high-order numerical methods give much less numerical errors compared to the low-order methods. For stability analysis, the high-order numerical methods could perfectly predict the stability map, while the low-order numerical methods failed to do so. For all theoretically unstable cases, the low-order methods predicted them to be stable. The result obtained in this paper is a strong evidence to show the benefits of using high-order numerical methods over the low-order ones, when they are applied to simulate natural circulation phenomenon that has already gain increasing interests in many future nuclear reactor designs.

  2. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    PubMed Central

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

    2013-01-01

    Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

  3. Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury

    SciTech Connect

    Bourdi, Mohammed Korrapati, Midhun C.; Chakraborty, Mala; Yee, Steven B.; Pohl, Lance R.

    2008-09-12

    Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2{sup -/-} mice were treated with 300 mg APAP/kg, 90% of JNK2{sup -/-} mice died of ALF compared to 20% of WT mice within 48 h. The high susceptibility of JNK2{sup -/-} mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair. Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Human antibody response to Campylobacter jejuni flagellin protein and a synthetic N-terminal flagellin peptide.

    PubMed Central

    Nachamkin, I; Yang, X H

    1989-01-01

    We measured isotype-specific human antibodies directed against Campylobacter jejuni native flagellin and a synthetic peptide derived from the N-terminal amino acid sequence of the protein by using a microdilution enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with gastrointestinal infection caused by C. jejuni (n = 20) and control samples (number from normal subjects = 20; number from patients with diarrhea other than campylobacter = 20) were tested in this assay. Serum specimens from patients with campylobacter infection showed statistically significant higher isotype-specific antiflagellin antibody titers than control samples did. Detection of immunoglobulin G (IgG) antibodies was less specific (70%) than detection of either IgA or IgM antibodies in infected patients (95%). The sensitivity of testing for any of the isotypes ranged from 64 to 100% in acute-phase serum specimens and 85 to 95% in convalescent-phase serum specimens. An ELISA with an N-terminal synthetic peptide derived from the flagellin protein as antigen was not sensitive (60%) for detecting campylobacter infection but was very specific (97.5%). In conclusion, detection of serum IgA or IgM against C. jejuni flagellin may be a useful marker of infection. Although the N-terminal synthetic peptide was antigenic in a few patients with infection and showed good specificity in the ELISA, additional amino acid sequences with better sensitivity for detecting infection need to be identified. PMID:2584372

  9. 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.

  10. Proline-directed phosphorylation of the dopamine transporter N-terminal domain

    PubMed Central

    Gorentla, Balachandra K.; Moritz, Amy E.; Foster, James D.; Vaughan, Roxanne A.

    2009-01-01

    Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with PKC- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCα, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCα-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses. PMID:19146407

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

    PubMed

    Kis, Mihaly; Burbridge, Emma; Brock, Ian W; Heggie, Laura; Dix, Philip J; Kavanagh, Tony A

    2004-03-01

    Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic

  17. 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.

  18. 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.

  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. Effects of Liquid Storage in a High pH Anticoagulant on the Circulation of Baboon Platelets.

    DTIC Science & Technology

    1982-04-23

    RD-Ri40 463 EFFECTS OF LIQUID STORAGE IN A HIGH PH ANTICOAGULANT ON /II THE CIRCULATION 0 .(U) BOSTON UNIV NAR SCHOOL OF MEDICINE R J MELARAGNO ET AL...EFFECTS OF LIQUID STORAGE IN A HIGH pH ANTICOAGULANT ON THE CIRCULATION OF BABOON PLATELETS by A. J. MELARAGNO, S. HALL, A. DOTY, J. G. WHITE, AND C. R...If neceeary and identify by block number) pH Liquid storage Anticoagulant Circulation Platelets Platelet-rich plasma Baboons Contamination 20

  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. Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: An efficient mechanism for the regulation of angiogenesis

    PubMed Central

    Struman, Ingrid; Bentzien, Frauke; Lee, Hsinyu; Mainfroid, Véronique; D’Angelo, Gisela; Goffin, Vincent; Weiner, Richard I.; Martial, Joseph A.

    1999-01-01

    Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin/growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activation or inhibition of mitogen-activated protein kinase signaling pathway. In addition, the N-terminal fragments stimulate expression of type 1 plasminogen activator inhibitor whereas the intact molecules have no effect, an observation consistent with the fragments acting via separate receptors. The concept that a single molecule encodes both angiogenic and antiangiogenic peptides represents an efficient model for regulating the balance of positive and negative factors controlling angiogenesis. This hypothesis has potential physiological importance for the control of the vascular connection between the fetal and maternal circulations in the placenta, where human prolactin, human placental lactogen, and human growth hormone variant are expressed. PMID:9990009

  4. 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.

  5. Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm.

    PubMed

    Frazee, Richard W; Taylor, Jennifer A; Tullius, Thomas D

    2002-11-01

    The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm

  6. N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo

    PubMed Central

    Charneira, C; Grilo, NM; Pereira, SA; Godinho, ALA; Monteiro, EC; Marques, MM; Antunes, AMM

    2012-01-01

    BACKGROUND AND PURPOSE The aim of this study was to obtain evidence for the activation of the nucleoside reverse transcriptase inhibitor abacavir to reactive aldehyde metabolites in vivo. Protein haptenation by these reactive metabolites may be a factor in abacavir-induced toxic events. EXPERIMENTAL APPROACH The formation of N-terminal valine adducts from the abacavir-derived aldehydes was investigated in the haemoglobin of Wistar rats treated with eight daily doses (120 mg·kg−1) of abacavir. The analyses were conducted by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry upon comparison with synthetic standards. KEY RESULTS An N-terminal valine haemoglobin adduct derived from an α,β-unsaturated aldehyde metabolite of abacavir was identified in vivo for the first time. CONCLUSIONS AND IMPLICATIONS This preliminary work on abacavir metabolism provides the first unequivocal evidence for the formation of an α,β-unsaturated aldehyde metabolite in vivo and of its ability to form haptens with proteins. The methodology described herein can be used to assess the formation of this metabolite in human samples and has the potential to become a valuable pharmacological tool for mechanistic studies of abacavir toxicity. In fact, the simplicity of the method suggests that the abacavir adduct with the N-terminal valine of haemoglobin could be used to investigate abacavir-induced toxicity for accurate risk/benefit estimations. PMID:22725138

  7. Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

    PubMed Central

    Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-01-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

  8. N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo.

    PubMed

    Charneira, C; Grilo, N M; Pereira, S A; Godinho, A L A; Monteiro, E C; Marques, M M; Antunes, A M M

    2012-11-01

    The aim of this study was to obtain evidence for the activation of the nucleoside reverse transcriptase inhibitor abacavir to reactive aldehyde metabolites in vivo. Protein haptenation by these reactive metabolites may be a factor in abacavir-induced toxic events. The formation of N-terminal valine adducts from the abacavir-derived aldehydes was investigated in the haemoglobin of Wistar rats treated with eight daily doses (120 mg·kg(-1)) of abacavir. The analyses were conducted by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry upon comparison with synthetic standards. An N-terminal valine haemoglobin adduct derived from an α,β-unsaturated aldehyde metabolite of abacavir was identified in vivo for the first time. This preliminary work on abacavir metabolism provides the first unequivocal evidence for the formation of an α,β-unsaturated aldehyde metabolite in vivo and of its ability to form haptens with proteins. The methodology described herein can be used to assess the formation of this metabolite in human samples and has the potential to become a valuable pharmacological tool for mechanistic studies of abacavir toxicity. In fact, the simplicity of the method suggests that the abacavir adduct with the N-terminal valine of haemoglobin could be used to investigate abacavir-induced toxicity for accurate risk/benefit estimations. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  9. 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

  10. 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.

  11. Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

    PubMed

    Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-03-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

  12. The N-terminal Capping Propensities of the D-helix Modulate the Allosteric Activation of the Escherichia coli cAMP Receptor Protein*

    PubMed Central

    Yu, Shaoning; Maillard, Rodrigo A.; Gribenko, Alexey V.; Lee, J. Ching

    2012-01-01

    Transduction of biological signals at the molecular level involves the activation and/or inhibition of allosteric proteins. In the transcription factor cAMP receptor protein (CRP) from Escherichia coli, the allosteric activation, or apo-holo transition, involves rigid body motions of domains and structural rearrangements within the hinge region connecting the cAMP- and DNA-binding domains. During this apo-holo transition, residue 138 is converted as part of the elongated D-helix to the position of the N-terminal capping residue of a shorter D-helix. The goal of the current study is to elucidate the role of residue 138 in modulating the allostery between cAMP and DNA binding. By systematically mutating residue 138, we found that mutants with higher N-terminal capping propensities lead to increased cooperativity of cAMP binding and a concomitant increase in affinity for lac-DNA. Furthermore, mutants with higher N-terminal capping propensity correlate with properties characteristic of holo-CRP, particularly, increase in protein structural dynamics. Overall, our results provide a quantitative characterization of the role of residue 138 in the isomerization equilibrium between the apo and holo forms of CRP, and in turn the thermodynamic underpin to the molecular model of allostery revealed by the high resolution structural studies. PMID:23035121

  13. The N-terminal domain of DDA3 regulates the spindle-association of the microtubule depolymerase Kif2a and controls the mitotic function of DDA3.

    PubMed

    Jang, Chang-Young; Fang, Guowei

    2009-10-01

    DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.

  14. Identification and characterization of a novel class of c-Jun N-terminal kinase inhibitors.

    PubMed

    Schepetkin, Igor A; Kirpotina, Liliya N; Khlebnikov, Andrei I; Hanks, Tracey S; Kochetkova, Irina; Pascual, David W; Jutila, Mark A; Quinn, Mark T

    2012-06-01

    In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

  15. 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

  16. Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Hanks, Tracey S.; Kochetkova, Irina; Pascual, David W.; Jutila, Mark A.

    2012-01-01

    In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs. PMID:22434859

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

    PubMed

    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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Surface Accessibility and Conformational Changes in the N-terminal Domain of Type I Inositol Trisphosphate Receptors

    PubMed Central

    Anyatonwu, Georgia; Joseph, Suresh K.

    2009-01-01

    To identify surface-accessible residues and monitor conformational changes of the type I inositol 1,4,5-trisphosphate receptor protein in membranes, we have introduced 10 cysteine substitutions into the N-terminal ligand-binding domain. The reactivity of these mutants with progressively larger maleimide-polyethylene glycol derivatives (MPEG) was measured using a gel shift assay of tryptic fragments. The results indicate that the mutations fall into four categories as follows: sites that are highly accessible based on reactivity with the largest 20-kDa MPEG (S2C); sites that are moderately accessible based on reactivity only with 5-kDa MPEG (S6C, S7C, A189C, and S277C); sites whose accessibility is markedly enhanced by Ca2+ (S171C, S277C, and A575C); and sites that are inaccessible irrespective of incubation conditions (S217C, A245C, and S436C). The stimulation of accessibility induced by Ca2+ at the S277C site occurred with an EC50 of 0.8 μm and was mimicked by Sr2+ but not Ba2+. Inositol 1,4,5-trisphosphate alone did not affect reactivity of any of the mutants in the presence or absence of Ca2+. The data are interpreted using crystal structures and EM reconstructions of the receptor. Our data identify N-terminal regions of the protein that become exposed upon Ca2+ binding and suggest possible orientations of the suppressor and ligand-binding domains that have implications for the mechanism of gating of the channel. PMID:19141613

  19. 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

  20. 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.

  1. N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity.

    PubMed

    Jehle, Stefan; Vollmar, Breanna S; Bardiaux, Benjamin; Dove, Katja K; Rajagopal, Ponni; Gonen, Tamir; Oschkinat, Hartmut; Klevit, Rachel E

    2011-04-19

    The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

  2. 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

  3. Large-scale identification of N-terminal peptides in the halophilic archaea Halobacterium salinarum and Natronomonas pharaonis.

    PubMed

    Aivaliotis, Michalis; Gevaert, Kris; Falb, Michaela; Tebbe, Andreas; Konstantinidis, Kosta; Bisle, Birgit; Klein, Christian; Martens, Lennart; Staes, An; Timmerman, Evy; Van Damme, Jozef; Siedler, Frank; Pfeiffer, Friedhelm; Vandekerckhove, Joël; Oesterhelt, Dieter

    2007-06-01

    Characterization of protein N-terminal peptides supports the quality assessment of data derived from genomic sequences (e.g., the correct assignment of start codons) and hints to in vivo N-terminal modifications such as N-terminal acetylation and removal of the initiator methionine. The current work represents the first large-scale identification of N-terminal peptides from prokaryotes, of the two halophilic euryarchaeota Halobacterium salinarum and Natronomonas pharaonis. Two methods were used that specifically allow the characterization of protein N-terminal peptides: combined fractional diagonal chromatography (COFRADIC) and strong cation exchange chromatography (SCX), both known to enrich for N-terminally blocked peptides. In addition to these specific methods, N-terminal peptide identifications were extracted from our previous genome-wide proteomic data. Combining all data, 606 N-terminal peptides from Hbt. salinarum and 328 from Nmn. pharaonis were reliably identified. These results constitute the largest available dataset holding identified and characterized protein N-termini for prokaryotes (archaea and bacteria). They allowed the validation/improvement of start codon assignments as automatic gene finders tend to misassign start codons for GC-rich genomes. In addition, the dataset allowed unravelling N-terminal protein maturation in archaea, showing that 60% of the proteins undergo methionine cleavage and that-in contrast to current knowledge-Nalpha-acetylation is common in the archaeal domain of life with 13-18% of the proteins being Nalpha-acetylated. The protein sets described in this paper are available by FTP and might be used as reference sets to test the performance of new gene finders.

  4. High Levels of Peripheral Blood Circulating Plasma Cells as a Specific Risk Factor for Progression of Smoldering Multiple Myeloma

    PubMed Central

    Bianchi, Giada; Kyle, Robert A.; Larson, Dirk R.; Witzig, Thomas E.; Kumar, Shaji; Dispenzieri, Angela; Morice, William G.; Rajkumar, S. Vincent

    2012-01-01

    Smoldering multiple myeloma (SMM) carries a 50% risk of progression to multiple myeloma (MM) or related malignancy within the first 5 years following diagnosis. The goal of this study was to determine if high levels of circulating plasma cells (PCs) are predictive of SMM transformation within the first 2–3 years from diagnosis. Ninety-one patients diagnosed with SMM at Mayo Clinic from January 1994 through January 2007 who had testing for circulating PCs using an immunofluorescent assay and adequate follow up to ascertain disease progression, were studied. High level of circulating PCs was defined as absolute peripheral blood PCs >5000 ×106/L and/or > 5% cytoplasmic immunoglobulin (Ig) positive PCs per 100 peripheral blood mononuclear cells. Patients with high circulating PCs (14 of 91 patients, 15%) were significantly more likely to progress to active disease within 2 years compared with patients without high circulating PCs, 71% versus 25%, respectively, P=0.001. Corresponding rates for progression within 3 years were 86% versus 35%, respectively, P<0.001. Overall survival (OS) after both SMM diagnosis and MM diagnosis was also significantly different. High levels of circulating PCs identify SMM patients with an elevated risk of progression within the first 2 to 3 years following diagnosis. PMID:22902364

  5. High levels of peripheral blood circulating plasma cells as a specific risk factor for progression of smoldering multiple myeloma.

    PubMed

    Bianchi, G; Kyle, R A; Larson, D R; Witzig, T E; Kumar, S; Dispenzieri, A; Morice, W G; Rajkumar, S V

    2013-03-01

    Smoldering multiple myeloma (SMM) carries a 50% risk of progression to multiple myeloma (MM) or related malignancy within the first 5 years following diagnosis. The goal of this study was to determine if high levels of circulating plasma cells (PCs) are predictive of SMM transformation within the first 2-3 years from diagnosis. Ninety-one patients diagnosed with SMM at Mayo Clinic from January 1994 through January 2007, who had testing for circulating PCs using an immunofluorescent assay and adequate follow-up to ascertain disease progression, were studied. High level of circulating PCs was defined as absolute peripheral blood PCs >5 × 10(6)/l and/or >5% PCs per 100 cytoplasmic immunoglobulin (Ig)-positive peripheral blood mononuclear cells. Patients with high circulating PCs (14 of 91 patients, 15%) were significantly more likely to progress to active disease within 2 years compared with patients without high circulating PCs, 71% versus 24%, respectively, P=0.001. Corresponding rates for progression within 3 years were 86% versus 34%, respectively, P<0.001. Overall survival (OS) after both SMM diagnosis and MM diagnosis was also significantly different. High levels of circulating PCs identify SMM patients with an elevated risk of progression within the first 2-3 years following diagnosis.

  6. 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.

  7. High-Resolution Simulation of Sea Ice and circulation in the West Antarctic Peninsula (WAP)

    NASA Astrophysics Data System (ADS)

    Schultz, C.; Doney, S. C.

    2016-02-01

    Over the last decades, the West Antarctic Peninsula (WAP) has undergone physical and ecological changes at a rapid pace, with winter air temperatures warming up to 4.8 times the global average rate. The effects of this warming can be felt in the ecosystem; with changes in the chlorophyll patterns and a poleward shift of ice dependent species. These fluctuations are associated with the sea ice cover in the region, which influences the upper ocean mixed layer depth, heat exchanges and local circulation. Recent research has found a consistent trend of a shortening in the sea ice season in the WAP, associated with changes in the wind pattern. The mechanisms behind these drastic climate changes are not fully understood and have been investigated by the Palmer-LTER (Long Term Ecological Research) over the last two decades. In this context, numerical modeling is a powerful tool, given the seasonal and sea ice constraints on data acquisition in the region. A high-resolution circulation model, coupled to a sea ice model, was implemented in the WAP to simulate the sea ice advance and retreat, and reproduce the annual cycle of sea ice and mixed layer depth in different climate variability scenarios. A successful sea ice model with high vertical and horizontal resolution is a necessary first step towards the implementation of a biogeochemical model that could allow a better understanding of the underlying water chemistry changes behind such drastic ecosystem shifts.

  8. Posterior circulation cerebral hyperperfusion syndrome after high flow external carotid artery to middle cerebral artery bypass.

    PubMed

    Quach, Eric T; Gonzalez, Andres A; Shilian, Parastou; Russin, Jonathan J

    2015-09-01

    We present the first report, to our knowledge, in which revascularization of the middle cerebral artery (MCA) with a high flow extracranial-intracranial procedure resulted in symptomatic hyperemia of the posterior circulation. Cerebral hyperperfusion syndrome (CHS) is a poorly understood phenomenon that is classically seen in the distribution of a revascularized artery. A 37-year-old woman presented with a 3 month history of cognitive and speech difficulties, persistent headaches, weakness, numbness, and paresthesia which was worse in the right extremities and face. She was found to have bilateral watershed infarcts worse in the left cerebral hemisphere, severe bilateral stenosis of the supraclinoid internal carotid artery, and a small left superior hypophyseal aneurysm. The patient underwent left cerebral hemisphere revascularization with a high flow external carotid artery to MCA bypass with aneurysm trapping. During skin closure, significant changes were seen in her bilateral upper extremity motor-evoked potentials. The patient's postoperative exam was noted for an intermittent inability to follow commands, bilateral upper extremity weakness, vertical nystagmus, and alogia that all dramatically improved with strict blood pressure control. Postoperative perfusion imaging revealed posterior circulation hyperemia. This patient highlights the potential for hyperemic complications outside the revascularized arterial territory. Strict blood pressure control is recommended in order to prevent and manage hyperemia-associated symptoms. Improving our understanding of CHS may assist in identifying at risk patients and at risk arterial territories in order to optimize CHS prevention and management strategies.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women.

    PubMed

    Cheng, Wern-Cherng; Lo, Shyi-Chyi; Tsai, Keh-Sung; Tu, Shih-Te; Wu, Jin-Shan; Chang, Ching-I; Chen, Chi-Ling; Shaw, Ning-Sing; Peng, Hui-Yu; Wang, Shu-Yi; Wu, Chih-Hsing; Jan, I-Shaw; Hsu, Ssu-Chun; Liu, Chao-Wei; Lee, Li-Na; Tai, Tong-Yuan

    2015-08-01

    Estrogen in hormone replacement therapy causes homeostatic changes. However, little is known regarding the safety of high-dose phytoestrogen on coagulation and hematological parameters in healthy postmenopausal women. This study evaluated the effects of high-dose soy isoflavone (300 mg/day) on blood pressure, hematological parameters, and coagulation functions including circulating microparticles in healthy postmenopausal women. The original study is a 2-year prospective, double-blind, placebo-controlled study. In total, 431 postmenopausal women (from 3 medical centers) were randomly assigned to receive either high-dose isoflavone or placebo for 2 years. At baseline, 6 months, 1 year, and 2 years after treatment, blood pressure, body weight, liver function tests, hematological parameters, and lipid profiles were measured. The 1(st) year blood specimens of 85 cases of 144 eligible participants (from one of the three centers) were analyzed as D-dimer, von Willebrand factor antigen, factor VII, plasminogen activator inhibitor type 1, and circulating cellular microparticles, including the measurement of monocyte, platelet, and endothelial microparticles. In the isoflavone group, after 1 year, the changes in liver function tests, hematological parameters, and coagulation tests were not different from those of the control. Triglyceride levels were significantly lower after 6 months of isoflavone treatment than the placebo group, but the difference did not persist after 1 year. Endothelial microparticles increased steadily in both groups during the 1-year period but the trend was not affected by treatment. The results of the present study indicate that high-dose isoflavone treatment (300 mg/day) does not cause hematological abnormalities or activate coagulation factors. Copyright © 2013. Published by Elsevier B.V.

  16. Establishment of a Cre/loxP recombination system for N-terminal epitope tagging of genes in Tetrahymena.

    PubMed

    Busch, Clara Jana-Lui; Vogt, Alexander; Mochizuki, Kazufumi

    2010-07-13

    Epitope tagging is a powerful strategy to study the function of proteins. Although tools for C-terminal protein tagging in the ciliated protozoan Tetrahymena thermophila have been developed, N-terminal protein tagging in this organism is still technically demanding. In this study, we have established a Cre/loxP recombination system in Tetrahymena and have applied this system for the N-terminal epitope tagging of Tetrahymena genes. Cre can be expressed in Tetrahymena and localizes to the macronucleus where it induces precise recombination at two loxP sequences in direct orientation in the Tetrahymena macronuclear chromosome. This Cre/loxP recombination can be used to remove a loxP-flanked drug-resistance marker from an N-terminal tagging construct after it is integrated into the macronucleus. The system established in this study allows us to express an N-terminal epitope tagged gene from its own endogenous promoter in Tetrahymena.

  17. Purification and N-terminal amino acid sequence of dextranicin 24, a bacteriocin of Leuconostoc sp.

    PubMed

    Revol-Junelles, A M; Lefebvre, G

    1996-08-01

    Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH2(-) K G V L G W L S M A S S A L T G P Q Q . . .

  18. 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.

  19. 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

  20. Effect of Increased Blood Flow on Pulmonary Circulation Before and During High Altitude Acclimatization.

    PubMed

    Hilty, Matthias Peter; Müller, Andrea; Flück, Daniela; Siebenmann, Christoph; Rasmussen, Peter; Keiser, Stefanie; Auinger, Katja; Lundby, Carsten; Maggiorini, Marco

    2016-12-01

    Matthias Peter Hilty, Andrea Mueller, Daniela Flück, Christoph Siebenmann, Peter Rasmussen, Stefanie Keiser, Katja Auinger, Carsten Lundby, and Marco Maggiorini. Effect of increased blood flow on the pulmonary circulation before and during high altitude acclimatization. High Alt Med Biol. 17:305-314, 2016.-Introduction and Methods: Acute exposure to high altitude increases pulmonary artery pressure (Ppa) and pulmonary vascular resistance (PVR). The evolution of Ppa and PVR with continuous hypoxic exposure remains, however, elusive. To test the hypothesis that altitude exposure leads to a persistent elevation in Ppa and PVR throughout acclimatization in seven healthy male subjects, echocardiography was performed at sea level (SL; 488 m) weekly during a 4-week sojourn at 3454 m (HA1-HA4) and upon return (SL2). Pulmonary artery catheterization and bilateral thigh cuff release maneuver were performed at SL and HA3 to study the properties of pulmonary circulation after 3 weeks of acclimatization. Pulmonary artery catheter determined that systolic Ppa (mean ± SEM) was increased from 20 ± 1 at SL to 27 ± 2 mmHg at HA3 (p < 0.01). Echocardiography assessed that systolic Ppa remained equally increased throughout acclimatization (26 ± 2, 25 ± 2, 25 ± 2, and 24 ± 2 mmHg at HA1-HA4; p = 0.93) and returned to baseline upon return (17 ± 2, 18 ± 1 mmHg at SL, SL2; p = 0.3). The same was shown for PVR. Right heart function remained unaffected. Thigh cuff release maneuvers at SL and HA3 resulted in similar increase in cardiac output (2.5 ± 0.5 and 2.2 ± 0.4 L/min; p = 0.61) without affecting mean Ppa. Prolonged altitude exposure leads to a persistent increase in Ppa and PVR without affecting right heart function and is fully reversible within 1 week after return to SL. The thigh cuff release maneuver-induced increase in cardiac output suggests a preserved ability of pulmonary circulation to cope with

  1. 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

  2. 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.

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

    PubMed

    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

    2016-03-15

    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.

  4. 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

  5. Membrane binding and self-association of the epsin N-terminal homology domain.

    PubMed

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

    2012-11-09

    Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate-lipid-targeting and membrane-curvature-generating element. Upon binding phosphatidylinositol 4,5-bisphosphate, the N-terminal helix (H(0)) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher-order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH-domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    SciTech Connect

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

    2007-08-10

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

  7. The N-terminal dimerization is required for TDP-43 splicing activity.

    PubMed

    Jiang, Lei-Lei; Xue, Wei; Hong, Jun-Ye; Zhang, Jun-Ting; Li, Min-Jun; Yu, Shao-Ning; He, Jian-Hua; Hu, Hong-Yu

    2017-07-21

    TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the β7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.

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

    PubMed

    Motomura, Shou; Suzuki, Tomohiko

    2016-06-01

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

  9. 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

  10. 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.

  11. The AAA+ ATPase TRIP13 remodels HORMA domains through N-terminal engagement and unfolding.

    PubMed

    Ye, Qiaozhen; Kim, Dong Hyun; Dereli, Ihsan; Rosenberg, Scott C; Hagemann, Goetz; Herzog, Franz; Tóth, Attila; Cleveland, Don W; Corbett, Kevin D

    2017-08-15

    Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed "closure motifs". The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain-closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31(comet) We show that p31(comet) binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 "pore loops", which then unfold MAD2 in the presence of ATP N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain-closure motif complexes by TRIP13. © 2017 The Authors.

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

    PubMed

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

    2002-08-01

    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.

  13. N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization

    PubMed Central

    De Napoli, MG; de Miguel, N; Lebrun, M; Moreno, SNJ; Angel, SO; Corvi, MM

    2013-01-01

    Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii. PMID:23485398

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    PubMed

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis. Copyright © 2016 by the American Society of Nephrology.

  2. 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

  3. 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

  4. 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.

  5. Associations Between Circulating Carotenoids, Genomic Instability and the Risk of High-Grade Prostate Cancer

    PubMed Central

    Nordström, Tobias; Van Blarigan, Erin L.; Ngo, Vy; Roy, Ritu; Weinberg, Vivian; Song, Xiaoling; Simko, Jeffry; Carroll, Peter R.; Chan, June M.; Paris, Pamela L.

    2017-01-01

    BACKGROUND Carotenoids are a class of nutrients with antioxidant properties that have been purported to protect against cancer. However, the reported associations between carotenoids and prostate cancer have been heterogeneous and lacking data on interactions with nucleotide sequence variations and genomic biomarkers. OBJECTIVE To examine the associations between carotenoid levels and the risk of high-grade prostate cancer, also considering antioxidant-related genes and tumor instability. METHODS We measured plasma levels of carotenoids and genotyped 20 single nucleotide polymorphisms (SNP) in SOD1, SOD2, SOD3, XRCC1, and OGG1 among 559 men with non-metastatic prostate cancer undergoing radical prostatectomy. We performed copy number analysis in a subset of these men (n =67) to study tumor instability assessed as Fraction of the Genome Altered (FGA). We examined associations between carotenoids, genotypes, tumor instability and risk of high-grade prostate cancer (Gleason grade ≥4 +3) using logistic and linear regression. RESULTS Circulating carotenoid levels were inversely associated with the risk of high-grade prostate cancer; odds ratios (OR) and 95% confidence intervals (CI) comparing highest versus lowest quartiles were: 0.34 (95% CI: 0.18–0.66) for α-carotene, 0.31 (95% CI: 0.15–0.63) for β-carotene, 0.55 (0.28–1.08) for lycopene and 0.37 (0.18–0.75) for total carotenoids. SNPs rs25489 in XRCC1, rs699473 in SOD3 and rs1052133 in OGG1 modified these associations for α-carotene, β-carotene and lycopene, respectively (P ≤0.05). The proportion of men with a high degree of FGA increased with Gleason Score (P <0.001). Among men with Gleason score ≤3 +4, higher lycopene levels were associated with lower FGA (P =0.04). CONCLUSION Circulating carotenoids at diagnosis, particularly among men carrying specific somatic variations, were inversely associated with risk of high-grade prostate cancer. In exploratory analyses, higher lycopene level was

  6. Associations between circulating carotenoids, genomic instability and the risk of high-grade prostate cancer.

    PubMed

    Nordström, Tobias; Van Blarigan, Erin L; Ngo, Vy; Roy, Ritu; Weinberg, Vivian; Song, Xiaoling; Simko, Jeffry; Carroll, Peter R; Chan, June M; Paris, Pamela L

    2016-03-01

    Carotenoids are a class of nutrients with antioxidant properties that have been purported to protect against cancer. However, the reported associations between carotenoids and prostate cancer have been heterogeneous and lacking data on interactions with nucleotide sequence variations and genomic biomarkers. To examine the associations between carotenoid levels and the risk of high-grade prostate cancer, also considering antioxidant-related genes and tumor instability. We measured plasma levels of carotenoids and genotyped 20 single nucleotide polymorphisms (SNP) in SOD1, SOD2, SOD3, XRCC1, and OGG1 among 559 men with non-metastatic prostate cancer undergoing radical prostatectomy. We performed copy number analysis in a subset of these men (n = 67) to study tumor instability assessed as Fraction of the Genome Altered (FGA). We examined associations between carotenoids, genotypes, tumor instability and risk of high-grade prostate cancer (Gleason grade ≥ 4 + 3) using logistic and linear regression. Circulating carotenoid levels were inversely associated with the risk of high-grade prostate cancer; odds ratios (OR) and 95% confidence intervals (CI) comparing highest versus lowest quartiles were: 0.34 (95% CI: 0.18-0.66) for α-carotene, 0.31 (95% CI: 0.15-0.63) for β-carotene, 0.55 (0.28-1.08) for lycopene and 0.37 (0.18-0.75) for total carotenoids. SNPs rs25489 in XRCC1, rs699473 in SOD3 and rs1052133 in OGG1 modified these associations for α-carotene, β-carotene and lycopene, respectively (P ≤ 0.05). The proportion of men with a high degree of FGA increased with Gleason Score (P < 0.001). Among men with Gleason score ≤ 3 + 4, higher lycopene levels were associated with lower FGA (P = 0.04). Circulating carotenoids at diagnosis, particularly among men carrying specific somatic variations, were inversely associated with risk of high-grade prostate cancer. In exploratory analyses, higher lycopene level was associated with less

  7. 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.

  8. 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.

  9. High-resolution 3D MR microangiography of the rat ocular circulation.

    PubMed

    Shih, Yen-Yu I; Muir, Eric R; Li, Guang; De La Garza, Bryan H; Duong, Timothy Q

    2012-07-01

    To develop high-spatial-resolution magnetic resonance (MR) microangiography techniques to image the rat ocular circulation. Animal experiments were performed with institutional Animal Care Committee approval. MR microangiography (resolution, 84×84×84 μm or 42×42×84 μm) of the rat eye (eight rats) was performed by using a custom-made small circular surface coil with an 11.7-T MR unit before and after monocrystalline iron oxide nanoparticle (MION) injection. MR microangiography measurements were made during air, oxygen, and carbogen inhalation. From three-dimensional MR microangiography, the retina was virtually flattened to enable en face views of various retinal depths, including the retinal and choroidal vascular layers. Signal intensity changes within the retinal or choroidal arteries and veins associated with gas challenges were analyzed. Statistical analysis was performed by using paired t tests, with P<.05 considered to indicate a significant difference. Bonferroni correction was used to adjust for multiple comparisons. The central retinal artery, long posterior ciliary arteries, and choroidal vasculature could be distinguished on MR microangiograms of the eye. With MR microangiography, retinal arteries and veins could be distinguished on the basis of blood oxygen level-dependent contrast. Carbogen inhalation-enhanced MR microangiography signal intensity in both the retina (P=.001) and choroid (P=.027) compared with oxygen inhalation. Carbogen inhalation showed significantly higher signal intensity changes in the retinal arteries (P=.001, compared with oxygen inhalation), but not in the veins (P=.549). With MION administration, MR microangiography depicted retinal arterial vasoconstriction when the animals were breathing oxygen (P=.02, compared with animals breathing air). MR microangiography of the eye allows depth-resolved imaging of small angiographic details of the ocular circulation. This approach may prove useful in studying microvascular

  10. Increased caloric intake after a high-fat preload: relation to circulating triglycerides and orexigenic peptides.

    PubMed

    Gaysinskaya, V A; Karatayev, O; Chang, G-Q; Leibowitz, S F

    2007-05-16

    To investigate mechanisms that mediate the greater food intake induced by a fat-rich diet, the present study tested an acute "preload-to-test meal" paradigm in normal-weight rats. In this paradigm, the rats were given a small high-fat (HF) compared to low-fat (LF) preload and, after an intermeal interval, allowed to consume freely on a subsequent test meal. Modified versions of this paradigm were tested to determine the robustness of the greater caloric intake induced by the HF preload while standardizing the test protocol. A HF preload of 10-15 kcals, compared to an equicaloric LF preload, significantly increased food intake by 40-50% in the subsequent test meal. This effect, a 4-6 kcal increase, was observed with HF preloads equal in energy density and palatability to the LF preloads. It was evident with preloads or test meals that were liquid or solid, preloads that were injected, test meals that had variable fat content, and natural intermeal intervals of 60-120 min. This overeating after a HF preload was invariably associated with a 2- to 3-fold increase in circulating levels of triglycerides (TG), with no change in leptin or insulin. It was also accompanied by increased expression of the orexigenic peptides, galanin in the paraventricular nucleus and orexin in the perifornical lateral hypothalamus. Moreover, if given repeatedly over several days, the HF compared to equicaloric LF preload significantly increased 24-h food intake. These results establish a protocol for studying the phenomenon of increased feeding on a HF diet under controlled conditions and suggest possible underlying mechanisms involving circulating lipids and orexigenic peptides.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. N-Terminal Fragments of Huntingtin Longer than Residue 170 form Visible Aggregates Independently to Polyglutamine Expansion.

    PubMed

    Chen, Moore Z; Mok, Sue-Ann; Ormsby, Angelique R; Muchowski, Paul J; Hatters, Danny M

    2017-01-01

    A hallmark of Huntington's disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. It remains unclear how the length of the N-terminal fragments relates to polyQ - mediated aggregation. We investigated the fundamental intracellular aggregation process of eight different-length N-terminal fragments of Htt in both short (25Q) and long polyQ (97Q). N-terminal fragments were fused to fluorescent proteins and transiently expressed in mammalian cell culture models. These included the classic exon 1 fragment (90 amino acids) and longer forms of 105, 117, 171, 513, 536, 552, and 586 amino acids based on wild-type Htt (of 23Q) sequence length nomenclature. N-terminal fragments of less than 171 amino acids only formed inclusions in polyQ-expanded form. By contrast the longer fragments formed inclusions irrespective of Q-length, with Q-length playing a negligible role in extent of aggregation. The inclusions could be classified into 3 distinct morphological categories. One type (Type A) was universally associated with polyQ expansions whereas the other two types (Types B and C) formed independently of polyQ length expansion. PolyQ-expansion was only required for fragments of less than 171 amino acids to aggregate. Longer fragments aggregated predominately through a non-polyQ mechanism, involving at least one, and probably more distinct clustering mechanisms.

  17. 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. Copyright © 2015 by the Genetics Society of America.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. 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

  4. 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

  5. Arctic storms simulated in atmospheric general circulation models under uniform high, uniform low, and variable resolutions

    NASA Astrophysics Data System (ADS)

    Roesler, E. L.; Bosler, P. A.; Taylor, M.

    2016-12-01

    The impact of strong extratropical storms on coastal communities is large, and the extent to which storms will change with a warming Arctic is unknown. Understanding storms in reanalysis and in climate models is important for future predictions. We know that the number of detected Arctic storms in reanalysis is sensitive to grid resolution. To understand Arctic storm sensitivity to resolution in climate models, we describe simulations designed to identify and compare Arctic storms at uniform low resolution (1 degree), at uniform high resolution (1/8 degree), and at variable resolution (1 degree to 1/8 degree). High-resolution simulations resolve more fine-scale structure and extremes, such as storms, in the atmosphere than a uniform low-resolution simulation. However, the computational cost of running a globally uniform high-resolution simulation is often prohibitive. The variable resolution tool in atmospheric general circulation models permits regional high-resolution solutions at a fraction of the computational cost. The storms are identified using the open-source search algorithm, Stride Search. The uniform high-resolution simulation has over 50% more storms than the uniform low-resolution and over 25% more storms than the variable resolution simulations. Storm statistics from each of the simulations is presented and compared with reanalysis. We propose variable resolution as a cost-effective means of investigating physics/dynamics coupling in the Arctic environment. Future work will include comparisons with observed storms to investigate tuning parameters for high resolution models. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000. SAND2016-7402 A

  6. 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

  7. N-terminal pro-B-type natriuretic peptide in amniotic fluid of fetuses with known or suspected cardiac load.

    PubMed

    Leufgen, Christina; Gembruch, Ulrich; Stoffel-Wagner, Birgit; Fimmers, Rolf; Merz, Waltraut M

    2017-01-01

    Myocardial dysfunction occurs in a variety of fetal disorders. Findings from adult cardiology, where n-terminal pro-B-type natriuretic peptide (nt-proBNP) is an established biomarker of left ventricular dysfunction have been extended to fetal life. Since fetal blood sampling is technically challenging we investigated amniotic fluid nt-proBNP for its suitability to diagnose fetal myocardial dysfunction. Ultrasound, Doppler examination and echocardiography was applied to classify cases and controls. Amniotic fluid nt-proBNP to amniotic fluid total protein ratio was calculated and compared to the gestational age-dependent reference intervals. In a subset of cases, fetal and maternal plasma nt-proBNP levels were determined. Specimen from 391 fetuses could be analyzed (171 cases, 220 controls). There was a high correlation between amniotic fluid and fetal blood nt-proBNP levels (r = 0.441 for cases; r = 0.515 for controls), whereas no correlation could be detected between maternal and fetal (blood and amniotic fluid) nt-proBNP concentrations. Specificity and positive likelihood ratio of amniotic fluid nt-proBNP to amniotic fluid total protein ratio were high (0.97 and 4.3, respectively). Amniotic fluid nt-proBNP measurement allows diagnostic confirmation of fetal myocardial dysfunction. It may serve as a useful adjunct in addition and correlation to existing tests of myocardial function, particularly in the context of invasive fetal therapy, where access to the amniotic cavity is part of the procedure.

  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. Insights into the Maturation of Hyperthermophilic Pyrolysin and the Roles of Its N-Terminal Propeptide and Long C-Terminal Extension

    PubMed Central

    Dai, Zheng; Fu, Heting; Zhang, Yufeng; Zeng, Jing; Tang, Bing

    2012-01-01

    Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform of Pyrococcus furiosus pyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both the N-terminal propeptide and the C-terminal portion of the long CTE, indicating that the long CTE actually consists of the C-terminal propeptide and the C-terminal extension (CTEm), which remains attached to the catalytic domain in the mature enzyme. Although the N-terminal propeptide deletion mutant PlsΔN displayed weak activity, this mutant was highly susceptible to autoproteolysis and/or thermogenic hydrolysis. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of pyrolysin into its thermostable conformation. In contrast, the C-terminal propeptide deletion mutant PlsΔC199 was converted to a mature form (mPlsΔC199), which is the same size as but less stable than mPls, suggesting that the C-terminal propeptide is not essential for folding but is important for pyrolysin hyperthermostability. Characterization of the full-length (mPls) and CTEm deletion (mPlsΔC740) mature forms demonstrated that CTEm not only confers additional stability to the enzyme but also improves its catalytic efficiency for both proteineous and small synthetic peptide substrates. Our results may provide important clues about the roles of propeptides and CTEs in the adaptation of hyperthermophilic proteases to hyperthermal environments. PMID:22504813

  10. Insights into the maturation of hyperthermophilic pyrolysin and the roles of its N-terminal propeptide and long C-terminal extension.

    PubMed

    Dai, Zheng; Fu, Heting; Zhang, Yufeng; Zeng, Jing; Tang, Bing; Tang, Xiao-Feng

    2012-06-01

    Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform of Pyrococcus furiosus pyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both the N-terminal propeptide and the C-terminal portion of the long CTE, indicating that the long CTE actually consists of the C-terminal propeptide and the C-terminal extension (CTEm), which remains attached to the catalytic domain in the mature enzyme. Although the N-terminal propeptide deletion mutant PlsΔN displayed weak activity, this mutant was highly susceptible to autoproteolysis and/or thermogenic hydrolysis. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of pyrolysin into its thermostable conformation. In contrast, the C-terminal propeptide deletion mutant PlsΔC199 was converted to a mature form (mPlsΔC199), which is the same size as but less stable than mPls, suggesting that the C-terminal propeptide is not essential for folding but is important for pyrolysin hyperthermostability. Characterization of the full-length (mPls) and CTEm deletion (mPlsΔC740) mature forms demonstrated that CTEm not only confers additional stability to the enzyme but also improves its catalytic efficiency for both proteineous and small synthetic peptide substrates. Our results may provide important clues about the roles of propeptides and CTEs in the adaptation of hyperthermophilic proteases to hyperthermal environments.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

    PubMed Central

    Chiba, Kunitoshi; Vogan, Jacob M.; Wu, Robert A.; Gill, Manraj S.; Zhang, Xiaozhu; Collins, Kathleen

    2016-01-01

    ABSTRACT Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo. PMID:27872149

  18. 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.

  19. [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.

  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. Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo.

    PubMed

    Chiba, Kunitoshi; Vogan, Jacob M; Wu, Robert A; Gill, Manraj S; Zhang, Xiaozhu; Collins, Kathleen; Hockemeyer, Dirk

    2017-02-01

    Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo. Copyright © 2017 American Society for Microbiology.

  3. Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

    PubMed Central

    Kwong, M. Y.; Harris, R. J.

    1994-01-01

    Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

  4. 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. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. 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.

  6. Human T-cells recognise N-terminally Fmoc-modified peptide.

    PubMed

    Mannering, Stuart I; Purcell, Anthony W; Honeyman, Margo C; McCluskey, James; Harrison, Leonard C

    2003-09-08

    We aimed to generate T-cell clones specific for human pre-proinsulin. An HLA DQ8, CD4+ T-cell clone that recognised a 10mer (C65-A9) peptide from pre-proinsulin was isolated. Further analysis revealed that the clone responded neither to recombinant proinsulin nor to re-synthesised C65-A9 peptide. Analysis of the original peptide revealed minor contamination (<0.5%) with an N-terminal Fmoc adduct. This peptide was synthesised and shown to stimulate the clone. Thus, Fmoc-modified peptides, which are common contaminants in synthetic peptides, can stimulate human CD4+ T-cells. This finding has important implications for the use of synthetic peptides in screening and epitope mapping studies and their use as vaccines in humans.

  7. 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

  8. Structure-Function Study of the N-terminal Domain of Exocyst Subunit Sec3

    SciTech Connect

    Baek, Kyuwon; Knödler, Andreas; Lee, Sung Haeng; Zhang, Xiaoyu; Orlando, Kelly; Zhang, Jian; Foskett, Trevor J.; Guo, Wei; Dominguez, Roberto

    2010-04-19

    The exocyst is an evolutionarily conserved octameric complex involved in polarized exocytosis from yeast to humans. The Sec3 subunit of the exocyst acts as a spatial landmark for exocytosis through its ability to bind phospholipids and small GTPases. The structure of the N-terminal domain of Sec3 (Sec3N) was determined ab initio and defines a new subclass of pleckstrin homology (PH) domains along with a new family of proteins carrying this domain. Respectively, N- and C-terminal to the PH domain Sec3N presents an additional {alpha}-helix and two {beta}-strands that mediate dimerization through domain swapping. The structure identifies residues responsible for phospholipid binding, which when mutated in cells impair the localization of exocyst components at the plasma membrane and lead to defects in exocytosis. Through its ability to bind the small GTPase Cdc42 and phospholipids, the PH domain of Sec3 functions as a coincidence detector at the plasma membrane.

  9. 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.

  10. Effect of AMOC collapse on ENSO in a high resolution general circulation model

    NASA Astrophysics Data System (ADS)

    Williamson, Mark S.; Collins, Mat; Drijfhout, Sybren S.; Kahana, Ron; Mecking, Jennifer V.; Lenton, Timothy M.

    2017-06-01

    We look at changes in the El Niño Southern Oscillation (ENSO) in a high-resolution eddy-permitting climate model experiment in which the Atlantic Meridional Circulation (AMOC) is switched off using freshwater hosing. The ENSO mode is shifted eastward and its period becomes longer and more regular when the AMOC is off. The eastward shift can be attributed to an anomalous eastern Ekman transport in the mean equatorial Pacific ocean state. Convergence of this transport deepens the thermocline in the eastern tropical Pacific and increases the temperature anomaly relaxation time, causing increased ENSO period. The anomalous Ekman transport is caused by a surface northerly wind anomaly in response to the meridional sea surface temperature dipole that results from switching the AMOC off. In contrast to a previous study with an earlier version of the model, which showed an increase in ENSO amplitude in an AMOC off experiment, here the amplitude remains the same as in the AMOC on control state. We attribute this difference to variations in the response of decreased stochastic forcing in the different models, which competes with the reduced damping of temperature anomalies. In the new high-resolution model, these effects approximately cancel resulting in no change in amplitude.

  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. 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.

  13. N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

    PubMed Central

    Rada, Petr; Makki, Abhijith Radhakrishna; Zimorski, Verena; Garg, Sriram; Hampl, Vladimír; Hrdý, Ivan; Gould, Sven B.

    2015-01-01

    Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution. PMID:26475173

  14. 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

  15. 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. Copyright 2004 Wiley Periodicals, Inc.

  16. 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.

  17. 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.

  18. 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.

  19. Angiotensin-Converting Enzyme N-Terminal Inactivation Alleviates Bleomycin-Induced Lung Injury

    PubMed Central

    Li, Ping; Xiao, Hong D.; Xu, Jianguo; Ong, Frank S.; Kwon, Mike; Roman, Jesse; Gal, Anthony; Bernstein, Kenneth E.; Fuchs, Sebastien

    2010-01-01

    Bleomycin has potent anti-oncogenic properties for several neoplasms, but drug administration is limited by bleomycin-induced lung fibrosis. Inhibition of the renin-angiotensin system has been suggested to decrease bleomycin toxicity, but the efficacy of such strategies remains uncertain and somewhat contradictory. Our hypothesis is that, besides angiotensin II, other substrates of angiotensin-converting enzyme (ACE), such as the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), play a significant role in controlling fibrosis. We studied bleomycin-induced lung injury in normotensive mice, termed N-KO and C-KO, which have point mutations inactivating either the N- or C-terminal catalytic sites of ACE, respectively. N-KO, but not C-KO mice, have a marked resistance to bleomycin lung injury as assessed by lung histology and hydroxyproline content. To determine the importance of the ACE N-terminal peptide substrate AcSDKP in the resistance to bleomycin injury, N-KO mice were treated with S-17092, a prolyl-oligopeptidase inhibitor that inhibits the formation of AcSDKP. In response to bleomycin injection, S-17092-treated N-KO mice developed lung fibrosis similar to wild-type mice. In contrast, the administration of AcSDKP to wild-type mice reduced lung fibrosis due to bleomycin administration. This study shows that the inactivation of the N-terminal catalytic site of ACE significantly reduced bleomycin-induced lung fibrosis and implicates AcSDKP in the mechanism of protection. These data suggest a possible means to increase tolerance to bleomycin and to treat fibrosing lung diseases. PMID:20651228

  20. 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

  1. 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.

  2. 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

  3. PERSPECTIVE: Intra-molecular chaperone: the role of the N-terminal in conformational selection and kinetic control

    NASA Astrophysics Data System (ADS)

    Tsai, Chung-Jung; Ma, Buyong; Nussinov, Ruth

    2009-03-01

    The vast majority of the proteins in nature are under thermodynamic control, consistent with the universally accepted notion that proteins exist in their thermodynamically most stable state. Yet, recently a number of examples of proteins whose fold is under kinetic control have come to light. Their functions and environments vary. The first among these are some proteases, discovered in the early 1990s. There, an N-terminal proregion is self-cleaved after the protein folded, leaving the remainder of the chain in a kinetically trapped state. A related scenario was observed for microcin J25, an antibacterial peptide. This peptide presents a trapped covalently knotted conformation. The third and the most recently discovered case is the multidrug-resistant transporter protein, P-glycoprotein. There, a synonymous 'silent' mutation leads to ribosome stalling with a consequent altered kinetically trapped state. Here we argue that in all three examples, the N-terminal plays the role of an intra-molecular chaperone, that is, the N-terminal conformation selects among all competing local conformations of a downstream segment. By providing a pattern, the N-terminal chaperone segment assists the protein folding process. If the N-terminal is subsequently cleaved, the protein can be under kinetic control, since it is trapped in a thermodynamically less-stable state.

  4. 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

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

    PubMed

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

    2016-12-12

    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 Ni(2+). The Ni(2+) ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni(2+) 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Mutagenesis of N-terminal residues of feline foamy virus Gag reveals entirely distinct functions during capsid formation, particle assembly, Gag processing and budding.

    PubMed

    Liu, Yang; Betts, Matthew J; Lei, Janet; Wei, Guochao; Bao, Qiuying; Kehl, Timo; Russell, Robert B; Löchelt, Martin

    2016-08-22

    Foamy viruses (FVs) of the Spumaretrovirinae subfamily are distinct retroviruses, with many features of their molecular biology and replication strategy clearly different from those of the Orthoretroviruses, such as human immunodeficiency, murine leukemia, and human T cell lymphotropic viruses. The FV Gag N-terminal region is responsible for capsid formation and particle budding via interaction with Env. However, the critical residues or motifs in this region and their functional interaction are currently ill-defined, especially in non-primate FVs. Mutagenesis of N-terminal Gag residues of feline FV (FFV) reveals key residues essential for either capsid assembly and/or viral budding via interaction with the FFV Env leader protein (Elp). In an in vitro Gag-Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Gradient sedimentation analyses of cytosolic proteins indicate that wild-type Gag is mostly assembled into virus capsids. Moreover, proteolytic processing of Gag correlates with capsid assembly and is mostly, if not completely, independent from particle budding. In addition, Gag processing correlates with the presence of packaging-competent FFV genomic RNA suggesting that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. Though an appended heterogeneous myristoylation signal rescues Gag particle budding of mutants unable to form capsids or defective in interacting with Elp, it fails to generate infectious particles that co-package Pol, as evidenced by a lack of Gag processing. Changes in proteolytic Gag processing, intracellular capsid assembly, particle budding and infectivity of defined N-terminal Gag mutants highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. Discussion of these findings will be based on a recent model developed for Gag

  15. 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.

  16. Raman optical activity demonstrates poly(L-proline) II helix in the N-terminal region of the ovine prion protein: implications for function and misfunction.

    PubMed

    Blanch, Ewan W; Gill, Andrew C; Rhie, Alexandre G O; Hope, James; Hecht, Lutz; Nielsen, Kurt; Barron, Laurence D

    2004-10-15

    The aqueous solution structure of the full-length recombinant ovine prion protein PrP(25-233), together with that of the N-terminal truncated version PrP(94-233), have been studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UVCD). A sharp positive band at approximately 1315 cm(-1) characteristic of poly(L-proline) II (PPII) helix that is present in the ROA spectrum of the full-length protein is absent from that of the truncated protein, together with bands characteristic of beta-turns. Although it is not possible similarly to identify PPII helix in the full-length protein directly from its UVCD spectrum, subtraction of the UVCD spectrum of PrP(94-233) from that of PrP(25-233) yields a difference UVCD spectrum also characteristic of PPII structure and very similar to the UVCD spectrum of murine PrP(25-113). These results provide confirmation that a major conformational element in the N-terminal region is PPII helix, but in addition show that the PPII structure is interspersed with beta-turns and that little PPII structure is present in PrP(94-233). A principal component analysis of the ROA data indicates that the alpha-helix and beta-sheet content, located in the structured C-terminal domain, of the full-length and truncated proteins are similar. The flexibility imparted by the high PPII content of the N-terminal domain region may be an essential factor in the function and possibly also the misfunction of prion proteins.

  17. 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

  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. Dissecting the Disulfide Linkage of the N-Terminal Domain of HMW 1Dx5 and Its Contributions to Dough Functionality.

    PubMed

    Wang, Jing Jing; Liu, Guang; Huang, Yan-Bo; Zeng, Qiao-Hui; Hou, Yi; Li, Lin; Ou, Shiyi; Zhang, Min; Hu, Song-Qing

    2017-08-02

    The N-terminal domain of HMW-GS 1Dx5 (1Dx5-N) contains three cysteine residues (Cys10, Cys25, Cys40), which are the basis of gluten network formation through disulfide bonds. Disulfide linkage in 1Dx5-N was dissected by site-directed mutagenesis and LC-MS/MS, and its contributions to structural and conformational stability of 1Dx5-N and dough functionality were investigated by circular dichroism, intrinsic fluorescence, surface hydrophobicity determination, size exclusion chromatography, nonreducing/reducing SDS-PAGE, atomic force microscopy, and farinographic analysis. Results showed that Cys10 and Cys40 of 1Dx5-N were the active sites for intermolecular linkage. Meanwhile, Cys40 also exhibited the ability to form intrachain disulfide linkage with Cys25. Moreover, Cys10 and Cys40 played a functionally important role in maintaining the structural and conformational stability and high surface hydrophobicity of the N-terminal domain of HMW-GS, which in turn facilitated the formation of HMW polymers and massive disulfide linkage of HMW-GS through hydrophobic interaction. Additionally, the 1Dx5-N mutants in which Cys were replaced by serine (Ser) presented different effects on dough functionality, while only the C25S mutant produced positive effects compared with wild type 1Dx5-N. Na2CO3-induced β-elimination of cystine might occur in glutenin without heating, which would make it much easier to reduce the nutritional quality of flour products by the cost of lysine. Therefore, these results give a deep understanding of the disulfide linkage of the N-terminal domain of HMW-GS and its functional importance, which will provide a practical guide to effectively generate a superior HMW-GS allele by artificial mutagenesis.

  20. 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

  1. 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

  2. Regulation of C-type Lectin Antimicrobial Activity by a Flexible N-terminal Prosegment*S⃞

    PubMed Central

    Mukherjee, Sohini; Partch, Carrie L.; Lehotzky, Rebecca E.; Whitham, Cecilia V.; Chu, Hiutung; Bevins, Charles L.; Gardner, Kevin H.; Hooper, Lora V.

    2009-01-01

    Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIγ and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity. PMID:19095652

  3. A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

    PubMed

    Jia, Haiwei; Zhang, Xiaojuan; Wang, Wenjun; Bai, Yuanyuan; Ling, Youguo; Cao, Cheng; Ma, Runlin Z; Zhong, Hui; Wang, Xue; Xu, Quanbin

    2015-02-27

    Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear. In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm. Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  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. 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.

  11. 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.

  12. 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.

  13. 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

  14. [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.

  15. 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.

  16. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. 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.

  18. The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection.

    PubMed

    Vihinen-Ranta, Maija; Wang, Dai; Weichert, Wendy S; Parrish, Colin R

    2002-02-01

    The unique N-terminal region of the parvovirus VP1 capsid protein is required for infectivity by the capsids but is not required for capsid assembly. The VP1 N terminus contains a number of groups of basic amino acids which resemble classical nuclear localization sequences, including a conserved sequence near the N terminus comprised of four basic amino acids, which in a peptide can act to transport other proteins into the cell nucleus. Testing with a monoclonal antibody recognizing residues 2 to 13 of VP1 (anti-VP1-2-13) and with a rabbit polyclonal serum against the entire VP1 unique region showed that the VP1 unique region was not exposed on purified capsids but that it became exposed after treatment of the capsids with heat (55 to 75 degrees C), or urea (3 to 5 M). A high concentration of anti-VP1-2-13 neutralized canine parvovirus (CPV) when it was incubated with the virus prior to inoculation of cells. Both antibodies blocked infection when injected into cells prior to virus inoculation, but neither prevented infection by coinjected infectious plasmid DNA. The VP1 unique region could be detected 4 and 8 h after the virus capsids were injected into cells, and that sequence exposure appeared to be correlated with nuclear transport of the capsids. To examine the role of the VP1 N terminus in infection, we altered that sequence in CPV, and some of those changes made the capsids inefficient at cell infection.

  19. 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

  20. 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.

  1. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. High density lipoprotein is positively correlated with the changes in circulating total adiponectin and high molecular weight adiponectin during dietary and fenofibrate treatment.

    PubMed

    Christou, Georgios A; Tellis, Konstantinos C; Elisaf, Moses C; Tselepis, Alexandros D; Kiortsis, Dimitrios N

    2012-01-01

    The investigation of the relationship between high density lipoprotein (HDL) and adiponectin. Thirty-seven obese or overweight [body mass index ≥27 Kg/m(2)], hypertriglyceridemic patients underwent one of the following interventions for 3 months: 1) Low-calorie diet (n=19), 2) Low-calorie diet plus fenofibrate (n=18). Circulating total adiponectin did not change significantly in the low-calorie diet group. However, in the subgroup of patients whose high density lipoprotein cholesterol (HDL-C) decreased over the first month of diet, a statistically significant reduction of the circulating total adiponectin was observed (p=0.010), while in the subgroup of patients whose HDL-C increased over the latter 2 months of the diet, an increase in circulating total adiponectin over the 2 months was found (p=0.043). The percentage change of HDL-C over the first month of diet was positively correlated with the percentage change of circulating total adiponectin (r=0.579, p=0.019). The percentage change of HDL-C over the 3 months of diet was positively correlated with the percentage changes of circulating total adiponectin (r=0.527, p=0.030) and high molecular weight (HMW) adiponectin (r=0.524, p=0.031). The change in circulating total adiponectin over the first month of diet was positively correlated with the HDL-C at 1 month (r=0.606, p=0.013). The change in HMW adiponectin over the 3 months of diet was positively correlated with the HDL-C at 3 months (r=0.602, p=0.011). The percentage change of circulating HMW adiponectin over the first month of fenofibrate treatment was positively correlated with the percentage change of HDL-C (r=0.594, p=0.012). HDL is positively correlated with the changes in circulating adiponectin during dietary and fenofibrate treatment.

  4. 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.

  5. 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.

  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. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling★

    PubMed Central

    Ma, Yinghuan; Bao, Yongxin; Li, Chenghao; Jiao, Fubin; Xin, Hongjie; Yuan, Zhengwei

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway. PMID:25337099

  8. Modifications in the purification protocol of Celosia cristata antiviral proteins lead to protein that can be N-terminally sequenced.

    PubMed

    Gholizadeh, Ashraf; Kapoor, H C

    2004-12-01

    Plants antiviral proteins are being used as anticancer agents and inhibit other viral diseases in humans. We modified the purification protocol of the two N-terminally blocked antiviral glycoproteins, CCP-25 and CCP-27, purified from the leaves of Celosia cristata. This not only gave rise to single pure samples with few steps of purification but also resulted in N-terminally free proteins. The extra purity of the samples was analyzed by reverse phase HPLC. Deglycosylation studies of CCP-25 with PNGase F enzyme revealed that its asparagine or asparagine-linked glycon contents are negligible. Partial N-terminal sequence of the CCP-25 showed the sequence (ANDIS), which seems to be conserved among plant antiviral proteins.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Nitrone protecting groups for enantiopure N-hydroxyamino acids: synthesis of N-terminal peptide hydroxylamines for chemoselective ligations.

    PubMed

    Medina, S Irene; Wu, Jian; Bode, Jeffrey W

    2010-08-07

    The synthesis of enantiopure N-benzylidene nitrones of N-hydroxy-alpha-amino acids and their incorporation using standard Fmoc-based peptide chemistry into solid-supported peptide chains is described. Deprotection and resin cleavage affords N-terminal peptide hydroxylamines, which are the key substrates for chemoselective ligations with C-terminal peptide alpha-ketoacids. This general route is applicable to a variety of different N-terminal residues and provides a general approach to the solid phase synthesis of peptide hydroxylamines.

  17. 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.

  18. The N-terminal fragment of Reelin is generated after endocytosis and released through the pathway regulated by Rab11.

    PubMed

    Hibi, Terumasa; Hattori, Mitsuharu

    2009-04-17

    Reelin is a large secreted glycoprotein essential for brain formation, but its trafficking and function at the molecular level remain incompletely understood. After binding to its receptor, Reelin is internalized by endocytosis. Here we show that internalized Reelin is subject to specific proteolysis within the cell and its N-terminal fragment is re-secreted. This re-secretion is inhibited by bafilomycin A(1) or by expression of a mutant of Rab11, a regulator of the recycling pathway. As the N-terminal fragment does not bind to Reelin receptor but has homology to F-spondin, its recycling may be involved in the regulation of extracellular matrix.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Tapered-slit membrane filters for high-throughput viable circulating tumor cell isolation.

    PubMed

    Kang, Yoon-Tae; Doh, Il; Cho, Young-Ho

    2015-04-01

    This paper presents tapered-slit membrane filters for high-throughput viable circulating tumor cell (CTC) isolation. The membrane filter with a 2D array of vertical tapered slits with a gap that is wide at the entrance and gradually decreases with depth, provide minimal cell stress and reduce 82.14% of the stress generated in conventional straight-hole filters. We designed two types of tapered-slit filters, Filters 6 and 8, respectively, containing the tapered slits with outlet widths of 6 μm and 8 μm at a slit density of 34,445/cm(2) on the membrane. We fabricated the vertical slits with a tapered angle of 2 ° on a SU8 membrane by adjusting the UV expose dose and the air gap between the membrane and the photomask during lithography. In the experimental study, the proposed tapered-slit filter captured 89.87% and 82.44% of the cancer cells spiked in phosphate buffered saline (PBS) and diluted blood (blood: PBS = 1:4), respectively, at a sample flow rate of 5 ml per hour, which is 33.3 times faster than previous lateral tapered-slit filters. We further verified the capability to culture on chip after capturing: 72.33% of cells among the captured cells still remained viable after a 5-day culture. The proposed tapered-slit membrane filters verified high-throughput viable CTC isolation capability, thereby inaugurating further advanced CTC research for cancer diagnosis and prognosis.

  4. 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

  5. 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.

  6. Role of N-Terminal Hydrophobic Region in Modulating the Subcellular Localization and Enzyme Activity of the Bisphosphate Nucleotidase from Debaryomyces hansenii

    PubMed Central

    Aggarwal, Monika; Mondal, Alok K.

    2006-01-01

    3′, 5′-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3′-phosphoadenosine-5′-phosphate to adenosine-5′-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (DHAL2) from the halotolerant yeast Debaryomyces hansenii. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism. PMID:16467467

  7. Simultaneous Rho kinase inhibition in circulating leukocytes and in cardiovascular tissue in rats with high angiotensin converting enzyme levels.

    PubMed

    Fierro, Camila; Novoa, Ulises; González, Veronica; Ocaranza, María Paz; Jalil, Jorge E

    2016-07-15

    The small guanosine triphosphatase RhoA and its direct target Rho kinase (ROCK) play important roles in cardiovascular pathophysiology. Activated ROCK phosphorylates intracellular proteins with detrimental effects on cardiovascular remodeling. Increased ROCK activity in circulating leukocytes is observed in hypertension and in heart failure, but its relationship with ROCK activation in the myocardium and vessels is unknown. We hypothesized that ROCK activation and phosphorylation/activation of some of its key downstream molecules in the heart and arterial wall are reflected in circulating leukocytes. Phosphorylation of MYPT1, ERM and p38-MAPK and levels of p65-NF-κB were determined in the left ventricle (LV), aortic wall and circulating leukocytes in rats with high (Brown Norway, BN) and low (Lewis) angiotensin converting enzyme. A group of BN rats received the ROCK inhibitor fasudil (7days). Compared to Lewis rats, in the BN group phosphorylated levels of MYPT1, ERM and p38-MAPK and levels of p65-NF-κB were increased (P<0.05) in the LV (67%, 92%, 52% and 98%, respectively); in the aortic wall (57%, 51%, 68% and 66%, respectively) and in circulating leukocytes (61%, 72%, 49% and 105%, respectively). Fasudil reduced all these levels to those observed in Lewis rats. Phosphorylated MYPT1, ERM, and p38-MAPK and levels of p65-NF-κB in circulating leukocytes were significantly correlated with their respective LV and aortic wall levels (excepting p65-NF-κB in aorta). ROCK activity in circulating leukocytes reflects activation of this signaling pathway in the myocardium and aortic wall in this model, and supports its value as a potential cardiovascular remodeling marker. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Structural analysis of the predicted coiled-coil rod domain of the cytoplasmic bullous pemphigoid antigen (BPAG1). Empirical localization of the N-terminal globular domain-rod boundary.

    PubMed

    Tang, H Y; Chaffotte, A F; Thacher, S M

    1996-04-19

    The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells. BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin. All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology. To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled-coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991) J. Biol. Chem. 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified. BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration. Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing. BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled-coil formation in the rod-like tails. The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin. These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family.

  9. Identification of the N-terminal region of TjZNT2, a Zrt/Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity.

    PubMed

    Nishida, Sho; Morinaga, Yasuhiro; Obata, Hitoshi; Mizuno, Takafumi

    2011-03-01

    The Zrt/Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn(2+) and Mn(2+) transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn(2+) uptake of yeast cells expressing them. As a result, the extracellular N-terminal ends were identified as regions involved in Zn(2+) selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn(2+) uptake activity. This suggests that the extended N-terminal region inhibits Zn(2+) transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn(2+) transport. Furthermore, the N-terminal truncated TjZNT2 lacked Mn(2+) uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters. © 2011 The Authors Journal compilation © 2011 FEBS.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

    PubMed

    Laurent, Clémentine; Lekeux, Gilles; Ukuwela, Ashwinie A; Xiao, Zhiguang; Charlier, Jean-Benoit; Bosman, Bernard; Carnol, Monique; Motte, Patrick; Damblon, Christian; Galleni, Moreno; Hanikenne, Marc

    2016-03-01

    PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

  18. 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.

  19. An N-terminal fragment of yeast ribosomal protein L3 inhibits the cytotoxicity of pokeweed antiviral protein in Saccharomyces cerevisiae.

    PubMed

    Di, Rong; Tumer, Nilgun E

    2014-04-11

    We have previously shown that ribosomal protein L3 is required for pokeweed antiviral protein (PAP), a type I ribosome inactivating protein, to bind to ribosomes and depurinate the α-sarcin/ricin loop (SRL) in yeast. Co-expression of the N-terminal 99 amino acids of yeast L3 (L3Δ99) with PAP in transgenic tobacco plants completely abolished the toxicity of PAP. In this study, we investigated the interaction between PAP and L3Δ99 in Saccharomyces cerevisiae. Yeast cells co-transformed with PAP and L3Δ99 showed markedly reduced growth inhibition and reduced rRNA depurination by PAP, compared to cells transformed with PAP alone. Co-transformation of yeast with PAP and L3Δ21 corresponding to the highly conserved N-terminal 21 amino acids of L3Δ99, reduced the cytotoxicity of PAP. PAP mRNA and protein levels were elevated and L3Δ99 or L3Δ21 mRNA and protein levels were reduced in yeast co-transformed with PAP and L3Δ99 or with PAP and L3Δ21, respectively. PAP interacted with L3Δ21 in yeast cells in vivo and by Biacore analysis in vitro, suggesting that the interaction between L3Δ21 and PAP may inhibit PAP-mediated depurination of the SRL, leading to a reduction in the cytotoxicity of PAP.

  20. 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-te