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Sample records for neuraminidase cysteine residues

  1. Identifying Functional Cysteine Residues in the Mitochondria.

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    Bak, Daniel W; Pizzagalli, Mattia D; Weerapana, Eranthie

    2017-04-21

    The mitochondria are dynamic organelles that regulate oxidative metabolism and mediate cellular redox homeostasis. Proteins within the mitochondria are exposed to large fluxes in the surrounding redox environment. In particular, cysteine residues within mitochondrial proteins sense and respond to these redox changes through oxidative modifications of the cysteine thiol group. These oxidative modifications result in a loss in cysteine reactivity, which can be monitored using cysteine-reactive chemical probes and quantitative mass spectrometry (MS). Analysis of cell lysates treated with cysteine-reactive probes enable the identification of hundreds of cysteine residues, however, the mitochondrial proteome is poorly represented (proteins and suppression of mitochondrial peptide MS signals by highly abundant cytosolic peptides. Here, we apply a mitochondrial isolation and purification protocol to substantially increase coverage of the mitochondrial cysteine proteome. Over 1500 cysteine residues from ∼450 mitochondrial proteins were identified, thereby enabling interrogation of an unprecedented number of mitochondrial cysteines. Specifically, these mitochondrial cysteines were ranked by reactivity to identify hyper-reactive cysteines with potential catalytic and regulatory functional roles. Furthermore, analyses of mitochondria exposed to nitrosative stress revealed previously uncharacterized sites of protein S-nitrosation on mitochondrial proteins. Together, the mitochondrial cysteine enrichment strategy presented herein enables detailed characterization of protein modifications that occur within the mitochondria during (patho)physiological fluxes in the redox environment.

  2. Fluorescent labeling of specific cysteine residues using CyMPL

    Science.gov (United States)

    Puljung, Michael C.

    2012-01-01

    The unique reactivity and relative scarcity of cysteine among amino acids makes it a convenient target for the site-specific chemical modification of proteins. Commercially available fluorophores and modifiers react with cysteine through a variety of electrophilic functional groups. However, it can be difficult to obtain specific labeling of a desired cysteine residue in a protein with multiple cysteines, in a mixture of proteins, or in a protein's native environment. CyMPL (Cysteine Metal Protection and Labeling) enables specific labeling by incorporating a cysteine of interest into a minimal binding site for group 12 metal ions (e.g. Cd2+ and Zn2+). These sites can be inserted into any region of known secondary structure in virtually any protein and cause minimal structural perturbation. Bound metal ions protect the cysteine from reaction while background cysteines are blocked with non-fluorescent modifiers. The metal ions are subsequently removed and the deprotected cysteine is labeled specifically. PMID:23151742

  3. Modification of Keap1 Cysteine Residues by Sulforaphane

    Science.gov (United States)

    Hu, Chenqi; Eggler, Aimee L.; Mesecar, Andrew D.; van Breemen, Richard B.

    2011-01-01

    Activation of the transcription factor NF-E2-related factor-2 (Nrf2) through modification of Kelch-like ECH-associated protein 1 (Keap1) cysteines, leading to up-regulation of the antioxidant response element (ARE), is an important mechanism of cellular defense against reactive oxygen species and xenobiotic electrophiles. Sulforaphane, occurring in cruciferous vegetables such as broccoli, is a potent natural ARE activator that functions by modifying Keap1 cysteine residues, but there are conflicting in vitro and in vivo data regarding which of these cysteine residues react. Although most biological data indicate that modification of C151 is essential for sulforaphane action, some recent studies using mass spectrometry have failed to identify C151 as a site of Keap1 sulforaphane reaction. We have reconciled these conflicting data using mass spectrometry with a revised sample preparation protocol and confirmed that C151 is indeed among the most readily modified cysteines of Keap1 by sulforaphane. Previous mass spectrometry-based studies used iodoacetamide during sample preparation to derivatize free cysteine sulfhydryl groups causing loss of sulforaphane from highly reactive and reversible cysteine residues on Keap1 including C151. By omitting iodoacetamide from the protocol and reducing sample preparation time, our mass spectrometry-based studies now confirm previous cell-based studies which showed that sulforaphane reacts with at least four cysteine residues of Keap1 including C151. PMID:21391649

  4. Chemical Protein Ubiquitylation with Preservation of the Native Cysteine Residues.

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    Yang, Kun; Li, Guorui; Gong, Ping; Gui, Weijun; Yuan, Libo; Zhuang, Zhihao

    2016-06-02

    We report a cysteine-based ligation strategy for generating a monoubiquitylated protein while preserving the native cysteine residues on the acceptor protein. In monoubiquitylation of proliferating cell nuclear antigen (PCNA) this method circumvents the need to mutate the native cysteine residues on PCNA. The chemically ubiquitylated PCNA contains a noncleavable linkage of the same length as the native isopeptide linkage. It also retains the normal function of the native Ub-PCNA in stimulating the ATPase activity of replication factor C (RFC) and lesion bypass synthesis by Polη. This method may be adapted for chemical ubiquitylation of other proteins and for site-specific modification of a target protein at a specific site through sulfhydryl chemistry. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

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    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  6. Carbon Nanotubes Facilitate Oxidation of Cysteine Residues of Proteins.

    Science.gov (United States)

    Hirano, Atsushi; Kameda, Tomoshi; Wada, Momoyo; Tanaka, Takeshi; Kataura, Hiromichi

    2017-10-19

    The adsorption of proteins onto nanoparticles such as carbon nanotubes (CNTs) governs the early stages of nanoparticle uptake into biological systems. Previous studies regarding these adsorption processes have primarily focused on the physical interactions between proteins and nanoparticles. In this study, using reduced lysozyme and intact human serum albumin in aqueous solutions, we demonstrated that CNTs interact chemically with proteins. The CNTs induce the oxidation of cysteine residues of the proteins, which is accounted for by charge transfer from the sulfhydryl groups of the cysteine residues to the CNTs. The redox reaction simultaneously suppresses the intermolecular association of proteins via disulfide bonds. These results suggest that CNTs can affect the folding and oxidation degree of proteins in biological systems such as blood and cytosol.

  7. The role of cysteine residues in the sulphate transporter, SHST1: construction of a functional cysteine-less transporter.

    Science.gov (United States)

    Howitt, Susan M

    2005-05-20

    We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulphate transport by C205 mutants was dependent on the size of the residue at this position. Alanine at position 205 resulted in a complete loss of function whereas leucine resulted in a 3-fold increase in sulphate transport relative to wild type SHST1. C205 is located in a putative intracellular loop and our results suggest that this loop may be important for sulphate transport. By replacing C205 with leucine and the other four cysteine residues with alanine, we constructed a cysteine-less variant of SHST1 that has transport characteristics indistinguishable from wild type. This construct will be useful for further structure and function studies of SHST1.

  8. Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model

    Science.gov (United States)

    Salsbury, Freddie R.; Poole, Leslie B.; Fetrow, Jacquelyn S.

    2013-01-01

    One of the most popular and simple models for the calculation of pKas from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pKas. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pKas; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pKas. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pKa values (where the calculation should reproduce the pKa within experimental error). Both the general behavior of cysteines in proteins and the perturbed pKa in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pKa should be shifted, and validation of force field parameters for cysteine residues. PMID:22777874

  9. Site-Specific Dual Functionalization of Cysteine Residue in Peptides and Proteins with 2-Azidoacrylates.

    Science.gov (United States)

    Ariyasu, Shinya; Hayashi, Hirohito; Xing, Bengang; Chiba, Shunsuke

    2017-04-19

    Herein, we report use of 2-azidoacrylates to perform site-specific dual functionalization of the cysteine residue of peptides and bovine serum albumin (BSA), a native protein containing one free cysteine residue. The sulfhydryl group of the cysteine residue could be conjugated with 2-azidoacrylates bearing various functionalities, such as fluorescent dyes under physiological aqueous buffer conditions, to afford peptide and protein conjugates anchoring an azide moiety. Successive azide-alkyne cycloaddition enables installation of the second functionality, thus affording dual-functionalized peptide- and protein-based materials.

  10. Electrostatics of cysteine residues in proteins: parameterization and validation of a simple model.

    Science.gov (United States)

    Salsbury, Freddie R; Poole, Leslie B; Fetrow, Jacquelyn S

    2012-11-01

    One of the most popular and simple models for the calculation of pK(a) s from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK(a) s. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK(a) s; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK(a) s. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK(a) values (where the calculation should reproduce the pK(a) within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK(a) in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK(a) should be shifted, and validation of force field parameters for cysteine residues. Copyright © 2012 Wiley Periodicals, Inc.

  11. Functional role and analysis of cysteine residues of the salt tolerance protein Sod2.

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    Ullah, Asad; El-Magd, Rabab Abou; Fliegel, Larry

    2014-01-01

    Sod2 is the major salt tolerance plasma membrane protein of Schizosaccharomyces pombe. It functions to remove excess intracellular sodium (or lithium) in exchange for protons. We investigated the role of cysteine residues and created a cysteine-free Sod2 protein. Each cysteine residue of the ten present was individually mutated to serine and the different proteins expressed and characterized in S. pombe. Western blotting revealed that all the individual mutant proteins were expressed. We examined the ability of the mutant proteins to confer salt tolerance to S. pombe with the endogenous Sod2 protein deleted. Only proteins with C26S and C374S mutations were partially reduced in their ability to confer salt tolerance. Additionally, they showed a change in conformation in comparison to the wild-type protein, indicated by differential sensitivity to trypsin. Deletion of all the cysteine residues of Sod2 resulted in a functional protein that was expressed in S. pombe at levels similar to the wild type and also conferred salt tolerance. The conformation of the cysteine-free Sod2 protein was not altered relative to the wild-type protein. We examined the accessibility of amino acids of the cysteineless protein present on putative extracellular loop 2. A cysteine placed at position Ala119 was accessible to externally applied [2-(trimethylammonium)ethyl] methane thiosulfonate bromide. The results demonstrate that cysteines in the Sod2 protein can be changed to serine residues resulting in an expressed, functional protein. The utility of the cysteine-free Sod2 protein for determination of topology and amino acid accessibility is demonstrated.

  12. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

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    Katja E. Menger

    2015-06-01

    Full Text Available Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT, to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

  13. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

    Science.gov (United States)

    Menger, Katja E.; James, Andrew M.; Cochemé, Helena M.; Harbour, Michael E.; Chouchani, Edward T.; Ding, Shujing; Fearnley, Ian M.; Partridge, Linda; Murphy, Michael P.

    2015-01-01

    Summary Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster. PMID:26095360

  14. Two Japanese CADASIL families exhibiting Notch3 mutation R75P not involving cysteine residue.

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    Mizuno, Toshiki; Muranishi, Manabu; Torugun, Torusunjian; Tango, Hiromi; Nagakane, Yoshinari; Kudeken, Tukasa; Kawase, Yuji; Kawabe, Kiyokazu; Oshima, Fumiko; Yaoi, Takeshi; Itoh, Kyoko; Fushiki, Shinji; Nakagawa, Masanori

    2008-01-01

    Most previously reported mutations in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) result in an odd number of cysteine residues within the epidermal growth factor (EGF)-like repeats in Notch3. We report here R75P mutation in two Japanese CADASIL families not directly involving cysteine residues located within the first EGF-like repeats. Probands in both families had repeated episodes of stroke, depression, dementia as well as T2 high-intensity lesions in the basal ganglia and periventricular white matter, but fewer white matter lesions in the temporal pole on MRI. These families provide new insights into the diagnosis and pathomechanisms of CADASIL.

  15. Contribution of cysteine residue to the properties of Listeria monocytogenes listeriolysin O.

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    Stachowiak, Radosław; Wiśniewski, Jarosław; Osińska, Olga; Bielecki, Jacek

    2009-10-01

    Listeriolysin (LLO) is the key virulence factor critical for Listeria monocytogenes pathogenesis. Listerial cytolysin belongs to the family of cholesterol-dependent cytolysins (CDCs), a group of pore-forming toxins produced by related gram-positive bacteria. Most CDCs contain a cysteine residue in the conserved undecapeptide - a sequence that is highly preserved among this group of proteins. Substitutions of cysteine do not always lead to loss of hemolytic activity, questioning the purpose of such strong conservation of this amino acid in the sequence of CDC. The properties of 3 L. monocytogenes strains, a wild type and 2 mutants expressing modified LLO within the cysteine residue, were analyzed in this work. The first of these mutants producing a toxin with cysteine to alanine substitution showed similar features to the wild type except that a thiol-reducing agent was not necessary for hemolytic activity. Another strain secreting LLO containing serine instead of cysteine exhibited strikingly different properties than the wild type. Modified toxin is independent of the reducing reagents, less stable, and shows accelerated kinetics of cytolysis in comparison with the unchanged protein. However, both mutant strains are less invasive in the cell culture model showing the important role of cysteine in L. monocytogenes virulence.

  16. Evaluation of Methods for the Calculation of the pKa of Cysteine Residues in Proteins.

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    Awoonor-Williams, Ernest; Rowley, Christopher N

    2016-09-13

    Methods for the calculation of the pKa ionizable amino acids are valuable tools for understanding pH-dependent properties of proteins. Cysteine is unique among the amino acids because of the chemical reactivity of its thiol group (S-H), which plays an instrumental role in several biochemical and regulatory functions. The acidity of noncatalytic cysteine residues is a factor in their susceptibility to chemical modification. Despite the plethora of existing pKa computing methods, no definitive protocol exists for accurately calculating the pKa's of cysteine residues in proteins. A cysteine pKa test set was developed, which is comprised of 18 cysteine residues in 12 proteins where the pKa's have been determined experimentally and an experimental structure is available. The pKa's of these residues were calculated using three methods that use an implicit solvent model (H++, MCCE, and PROPKA) and an all-atom replica-exchange thermodynamic integration approach with the CHARMM36 and AMBER ff99SB-ILDNP force fields. The models that use implicit solvation methods were generally unreliable in predicting cysteine residue pKa's, with RMSDs between 3.41 and 4.72 pKa units. On average, the explicit solvent methods performed better than the implicit solvent methods. RMSD values of 2.40 and 3.20 were obtained for simulations with the CHARMM36 and AMBER ff99SB-ILDNP force fields, respectively. Further development of these methods is necessary because the performance of the best method is similar to that of the null-model (RMSD = 2.74) and these differences in RMSD are of limited statistical significance given the small size of our test set.

  17. Covalent binding of nitrogen mustards to the cysteine-34 residue in human serum albumin

    NARCIS (Netherlands)

    Noort, D.; Hulst, A.G.; Jansen, R.

    2002-01-01

    Covalent binding of various clinically important nitrogen mustards to the cysteine-34 residue of human serum albumin, in vitro and in vivo, is demonstrated. A rapid method for detection of these adducts is presented, based on liquid chromatography-tandem mass spectrometry analysis of the adducted

  18. CONSTRUCTION OF A FUNCTIONAL LACTOSE PERMEASE DEVOID OF CYSTEINE RESIDUES

    NARCIS (Netherlands)

    VANIWAARDEN, PR; PASTORE, JC; KONINGS, WN; KABACK, HR

    1991-01-01

    By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease). Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced

  19. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...

  20. Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

    DEFF Research Database (Denmark)

    Chen, Z. W.; Jiang, C. Y.; She, Qunxin

    2005-01-01

    ). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant......Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system...... proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have...

  1. Conserved cysteine residues provide a protein-protein interaction surface in dual oxidase (DUOX) proteins.

    Science.gov (United States)

    Meitzler, Jennifer L; Hinde, Sara; Bánfi, Botond; Nauseef, William M; Ortiz de Montellano, Paul R

    2013-03-08

    Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.

  2. The masked cysteine residues in methylmalonyl-CoA mutase from Propionibacterium shermanii are essential for catalytic activity.

    Science.gov (United States)

    Roy, I

    1996-09-30

    Two masked cysteine residues have been reported in methylmalonyl-CoA mutase from Propionibacterium shermanii, Cys-535 in the alpha-subunit and Cys-517 in the beta-subunit, which are revealed only after reduction of the denatured enzyme with dithiothreitol. It has been postulated that these residues are involved in disulphide linkages to unknown thiols of low M(r). These two masked cysteine residues have been changed to an alanine, individually. Both the mutants, C535alphaA and C517betaA, were inactive. This shows that both these residues are essential for catalytic activity.

  3. Vitamin B12 Inhibits Tau Fibrillization via Binding to Cysteine Residues of Tau.

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    Rafiee, Saharnaz; Asadollahi, Kazem; Riazi, Gholamhossein; Ahmadian, Shahin; Saboury, Ali Akbar

    2017-12-20

    Two mechanisms underlie the inhibitory/acceleratory action of chemical compounds on tau aggregation including the regulation of cellular kinases and phosphatases activity and direct binding to tau protein. Vitamin B12 is one of the tau polymerization inhibitors, and its deficiency is linked to inactivation of protein phosphatase 2A and subsequently hyperphosphorylation and aggregation of tau protein. Regarding the structure and function of vitamin B12 and tau protein, we assumed that vitamin B12 is also able to directly bind to tau protein. Hence, we investigated the interaction of vitamin B12 with tau protein in vitro using fluorometry and circular dichrosim. Interaction studies was followed by investigation into the effect of vitamin B12 on tau aggregation using ThT fluorescence, circular dichroism, transmission electron microscopy, and SDS-PAGE. The results indicated that vitamin B12 interacts with tau protein and prevents fibrillization of tau protein. Blocking the cysteine residues of tau confirmed the cysteine-mediated binding of vitamin B12 to tau and showed that binding to cysteine is essential for inhibitory effect of vitamin B12 on tau aggregation. SDS-PAGE analysis indicated that vitamin B12 inhibits tau aggregation and that tau oligomers formed in the presence of vitamin B12 are mostly SDS-soluble. We propose that direct binding of vitamin B12 is another mechanism underlying the inhibitory role of vitamin B12 on tau aggregation and neurodegeneration.

  4. Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability.

    Science.gov (United States)

    Sun, Lichang; Zhou, Yan; Liu, Runxia; Li, Yanhua; Gao, Fei; Wang, Xiaomin; Fan, Hongjie; Yuan, Shishan; Wei, Zuzhang; Tong, Guangzhi

    2015-02-02

    ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Modulation of the reactivity of the essential cysteine residue of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    González-Segura, Lilian; Velasco-García, Roberto; Muñoz-Clares, Rosario A

    2002-02-01

    Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible NAD(P)(+)-dependent oxidation of betaine aldehyde to glycine betaine. In the human opportunistic pathogen Pseudomonas aeruginosa this reaction is an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. As with every aldehyde dehydrogenase studied so far, BADH possesses an essential cysteine residue involved in the formation of the intermediate thiohemiacetal with the aldehyde substrate. We report here that the chemical modification of this residue is conveniently measured by the loss in enzyme activity, which allowed us to explore its reactivity in a pH range around neutrality. The pH dependence of the observed second-order rate constant of BADH inactivation by methyl methanethiosulphonate (MMTS) suggests that at low pH values the essential cysteine residue exists as thiolate by the formation of an ion pair with a positively charged residue. The estimated macroscopic pK values are 8.6 and 4.0 for the free and ion-pair-forming thiolate respectively. The reactivity towards MMTS of both thiolate forms is notably lower than that of model compounds of similar pK, suggesting a considerable steric inhibition by the structure of the protein. Binding of the dinucleotides rapidly induced a significant and transitory increment of thiolate reactivity, followed by a relatively slow change to an almost unreactive form. Thus it seems that to gain protection against oxidation without compromising catalytic efficiency, BADH from P. aeruginosa has evolved a complex and previously undescribed mechanism, involving several conformational rearrangements of the active site, to suit the reactivity of the essential thiol to the availability of coenzyme and substrate.

  6. S-nitrosoglutathione covalently modifies cysteine residues of human carbonyl reductase 1 and affects its activity.

    Science.gov (United States)

    Hartmanová, Tereza; Tambor, Vojtěch; Lenčo, Juraj; Staab-Weijnitz, Claudia A; Maser, Edmund; Wsól, Vladimír

    2013-02-25

    Carbonyl reductase 1 (CBR1 or SDR21C1) is a ubiquitously-expressed, cytosolic, monomeric, and NADPH-dependent enzyme. CBR1 participates in apoptosis, carcinogenesis and drug resistance, and has a protective role in oxidative stress, cancer and neurodegeneration. S-Nitrosoglutathione (GSNO) represents the newest addition to its diverse substrate spectrum, which includes a wide range of xenobiotics and endogenous substances. GSNO has also been shown to covalently modify and inhibit CBR1. The aim of the present study was to quantify and characterize the resulting modifications. Of five candidate cysteines for modification by 2 mM GSNO (positions 26, 122, 150, 226, 227), the last four were analyzed using MALDI-TOF/TOF mass spectrometry and then quantified using the Selected Reaction Monitoring Approach on hyphenated HPLC with a triple quadrupole mass spectrometer. The analysis confirmed GSNO concentration-dependent S-glutathionylation of cysteines at positions 122, 150, 226, 227 which was 2-700 times higher compared to wild-type CBR1 (WT-CBR1). Moreover, a disulfide bond between neighboring Cys-226 and Cys-227 was detected. We suggest a role of these two cysteines as a redox-sensitive cysteine pair. The catalytic properties of wild-type and enzyme modified with 2 mM GSNO were also investigated by steady state kinetic experiments with various substrates. GSNO treatment of CBR1 resulted in a 2-5-fold decrease in kcat with menadione, 4-benzoylpyridine, 2,3-hexanedione, daunorubicin and 1,4-naphthoquinone. In contrast, the same treatment increased kcat for substrates containing a 1,2-diketo group in a ring structure (1,2-naphthoquinone, 9,10-phenanthrenequinone, isatin). Except for 9,10-phenanthrenequinone, all changes in kcat were at least in part compensated for by a similar change in Km, overall yielding no drastic changes in catalytic efficiency. The findings indicate that GSNO-induced covalent modification of cysteine residues affects the kinetic mechanism of CBR1

  7. Modification of cysteine residues by cyclopentenone prostaglandins: interplay with redox regulation of protein function.

    Science.gov (United States)

    Oeste, Clara L; Pérez-Sala, Dolores

    2014-01-01

    Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling. Copyright © 2013 Wiley Periodicals, Inc.

  8. The Role of Cysteine Residues in Catalysis of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Iva Machová

    Full Text Available Mycobacterium tuberculosis (MTb, the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32 is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.

  9. Chemical Interaction of Protein Cysteine Residues with Reactive Metabolites of Methyleugenol.

    Science.gov (United States)

    Feng, Yukun; Wang, Hui; Wang, Qian; Huang, Wenlin; Peng, Ying; Zheng, Jiang

    2017-02-20

    Methyleugenol (ME), an alkenylbenzene compound, is a natural ingredient of several herbs and is used as flavoring agent in foodstuffs and fragrance in cosmetics. The hepatotoxicity, cytotoxicity, and carcinogenesis of ME have been well documented, and metabolic activation has been suggested to involve in ME-induced toxicities. The objective of this study was to identify chemical identity of interactions of protein with reactive metabolites of ME. Modification of cysteine residues of protein was observed in microsomal incubations and mice after exposure to ME. Three types of protein modification derived from the corresponding epoxide, α,β-unsaturated aldehyde, and carbonium ion of ME were detected in vitro and in vivo. The protein adduction took place in time- and dose-dependent manners. Dexamethasone, ketoconazole, and l-buthionine sulfoximine increased the protein modification induced by ME, which was proportional to the hepatotoxicity of ME. The findings facilitate the understanding of mechanism action of ME toxicities.

  10. Site-directed Mutagenesis of Cysteine Residues in Phi-class Glutathione S-transferase F3 from Oryza sativa

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Hyunjoo; Lee, Juwon; Noh, Jinseok; Kong, Kwanghoon [Chung-Ang Univ., Seoul (Korea, Republic of)

    2012-12-15

    To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the K{sub m}{sup CDNB} value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

  11. Model studies on protein glycation: influence of cysteine on the reactivity of arginine and lysine residues toward glyoxal.

    Science.gov (United States)

    Schwarzenbolz, Uwe; Mende, Susann; Henle, Thomas

    2008-04-01

    Mixtures of N alpha-hippurylarginin, N alpha-hippuryllysine, and glyoxal were incubated in the absence and presence of N alpha-acetylcysteine in order to assess the individual reactivity of these nucleophilic amino acid residues. The incubations were performed under atmospheric and high hydrostatic pressure (400 MPa), and, at the same time, beta-casein was reacted with glyoxal. The results showed that arginine is the main partner for glyoxal in the absence of cysteine, whereas a lysine derivatization was not apparent. In the presence of cysteine, however, arginine was almost completely protected from the reaction, whereas a noticeable formation of lysine derivatives, mainly carboxymethyllysine, was observed. Based on these findings, a reaction mechanism is proposed to explain the influence of cysteine on the reaction.

  12. Direct determination of the redox status of cysteine residues in proteins in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Hara, Satoshi [Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503 (Japan); Tatenaka, Yuki; Ohuchi, Yuya [Dojindo Laboratories, 2025-5 Tabaru, Mashiki-machi, Kumamoto 861-2202 (Japan); Hisabori, Toru, E-mail: thisabor@res.titech.ac.jp [Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075 (Japan)

    2015-01-02

    Highlights: • A new DNA-maleimide which is cleaved by UV irradiation, DNA-PCMal, was developed. • DNA-PCMal can be used like DNA-Mal to analyze the redox state of cysteine residues. • It is useful for detecting the thiol redox status of a protein in vivo by Western blotting method. • Thus, DNA-PCMal can be a powerful tool for redox proteomics analysis. - Abstract: The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

  13. Recombinant Staphylococcal protein A with cysteine residue for preparation of affinity chromatography stationary phase and immunosensor applications

    Directory of Open Access Journals (Sweden)

    Gorbatiuk O. B.

    2015-04-01

    Full Text Available Aim. Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys for preparation of affinity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affinity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purified and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affinity chromatography stationary phase was 10–12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purification procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affinity chromatography stationary phase and formation of the bioselective element of immunosensor.

  14. Control of the activity of Rubisco from Chlamydomonas reinhardtii through the redox state of its cysteine residues

    OpenAIRE

    Sudhani, Hemanth P. K.

    2012-01-01

    RESUMEN de la MEMORIA de TESIS DOCTORAL titulada: CONTROL OF THE ACTIVITY OF RUBISCO FROM CHLAMYDOMONAS REINHARDTII THROUGH THE REDOX STATE OF ITS CYSTEINE RESIDUES. presentada por HEMANTH PHANI KUMAR SUDHANI para optar al título de DOCTOR dentro del Programa Oficial de Posgrado en BIOTECNOLOGÍA (D030-01) de la Universitat de València. Tema: La actividad del enzima fijador de carbono en eucariotas fotosintéticos, la ribulosa 1,5-bisfosfato carboxilas/oxigenasa (Rubisco), puede re...

  15. Methylmalonyl-CoA mutase from Propionibacterium shermanii. Evidence for the presence of two masked cysteine residues.

    Science.gov (United States)

    Marsh, E N; Leadlay, P F

    1989-01-01

    Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii contains no intramolecular disulphide bridges, but two of the six thiol groups in the heterodimer are only revealed after reduction of the denatured enzyme with dithiothreitol. The available evidence suggests that they are present in disulphide linkages to unknown thiols of low Mr. The two specifically masked cysteine residues are Cys-535 in the alpha-subunit and Cys-517 in the beta-subunit, which occupy exactly homologous positions in each chain. Images Fig. 1. Fig. 3. PMID:2569860

  16. Improved stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by replacement of cysteine residues

    NARCIS (Netherlands)

    Tang, Lixia; van Hylckama Vlieg, Johan E.T.; Lutje Spelberg, Jeffrey H.; Fraaije, Marco W.; Janssen, DB

    2002-01-01

    Halohydrin dehalogenase from Agrobacterium radiobacter AD1 is a homo-tetrameric protein containing three cysteines per 28 kDa subunit. Under oxidizing conditions the enzyme was found to be susceptible to inactivation which could be prevented by the addition of beta-mercaptoethanol or glycerol. Gel

  17. A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death.

    Science.gov (United States)

    Yadeta, Koste A; Elmore, James M; Creer, Athena Y; Feng, Baomin; Franco, Jessica Y; Rufian, Jose Sebastian; He, Ping; Phinney, Brett; Coaker, Gitta

    2017-01-01

    Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses. © 2017 American Society of Plant Biologists. All Rights Reserved.

  18. The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

    Directory of Open Access Journals (Sweden)

    Katsiaryna Skryhan

    Full Text Available Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1 responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1 that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2 that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1, thioredoxin m4 (Trxm4 and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC. AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%. Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%. In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these

  19. The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

    Science.gov (United States)

    Skryhan, Katsiaryna; Cuesta-Seijo, Jose A; Nielsen, Morten M; Marri, Lucia; Mellor, Silas B; Glaring, Mikkel A; Jensen, Poul E; Palcic, Monica M; Blennow, Andreas

    2015-01-01

    Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is

  20. Cysteine residues mediate high‐affinity binding of thioredoxin to ASK1

    Czech Academy of Sciences Publication Activity Database

    Kylarová, Salome; Košek, Dalibor; Petrvalská, Olivia; Pšenáková, Katarína; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

    2016-01-01

    Roč. 283, č. 20 (2016), s. 3821-3838 ISSN 1742-464X R&D Projects: GA ČR(CZ) GA14-10061S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : ASK 1 * cysteine * disulfide bond * mass spectrometry * TRX Subject RIV: CE - Biochemistry; EE - Microbiology, Virology (MBU-M) Impact factor: 3.902, year: 2016

  1. Probing the effects of cysteine residues on protein adsorption onto gold nanoparticles using wild-type and mutated GB3 proteins.

    Science.gov (United States)

    Siriwardana, Kumudu; Wang, Ailin; Vangala, Karthikeshwar; Fitzkee, Nicholas; Zhang, Dongmao

    2013-09-03

    The role of cysteine residues in the protein binding kinetics and stability on gold nanoparticles (AuNP) was studied using AuNP localized surface plasmon resonance (LSPR) in combination with an organothiol (OT) displacement method. GB3, the third IgG-binding domain of protein G, was used to model protein-AuNP adsorption. While wild-type GB3 (GB30) contains no cysteine residues, bioengineered GB3 variants containing one (GB31) and two (GB32) cysteine residues were also tested. The cysteine content has no significant effect on GB3 binding kinetics with AuNPs, and most protein adsorption occurs within the first few seconds upon protein/AuNP mixing. However, the stability of GB3 on the AuNP surface against OT displacement depends strongly on the cysteine content and the age of the AuNP/GB3 mixture. The GB30 covered AuNPs can be completely destabilized and aggregated by OTs, regardless of the age of the GB30/AuNP mixtures. Long-time incubation of GB31 or GB32 with AuNPs can stabilize AuNPs against the OT adsorption inducted aggregation. This study indicates that multiple forces involved in the GB3/AuNP interaction, and covalent binding between cysteine and AuNP is essential for a stable protein/AuNP complex.

  2. Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease.

    Science.gov (United States)

    Voss, J; Sun, J; Venkatesan, P; Kaback, H R

    1998-06-02

    To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.

  3. Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins.

    Science.gov (United States)

    Irvine, Gordon W; Stillman, Martin J

    2017-04-26

    Structural information regarding metallothioneins (MTs) has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure, and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent electrospray ionization mass spectrometry (ESI-MS), and step-wise "snapshot" ESI-MS, we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation.

  4. Pyroacm Resin: An Acetamidomethyl Derived Resin for Solid Phase Synthesis of Peptides through Side Chain Anchoring of C-Terminal Cysteine Residues.

    Science.gov (United States)

    Juvekar, Vinayak; Gong, Young Dae

    2016-02-19

    The design, synthesis and utilization of an efficient acetamidomethyl derived resin for the peptide synthesis is presented using established Fmoc and Boc protocols via side chain anchoring. Cleavage of the target peptide from the resin is performed using carboxymethylsulfenyl chloride under mild conditions which gave in situ thiol-sulfenyl protection of the cysteine residues. The utility of the resin is successfully demonstrated through applications to the syntheses of model peptides and natural products Riparin 1.1 and Riparin 1.2.

  5. The role of cysteine residues in redox regulation and protein stability of Arabidopsis thaliana starch synthase 1

    DEFF Research Database (Denmark)

    Skryhan, Katsiaryna; Cuesta-Seijo, Jose A.; Nielsen, Morten M

    2015-01-01

    Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influenc...... its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated...... is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis...... of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein...

  6. Phage display-mediated discovery of novel tyrosinase-targeting tetrapeptide inhibitors reveals the significance of N-terminal preference of cysteine residues and their functional sulfur atom.

    Science.gov (United States)

    Lee, Yu-Ching; Hsiao, Nai-Wan; Tseng, Tien-Sheng; Chen, Wang-Chuan; Lin, Hui-Hsiung; Leu, Sy-Jye; Yang, Ei-Wen; Tsai, Keng-Chang

    2015-02-01

    Tyrosinase, a key copper-containing enzyme involved in melanin biosynthesis, is closely associated with hyperpigmentation disorders, cancer, and neurodegenerative diseases, and as such, it is an essential target in medicine and cosmetics. Known tyrosinase inhibitors possess adverse side effects, and there are no safety regulations; therefore, it is necessary to develop new inhibitors with fewer side effects and less toxicity. Peptides are exquisitely specific to their in vivo targets, with high potencies and relatively few off-target side effects. Thus, we systematically and comprehensively investigated the tyrosinase-inhibitory abilities of N- and C-terminal cysteine/tyrosine-containing tetrapeptides by constructing a phage-display random tetrapeptide library and conducting computational molecular docking studies on novel tyrosinase tetrapeptide inhibitors. We found that N-terminal cysteine-containing tetrapeptides exhibited the most potent tyrosinase-inhibitory abilities. The positional preference of cysteine residues at the N terminus in the tetrapeptides significantly contributed to their tyrosinase-inhibitory function. The sulfur atom in cysteine moieties of N- and C-terminal cysteine-containing tetrapeptides coordinated with copper ions, which then tightly blocked substrate-binding sites. N- and C-terminal tyrosine-containing tetrapeptides functioned as competitive inhibitors against mushroom tyrosinase by using the phenol ring of tyrosine to stack with the imidazole ring of His263, thus competing for the substrate-binding site. The N-terminal cysteine-containing tetrapeptide CRVI exhibited the strongest tyrosinase-inhibitory potency (with an IC50 of 2.7 ± 0.5 μM), which was superior to those of the known tyrosinase inhibitors (arbutin and kojic acid) and outperformed kojic acid-tripeptides, mimosine-FFY, and short-sequence oligopeptides at inhibiting mushroom tyrosinase. Copyright © 2014 by The American Society for Pharmacology and Experimental

  7. Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

    Directory of Open Access Journals (Sweden)

    Yuanwei Zhang

    2016-04-01

    Full Text Available Finely tuned changes in cytosolic free calcium ([Ca2+]c mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS. The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs, a putative proton V-type proton ATPase (Vma5 homolog and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

  8. Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation.

    Science.gov (United States)

    Helgadóttir, Sunna; Sinapah, Sylvie; Söll, Dieter; Ling, Jiqiang

    2012-01-02

    In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions.

    Science.gov (United States)

    Petrushanko, Irina Yu; Mitkevich, Vladimir A; Lakunina, Valentina A; Anashkina, Anastasia A; Spirin, Pavel V; Rubtsov, Peter M; Prassolov, Vladimir S; Bogdanov, Nikolay B; Hänggi, Pascal; Fuller, William; Makarov, Alexander A; Bogdanova, Anna

    2017-10-01

    Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O 2 -sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na

  10. Cysteine residues 244 and 458–459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

    Directory of Open Access Journals (Sweden)

    Irina Yu. Petrushanko

    2017-10-01

    Full Text Available Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD and nucleotide binding domain (NBD of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG. Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459 were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor

  11. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    DEFF Research Database (Denmark)

    Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (S...

  12. Comparison of metal-binding strength between methionine and cysteine residues: Implications for the design of metal-binding motifs in proteins.

    Science.gov (United States)

    Krishna Deepak, R N V; Chandrakar, Brijesh; Sankararamakrishnan, Ramasubbu

    2017-05-01

    Metals play vital role in various physiological processes and are bound to biomolecules. Although cysteine sulfur is more frequently found as metal-binding ligand, methionine prefers to occur in copper-binding motifs of some proteins. To address methionine's lower preference in copper-binding sites in comparison to cysteine, we have considered copper-binding motifs (His-Cys-His-Met) from seven different high-resolution protein structures. We performed quantum chemical calculations to find out the strength of interactions between sulfur and metal ion in both Met and Cys residues. In the case of Cys, both neutral (CysH) and the deprotonated form (Cys - ) were considered. We used two different levels of theory (B3LYP and M06-2X) and the model compounds methyl propyl sulfide, ethanethiol and ethanethiolate were used to represent Met, CysH and Cys - respectively. To compare the metal-binding strength, we mutated Met in silico to CysH/Cys - and performed the calculations. We also carried out calculations with wild-type Cys present in the same metal-binding motif. On average, interactions of Met with copper ion are stronger by 13-35kcal/mol compared to CysH. However, Cys - interactions with copper is stronger than that of Met by ~250kcal/mol. We then considered the entire metal-binding motif with four residues and calculated the interaction energies with the copper ion. We also considered Met→Cys - mutation in the motif and repeated the calculations. Interaction of the wild-type motif with the copper ion is ~160kcal/mol weaker than that of mutated motif. Our studies suggest the factors that could explain why Met is not as frequently observed as Cys in the metal-binding motifs. Results of these studies will help in designing metal-binding motifs in proteins with varying interaction strengths. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Influence of protein-micelle ratios and cysteine residues on the kinetic stability and unfolding rates of human mitochondrial VDAC-2.

    Directory of Open Access Journals (Sweden)

    Svetlana Rajkumar Maurya

    Full Text Available Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2, one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 - 13,000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol(-1 for full-length hVDAC-2 and ∼23 kcal mol(-1 for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis.

  14. Herpes Simplex Virus Type 1 Glycoprotein B Requires a Cysteine Residue at Position 633 for Folding, Processing, and Incorporation into Mature Infectious Virus Particles

    Science.gov (United States)

    Laquerre, Sylvie; Anderson, Dina B.; Argnani, Rafaela; Glorioso, Joseph C.

    1998-01-01

    Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) resides in the virus envelope in an oligomeric form and plays an essential role in virus entry into susceptible host cells. The oligomerizing domain is a movable element consisting of amino acids 626 to 653 in the gB external domain. This domain contains a single cysteine residue at position 633 (Cys-633) that is predicted to form an intramolecular disulfide bridge with Cys-596. In this study, we examined gB oligomerization, processing, and incorporation into mature virus during infection by two mutant viruses in which either the gB Cys-633 [KgB(C633S)] or both Cys-633 and Cys-596 [KgB(C596S/C633S)] residues were mutated to serine. The result of immunofluorescence studies and analyses of released virus particles showed that the mutant gB molecules were not transported to the cell surface or incorporated into mature virus envelopes and thus infectious virus was not produced. Immunoprecipitation studies revealed that the mutant gB molecules were in an oligomeric configuration and that these mutants produced hetero-oligomers with a truncated form of gB consisting of residues 1 to 43 and 595 to 904, the latter containing the oligomerization domain. Pulse-chase experiments in combination with endoglycosidase H treatment determined that the mutant molecules were improperly processed, having been retained in the endoplasmic reticulum (ER). Coimmunoprecipitation experiments revealed that the cysteine mutations resulted in gB misfolding and retention by the molecular chaperones calnexin, calreticulin, and Grp78 in the ER. The altered conformation of the gB mutant glycoproteins was directly detected by a reduction in monoclonal antibody recognition of two previously defined distinct antigenic sites located within residues 381 to 441 and 595 to 737. The misfolded molecules were not transported to the cell surface as hetero-oligomers with wild-type gB, suggesting that the conformational change could not be corrected by

  15. Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Eun Jin Kim; Jian Feng; Matthew R. Bramlett; Paul A. Lindahl

    2004-05-18

    OAK-B135 Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was ''rescued'' by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also ''rescued'' by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster. Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

  16. Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

    International Nuclear Information System (INIS)

    Eun Jin Kim; Jian Feng; Bramlett, Matthew R.; Lindahl, Paul A.

    2004-01-01

    OAK-B135 Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was ''rescued'' by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also ''rescued'' by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster. Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity

  17. Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1.

    Science.gov (United States)

    Zhang, Xu; Lu, Jun; Ren, Xiaoyuan; Du, Yatao; Zheng, Yujuan; Ioannou, Panayiotis V; Holmgren, Arne

    2015-12-01

    Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)3, As(GS)3, As(Penicillamine)3, As(Mercaptoethanesulfonate)3, As(Mercaptopurine)3, As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)3 which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 showed stronger cytotoxicity than the others. As(2-mercaptopyridine)3 which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)3 oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Role of cysteine residues in the structure, stability, and alkane producing activity of cyanobacterial aldehyde deformylating oxygenase.

    Directory of Open Access Journals (Sweden)

    Yuuki Hayashi

    Full Text Available Aldehyde deformylating oxygenase (AD is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production in vitro and in vivo. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from Nostoc punctiforme PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

  19. Inhibition of neuraminidase by Ganoderma triterpenoids and implications for neuraminidase inhibitor design

    Science.gov (United States)

    Zhu, Qinchang; Bang, Tran Hai; Ohnuki, Koichiro; Sawai, Takashi; Sawai, Ken; Shimizu, Kuniyoshi

    2015-01-01

    Neuraminidase (NA) inhibitors are the dominant antiviral drugs for treating influenza in the clinic. Increasing prevalence of drug resistance makes the discovery of new NA inhibitors a high priority. Thirty-one triterpenoids from the medicinal mushroom Ganoderma lingzhi were analyzed in an in vitro NA inhibition assay, leading to the discovery of ganoderic acid T-Q and TR as two inhibitors of H5N1 and H1N1 NAs. Structure-activity relationship studies revealed that the corresponding triterpenoid structure is a potential scaffold for the design of NA inhibitors. Using these triterpenoids as probes we found, through further in silico docking and interaction analysis, that interactions with the amino-acid residues Arg292 and/or Glu119 of NA are critical for the inhibition of H5N1 and H1N1. These findings should prove valuable for the design and development of NA inhibitors. PMID:26307417

  20. Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.

    Science.gov (United States)

    Kobayashi, M; Izui, H; Nagasawa, T; Yamada, H

    1993-01-01

    Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163. Images PMID:8419930

  1. Dominant negative mutant of ionotropic glutamate receptor subunit GluR3: implications for the role of a cysteine residue for its channel activity and pharmacological properties.

    Science.gov (United States)

    Watase, K; Sekiguchi, M; Matsui, T A; Tagawa, Y; Wada, K

    1997-01-01

    We reported that a 33-amino-acid deletion (from tyrosine-715 to glycine-747) in a putative extracellular loop of GluR3 produced a mutant that exhibited dominant negative effects upon the functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [Sekiguchi et al. (1994) J. Biol. Chem. 269, 14559-14565]. In this study, we searched for a key residue in the dominant negative effects to explore the mechanism and examined the role of the residue in the function of the AMPA receptor. We prepared 20 GluR3 mutants with amino acid substitutions within the 33-amino-acid-region, and dominant negative effects were tested electrophysiologically in Xenopus oocytes co-expressing the mutant and normal subunits. Among the mutants, only a GluR3 mutant in which an original cysteine (Cys)-722 was replaced by alanine exhibited a dominant negative effect comparable with that of the original mutant in which the entire 33-amino-acid segment is deleted. The co-expression of the Cys-722 mutant did not inhibit the translation of normal subunits in oocytes. The Cys-722 mutant formed a functional homomeric receptor with significantly higher affinity for glutamate or kainate than a homomeric GluR3 receptor. The Cys-722 mutation greatly enhanced the sensitivity of GluR3 for aniracetam, which alters kinetic properties of AMPA receptors. The kainate-induced currents in oocytes expressing the Cys-722 mutant alone showed strong inward rectification. These results suggest that the Cys-722 in GluR3 is important for dominant negative effects and plays a crucial role in the determination of pharmacological properties in AMPA receptor function. PMID:9065754

  2. Three cysteine residues of SLC52A1, a receptor for the porcine endogenous retrovirus-A (PERV-A), play a critical role in cell surface expression and infectivity.

    Science.gov (United States)

    Colon-Moran, Winston; Argaw, Takele; Wilson, Carolyn A

    2017-07-01

    Porcine endogenous retrovirus-A (PERV-A), a gammaretrovirus, infects human cells in vitro, thus raising the potential risk of cross-species transmission in xenotransplantation. Two members of the solute carrier family 52 (SLC52A1 and SLC52A2) are PERV-A receptors. Site-directed mutagenesis of the cDNA encoding SLC52A1 identified that only one of two putative glycosylation signals is occupied by glycans. In addition, we showed that glycosylation of SLC52A1 is not necessary for PERV-A receptor function. We also identified that at a minimum, three cysteine residues are sufficient for SLC52A1 cell surface expression. Mutation of cysteine at position 365 and either of the two cysteine residues in the C-terminal tail at positions 442 or 446 reduced SLC52A1 surface expression and PERV-A infection suggesting that these residues may contribute to overall structural stability and receptor function. Understanding interactions between PERV-A and its cellular receptor may provide novel strategies to prevent zoonotic infection in the setting of xenotransplantation. Published by Elsevier Inc.

  3. Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

    2017-04-01

    Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  4. Computational study of the covalent bonding of microcystins to cysteine residues--a reaction involved in the inhibition of the PPP family of protein phosphatases.

    Science.gov (United States)

    Pereira, Susana R; Vasconcelos, Vítor M; Antunes, Agostinho

    2013-01-01

    Microcystins (MCs) are cyclic peptides, produced by cyanobacteria, that are hepatotoxic to mammals. The toxicity mechanism involves the potent inhibition of protein phosphatases, as the toxins bind the catalytic subunits of five enzymes of the phosphoprotein phosphatase (PPP) family of serine/threonine-specific phosphatases: Ppp1 (aka PP1), Ppp2 (aka PP2A), Ppp4, Ppp5 and Ppp6. The interaction with the proteins includes the formation of a covalent bond with a cysteine residue. Although this reaction seems to be accessory for the inhibition of PPP enzymes, it has been suggested to play an important part in the biological role of MCs and furthermore is involved in their nonenzymatic conjugation to glutathione. In this study, the molecular interaction of microcystins with their targeted PPP catalytic subunits is reviewed, including the relevance of the covalent bond for overall inhibition. The chemical reaction that leads to the formation of the covalent bond was evaluated in silico, both thermodynamically and kinetically, using quantum mechanical-based methods. As a result, it was confirmed to be a Michael-type addition, with simultaneous abstraction of the thiol hydrogen by a water molecule, transfer of hydrogen from the water to the α,β-unsaturated carbonyl group of the microcystin and addition of the sulfur to the β-carbon of the microcystin moiety. The calculated kinetics are in agreement with previous experimental results that had indicated the reaction to occur in a second step after a fast noncovalent interaction that inhibited the enzymes per se. © 2011 The Authors Journal compilation © 2011 FEBS.

  5. Chemical Protein Modification through Cysteine.

    Science.gov (United States)

    Gunnoo, Smita B; Madder, Annemieke

    2016-04-01

    The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance--both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Neuraminidase-1 contributes significantly to the degradation of neuronal B-series gangliosides but not to the bypass of the catabolic block in Tay–Sachs mouse models

    Directory of Open Access Journals (Sweden)

    Z.K. Timur

    2015-09-01

    Full Text Available Tay–Sachs disease is a severe lysosomal storage disorder caused by mutations in the HEXA gene coding for α subunit of lysosomal β-Hexosaminidase A enzyme, which converts GM2 to GM3 ganglioside. HexA−/− mice, depleted of the β-Hexosaminidase A iso-enzyme, remain asymptomatic up to 1 year of age because of a metabolic bypass by neuraminidase(s. These enzymes remove a sialic acid residue converting GM2 to GA2, which is further degraded by the still intact β-Hexosaminidase B iso-enzyme into lactosylceramide. A previously identified ganglioside metabolizing neuraminidase, Neu4, is abundantly expressed in the mouse brain and has activity against gangliosides like GM2 in vitro. Neu4−/− mice showed increased GD1a and decreased GM1 ganglioside in the brain suggesting the importance of the Neu4 in ganglioside catabolism. Mice with targeted disruption of both HexA and Neu4 genes showed accumulating GM2 ganglioside and epileptic seizures with 40% penetrance, indicating that the neuraminidase Neu4 is a modulatory gene, but may not be the only neuraminidase contributing to the metabolic bypass in HexA−/− mice. Therefore, we elucidated the biological role of neuraminidase-1 in ganglioside degradation in mouse. Analysis of HexA−/−Neu1−/− and HexA−/−Neu4−/−Neu1−/− mice models showed significant contribution of neuraminidase-1 on B-series ganglioside degradation in the brain. Therefore, we speculate that other neuraminidase/neuraminidases such as Neu2 and/or Neu3 might be also involved in the ganglioside degradation pathway in HexA−/− mice.

  7. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.

    Science.gov (United States)

    Pan, Xuefang; De Aragão, Camila De Britto Pará; Velasco-Martin, Juan P; Priestman, David A; Wu, Harry Y; Takahashi, Kohta; Yamaguchi, Kazunori; Sturiale, Luisella; Garozzo, Domenico; Platt, Frances M; Lamarche-Vane, Nathalie; Morales, Carlos R; Miyagi, Taeko; Pshezhetsky, Alexey V

    2017-08-01

    Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, G M3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of G M1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of G M2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides. © FASEB.

  8. Protein modification by acrolein: Formation and stability of cysteine adducts

    OpenAIRE

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2009-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

  9. Regulation of neuraminidase expression in Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Gualdi Luciana

    2012-09-01

    Full Text Available Abstract Background Sialic acid (N-acetylneuraminic acid; NeuNAc is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung. Results In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells. Conclusions The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.

  10. Zinc-Binding Cysteines: Diverse Functions and Structural Motifs

    Science.gov (United States)

    Pace, Nicholas J.; Weerapana, Eranthie

    2014-01-01

    Cysteine residues are known to perform essential functions within proteins, including binding to various metal ions. In particular, cysteine residues can display high affinity toward zinc ions (Zn2+), and these resulting Zn2+-cysteine complexes are critical mediators of protein structure, catalysis and regulation. Recent advances in both experimental and theoretical platforms have accelerated the identification and functional characterization of Zn2+-bound cysteines. Zn2+-cysteine complexes have been observed across diverse protein classes and are known to facilitate a variety of cellular processes. Here, we highlight the structural characteristics and diverse functional roles of Zn2+-cysteine complexes in proteins and describe structural, computational and chemical proteomic technologies that have enabled the global discovery of novel Zn2+-binding cysteines. PMID:24970223

  11. Quantitative reactivity profiling predicts functional cysteines in proteomes.

    Science.gov (United States)

    Weerapana, Eranthie; Wang, Chu; Simon, Gabriel M; Richter, Florian; Khare, Sagar; Dillon, Myles B D; Bachovchin, Daniel A; Mowen, Kerri; Baker, David; Cravatt, Benjamin F

    2010-12-09

    Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

  12. Homology modelling and docking studies on Neuraminidase enzyme as a natural product target for combating influenza

    Directory of Open Access Journals (Sweden)

    Nisha Singh

    2017-10-01

    Full Text Available Influenza remains to be dreadful with yearly epidemics and sudden pandemic outbreaks causing significant mortality, even in nations with the most advanced health care systems. Thus, there has been a long-standing interest to develop effective and safe antiviral agents to treat infected individuals. Attempt to identify suitable molecular targets as antiviral compounds have focused recently on the influenza virus neuraminidase (NA, a key enzyme in viral replication [1]. In this research, virtual screening was done on a total of 600 natural compounds from 22 ethno medicinal Indian herbs for activity against neuraminidase enzyme exploiting representative protein conformations selected from molecular dynamics simulations. Neuraminidase enzyme sequences from different existing strains available on National Center of Biotechnology Information [2] (NCBI protein database were aligned using Clustal W [3] and CLC workbench 10 [4] to find the conserved residues. Neuraminidase protein sequence from H1N1 strain available on NCBI was used to structure 3D target model predicted against dataset from Protein data bank using modeller [5]. The target model was validated on different parameter at SAVES Server [6]. Using this target model a pharmacophore model was developed using ligand based strategy exploiting the three known inhibitors. The docking parameters were validated by redocking Zanamivir to its co-complex 2009 H1N1 NA crystal structure (PDB ID: 3TI5 generating best pose with a RMSD value of 0.7543 A°. This model was then used for in silico analysis of a library of natural compounds from 22 ethno medicinal Indian herbs known to have antiviral activity taken downloaded from PubChem database and selected on the basis of drug likeliness. All the compounds were docked in the binding pocket of neuraminidase. Top compounds having binding affinity better than or comparable to the control drug Zanamivir were selected and analyzed for their ADME and toxicity

  13. Cysteine proteinases and cystatins

    Directory of Open Access Journals (Sweden)

    Adeliana S. Oliveira

    2003-01-01

    Full Text Available This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in the specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction.Nesta revisão foram descritas definições, localizações, funções e exemplos de proteinases cisteínicas e suas proteinas inibidoras em animais vertebrados e invertebrados e plantas. Tratamos principalmente com aqueles inibidores que são relatados com o mecanismo de defesa da planta contra pestes. Em adição, comentamos sobre recentes trabalhos que contribuíram para uma melhor compreenção dos fatores envolvidos na interação específica proteinase cisteínica-cistatina. Por outro lado, chamamos atenção para o alto grau de afinidade e grande especificidade na interação que não são apenas representadas pela compatibilidade entre os residuos de aminoácidos do sítio ativo envolvidos na catalise, mas também de todos os resíduos de aminoácidos que participam da interação enzima-inibidor.

  14. Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

    Science.gov (United States)

    Lórenz-Fonfría, Víctor A.; Muders, Vera; Schlesinger, Ramona; Heberle, Joachim

    2014-12-01

    Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of "dangling" water molecules were detected in two different states of the protein using H218O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm-1) and with the other O-H group medium (3440 cm-1) to moderately strongly (3300 cm-1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.

  15. The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection.

    Science.gov (United States)

    Ma, Hongfang; Jiang, Longguang; Qiao, Songlin; Zhi, Yubao; Chen, Xin-Xin; Yang, Yanyan; Huang, Xiaojing; Huang, Mingdong; Li, Rui; Zhang, Gai-Ping

    2017-02-01

    Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus. PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our

  16. Cloning of neuraminidase (NA) gene and identification of its antiviral ...

    African Journals Online (AJOL)

    Neuraminidase not only works as an antigen, inducing target-specific antibodies, but also plays a role of enzyme activity and destroys the sialic acid receptor required by virus infection of the host cell surface which protects the host from virus damage. In order to explore a new idea to use neuraminidase (NA) gene and ...

  17. Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.

    Science.gov (United States)

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-16

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    Science.gov (United States)

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

  19. Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.

    Science.gov (United States)

    Komaru, Keiichi; Ishida, Yoko; Amaya, Yoshihiro; Goseki-Sone, Masae; Orimo, Hideo; Oda, Kimimitsu

    2005-04-01

    In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

  20. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

    Directory of Open Access Journals (Sweden)

    Kun Zhang

    2017-01-01

    Full Text Available Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1 can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1 and three mutant SOD1s (mhSOD1s, in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

  1. Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro.

    Science.gov (United States)

    Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

    2014-12-06

    Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-L-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  2. New iodo-acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido-redox status.

    Science.gov (United States)

    Bruschi, Maurizio; Grilli, Stefano; Candiano, Giovanni; Fabbroni, Serena; Della Ciana, Leopoldo; Petretto, Andrea; Santucci, Laura; Urbani, Andrea; Gusmano, Rosanna; Scolari, Francesco; Ghiggeri, Gian Marco

    2009-01-01

    Two new iodoacetamide-substituted cyanines, C3NIASO3 and C5NIASO3, were synthesized starting from hemicyanine and were utilized for labeling plasma proteins. Specificity, sensitivity and feasibility for SH residues was tested utilizing an equimolar mixture of standard proteins and with normal plasma. Oxidized plasma proteins following H(2)O(2 )exposure and plasma from patients with focal glomerulosclerosis were analyzed as models of altered protein oxido-redox status. Following optimization of the assay (dye/protein ratio, pH), C3NIASO3 and C5NIASO3 gave a sensitivity slightly better than N-hydroxysuccinimidyl dyes for plasma proteins and were successfully employed for differential display electrophoresis (DIGE). Twenty-nine proteins were detected in normal plasma after 2-DE while less proteins were detected in plasma of patients with glomerulosclerosis. Following massive 'in vitro' oxidation with H(2)O(2), C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis of two new iodoacetamide cyanines that can be utilized for the analysis of plasma proteins with 2-DE and DIGE. They are also indicated for the definition of the oxido-redox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.

  3. Identification in the mu-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents.

    Science.gov (United States)

    Gaibelet, G; Capeyrou, R; Dietrich, G; Emorine, L J

    1997-05-19

    Inactivation by thiol reducing and alkylating agents of ligand binding to the human mu-opioid receptor was examined. Dithiothreitol reduced the number of [3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the mu receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N-ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC50 = 20 microM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC50 = 2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N-ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the mu-opioid receptor.

  4. Edwardsiella tarda Ivy, a lysozyme inhibitor that blocks the lytic effect of lysozyme and facilitates host infection in a manner that is dependent on the conserved cysteine residue.

    Science.gov (United States)

    Wang, Chong; Hu, Yong-hua; Sun, Bo-guang; Li, Jun; Sun, Li

    2013-10-01

    Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenic E. tarda strain TX01. IvyEt possesses the Ivy signature motif CKPHDC in the form of (82)CQPHNC(87) and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of the ivyEt gene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt (rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared. In vitro studies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme on E. tarda. In vivo analysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEt is a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

  5. Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

    Science.gov (United States)

    Parker, W. Ryan; Brodbelt, Jennifer S.

    2016-08-01

    Characterization of the cysteine content of proteins is a key aspect of proteomics. By defining both the total number of cysteines and their bound/unbound state, the number of candidate proteins considered in database searches is significantly constrained. Herein we present a methodology that utilizes 266 nm UVPD to count the number of free and bound cysteines in intact proteins. In order to attain this goal, proteins were derivatized with N-(phenylseleno)phthalimide (NPSP) to install a selectively cleavable Se-S bond upon 266 UVPD. The number of Se-S bonds cleaved upon UVPD, a process that releases SePh moieties, corresponds to the number of cysteine residues per protein.

  6. Neuraminidase Inhibitor Susceptibility Testing in Human Influenza Viruses: A Laboratory Surveillance Perspective

    Science.gov (United States)

    Okomo-Adhiambo, Margaret; Sleeman, Katrina; Ballenger, Kristina; Nguyen, Ha T.; Mishin, Vasiliy P.; Sheu, Tiffany G.; Smagala, James; Li, Yan; Klimov, Alexander I.; Gubareva, Larisa V.

    2010-01-01

    Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008–2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC50s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC50 >3 interquartile ranges (IQR) from the 75th percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance. PMID:21994620

  7. Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans.

    Science.gov (United States)

    Kilbourne, E D

    1976-10-01

    Groups of college students received either conventional A/England/42/72 (H3N2) vaccine (X-37), an antigenic hybrid (Heq1N2) vaccine (X-38) containing the same neuraminidase (and thus effectively neuraminidase-monospecific), or a placebo injection. The vaccines contained 798 and 643 chick cell-agglutinating units per dose, respectively, and equivalent immunogenic units of N2 as defined in antigenic extinction tests in rabbits. All subjects had antibody to N2 before immunization, and mean initial titers were comparable in both vaccine groups. Homotypic hemagglutination-inhibition response to vaccine hemagglutinin was slightly more frequent (77%) but of lower magnitude in the students vaccinated with X-38 than in those vaccinated with X-37. Significant antibody response to N2 was observed in 25% of those vaccinated with X-37 and in 69% of those vaccinated with X-38. Mean antibody response to N2 was twofold greater in those vaccinated with X-38. Heterotypic hemagglutination-inhibition was seen in 56% of those receiving X-38 vaccine. In preliminary plaque-inhibition titrations this heterotypic antibody did not have neuralizing activity. Testing of antibody response to N2 with earlier neuraminidase antigens demonstrated "original antigenic sin" from earlier priming. The superiority of the "neuraminidase-specific" X-38 (Heq1N2) vaccine as an immunogen for antibody to neuraminidase may reflect different processing of N2 when it is associated with a hemagglutinin to which the study population has not been previously exposed.

  8. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation.

    Directory of Open Access Journals (Sweden)

    Aerin Yoon

    Full Text Available For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv, we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7 and two VH (H13 and H16 mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

  9. Neuraminidase-Mediated, NKp46-Dependent Immune-Evasion Mechanism of Influenza Viruses

    Directory of Open Access Journals (Sweden)

    Yotam Bar-On

    2013-04-01

    Full Text Available Natural killer (NK cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA protein. By using various NA blockers, we show that NA removes sialic acid residues from NKp46 and that this leads to reduced recognition of HA. Furthermore, we provide in vivo and in vitro evidence for the existence of this NA-mediated, NKp46-dependent immune-evasion mechanism and demonstrate that NA inhibitors, which are commonly used for the treatment of influenza infections, are useful not only as blockers of virus budding but also as boosters of NKp46 recognition.

  10. Cysteine-rich mini-proteins in human biology.

    Science.gov (United States)

    Lavergne, Vincent; Taft, Ryan J; Alewood, Paul F

    2012-01-01

    Understanding the relationship between structure and function underpins both biochemistry and chemical biology, and has enabled the discovery of numerous agricultural and therapeutic agents. Small cysteine-rich proteins, which form a unique set of protein frameworks and folds, are found in all living organisms and often play crucial roles as hormones, growth factors, ion channel modulators and enzyme inhibitors in various biological pathways. Here we review secreted human cysteine-rich mini-proteins, classify them into broad families and briefly describe their structure and function. To systematically investigate this protein sub-class we designed a step-wise high throughput algorithm that is able to isolate the mature and active forms of human secreted cysteine-rich proteins (up to 200 amino acids in length) and extract their cysteine scaffolds. We limited our search to frameworks that contain an even number of cysteine residues (cysteine-rich frameworks spread over 378 secreted cysteine-rich mini-proteins. Restricting our search to those that contain >5% cysteine residues led to the identification of 22 cysteine-rich frameworks representing 21 protein families. Analysis of their molecular targets showed that these mini-proteins are frequently ligands for G protein- and enzyme-coupled receptors, transporters, extracellular enzyme inhibitors, and antimicrobial peptides. It is clear that these human secreted mini-proteins possess a wide diversity of frameworks and folds, some of which are conserved across the phylogenetic spectrum. Further study of these proteins will undoubtedly lead to insights into unresolved questions of basic biology, and the development of system-specific human therapeutics.

  11. Electrostatic influence of local cysteine environments on disulfide exchange kinetics.

    Science.gov (United States)

    Snyder, G H; Cennerazzo, M J; Karalis, A J; Field, D

    1981-11-10

    The ionic strength dependence of the bimolecular rate constant for reaction of the negative disulfide 5,5'-dithiobis (2-nitrobenzoic acid) with cysteines in fragments of naturally occurring proteins was determined by stopped-flow spectroscopy. The Debye-Hückel relationship was applied to determine the effective charge at the cysteine and thereby determine the extent to which nearby neighbors in the primary sequence influence the kinetics. Corrections for the secondary salt effect on cysteine pKs were determined by direct spectrometric pH titration of sulfhydryl groups or by observation of the ionic strength dependence of kinetics of cysteine reaction with the neutral disulfide 2,2'-dithiodipyridine. Quantitative expressions was verified by model studies with N-acetyl-cystein. At ionic strengths equal to or greater than 20 mM, the net charge at the polypeptide cysteine site is the sum of the single negative charge of the thiolate anion and the charges of the amino acids immediately preceding and following the cysteine in the primary sequence. At lower ionic strengths, more distant residues influence kinetics. At pH 7.0, 23 degree C, and an ionic strength of 20 mM, rate constants for reaction of the negative disulfide with a cysteine having two positive neighbors, one positive and one neutral neighbor, or two neutral neighbors are 132000, 3350, and 367 s-1 M-1, respectively. This corresponds to a contribution to the activation energy of 0.65- 1.1 kcal/mol per ion pair involved in collision between the cysteine and disulfide regions. The results permit the estimation that cysteine local environments may provide a means of achieving a 10(6)-fold range in rate constants in disulfide exchange reactions in random-coil proteins. This range may prove useful in developing strategies for directing disulfide pairing in synthetic proteins.

  12. Neuraminidase Inhibitory Activity and Constituent Characterization of Fagopyrum dibotrys

    Directory of Open Access Journals (Sweden)

    Xiang Zhang

    2017-11-01

    Full Text Available This study aimed to identify a new biological activity of the widely distributed species Fagopyrum dibotrys. Four F. dibotrys extracts (ethyl acetate (EA, petroleum ether (P, ethanol (E, and water (W were explored for their anti-neuraminidase (NA activity. A total of 32 compounds were identified using UHPLC-Q-Exactive Orbitrap HRMS in the EA extract, which had the best NA inhibitory effects. We used the docking data for supporting compounds’ anti-neuraminidase activity. Among them, five compounds including one flavonoid, three organic acids, and one glucoside were discovered for the first time in F. dibotrys. Docking studies and NA activity assay revealed the remarkable NA inhibitory activity of eight components in EA extract, especially rutin, hesperidin, procyanidin B2, and quercitrin. Therefore, F. dibotrys could be used to develop anti-influenza drugs.

  13. Neuraminidase Inhibitory Activity and Constituent Characterization of Fagopyrum dibotrys.

    Science.gov (United States)

    Zhang, Xiang; Cao, Yu; Li, Jinhua; Liu, Ailin; Liu, Haibo; Huang, Linfang

    2017-11-18

    This study aimed to identify a new biological activity of the widely distributed species Fagopyrum dibotrys . Four F. dibotrys extracts (ethyl acetate (EA), petroleum ether (P), ethanol (E), and water (W)) were explored for their anti-neuraminidase (NA) activity. A total of 32 compounds were identified using UHPLC-Q-Exactive Orbitrap HRMS in the EA extract, which had the best NA inhibitory effects. We used the docking data for supporting compounds' anti-neuraminidase activity. Among them, five compounds including one flavonoid, three organic acids, and one glucoside were discovered for the first time in F. dibotrys . Docking studies and NA activity assay revealed the remarkable NA inhibitory activity of eight components in EA extract, especially rutin, hesperidin, procyanidin B₂, and quercitrin. Therefore, F. dibotrys could be used to develop anti-influenza drugs.

  14. Exploring the chemical space of influenza neuraminidase inhibitors

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    Nuttapat Anuwongcharoen

    2016-04-01

    Full Text Available The fight against the emergence of mutant influenza strains has led to the screening of an increasing number of compounds for inhibitory activity against influenza neuraminidase. This study explores the chemical space of neuraminidase inhibitors (NAIs, which provides an opportunity to obtain further molecular insights regarding the underlying basis of their bioactivity. In particular, a large set of 347 and 175 NAIs against influenza A and B, respectively, was compiled from the literature. Molecular and quantum chemical descriptors were obtained from low-energy conformational structures geometrically optimized at the PM6 level. The bioactivities of NAIs were classified as active or inactive according to their half maximum inhibitory concentration (IC50 value in which IC50 < 1µM and ≥ 10µM were defined as active and inactive compounds, respectively. Interpretable decision rules were derived from a quantitative structure–activity relationship (QSAR model established using a set of substructure descriptors via decision tree analysis. Univariate analysis, feature importance analysis from decision tree modeling and molecular scaffold analysis were performed on both data sets for discriminating important structural features amongst active and inactive NAIs. Good predictive performance was achieved as deduced from accuracy and Matthews correlation coefficient values in excess of 81% and 0.58, respectively, for both influenza A and B NAIs. Furthermore, molecular docking was employed to investigate the binding modes and their moiety preferences of active NAIs against both influenza A and B neuraminidases. Moreover, novel NAIs with robust binding fitness towards influenza A and B neuraminidase were generated via combinatorial library enumeration and their binding fitness was on par or better than FDA-approved drugs. The results from this study are anticipated to be beneficial for guiding the rational drug design of novel NAIs for treating influenza

  15. Using Common Spatial Distributions of Atoms to Relate Functionally Divergent Influenza Virus N10 and N11 Protein Structures to Functionally Characterized Neuraminidase Structures, Toxin Cell Entry Domains, and Non-Influenza Virus Cell Entry Domains

    Science.gov (United States)

    Weininger, Arthur; Weininger, Susan

    2015-01-01

    The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell

  16. Cysteine-functional polymers via thiol-ene conjugation.

    Science.gov (United States)

    Kuhlmann, Matthias; Reimann, Oliver; Hackenberger, Christian P R; Groll, Jürgen

    2015-03-01

    A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Detection of Cysteine Redox States in Mitochondrial Proteins in Intact Mammalian Cells.

    Science.gov (United States)

    Habich, Markus; Riemer, Jan

    2017-01-01

    Import, folding, and activity regulation of mitochondrial proteins are important for mitochondrial function. Cysteine residues play crucial roles in these processes as their thiol groups can undergo (reversible) oxidation reactions. For example, during import of many intermembrane space (IMS) proteins, cysteine oxidation drives protein folding and translocation over the outer membrane. Mature mitochondrial proteins can undergo changes in the redox state of specific cysteine residues, for example, as part of their enzymatic reaction cycle or as adaptations to changes of the local redox environment which might influence their activity. Here we describe methods to study changes in cysteine residue redox states in intact cells. These approaches allow to monitor oxidation-driven protein import as well as changes of cysteine redox states in mature proteins during oxidative stress or during the reaction cycle of thiol-dependent enzymes like oxidoreductases.

  18. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  19. Measuring site occupancy: a new perspective on cysteine oxidation.

    Science.gov (United States)

    Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna; Williamson, James; Roepstorff, Peter

    2014-10-01

    Site occupancy is an extremely important aspect of quantification of protein modifications. Knowing the degree of modification of each oxidised cysteine residue is critical to understanding the biological role of these modifications. Yet modification site occupancy is very often overlooked, in part because there are very few analytical tools that allow such measurements. Here we present a new strategy, which provides quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) simultaneously at the resolution of single cysteine and allows for determination of relative oxidation occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass spectrometry. The method relies on differential reduction of "total" cysteines, SNO cysteines and SOH cysteines with TCEP, sodium ascorbate and sodium arsenite respectively followed by iodoTMT(TM) alkylation. Enrichment of iodoTMT(TM)-containing peptides is performed using anti-TMT antibody. In vivo model of mild oxidative stress in Escherichia coli is used. To induce endogenous SNO bacteria were grown anaerobically in minimal media supplemented with fumarate or nitrate. Short-term treatment with submilimolar levels of hydrogen peroxide were used to induce SOH. We have quantified 114 SNO/SOH modified peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site

  20. Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

    Science.gov (United States)

    Mögling, Ramona; Richard, Mathilde J; Vliet, Stefan van der; Beek, Ruud van; Schrauwen, Eefje J A; Spronken, Monique I; Rimmelzwaan, Guus F; Fouchier, Ron A M

    2017-06-01

    Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

  1. Purification of neuraminidase from Influenza virus subtype H5N1

    Directory of Open Access Journals (Sweden)

    Simson Tariga

    2009-03-01

    Full Text Available Influenza-virus neuraminidase plays vital role in the survival of the organisms. Vaccination of animals with this glycoprotein confers immune responses so that enable it to protect the animals from incoming infection. Supplementation of conventional vaccines with this glycoprotein increases the protection and longevity of the vaccine. Purified neuraminidase can also be used to develop serological tests for differentiation of serologically positive animals due to infection or to vaccination. In this study purification of neuraminidase from influenza virus subtype H5N1 was described. Triton x-100 and Octyl β-D-glucopyranoside were used to extract and diluted the glycoprotein membrane. The enzymatic activity of the neuraminidase was assayed using a fluorochrome substrate, 4-methylumbelliferyl-a-D-N-acetyl neuraminic acid, which was found to be simple, sensitive and suitable for the purification purpose. The neuraminidase was absorbed selectively on an oxamic-acid agarose column. The purity of neuraminidase eluted from this affinity column was high. A higher purity of the neuraminidase was obtained by further separation with gel filtration on Superdex-200. The purified neuraminidase was enzymatically active and did not contain any detectable haemagglutinin, either by haemagglutination assay or by monospecific antibodies raised against H5N1 hemagglutinin. The purified neuraminidase was recognized strongly by antibodies raised against an internal but only weakly by that against C-terminal regions of the neuraminidase protein of H5N1-influenza virus. The purified neuraminidase was in tetrameric forms but dissociated into monomeric form on reducing condition, or mostly dimeric form on non-reducing SDS-PAGE.

  2. Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

    Science.gov (United States)

    Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

    2010-06-01

    Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

  3. Mouse Saliva Inhibits Transit of Influenza Virus to the Lower Respiratory Tract by Efficiently Blocking Influenza Virus Neuraminidase Activity.

    Science.gov (United States)

    Gilbertson, Brad; Ng, Wy Ching; Crawford, Simon; McKimm-Breschkin, Jenny L; Brown, Lorena E

    2017-07-15

    We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein, was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibitor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location, and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus. IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge, mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a

  4. The influence of 150-cavity binders on the dynamics of influenza A neuraminidases as revealed by molecular dynamics simulations and combined clustering.

    Directory of Open Access Journals (Sweden)

    Kyle T Greenway

    Full Text Available Neuraminidase inhibitors are the main pharmaceutical agents employed for treatments of influenza infections. The neuraminidase structures typically exhibit a 150-cavity, an exposed pocket that is adjacent to the catalytic site. This site offers promising additional contact points for improving potency of existing pharmaceuticals, as well as generating entirely new candidate inhibitors. Several inhibitors based on known compounds and designed to interact with 150-cavity residues have been reported. However, the dynamics of any of these inhibitors remains unstudied and their viability remains unknown. This work reports the outcome of long-term, all-atom molecular dynamics simulations of four such inhibitors, along with three standard inhibitors for comparison. Each is studied in complex with four representative neuraminidase structures, which are also simulated in the absence of ligands for comparison, resulting in a total simulation time of 9.6 µs. Our results demonstrate that standard inhibitors characteristically reduce the mobility of these dynamic proteins, while the 150-binders do not, instead giving rise to many unique conformations. We further describe an improved RMSD-based clustering technique that isolates these conformations--the structures of which are provided to facilitate future molecular docking studies--and reveals their interdependence. We find that this approach confers many advantages over previously described techniques, and the implications for rational drug design are discussed.

  5. Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

    Science.gov (United States)

    Li, R; Kast, J

    2017-01-01

    Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

  6. Neuraminidase-mediated, NKp46-dependent immune-evasion mechanism of influenza viruses.

    Science.gov (United States)

    Bar-On, Yotam; Glasner, Ariella; Meningher, Tal; Achdout, Hagit; Gur, Chamutal; Lankry, Dikla; Vitenshtein, Alon; Meyers, Adrienne F A; Mandelboim, Michal; Mandelboim, Ofer

    2013-04-25

    Natural killer (NK) cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA) protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA) protein. By using various NA blockers, we show that NA removes sialic acid residues from NKp46 and that this leads to reduced recognition of HA. Furthermore, we provide in vivo and in vitro evidence for the existence of this NA-mediated, NKp46-dependent immune-evasion mechanism and demonstrate that NA inhibitors, which are commonly used for the treatment of influenza infections, are useful not only as blockers of virus budding but also as boosters of NKp46 recognition. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    Science.gov (United States)

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-12-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  8. The cysteine residues at the C-terminal tail of Bamboo mosaic virus triple gene block protein 2 are critical for efficient plasmodesmata localization of protein 1 in the same block.

    Science.gov (United States)

    Ho, Tsai-Ling; Lee, Hsiang-Chi; Chou, Yuan-Lin; Tseng, Yang-Hao; Huang, Wei-Cheng; Wung, Chiung-Hua; Lin, Na-Sheng; Hsu, Yau-Heiu; Chang, Ban-Yang

    2017-01-15

    The movement of some plant viruses are accomplished by three proteins encoded by a triple gene block (TGB). The second protein (TGBp2) in the block is a transmembrane protein. This study was aimed to unravel the mechanism underlying the relatively inefficient cell-to-cell movement of Bamboo mosaic virus (BaMV) caused by amino acid substitutions for the three Cys residues, Cys-109, Cys-112 and Cys-119, at the C-terminal tail of TGBp2. Results from confocal microscopy revealed that substitutions of the three Cys residues of TGBp2, especially Cys-109 and Cys-112, would reduce the efficiency of TGBp2- and TGBp3-dependent PD localization of TGBp1. Moreover, there is an additive effect of the substitutions on reducing the efficiency of PD localization of TGBp1. These results indicate that the Cys residues in the C-terminal tail region of TGBp2 participate in the TGBp2- and TGBp3-dependent PD localization of TGBp1, and thus influence the cell-to-cell movement capability of BaMV. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Promising Anti-influenza Properties of Active Constituent of Withania somnifera Ayurvedic Herb in Targeting Neuraminidase of H1N1 Influenza: Computational Study.

    Science.gov (United States)

    Cai, Zhi; Zhang, Guoyin; Tang, Bin; Liu, Yan; Fu, Xiaojing; Zhang, Xuejin

    2015-07-01

    Neuraminidase (NA) is a membrane surface antigen which helps in the release of influenza viruses from the host cells after replication. Anti-influenza drugs such as zanamivir bind with eight highly conserved functional residues (R118, D151, R152, R224, E276, R292, R371, and Y406) in the active site of NA, thus restricting the viral release the from host cells. Binding of the drug in active site inhibits the ability of enzyme to cleave sialic acid residues on the cell membrane. Reports on the emergence of zanamivir-resistant strains of H1N1 Influenza virus necessitated a search for alternative drug candidates, preferably from plant source due to their known benefits such as less or no side effects, availability, and low cost. Withaferin A (WA), an active constituent of Withania somnifera ayurvedic herb, has been shown to have a broad range of medicinal properties including its anti-viral activity. The present study demonstrated that WA has the potential to attenuate the neuraminidase of H1N1 influenza. Our docking and simulation results predicted high binding affinity of the WA toward NA and revealed several interesting molecular interactions with the residues which are catalytically important during molecular dynamic simulations. The results presented in the article could be of high importance for further designing of target-specific anti-influenza drug candidates.

  10. Cysteine Protease Zymography: Brief Review.

    Science.gov (United States)

    Wilkesman, Jeff

    2017-01-01

    Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

  11. Mapping substrate interactions of the human membrane-associated neuraminidase, NEU3, using STD NMR.

    Science.gov (United States)

    Albohy, Amgad; Richards, Michele R; Cairo, Christopher W

    2015-03-01

    Saturation transfer difference (STD) nuclear magnetic resonance (NMR) is a powerful technique which can be used to investigate interactions between proteins and their substrates. The method identifies specific sites of interaction found on a small molecule ligand when in complex with a protein. The ability of STD NMR to provide specific insight into binding interactions in the absence of other structural data is an attractive feature for its use with membrane proteins. We chose to employ STD NMR in our ongoing investigations of the human membrane-associated neuraminidase NEU3 and its interaction with glycolipid substrates (e.g., GM3). In order to identify critical substrate-enzyme interactions, we performed STD NMR with a catalytically inactive form of the enzyme, NEU3(Y370F), containing an N-terminal maltose-binding protein (MBP)-affinity tag. In the absence of crystallographic data on the enzyme, these data represent a critical experimental test of proposed homology models, as well as valuable new structural data. To aid interpretation of the STD NMR data, we compared the results with molecular dynamics (MD) simulations of the enzyme-substrate complexes. We find that the homology model is able to predict essential features of the experimental data, including close contact of the hydrophobic aglycone and the Neu5Ac residue with the enzyme. Additionally, the model and STD NMR data agree on the facial recognition of the galactose and glucose residues of the GM3-analog studied. We conclude that the homology model of NEU3 can be used to predict substrate recognition, but our data indicate that unstructured portions of the NEU3 model may require further refinement. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Screening for Neuraminidase Inhibitory Activity in Traditional Chinese Medicines Used to Treat Influenza.

    Science.gov (United States)

    Yang, Xian-Ying; Liu, Ai-Lin; Liu, Shu-Jing; Xu, Xiao-Wei; Huang, Lin-Fang

    2016-08-27

    To screen for influenza virus neuraminidase inhibition and to provide a reference for the clinical treatment of influenza using traditional Chinese medicines (TCM). In this study, 421 crude extracts (solubilized with petroleum ether, ethanol, ethyl acetate, and aqueous solvents) were obtained from 113 TCM. The medicine extracts were then reacted with oseltamivir, using 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) as the substrate, to determine influenza virus neuraminidase activity using a standard fluorimetric assay. It was found that Chinese medicine extracts from Pyrola calliantha, Cynanchum wilfordii, Balanophora involucrata and Paeonia delavayi significantly inhibited neuraminidase activity at a concentration of 40 μg/mL. Dose-dependent inhibitory assays also revealed significant inhibition. The IC50 range of the TCM extracts for influenza virus neuraminidase was approximately 12.66-34.85 μg/mL, respectively. Some Chinese medicines have clear anti-influenza viral effects that may play an important role in the treatment of influenza through the inhibition of viral neuraminidase. The results of this study demonstrated that plant medicines can serve as a useful source of neuraminidase (NA) inhibitors and further investigation into the pharmacologic activities of these extracts is warranted.

  13. The use of a rapid assay to detect the neuraminidase production in oral Porphyromonas spp. isolated from dogs and humans.

    Science.gov (United States)

    de Assis, Paulo R G R; Nakano, Viviane; Senhorinho, Gerusa N A; Avila-Campos, Mario J

    2013-09-01

    Neuraminidase was produced by 32.1% and 28.5% of Porphyromonas from dogs with and without periodontitis, respectively; and by 31.8% of bacteria from humans. The presence of neuraminidase in Porphyromonas spp. suggests that this enzyme can be involved with the pathogenesis of the periodontal disease, and the use of this assay to detect the neuraminidase production in oral Porphyromonas species is suggested. © 2013.

  14. 35S cystein chlorhydrate preparation

    International Nuclear Information System (INIS)

    Emiliozzi, R.; Pichat, P.; Herbert, M.

    1960-01-01

    35 S cystein chlorhydrate has been prepared with a quantitative yield by electrolytic reduction of 35 S cystin in hydrochloric medium on a vibrating mercury cathode. Reprint of a paper published in Bulletin de la Societe chimique de France, no. 2653, 4. quarter 1959, p. 1544-1545 [fr

  15. Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Ping; Swanson, Kurt A.; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S. (Stanford-MED); (NWU)

    2014-10-02

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

  16. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

    Science.gov (United States)

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C

    2015-09-24

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

  17. Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

    Science.gov (United States)

    Mason, Alexander F; Thordarson, Pall

    2016-07-20

    The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

  18. Influenza neuraminidase: a druggable target for natural products.

    Science.gov (United States)

    Grienke, Ulrike; Schmidtke, Michaela; von Grafenstein, Susanne; Kirchmair, Johannes; Liedl, Klaus R; Rollinger, Judith M

    2012-01-01

    The imminent threat of influenza pandemics and repeatedly reported emergence of new drug-resistant influenza virus strains demonstrate the urgent need for developing innovative and effective antiviral agents for prevention and treatment. At present, influenza neuraminidase (NA), a key enzyme in viral replication, spread, and pathogenesis, is considered to be one of the most promising targets for combating influenza. Despite the substantial medical potential of NA inhibitors (NAIs), only three of these drugs are currently on the market (zanamivir, oseltamivir, and peramivir). Moreover, sudden changes in NAI susceptibility revealed the urgent need in the discovery/identification of novel inhibitors. Nature offers an abundance of biosynthesized compounds comprising chemical scaffolds of high diversity, which present an infinite pool of chemical entities for target-oriented drug discovery in the battle against this highly contagious pathogen. This review illuminates the increasing research efforts of the past decade (2000-2011), focusing on the structure, function and druggability of influenza NA, as well as its inhibition by natural products. Following a critical discussion of publications describing some 150 secondary plant metabolites tested for their inhibitory potential against influenza NA, the impact of three different strategies to identify and develop novel NAIs is presented: (i) bioactivity screening of herbal extracts, (ii) exploitation of empirical knowledge, and (iii) computational approaches. This work addresses the latest developments in theoretical and experimental research on properties of NA that are and will be driving anti-influenza drug development now and in the near future.

  19. Influenza virus neuraminidase (NA): a target for antivirals and vaccines.

    Science.gov (United States)

    Jagadesh, Anitha; Salam, Abdul Ajees Abdul; Mudgal, Piya Paul; Arunkumar, Govindakarnavar

    2016-08-01

    Influenza, the most common infectious disease, poses a great threat to human health because of its highly contagious nature and fast transmissibility, often leading to high morbidity and mortality. Effective vaccination strategies may aid in the prevention and control of recurring epidemics and pandemics associated with this infectious disease. However, antigenic shifts and drifts are major concerns with influenza virus, requiring effective global monitoring and updating of vaccines. Current vaccines are standardized primarily based on the amount of hemagglutinin, a major surface antigen, which chiefly constitutes these preparations along with the varying amounts of neuraminidase (NA). Anti-influenza drugs targeting the active site of NA have been in use for more than a decade now. However, NA has not been approved as an effective antigenic component of the influenza vaccine because of standardization issues. Although some studies have suggested that NA antibodies are able to reduce the severity of the disease and induce a long-term and cross-protective immunity, a few major scientific issues need to be addressed prior to launching NA-based vaccines. Interestingly, an increasing number of studies have shown NA to be a promising target for future influenza vaccines. This review is an attempt to consolidate studies that reflect the strength of NA as a suitable vaccine target. The studies discussed in this article highlight NA as a potential influenza vaccine candidate and support taking the process of developing NA vaccines to the next stage.

  20. Regulation of Protein Function and Signaling by Reversible Cysteine S-Nitrosylation*

    Science.gov (United States)

    Gould, Neal; Doulias, Paschalis-Thomas; Tenopoulou, Margarita; Raju, Karthik; Ischiropoulos, Harry

    2013-01-01

    NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications. PMID:23861393

  1. Regulation of protein function and signaling by reversible cysteine S-nitrosylation.

    Science.gov (United States)

    Gould, Neal; Doulias, Paschalis-Thomas; Tenopoulou, Margarita; Raju, Karthik; Ischiropoulos, Harry

    2013-09-13

    NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications.

  2. Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

    Science.gov (United States)

    Blanchette, Krystle A.; Shenoy, Anukul T.; Milner, Jeffrey; Gilley, Ryan P.; McClure, Erin; Hinojosa, Cecilia A.; Kumar, Nikhil; Daugherty, Sean C.; Tallon, Luke J.; Ott, Sandra; King, Samantha J.; Ferreira, Daniela M.; Gordon, Stephen B.; Tettelin, Hervé

    2016-01-01

    Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro. Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo. Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. PMID:27481242

  3. Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization.

    Science.gov (United States)

    Blanchette, Krystle A; Shenoy, Anukul T; Milner, Jeffrey; Gilley, Ryan P; McClure, Erin; Hinojosa, Cecilia A; Kumar, Nikhil; Daugherty, Sean C; Tallon, Luke J; Ott, Sandra; King, Samantha J; Ferreira, Daniela M; Gordon, Stephen B; Tettelin, Hervé; Orihuela, Carlos J

    2016-10-01

    Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Protein topology determines cysteine oxidation fate: the case of sulfenyl amide formation among protein families.

    Science.gov (United States)

    Defelipe, Lucas A; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A; Turjanski, Adrián G

    2015-03-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function.

  5. Contribution of neuraminidase 3 to the differentiation of induced regulatory T cells.

    Science.gov (United States)

    Kaminuma, Osamu; Katoh, Shigeki; Miyagi, Taeko; Watanabe, Nobumasa; Kitamura, Noriko; Nishimura, Tomoe; Saeki, Mayumi; Mori, Akio; Hiroi, Takachika

    2018-02-01

    Neuraminidase family enzymes that hydrolyze the terminal sialic acid linkage in biomolecules are involved in various immune responses. We previously showed that Th1 and Th2 cells differentially express several neuraminidases. Herein, the expression of neuraminidases in induced regulatory T (iTreg) cells was investigated in comparison with that in other T-cell subsets. Contrary to the tendency toward higher neuraminidase 1 mRNA expression in in vitro-differentiated Th2 cells, compared to Th1, Th17 and iTreg cells, we observed significantly higher expression of neuraminidase 3 (Neu3) in iTreg cells. Furthermore, the expression of Neu3 in FoxP3 + CD62L - spleen cells was higher than that in FoxP3 + CD62L + and FoxP3 - cells. Lentiviral expression of Neu3 in naïve CD4 + T cells during the stimulation culture led to upregulation of FoxP3 expression. On the basis of these findings, we conclude that Neu3 contributes to the differentiation of iTreg cells by upregulation of FoxP3. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  6. Baculovirus Surface Display Using Infuenza Neuraminidase (NA Transmembrane Anchor

    Directory of Open Access Journals (Sweden)

    Irisa Trianti

    2016-11-01

    Full Text Available Baculovirus surface display has been employed as an excellent tools for presentation of foreign peptides and proteins on virus surface with native conformation, functions and immunogenicity. A baculovirus major envelope protein, gp64, or a capsid protein, vp39 are generally used as fusion partners for displaying of polypeptides on the surface of virions. Alternatively, a membrane anchoring domain of vesicular stomatitis virus G protein (VSV-G can also be used. In this study, an influenza neuraminidase (NA was proposed as a new membrane anchor for the display of Angiotensin II (AngII, DRVYIHPFHL, peptides. The AngII peptides were inserted into NA by replacing NA amino acid number 60-67 with AngII, and then integrated into a baculovirus genome. A recombinant baculovirus expressing the NA fusion-AngII peptides was generated from infected insect cells. Those peptides were found to express and translocated on the membrane of the baculovirus infected insect cell (Sf9 cell as detected by immunocytochemistry using anti-AngII monoclonal antibody. Upon budding of the recombinant baculovirus progenies through the insect cells membrane, the recombinant NA-AngII peptides was acquired to envelopes of the new baculovirus progenies. The conformation of NA on baculovirus surface was not affected by the deletion, as the 55 kDa band of NA can be detected from Western Blotting analysis by specific anti-NA monoclonal antibody. In addition, the same protein was also found by anti-AngII antibody indicating that the AngII peptides had been successfully fused with the recombinant NA. Interestingly, electron microscopy analysis demonstrated that not only the recombinant baculovirus displaying AngII peptides were generated by infected insect cells, but also the NA virus-like-particle displaying AngII peptides.

  7. Antiviral Drug-Resistant Influenza B Viruses Carrying H134N Substitution in Neuraminidase, Laos, February 2016.

    Science.gov (United States)

    Baranovich, Tatiana; Vongphrachanh, Phengta; Ketmayoon, Pakapak; Sisouk, Thongchanh; Chomlasack, Khampheng; Khanthamaly, Viengphone; Nguyen, Ha Thuy; Mishin, Vasiliy P; Marjuki, Henju; Barnes, John R; Garten, Rebecca J; Stevens, James; Wentworth, David E; Gubareva, Larisa V

    2017-04-01

    In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused by viruses harboring this mutation.

  8. Antiviral Drug–Resistant Influenza B Viruses Carrying H134N Substitution in Neuraminidase, Laos, February 2016

    Science.gov (United States)

    Baranovich, Tatiana; Vongphrachanh, Phengta; Ketmayoon, Pakapak; Sisouk, Thongchanh; Chomlasack, Khampheng; Khanthamaly, Viengphone; Nguyen, Ha Thuy; Mishin, Vasiliy P.; Marjuki, Henju; Barnes, John R.; Garten, Rebecca J.; Stevens, James; Wentworth, David E.

    2017-01-01

    In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused by viruses harboring this mutation. PMID:28322707

  9. Mutation effects of neuraminidases and their docking with ligands: a molecular dynamics and free energy calculation study

    Science.gov (United States)

    Yang, Zhiwei; Yang, Gang; Zhou, Lijun

    2013-11-01

    A systematic study has been performed on neuraminidase (NA) mutations and NA-inhibitor docked complexes, with the aim to understand protein-ligand interactions and design broad-spectrum antiviral drugs with minimal resistances. The catalytic D151 residue is likely to mutate while others are relatively conserved. The NA active-site conformations are altered by mutations, but more alterations do not necessarily result in larger deviations to the binding properties. The effects of all related mutations have been discussed; e.g., for the arginine triad (R118, R292 and R371), it is found that residue R118 plays the most significant role during ligand binding. Generally, the calculated binding free energies agree well with the experimental observations. Susceptibility of influenza virus to NA inhibitors can be reinforced by some mutations; e.g., the binding free energies of ligands with N2 subtype increase from -18.0 to -42.1 kcal mol-1 by the E119D mutation. Mutations of the various NA subtypes often cause similar conformational and binding changes, explaining the occurrence of cross resistances; nonetheless, differences can be detected in some cases that correspond to subtype-specific resistances. For all NA subtypes, the electrostatic contributions are the major driving force for ligand binding and largely responsible for the binding differences between the wild-type and mutated NA proteins.

  10. Accessibility of Myofilament Cysteines and Effects on ATPase Depend on the Activation State during Exposure to Oxidants

    Science.gov (United States)

    Gross, Sean M.; Lehman, Steven L.

    2013-01-01

    Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types. PMID:23894416

  11. A new technique of tritium labelling of neuraminidase from Vibrio cholerae

    International Nuclear Information System (INIS)

    Keune, D.

    1981-01-01

    By acylation of the free amino groups of the enzyme neuraminidase from Vibrio cholerae using N-[(2,3 - 3 H)-propionyloxy]-succinimide it was possible to transfer tritium-labelled propionyl groups to free amino groups of the enzyme glycoprotein. It was established by preliminary trials that a certain minimum concentration of protein was necessary to achieve a satisfactory degree of acylation. After the various processing stages, the acylation of neuraminidase with N-[(2,3 - 3 H)-propionyloxy]-succinimide led to the incorporation of 5.26 μCi radioactivity per mg enzymal protein. Comparison with a known method for neuraminidase labelling showed that the new process is more effective in terms of incorporation of radioactivity. Enzyme activity is inhibited by both methods. (orig./MG) [de

  12. Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding

    Science.gov (United States)

    Hagelueken, Gregor; Naismith, James H

    2013-01-01

    Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses proteins. PMID:24091556

  13. Degenerate cysteine patterns mediate two redox sensing mechanisms in the papillomavirus E7 oncoprotein.

    Science.gov (United States)

    Camporeale, Gabriela; Lorenzo, Juan R; Thomas, Maria G; Salvatierra, Edgardo; Borkosky, Silvia S; Risso, Marikena G; Sánchez, Ignacio E; de Prat Gay, Gonzalo; Alonso, Leonardo G

    2017-04-01

    Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn +2 binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Extending the scope of site-specific cysteine bioconjugation by appending a prelabeled cysteine tag to proteins using protein trans-splicing.

    Science.gov (United States)

    Dhar, Tulika; Kurpiers, Thomas; Mootz, Henning D

    2011-01-01

    Incorporating synthetic probes site-specifically into proteins is of central interest in several areas of biotechnology and protein chemistry. Bioconjugation techniques provide a simple and effective means of chemically modifying a protein. In particular, covalent chemical modifications of cysteine residues belong to one of the most important reactions due to the unique reactivity of its thiol moiety and the relatively low abundance of this amino acid in proteins. However, such types of modifications cannot be performed in a regioselective fashion when one or more additional cysteines are present. To address this limitation, we have developed an approach where a short cysteine-containing tag (Cys-Tag) fused to one part of a split intein and modified at its sulfhydryl group can be used to label proteins by trans-splicing with a protein of interest (POI) fused to the other half of the split intein. In this way, it is possible to selectively label a protein containing multiple cysteines. The artificially split Mycobacterium xenopi GyrA intein and the Synechocystis sp. DnaB intein were highly suitable for this purpose and were successfully used for the labeling of several proteins. This approach enables a simple route for labeling proteins by site-specific cysteine bioconjugation with any one of several commercially available cysteine-modifying probes.

  15. The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

    Science.gov (United States)

    Calce, Enrica; De Luca, Stefania

    2017-01-05

    Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Allenamides as orthogonal handles for selective modification of cysteine in peptides and proteins.

    Science.gov (United States)

    Abbas, Ata; Xing, Bengang; Loh, Teck-Peng

    2014-07-14

    In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent-labeling studies demonstrated that the installation of a C-terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site-specific labeling of cysteine-containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The tumour metabolism inhibitors GSAO and PENAO react with cysteines 57 and 257 of mitochondrial adenine nucleotide translocase

    Directory of Open Access Journals (Sweden)

    Park Danielle

    2012-03-01

    Full Text Available Abstract Background GSAO (4-(N-(S-glutathionylacetylamino phenylarsonous acid and PENAO (4-(N-(S-penicillaminylacetylamino phenylarsonous acid are tumour metabolism inhibitors that target adenine nucleotide translocase (ANT of the inner-mitochondrial membrane. Both compounds are currently being trialled in patients with solid tumours. The trivalent arsenical moiety of GSAO and PENAO reacts with two matrix facing cysteine residues of ANT, inactivating the transporter. This leads to proliferation arrest and death of tumour and tumour-supporting cells. Results The two reactive ANT cysteine residues have been identified in this study by expressing cysteine mutants of human ANT1 in Saccharomyces cerevisiae and measuring interaction with the arsenical moiety of GSAO and PENAO. The arsenic atom of both compounds cross-links cysteine residues 57 and 257 of human ANT1. Conclusions The sulphur atoms of these two cysteines are 20 Å apart in the crystal structures of ANT and the optimal spacing of cysteine thiolates for reaction with As (III is 3-4 Å. This implies that a significant conformational change in ANT is required for the organoarsenicals to react with cysteines 57 and 257. This conformational change may relate to the selectivity of the compounds for proliferating cells.

  18. CMD: A Database to Store the Bonding States of Cysteine Motifs with Secondary Structures

    Directory of Open Access Journals (Sweden)

    Hamed Bostan

    2012-01-01

    Full Text Available Computational approaches to the disulphide bonding state and its connectivity pattern prediction are based on various descriptors. One descriptor is the amino acid sequence motifs flanking the cysteine residue motifs. Despite the existence of disulphide bonding information in many databases and applications, there is no complete reference and motif query available at the moment. Cysteine motif database (CMD is the first online resource that stores all cysteine residues, their flanking motifs with their secondary structure, and propensity values assignment derived from the laboratory data. We extracted more than 3 million cysteine motifs from PDB and UniProt data, annotated with secondary structure assignment, propensity value assignment, and frequency of occurrence and coefficiency of their bonding status. Removal of redundancies generated 15875 unique flanking motifs that are always bonded and 41577 unique patterns that are always nonbonded. Queries are based on the protein ID, FASTA sequence, sequence motif, and secondary structure individually or in batch format using the provided APIs that allow remote users to query our database via third party software and/or high throughput screening/querying. The CMD offers extensive information about the bonded, free cysteine residues, and their motifs that allows in-depth characterization of the sequence motif composition.

  19. Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry.

    Science.gov (United States)

    García-Santamarina, Sarela; Boronat, Susanna; Domènech, Alba; Ayté, José; Molina, Henrik; Hidalgo, Elena

    2014-05-01

    Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labeling.

  20. Systematic review of influenza resistance to the neuraminidase inhibitors

    Directory of Open Access Journals (Sweden)

    Boivin Guy

    2011-05-01

    Full Text Available Abstract Background Antivirals play a critical role in the prevention and the management of influenza. One class of antivirals, neuraminidase inhibitors (NAIs, is effective against all human influenza viruses. Currently there are two NAI drugs which are licensed worldwide: oseltamivir (Tamiflu® and zanamivir (Relenza®; and two drugs which have received recent approval in Japan: peramivir and laninamivir. Until recently, the prevalence of antiviral resistance has been relatively low. However, almost all seasonal H1N1 strains that circulated in 2008-09 were resistant to oseltamivir whereas about 1% of tested 2009 pandemic H1N1 viruses were found to be resistant to oseltamivir. To date, no studies have demonstrated widespread resistance to zanamivir. It seems likely that the literature on antiviral resistance associated with oseltamivir as well as zanamivir is now sufficiently comprehensive to warrant a systematic review. The primary objectives were to systematically review the literature to determine the incidence of resistance to oseltamivir, zanamivir, and peramivir in different population groups as well as assess the clinical consequences of antiviral resistance. Methods We searched MEDLINE and EMBASE without language restrictions in September 2010 to identify studies reporting incidence of resistance to oseltamivir, zanamivir, and peramivir. We used forest plots and meta-analysis of incidence of antiviral resistance associated with the three NAIs. Subgroup analyses were done across a number of population groups. Meta-analysis was also performed to evaluate associations between antiviral resistance and clinical complications and symptoms. Results We identified 19 studies reporting incidence of antiviral resistance. Meta-analysis of 15 studies yielded a pooled incidence rate for oseltamivir resistance of 2.6% (95%CI 0.7% to 5.5%. The incidence rate for all zanamivir resistance studies was 0%. Only one study measured incidence of antiviral

  1. Chalcones as novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Nguyen, Phi Hung; Lee, Hong Sik

    2011-01-01

    -8) chalcones were isolated as active principles from the acetone extract of Glycyrrhiza inflata. Compounds 3 and 6 without prenyl group showed strong inhibitory effects on various neuraminidases from influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed...

  2. Oseltamivir analogues bearing N-substituted guanidines as potent neuraminidase inhibitors

    NARCIS (Netherlands)

    Mooney, Caitlin A.; Johnson, Stuart A.; 'T Hart, Peter; Quarles Van Ufford, Linda; De Haan, Cornelis A M; Moret, Ed E.; Martin, Nathaniel I.

    2014-01-01

    A series of oseltamivir analogues bearing an N-substituted guanidine unit were prepared and evaluated as inhibitors of neuraminidases from four strains of influenza the two most potent analogues identified contain relatively small N-guanidine substituents (N-methyl and N-hydroxyl) and display

  3. Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin

    DEFF Research Database (Denmark)

    Nielsen, Line R.; Lund, Marianne N.; Davies, Michael J.

    2018-01-01

    α-Lactalbumin (α-LA) is a key commercial whey protein for nutritional purposes. The holo protein (calcium saturated) is considered the most heat stable whey protein, capable of refolding from unfolded states under many conditions. This is due to the absence of free thiols (cysteine residues...

  4. Neuraminidase inhibitor R-125489 - A promising drug for treating influenza virus: Steered molecular dynamics approach

    International Nuclear Information System (INIS)

    Mai, Binh Khanh; Li, Mai Suan

    2011-01-01

    Highlights: → We study binding affinity of R-125489 and its prodrug CS-8958 to neuraminidase of pathogenic influenza viruses by molecular dynamics simulations. → It is shown that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. → We predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus. → The high correlation between theoretical and experimental data implies that SMD is a very promising tool for drug design. -- Abstract: Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenza virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson-Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.

  5. [Inhibition by cysteine of the carbohydrate-binding activity of lectins from Ricinus communis, Canavalia ensiformis and Euonymus europaeus].

    Science.gov (United States)

    Dvorkin, V M

    1985-10-01

    Precipitation induced by different lectins has been studied in the presence of some aminoacids. It was shown that precipitates formed by lectins from Ricinus communis (RCA1), Canavalia ensiformis (Con A), Euonymus europaeus (Eel) in the presence of appropriate carbohydrate-containing molecules disappeared after cysteine addition, like after addition of specific carbohydrate precipitation inhibitors. It is assumed that cysteine residues of RCA1, Con A and Eel lectins are essential for their carbohydrate binding activity.

  6. Blockade of endogenous neuraminidase leads to an increase of neuronal excitability and activity-dependent synaptogenesis in the rat hippocampus.

    Science.gov (United States)

    Isaeva, Elena; Lushnikova, Irina; Savrasova, Alina; Skibo, Galina; Holmes, Gregory L; Isaev, Dmytro

    2010-12-01

    Polysialic acids are widely distributed in neuronal tissue. Due to their position on glycoproteins and gangliosides on the outer cell membranes and anionic nature, polysialic acids are involved in multiple cell signaling events. The level of sialylation of the cellular surface is regulated by endogenous neuraminidase (NEU), which catalyses the hydrolysis of terminal sialic acid residues. Using the specific blocker of endogenous NEU, N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), we show that downregulation of the endogenous NEU activity causes a significant increase in the level of hippocampal tissue sialylation. Acute application of NADNA increased the firing frequency and amplitude of spontaneous synchronous oscillations, and frequency of multiple unit activity in cultured hippocampal slices. The tonic phase of seizure-like activity in the low-magnesium model of ictogenesis was significantly increased in slices pretreated with NADNA. These data indicate that the degree of synchronization is influenced by the amount of active NEU in cultured hippocampal slices. Pretreatment with NADNA led to an increase of the density of simple and perforated synapses in the hippocampal CA1 stratum radiatum region. Co-incubation of slices with NADNA and high concentrations of calcium eliminated the effect of the NEU blocker on synaptic density, suggesting that synaptogenesis observed following downregulation of the endogenous NEU activity is an activity-dependent process. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  7. Identification of Suitable Natural Inhibitor against Influenza A (H1N1 Neuraminidase Protein by Molecular Docking

    Directory of Open Access Journals (Sweden)

    Maheswata Sahoo

    2016-09-01

    Full Text Available The influenza A (H1N1 virus, also known as swine flu is a leading cause of morbidity and mortality since 2009. There is a need to explore novel anti-viral drugs for overcoming the epidemics. Traditionally, different plant extracts of garlic, ginger, kalmegh, ajwain, green tea, turmeric, menthe, tulsi, etc. have been used as hopeful source of prevention and treatment of human influenza. The H1N1 virus contains an important glycoprotein, known as neuraminidase (NA that is mainly responsible for initiation of viral infection and is essential for the life cycle of H1N1. It is responsible for sialic acid cleavage from glycans of the infected cell. We employed amino acid sequence of H1N1 NA to predict the tertiary structure using Phyre2 server and validated using ProCheck, ProSA, ProQ, and ERRAT server. Further, the modelled structure was docked with thirteen natural compounds of plant origin using AutoDock4.2. Most of the natural compounds showed effective inhibitory activity against H1N1 NA in binding condition. This study also highlights interaction of these natural inhibitors with amino residues of NA protein. Furthermore, among 13 natural compounds, theaflavin, found in green tea, was observed to inhibit H1N1 NA proteins strongly supported by lowest docking energy. Hence, it may be of interest to consider theaflavin for further in vitro and in vivo evaluation.

  8. Parallel screening of wild-type and drug-resistant targets for anti-resistance neuraminidase inhibitors.

    Directory of Open Access Journals (Sweden)

    Kai-Cheng Hsu

    Full Text Available Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye, which inhibited WT NA and MDR NA with IC(50 values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high

  9. Redox Signaling Regulated by Cysteine Persulfide and Protein Polysulfidation.

    Science.gov (United States)

    Kasamatsu, Shingo; Nishimura, Akira; Morita, Masanobu; Matsunaga, Tetsuro; Abdul Hamid, Hisyam; Akaike, Takaaki

    2016-12-15

    For decades, reactive persulfide species including cysteine persulfide (CysSSH) have been known to exist endogenously in organisms. However, the physiological significance of endogenous persulfides remains poorly understood. That cystathionine β-synthase and cystathionine γ-lyase produced CysSSH from cystine was recently demonstrated. An endogenous sulfur transfer system involving CysSSH evidently generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo. Because reactive persulfide species such as CysSSH and GSSH have higher nucleophilicity than parental cysteine (Cys) and glutathione do, these reactive species exhibit strong scavenging activities against oxidants, e.g., hydrogen peroxide, and electrophiles, which contributes to redox signaling regulation. Also, several papers indicated that various proteins and enzymes have Cys polysulfides including CysSSH at their specific Cys residues, which is called protein polysulfidation. Apart from the redox signaling regulatory mechanism, another plausible function of protein polysulfidation is providing protection for protein thiol residues against irreversible chemical modification caused by oxidants and electrophiles. Elucidation of the redox signaling regulatory mechanism of reactive persulfide species including small thiol molecules and thiol-containing proteins should lead to the development of new therapeutic strategies and drug discoveries for oxidative and electrophilic stress-related diseases.

  10. Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

    Directory of Open Access Journals (Sweden)

    Gustavo Coelho Correa

    2001-12-01

    Full Text Available Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB como pertencentes à sub

  11. Curcuminoids from Curcuma longa and their inhibitory activities on influenza A neuraminidases

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Won, Ho Keun; Kim, Eun Hee

    2012-01-01

    inhibitory effects on the neuraminidases from two influenza viral strains, H1N1 and H9N2, as noncompetitive inhibitors with IC50 values ranging from 6.18 ± 0.64 to 40.17 ± 0.79 μg/ml and 3.77 ± 0.75 to 31.82 ± 1.33 μg/ml, respectively. Compounds 4, 5, and 13 also exhibited significant inhibitory activity...... against the neuraminidases from novel influenza H1N1 (WT) and oseltamivir-resistant novel H1N1 (H274Y mutant) expressed in 293T cells. Our results suggest that the curcuminoids from C. longa may be potential supplemental molecules in the prevention and treatment of disease by influenza viruses....

  12. A new method for tritium labelling of neuraminidase from Vibrio cholerae

    International Nuclear Information System (INIS)

    Keune, D.

    1981-01-01

    This research work related to the radioactive labelling with tritium of the enzyme neuraminidase from Vibrio cholerae by an easily handled method. The reactive compound N-propionyloxysuccinimide, the ester of propionic acid and N-hydroxysuccinimide, offered a suitable labelling reagent. For comparison purposes an already known method of labelling neuraminidase with tritium by the oxidation of hydroxyl groups of the hydrocarbon chain of the enzymal protein and subsequent reduction of the aldehyde groups formed with tritiated sodium borhydride, was also carried out. The advantages and disadvantages of both methods are described in detail, in particular with regard to yields of radioactivity and the influence on enzyme activity. The fact that only 1 mg enzymal protein was available for each modification of the enzyme molecule posed particular problems and, as a consequence, extensive preliminary experiments had to be carried with another protein (beef serum album) in the same concentration range. (orig./MG) [de

  13. Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein.

    Science.gov (United States)

    Yuan, Ping; Leser, George P; Demeler, Borries; Lamb, Robert A; Jardetzky, Theodore S

    2008-09-01

    The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

  14. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Zhengliang L., E-mail: Leon.wu@bio-techne.com [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Zhou, Hui [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States); Ethen, Cheryl M. [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Reinhold, Vernon N., E-mail: Vernon.Reinhold@unh.edu [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States)

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain

  15. Effectiveness of neuraminidase inhibitors for preventing staff absenteeism during pandemic influenza

    OpenAIRE

    Lee, VJ; Chen, MI

    2007-01-01

    We used a deterministic SEIR (susceptible-exposed-infectious-removed) meta-population model, together with scenario, sensitivity, and simulation analyses, to determine stockpiling strategies for neuraminidase inhibitors that would minimize absenteeism among healthcare workers. A pandemic with a basic reproductive number (R0) of 2.5 resulted in peak absenteeism of 10%. Treatment decreased peak absenteeism to 8%, while 8 weeks' prophylaxis reduced it to 2%. For pandemics with higher R0, peak ab...

  16. Financial conflicts of interest and conclusions about neuraminidase inhibitors for influenza: an analysis of systematic reviews.

    Science.gov (United States)

    Dunn, Adam G; Arachi, Diana; Hudgins, Joel; Tsafnat, Guy; Coiera, Enrico; Bourgeois, Florence T

    2014-10-07

    Industry funding and financial conflicts of interest may contribute to bias in the synthesis and interpretation of scientific evidence. To examine the association between financial conflicts of interest and characteristics of systematic reviews of neuraminidase inhibitors. Retrospective analysis. Reviews that examined the use of neuraminidase inhibitors in the prophylaxis or treatment of influenza, were published between January 2005 and May 2014, and used a systematic search protocol. Two investigators blinded to all information regarding the review authors independently assessed the presentation of evidence on the use of neuraminidase inhibitors as favorable or not favorable. Financial conflicts of interest were identified using the index reviews, other publications, and Web-based searches. Associations between financial conflicts of interest, favorability assessments, and presence of critical appraisals of evidence quality were analyzed. Twenty-six systematic reviews were identified, of which 13 examined prophylaxis and 24 examined treatment, accounting for 37 distinct assessments. Among assessments associated with a financial conflict of interest, 7 of 8 (88%) were classified as favorable, compared with 5 of 29 (17%) among those without a financial conflict of interest. Reviewers without financial conflicts of interest were more likely to include statements about the quality of the primary studies than those with financial conflicts of interest. The heterogeneity in populations and outcomes examined in the reviews precluded analysis of the contribution of selective inclusion of evidence on the discordance of the assessments made in the reviews. Many of the systematic reviews had overlapping authorship. Reviewers with financial conflicts of interest may be more likely to present evidence about neuraminidase inhibitors in a favorable manner and recommend the use of these drugs than reviewers without financial conflicts of interest. Australian National Health and

  17. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

    International Nuclear Information System (INIS)

    Wu, Zhengliang L.; Zhou, Hui; Ethen, Cheryl M.; Reinhold, Vernon N.

    2016-01-01

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and 3 H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain why

  18. Neuraminidase-Mediated, NKp46-Dependent Immune-Evasion Mechanism of Influenza Viruses

    OpenAIRE

    Bar-On, Yotam; Glasner, Ariella; Meningher, Tal; Achdout, Hagit; Gur, Chamutal; Lankry, Dikla; Vitenshtein, Alon; Meyers, Adrienne F.A.; Mandelboim, Michal; Mandelboim, Ofer

    2013-01-01

    Natural killer (NK) cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA) protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA) protein. By using various NA...

  19. Long-term cysteine fortification impacts cysteine/glutathione homeostasis and food intake in ageing rats

    OpenAIRE

    Vidal, Karine; Breuille, Denis; Serrant, Patrick; Denis, Philippe; Glomot, Francoise; Bechereau, Fabienne; Papet, Isabelle

    2014-01-01

    Healthy ageing is associated with higher levels of glutathione. The study aimed to determine whether long-term dietary fortification with cysteine increases cysteine and glutathione pools, thus alleviating age-associated low-grade inflammation and resulting in global physiological benefits. The effect of a 14-week dietary fortification with cysteine was studied in non-inflamed (NI, healthy at baseline) and in spontaneously age-related low-grade inflamed (LGI, prefrail at baseline) 21-month-ol...

  20. Protein cysteine oxidation in redox signaling

    DEFF Research Database (Denmark)

    Forman, Henry Jay; Davies, Michael J; Krämer, Anna C

    2017-01-01

    Oxidation of critical signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione...... of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids....

  1. Residual deposits (residual soil)

    International Nuclear Information System (INIS)

    Khasanov, A.Kh.

    1988-01-01

    Residual soil deposits is accumulation of new formate ore minerals on the earth surface, arise as a result of chemical decomposition of rocks. As is well known, at the hyper genes zone under the influence of different factors (water, carbonic acid, organic acids, oxygen, microorganism activity) passes chemical weathering of rocks. Residual soil deposits forming depends from complex of geologic and climatic factors and also from composition and physical and chemical properties of initial rocks

  2. Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures

    DEFF Research Database (Denmark)

    Hansen, Simon Boje; Laursen, Nick Stub; Andersen, Gregers Rom

    2017-01-01

    of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend......The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent...... the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally...

  3. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A.

    Science.gov (United States)

    Mallorquí-Fernández, Noemí; Manandhar, Surya P; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B; Enghild, Jan J; Potempa, Jan; Gomis-Rüth, F Xavier

    2008-02-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

  4. Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans

    OpenAIRE

    Hennicke, Florian; Grumbt, Maria; Lermann, Ulrich; Ueberschaar, Nico; Palige, Katja; Böttcher, Bettina; Jacobsen, Ilse D.; Staib, Claudia; Morschhäuser, Joachim; Monod, Michel; Hube, Bernhard; Hertweck, Christian; Staib, Peter

    2013-01-01

    The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions ...

  5. Synthesis and Application of Aurophilic Poly(Cysteine and Poly(Cysteine-Containing Copolymers

    Directory of Open Access Journals (Sweden)

    David Ulkoski

    2017-10-01

    Full Text Available The redox capacity, as well as the aurophilicity of the terminal thiol side groups, in poly(Cysteine lend a unique characteristic to this poly(amino acid or polypeptide. There are two major application fields for this polymer: (i biomedical applications in drug delivery and surface modification of biomedical devices and (ii as coating for electrodes to enhance their electrochemical sensitivity. The intended application determines the synthetic route for p(Cysteine. Polymers to be used in biomedical applications are typically polymerized from the cysteine N-carboxyanhydride by a ring-opening polymerization, where the thiol group needs to be protected during the polymerization. Advances in this methodology have led to conditions under which the polymerization progresses as living polymerization, which allows for a strict control of the molecular architecture, molecular weight and polydispersity and the formation of block copolymers, which eventually could display polyphilic properties. Poly(Cysteine used as electrode coating is typically polymerized onto the electrode by cyclic voltammetry, which actually produces a continuous, pinhole-free film on the electrode via the formation of covalent bonds between the amino group of Cysteine and the carbon of the electrode. This resulting coating is chemically very different from the well-defined poly(Cysteine obtained by ring-opening polymerizations. Based on the structure of cysteine a significant degree of cross-linking within the coating deposited by cyclic voltammetry can be assumed. This manuscript provides a detailed discussion of the ring-opening polymerization of cysteine, a brief consideration of the role of glutathione, a key cysteine-containing tripeptide, and examples for the utilization of poly(Cysteine and poly(Cysteine-containing copolymers, in both, the biomedical as well as electrochemical realm.

  6. [Genetic variation and clustal analysis of Trichomonas vaginalis cysteine proteases].

    Science.gov (United States)

    Jia, Wan-Zhong; Li, Zhi; Zhao, Liang; Lun, Zhao-Rong

    2008-06-30

    To clone the genes coding for cysteine proteases (CPs, TvCPs) from Trichomonas vaginalis and to analyze their genetic variations with the related sequences from NCBI database (GenBank) and T. vaginalis Genome Project database from The Institute for Genomic Research (TIGR). TvCP genes were amplified using PCR, and inserted into vector pET28b or pBS-T. The recombinant plasmids were then transformed to Escherichia coli BL21 or Topl0 strain. The recombinant plasmids were used for sequencing. Homologous TvCP genes were blasted based on NCBI GenBank and TIGR T. vaginalis Genome Project database. The sequences of cloned TvCP genes were aligned and clustered by Clustal X (1.83 version) with retrieved sequences. Comparisons of amino acids among cathepsin L-like TvCPs, human L-like cathepsins and papaya papain were performed using DNAstar software, and their phylogenic tree was constructed based on neighbor-joining method using Clustal X. Two TvCP3 clones and one TvCP2 had a high identity of more than 99% with their responding TvCPs. Three clones of TvCP4 genes, GZ-CP4-clone 1-3, belonged to two members of a family showing a high percentage identity of more than 97.5% with the sequences of TvCP4 genes from databases (GenBank and TIGR) both at amino acid and nucleotide levels. Nine homologous TvCP4 pro-enzymes with 304 amino acids and other two members with deletions of N-terminal sequence existed in T. vaginalis sharing a similarity of 62.3-96.7% amino acids, which may evolve by means of gene replication and deletion. TvCP1-4, TvCP12, TvCP25 and CP65 had an identity of 61-88.2% at amino acid levels. So far, all reported sequences of C1 family from T. vaginalis belonged to capanthesin L-like subfamily with the same enzymatic active sites, conserved cysteine residues and similar structural features such as ERFNIN-like motif in pro-enzyme region, suggesting that they might result from gene duplication and mutations. TvCPs belong to cathepsin L-like family with genetic diversity

  7. Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

    DEFF Research Database (Denmark)

    Loechel, F; Overgaard, M T; Oxvig, C

    1999-01-01

    The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prod......The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen......, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition...... of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity...

  8. ROSics: chemistry and proteomics of cysteine modifications in redox biology.

    Science.gov (United States)

    Kim, Hee-Jung; Ha, Sura; Lee, Hee Yoon; Lee, Kong-Joo

    2015-01-01

    Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1-2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, "ROSics," for the science which describes the principles of mode of action of ROS at molecular levels. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

  9. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    Directory of Open Access Journals (Sweden)

    Marian Dorcas Quain

    2013-08-01

    Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

  10. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

    2006-01-01

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

  11. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    Science.gov (United States)

    Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

    2016-01-01

    Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

  12. Cysteine-based redox regulation and signalling in plants

    Directory of Open Access Journals (Sweden)

    Jérémy eCouturier

    2013-04-01

    Full Text Available Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen (ROS, nitrogen (RNS and sulfur (RSS species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past years, besides their recognized catalytic and regulatory functions, the modification of cysteine thiol group was increasingly viewed as either protective or redox signalling mechanisms. The most physiologically relevant reversible redox post-translational modifications (PTMs are disulfide bonds, sulfenic acids, S-glutathionylated adducts, S-nitrosothiols and to a lesser extent S-sulfenylamides, thiosulfinates and S-persulfides. These redox PTMs are mostly controlled by two oxidoreductase families, thioredoxins and glutaredoxins. This review focuses on recent advances highlighting the variety and physiological roles of these PTMs and the proteomic strategies used for their detection.

  13. Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

    Science.gov (United States)

    Anderson, Mark T; Mitchell, Lindsay A; Mobley, Harry L T

    2017-08-15

    Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes ( cyaA , crp , fliJ , and fliP ) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O -acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O -acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescens cysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescens IMPORTANCE Serratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine

  14. Zanamivir immobilized magnetic beads for voltammetric measurement of neuraminidase at gold-modified boron doped diamond electrode

    Energy Technology Data Exchange (ETDEWEB)

    Wahyuni, Wulan Tri, E-mail: wulantriws@gmail.com [Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680 (Indonesia); Department of Chemistry, FMIPA, Universitas Indonesia, Kampus UI Depok (Indonesia); Ivandini, Tribidasari A.; Saepudin, Endang [Department of Chemistry, FMIPA, Universitas Indonesia, Kampus UI Depok (Indonesia); Einaga, Yasuaki [Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Yokohama 223-8522 (Japan); CREST, JST, 3-14-1 Hiyoshi, Yokohama 223-8522 (Japan)

    2016-04-19

    Biomolecule modified magnetic beads has been widely used in separation and sensing process. This study used streptavidin modified magnetic beads to immobilize biotin modified zanamivir. Biotin-streptavidin affinity facilitates immobilization of zanamivir on magnetic beads. Then interaction of zanamivir and neuraminidase was adopted as basic for enzyme detection. Detection of neuraminidase was performed at gold modified BDD using cyclic voltammetry technique. The measurement was carried out based on alteration of electrochemical signals of working electrode as neuraminidase response. The result showed that zanamivir was successfully immobilized on magnetic beads. The optimum amount of magnetic beads for zanamivir immobilization was 120 ug. Linear responses of neuraminidase were detected in concentration range of 0-15 mU. Detection limit (LOD) of measurement was 2.32 mU (R2 = 0.959) with precision as % RSD of 1.41%. Measurement of neuraminidase on magnetic beads could be also performed in the presence of mucin matrix. The linearity range was 0-8 mU with LOD of 0.64 mU (R2 = 0.950) and % RSD of 7.25%.

  15. Combining crystallographic information and an aspherical-atom data bank in the evaluation of the electrostatic interaction energy in an enzyme–substrate complex: influenza neuraminidase inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Dominiak, Paulina M., E-mail: pdomin@chem.uw.edu.pl [Department of Chemistry, State University of New York at Buffalo, NY 14260 (United States); Department of Chemistry, University of Warsaw, ul. Pasteura 1, 02-093 Warszawa (Poland); Volkov, Anatoliy; Dominiak, Adam P. [Department of Chemistry, State University of New York at Buffalo, NY 14260 (United States); Jarzembska, Katarzyna N. [Department of Chemistry, University of Warsaw, ul. Pasteura 1, 02-093 Warszawa (Poland); Coppens, Philip, E-mail: pdomin@chem.uw.edu.pl [Department of Chemistry, State University of New York at Buffalo, NY 14260 (United States)

    2009-05-01

    The electrostatic component of the enzyme/inhibitor interaction of a wide range influenza neuraminidases and inhibitors has been analyzed using transferable aspherical-atom densities from a recently compiled databank. Results are subdivided into the contributions of individual active-site residues and different functional groups of the inhibitors, and the effect of the Arg292→Lys mutation is considered. Although electrostatic interactions contribute only a part of the interaction energies between macromolecules, unlike dispersion forces they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes and contribute significantly to differences in inhibition strength among related enzyme inhibitors. In the reported study, a wide range of complexes of influenza neuraminidases with inhibitor molecules (sialic acid derivatives and others) have been analyzed using charge densities from a transferable aspherical-atom data bank. The strongest interactions of the residues are with the acidic group at the C2 position of the inhibitor (∼−300 kJ mol{sup −1} for —COO{sup −} in non-aromatic inhibitors, ∼−120–210 kJ mol{sup −1} for —COO{sup −} in aromatic inhibitors and ∼−450 kJ mol{sup −1} for —PO{sub 3}{sup 2−}) and with the amino and guanidine groups at C4 (∼−250 kJ mol{sup −1}). Other groups contribute less than ∼100 kJ mol{sup −1}. Residues Glu119, Asp151, Glu227, Glu276 and Arg371 show the largest variation in electrostatic energies of interaction with different groups of inhibitors, which points to their important role in the inhibitor recognition. The Arg292→Lys mutation reduces the electrostatic interactions of the enzyme with the acidic group at C2 for all inhibitors that have been studied (SIA, DAN, 4AM, ZMR, G20, G28, G39 and BCZ), but enhances the interactions with the glycerol group at C6 for inhibitors that contain it. This is in agreement with the lower level

  16. A functional fragment of Tau forms fibers without the need for an intermolecular cysteine bridge

    Energy Technology Data Exchange (ETDEWEB)

    Huvent, Isabelle; Kamah, Amina; Cantrelle, François-Xavier [CNRS UMR 8576, University of Lille1, 59655 Villeneuve d’Ascq (France); Barois, Nicolas [Plate-forme BICeL-IFR142, Institut Pasteur de Lille, Lille (France); Slomianny, Christian [Inserm U1003, Laboratoire de physiologie cellulaire, Université Lille 1, 59650 Villeneuve d’Ascq (France); Smet-Nocca, Caroline; Landrieu, Isabelle [CNRS UMR 8576, University of Lille1, 59655 Villeneuve d’Ascq (France); Lippens, Guy, E-mail: Guy.Lippens@univ-lille1.fr [CNRS UMR 8576, University of Lille1, 59655 Villeneuve d’Ascq (France)

    2014-03-07

    Highlights: • A functional fragment of Tau forms bundled ribbon-like fibrils. • Nucleation of its fibril formation is faster than for full-length Tau. • In contrast to full-length Tau, without cysteines, the fragment still forms fibers. - Abstract: We study the aggregation of a fragment of the neuronal protein Tau that contains part of the proline rich domain and of the microtubule binding repeats. When incubated at 37 °C with heparin, the fragment readily forms fibers as witnessed by Thioflavin T fluorescence. Electron microscopy and NMR spectroscopy show bundled ribbon like structures with most residues rigidly incorporated in the fibril. Without its cysteines, this fragment still forms fibers of a similar morphology, but with lesser Thioflavin T binding sites and more mobility for the C-terminal residues.

  17. Cysteine-independent activation/inhibition of heme oxygenase-2

    Directory of Open Access Journals (Sweden)

    Dragic Vukomanovic

    2016-01-01

    Full Text Available Reactive thiols of cysteine (cys residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2 isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2 and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

  18. Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin

    Directory of Open Access Journals (Sweden)

    Madhusudan Rajendran

    2017-03-01

    Full Text Available Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA, we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for “original antigenic sin.” Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.

  19. Sensitivity of molecular docking to induced fit effects in influenza virus neuraminidase

    Science.gov (United States)

    Birch, Louise; Murray, Christopher W.; Hartshorn, Michael J.; Tickle, Ian J.; Verdonk, Marcel L.

    2002-12-01

    Many proteins undergo small side chain or even backbone movements on binding of different ligands into the same protein structure. This is known as induced fit and is potentially problematic for virtual screening of databases against protein targets. In this report we investigate the limits of the rigid protein approximation used by the docking program, GOLD, through cross-docking using protein structures of influenza neuraminidase. Neuraminidase is known to exhibit small but significant induced fit effects on ligand binding. Some neuraminidase crystal structures caused concern due to the bound ligand conformation and GOLD performed poorly on these complexes. A `clean' set, which contained unique, unambiguous complexes, was defined. For this set, the lowest energy structure was correctly docked (i.e. RMSD < 1.5 Å away from the crystal reference structure) in 84% of proteins, and the most promiscuous protein (1mwe) was able to dock all 15 ligands accurately including those that normally required an induced fit movement. This is considerably better than the 70% success rate seen with GOLD against general validation sets. Inclusion of specific water molecules involved in water-mediated hydrogen bonds did not significantly improve the docking performance for ligands that formed water-mediated contacts but it did prevent docking of ligands that displaced these waters. Our data supports the use of a single protein structure for virtual screening with GOLD in some applications involving induced fit effects, although care must be taken to identify the protein structure that performs best against a wide variety of ligands. The performance of GOLD was significantly better than the GOLD implementation of ChemScore and the reasons for this are discussed. Overall, GOLD has shown itself to be an extremely good, robust docking program for this system.

  20. C-Methylated Flavonoids from Cleistocalyx operculatus and Their Inhibitory Effects on Novel Influenza A (H1N1) Neuraminidase

    DEFF Research Database (Denmark)

    Dao, Trong-Tuan; Tung, Bui-Thanh; Nguyen, Phi-Hung

    2010-01-01

    As part of an ongoing study focused on the discovery of anti-influenza agents from plants, four new (1-4) and 10 known (5-14) C-methylated flavonoids were isolated from a methanol extract of Cleistocalyx operculatus buds using an influenza H1N1 neuraminidase inhibition assay. Compounds 4, 7, 8......, and 14, with a chalcone skeleton, showed significant inhibitory effects on the viral neuraminidases from two influenza viral strains, H1N1 and H9N2. Compound 4 showed the strongest inhibitory activity against the neuraminidases from novel influenza H1N1 (WT) and oseltamivir-resistant novel H1N1 (H274Y...

  1. Structural Properties of Macrodontain I, a Cysteine Protease from Pseudananas macrodontes (Morr.) Harms (Bromeliaceae).

    Science.gov (United States)

    Errasti, María E; Natalucci, Claudia L; Caffini, Néstor O; Rotelli, Alejandra E; Brullo, Adriana; Maras, Bruno; Trejo, Sebastián A; López, Laura M I

    2018-03-15

    The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman's degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18 Da, pI value 6.99, and a molar extinction coefficient at 280 nm of 61,685 M -1  cm -1 . The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α + β class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153-Cys201). Further, we were able to establish that the cysteine peptidases from P. macrodontes are involved in the anti-inflammatory activity.

  2. Curcuminoids from Curcuma longa and their inhibitory activities on influenza A neuraminidases

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Won, Ho Keun; Kim, Eun Hee

    2012-01-01

    The emergence of drug-resistant influenza viruses and the threat of pandemics highlight the need for new and effective antiviral agents. In this study, we describe the isolation of 3 new (1–3) and 10 known (4−13) curcuminoids from a methanol extract of Curcuma longa L. All compounds had strong...... against the neuraminidases from novel influenza H1N1 (WT) and oseltamivir-resistant novel H1N1 (H274Y mutant) expressed in 293T cells. Our results suggest that the curcuminoids from C. longa may be potential supplemental molecules in the prevention and treatment of disease by influenza viruses....

  3. Influenza A (H1N1) neuraminidase inhibitors from Vitis amurensis

    DEFF Research Database (Denmark)

    Nguyen, Ngoc Anh; Dao, Trong Tuan; Tung, Bui Thanh

    2011-01-01

    Recently, a novel H1N1 influenza A virus (H1N1/09 virus) was identified and considered a strong candidate for a novel influenza pandemic. As part of an ongoing anti-influenza screening programme on natural products, eight oligostilbenes were isolated as active principles from the methanol extract...... of Vitis amurensis. This manuscript reports the isolation, structural elucidation, and anti-viral activities of eight compounds on various neuraminidases from influenza A/PR/8/34 (H1N1), novel swine-origin influenza A (H1N1), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells...

  4. Sample Multiplexing with Cysteine-Selective Approaches: cysDML and cPILOT

    Science.gov (United States)

    Gu, Liqing; Evans, Adam R.; Robinson, Renã A. S.

    2015-04-01

    Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

  5. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

    En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner.......En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner....

  6. Susceptibility of influenza viruses circulating in Western Saudi Arabia to neuraminidase inhibitors

    Directory of Open Access Journals (Sweden)

    Ahmed M. Tolah

    2016-04-01

    Full Text Available Objectives: To investigate the sensitivity of circulating influenza viruses in Western Saudi Arabia to neuraminidase inhibitors (NAIs; mainly, zanamivir and oseltamivir. Methods: Respiratory samples were collected from patients presenting with respiratory symptoms to King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia (KSA between September 2013 and October 2014. All samples were tested prospectively by real-time reverse-transcription polymerase chain reaction for influenza A and B viruses. Positive samples were then inoculated on Madin-Darby Canine Kidney (MDCK cells and isolated viruses were examined for their sensitivity to NAIs using fluorescent neuraminidase inhibition assay. Results: Out of 406 tested samples, 25 samples (6.2% were positive for influenza A/pdmH1N1 virus, one sample (0.25% was positive for influenza A/H3N2 virus, and 7 samples (1.7% were positive for influenza B Yamagata-like virus. Screening of isolated influenza A and B viruses (9 out of 33 for their sensitivity to NAIs showed no significant resistance to available NAIs. Conclusion: Our results show that circulating influenza viruses in Jeddah are still sensitive to NAIs.

  7. A novel role for methyl cysteinate, a cysteine derivative, in cesium accumulation in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Adams, Eri; Miyazaki, Takae; Hayaishi-Satoh, Aya

    2017-01-01

    Phytoaccumulation is a technique to extract metals from soil utilising ability of plants. Cesium is a valuable metal while radioactive isotopes of cesium can be hazardous. In order to establish a more efficient phytoaccumulation system, small molecules which promote plants to accumulate cesium were...... investigated. Through chemical library screening, 14 chemicals were isolated as 'cesium accumulators' in Arabidopsis thaliana. Of those, methyl cysteinate, a derivative of cysteine, was found to function within the plant to accumulate externally supplemented cesium. Moreover, metabolite profiling demonstrated...

  8. Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin.

    Science.gov (United States)

    Rajendran, Madhusudan; Nachbagauer, Raffael; Ermler, Megan E; Bunduc, Paul; Amanat, Fatima; Izikson, Ruvim; Cox, Manon; Palese, Peter; Eichelberger, Maryna; Krammer, Florian

    2017-03-21

    Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for "original antigenic sin." Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.) IMPORTANCE Anti-neuraminidase antibodies can afford broad protection from influenza virus infection in animal models and humans. However, little is known about the breadth and magnitude of the anti-neuraminidase response in the human population. Here we assessed antibody levels of children, adults, and the elderly against a panel of N1, N2, and type B influenza virus neuraminidases. We demonstrated that antibody levels measured by ELISA correlate well with functional

  9. Accurate disulfide-bonding network predictions improve ab initio structure prediction of cysteine-rich proteins.

    Science.gov (United States)

    Yang, Jing; He, Bao-Ji; Jang, Richard; Zhang, Yang; Shen, Hong-Bin

    2015-12-01

    Cysteine-rich proteins cover many important families in nature but there are currently no methods specifically designed for modeling the structure of these proteins. The accuracy of disulfide connectivity pattern prediction, particularly for the proteins of higher-order connections, e.g., >3 bonds, is too low to effectively assist structure assembly simulations. We propose a new hierarchical order reduction protocol called Cyscon for disulfide-bonding prediction. The most confident disulfide bonds are first identified and bonding prediction is then focused on the remaining cysteine residues based on SVR training. Compared with purely machine learning-based approaches, Cyscon improved the average accuracy of connectivity pattern prediction by 21.9%. For proteins with more than 5 disulfide bonds, Cyscon improved the accuracy by 585% on the benchmark set of PDBCYS. When applied to 158 non-redundant cysteine-rich proteins, Cyscon predictions helped increase (or decrease) the TM-score (or RMSD) of the ab initio QUARK modeling by 12.1% (or 14.4%). This result demonstrates a new avenue to improve the ab initio structure modeling for cysteine-rich proteins. http://www.csbio.sjtu.edu.cn/bioinf/Cyscon/ zhng@umich.edu or hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    International Nuclear Information System (INIS)

    Manne, V.; Roberts, D.; Tobin, A.; O'Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, Hsiang-Fu

    1990-01-01

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2 Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

  11. Effectiveness of cysteine proteases on protein/pigment film removal.

    Science.gov (United States)

    Yao, Jiang-Wu; Xiao, Yin; Zuo, Qi-liang; Zhang, Yi; Tao, Tao; Lin, Chang-Jian

    2013-11-01

    Theaflavin (TF) from the black tea can react to human salivary proline-rich proteins (PRPs) to form stains on exposed dental surfaces. Here, we employed a model of protein/pigment film using TF and dephosphorylated bovine β-casein (Dβ-CN), which has an extended conformation, similar to that of salivary PRPs, on a sensor surface to assess the efficacy of cysteine proteases (CPs) including papain, stem bromelain, and ficin, on removing TF bound to Dβ-CN and the control TF readsorption on the residual substrate surfaces was also measured. The protein/pigment complex film was built by using a quartz crystal microbalance with dissipation (QCM-D). The efficacies of CPs were assessed by Boltzman equation model. The surface details were detected by grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles. The efficacy order of CPs on hydrolyzing protein/pigment complex film is ficin>papain>bromelain. The results from grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles demonstrated that TF bound on the Dβ-CN was effectively removed by the CPs, and the amount of TF readsorption on both the residual film of the Dβ-CN/TF and the Dβ-CN was markedly decreased after hydrolysis. This study indicates the potential application of the CPs for tooth stain removal and suggests that these enzymes are worthy of further investigation for use in oral healthcare. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

    Science.gov (United States)

    Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

  13. L-Cysteine Metabolism and Fermentation in Microorganisms.

    Science.gov (United States)

    Takagi, Hiroshi; Ohtsu, Iwao

    L-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are industrially produced by microbial fermentation, L-cysteine has been mainly produced by protein hydrolysis. Due to environmental and safety problems, synthetic or biotechnological products have been preferred in the market. Here, we reviewed L-cysteine metabolism, including biosynthesis, degradation, and transport, and biotechnological production (including both enzymatic and fermentation processes) of L-cysteine. The metabolic regulation of L-cysteine including novel sulfur metabolic pathways found in microorganisms is also discussed. Recent advancement in biochemical studies, genome sequencing, structural biology, and metabolome analysis has enabled us to use various approaches to achieve direct fermentation of L-cysteine from glucose. For example, worldwide companies began to supply L-cysteine and its derivatives produced by bacterial fermentation. These companies successfully optimized the original metabolism of their private strains. Basically, a combination of three factors should be required for improving L-cysteine fermentation: that is, (1) enhancing biosynthesis: overexpression of the altered cysE gene encoding feedback inhibition-insensitive L-serine O-acetyltransferase (SAT), (2) weakening degradation: knockout of the genes encoding L-cysteine desulfhydrases, and (3) exploiting export system: overexpression of the gene involved in L-cysteine transport. Moreover, we found that "thiosulfate" is much more effective sulfur source than commonly used "sulfate" for L-cysteine production in Escherichia coli, because thiosulfate is advantageous for saving consumption of NADPH and relating energy molecules.

  14. Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; End, Caroline

    2004-01-01

    The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight...... acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical...

  15. Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.

    Science.gov (United States)

    Astafieva, A A; Rogozhin, E A; Odintsova, T I; Khadeeva, N V; Grishin, E V; Egorov, Ts A

    2012-08-01

    Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  16. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

    2013-06-15

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

  17. Life-Threatening Abnormal Behavior Incidence in 10-19 Year Old Patients Administered Neuraminidase Inhibitors.

    Directory of Open Access Journals (Sweden)

    Yuuki Nakamura

    Full Text Available Much discussion has surrounded the association between the administration of neuraminidase inhibitors (NI and severe abnormal behaviors, including sudden running away and jumping from a high place, which can be life-threatening if no one intervenes. Using data on the number of abnormal behaviors and patients who had been prescribed NI in Japan, we calculated the incidence rate of severe abnormal behaviors among influenza patients who had been prescribed NI. Then, we evaluated the relative risk between the four types of NI on severe abnormal behavior. We found no significant difference in the incidence rates of abnormal behavior by the type of NI. Results implicate that the current policy of package inserts, which warn physicians that patients who were administered ANY type of NI might exhibit abnormal behavior, seems to be appropriate.

  18. Virtual screening of Indonesian flavonoid as neuraminidase inhibitor of influenza a subtype H5N1

    Science.gov (United States)

    Parikesit, A. A.; Ardiansah, B.; Handayani, D. M.; Tambunan, U. S. F.; Kerami, D.

    2016-02-01

    Highly Pathogenic Avian Influenza (HPAI) H5N1 poses a significant threat to animal and human health worldwide. The number of H5N1 infection in Indonesia is the highest during 2005-2013, with a mortality rate up to 83%. A mutation that occurred in H5N1 strain made it resistant to commercial antiviral agents such as oseltamivir and zanamivir, so the more potent antiviral agent is needed. In this study, virtual screening of Indonesian flavonoid as neuraminidase inhibitor of H5N1 was conducted. Total 491 flavonoid compound obtained from HerbalDB were screened. Molecular docking was performed using MOE 2008.10. This research resulted in Guajavin B as the best ligand.

  19. Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

    International Nuclear Information System (INIS)

    Doinel, C.; Ropars, C.; Salmon, C.

    1978-01-01

    Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

  20. Polyphenolic glycosides isolated from Pogostemon cablin (Blanco) Benth. as novel influenza neuraminidase inhibitors.

    Science.gov (United States)

    Liu, Fang; Cao, Wei; Deng, Chao; Wu, Zhaoquan; Zeng, Guangyao; Zhou, Yingjun

    2016-01-01

    Influenza is historically an ancient disease that causes annual epidemics and, at irregular intervals, pandemics. At present, the first-line drugs (oseltamivir and zanamivir) don't seem to be optimistic due to the spontaneously arising and spreading of oseltamivir resistance among influenza virus. Pogostemon cablin (Blanco) Benth. (P. cablin) is an important traditional Chinese medicine herb that has been widely used for treatment on common cold, nausea and fever. In our previous study, we have identified an extract derived from P. cablin as a novel selective neuraminidase (NA) inhibitor. A series of polyphenolic compounds were isolated from P. cablin for their potential ability to inhibit neuraminidase of influenza A virus. Two new octaketides (1, 2), together with other twenty compounds were isolated from P. cablin. These compounds showed better inhibitory activity against NA. The significant potent compounds of this series were compounds 2 (IC50 = 3.87 ± 0.19 μ mol/ml), 11, 12, 14, 15, 19 and 20 (IC50 was in 2.12 to 3.87 μ mol/ml), which were about fourfold to doubled less potent than zanamivir and could be used to design novel influenza NA inhibitors, especially compound 2, that exhibit increased activity based on these compounds. With the help of molecular docking, we had a preliminary understanding of the mechanism of the two new compounds (1-2)' NA inhibitory activity. Fractions 6 and polyphenolic compounds isolated from fractions 6 showed higher NA inhibition than that of the initial plant exacts. The findings of this study indicate that polyphenolic compounds and fractions 6 derived from P. cablin are potential NA inhibitors. This work is one of the evidence that P. cablin has better inhibitory activity against influenza, which not only enriches the compound library of P. cablin, but also facilitates further development and promises its therapeutic potential for the rising challenge of influenza diseases.

  1. Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hua; Carney, Paul J.; Mishin, Vasiliy P.; Guo, Zhu; Chang, Jessie C.; Wentworth, David E.; Gubareva, Larisa V.; Stevens, James; Schultz-Cherry, S.

    2016-04-06

    ABSTRACT

    During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (HPAI) A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015, the virus was detected in wild birds in Canada and the United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA-approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses and to continually assess future changes that may increase their pandemic potential.

    IMPORTANCEThe H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.

  2. Enzyme II/sup Mtl/ of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

    International Nuclear Information System (INIS)

    Pas, H.H.; Robillard, G.T.

    1988-01-01

    The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [ 14 C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing

  3. Could a deletion in neuraminidase stalk strengthen human tropism of the novel avian influenza virus H7N9 in China, 2013?

    Science.gov (United States)

    Chen, Liang; Zhu, Feng; Xiong, Chenglong; Zhang, Zhijie; Jiang, Lufang; Chen, Yue; Zhao, Genming; Jiang, Qingwu

    2015-01-20

    Objective. A novel avian influenza A virus (AIV) H7N9 subtype which emerged in China in 2013 caused worldwide concern. Deletion of amino-acids 69 to 73 in the neuraminidase stalk was its most notable characteristic. This study is aimed to discuss the tropism and virulence effects of this deletion. Neuraminidase gene sequences of N9 subtype were collected from NCBI and GISAID. MEGA6.0, Stata12.0, and UCSF Chimera were employed for sequence aligning, significance testing, and protein tertiary structure homology modeling. A total of 736 sequences were obtained; there were 81 human isolates of the novel AIV H7N9, of which 79 had the deletion. Among all the 654 avian origin sequences, only 43 had the deletion (p deletion obviously changed the spatial direction of neuraminidase. The deletion in neuraminidase stalk could have strengthened human tropism of the novel AIV H7N9, as well as its virulence.

  4. Neuraminidase Activity in Streptococcus sanguis and in the Viridans Group, and Occurrence of Acylneuraminate Lyase in Viridans Organisms Isolated from Patients with Septicemia

    Science.gov (United States)

    Müller, H. E.

    1974-01-01

    The enzyme neuraminidase (EC 3.2.1.18) was found to be strongly active in different types of Streptococcus sanguis and S. viridans, and, in addition, the occurrence of the enzyme acylneuraminate pyruvate lyase (EC 4.1.3.3) was described in S. viridans. The enzyme-active bacteria strains were isolated from blood cultures of patients with septicemia. Whereas S. sanguis lost its strong neuraminidase activity after some weeks, S. viridans retained its enzyme activity for a long time in culture. Immunoelectrophoretic studies of the blood cultures of patients with streptococcal infections showed the loss of neuraminic acid in most glycoproteins of the serum, proving the in vivo action of neuraminidase. The pathogenic role of neuraminidase is discussed in streptococcal septicemia from the viewpoint of present knowledge. Images PMID:4816461

  5. Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

    Science.gov (United States)

    Sengupta, Ishita; Udgaonkar, Jayant B

    2017-12-01

    The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The β-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Differential expression of cysteine desulfurases in soybean

    Directory of Open Access Journals (Sweden)

    Heis Marta D

    2011-11-01

    Full Text Available Abstract Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11

  7. Factors supporting cysteine tolerance and sulfite production in Candida albicans.

    Science.gov (United States)

    Hennicke, Florian; Grumbt, Maria; Lermann, Ulrich; Ueberschaar, Nico; Palige, Katja; Böttcher, Bettina; Jacobsen, Ilse D; Staib, Claudia; Morschhäuser, Joachim; Monod, Michel; Hube, Bernhard; Hertweck, Christian; Staib, Peter

    2013-04-01

    The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

  8. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K [Castro Valley, CA

    2008-10-21

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  9. Residuation theory

    CERN Document Server

    Blyth, T S; Sneddon, I N; Stark, M

    1972-01-01

    Residuation Theory aims to contribute to literature in the field of ordered algebraic structures, especially on the subject of residual mappings. The book is divided into three chapters. Chapter 1 focuses on ordered sets; directed sets; semilattices; lattices; and complete lattices. Chapter 2 tackles Baer rings; Baer semigroups; Foulis semigroups; residual mappings; the notion of involution; and Boolean algebras. Chapter 3 covers residuated groupoids and semigroups; group homomorphic and isotone homomorphic Boolean images of ordered semigroups; Dubreil-Jacotin and Brouwer semigroups; and loli

  10. Kinetic, thermodynamic and structural analysis of tamiphosphor binding to neuraminidase of H1N1 (2009) pandemic influenza

    Czech Academy of Sciences Publication Activity Database

    Albinana, C. B.; Machara, A.; Řezáčová, Pavlína; Pachl, Petr; Konvalinka, Jan; Kožíšek, Milan

    2016-01-01

    Roč. 121, Oct 4 (2016), s. 100-109 ISSN 0223-5234 R&D Projects: GA ČR GA13-19561S; GA MŠk LO1302; GA MŠk(CZ) LO1304 Institutional support: RVO:61388963 Keywords : influenza neuraminidase * oseltamivir * tamiphosphor * isothermal titration calorimetry * crystal structure * lattice-translocation defect Subject RIV: CE - Biochemistry Impact factor: 4.519, year: 2016

  11. Kinetic, thermodynamic and structural analysis of tamiphosphor binding to neuraminidase of H1N1 (2009) pandemic influenza

    Czech Academy of Sciences Publication Activity Database

    Albinana, C. B.; Machara, A.; Řezáčová, Pavlína; Pachl, P.; Konvalinka, J.; Kožíšek, M.

    2016-01-01

    Roč. 121, OCT 4 (2016), s. 100-109 ISSN 0223-5234 R&D Projects: GA ČR GA13-19561S; GA MŠk LO1302; GA MŠk(CZ) LO1304 Institutional support: RVO:68378050 Keywords : Influenza neuraminidase * Oseltamivir * Tamiphosphor * Isothermal titration calorimetry * Crystal structure * Lattice-translocation defect Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.519, year: 2016

  12. [Susceptibility of Influenza B Viruses to Neuraminidase Inhibitors Isolated during 2013-2014 Influenza Season in Mainland China].

    Science.gov (United States)

    Huang, Weijuang; Li, Xiyan; Tan, Minju; Wei, Hejiang; Cheng, Yanhui; Guo, Junfeng; Wang, Zhao; Xiao, Ning; Wang, Dayan; Shu, Yuelong

    2015-03-01

    Data based on the antiviral-resistant phenotyping characteristics of 884 influenza B viruses circulating in mainland China from October 2013 to March 2014 were analyzed to assess the susceptibility of influenza B viruses to neuraminidase inhibitors. All 884 viruses were sensitive to oseltamivir; two viruses (0.23%) had reduced sensitivity to zanamivir and all other viruses were sensitive to zanamivir. Among the 38 viruses with a B/Victoria lineage, B/Shandong-Kuiwen/1195/2014 exhibited a half-maximal inhibitory concentration (IC50) for zanamivir that was elevated by 5. 12-fold (1.78 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D35G, N59D and S402T (39, 64 and 399 with N2 number) amino-acid substitutions in the NA gene were detected with no previously reported antiviral-resistant substitutions. Among viruses with the 846 B/Yamagata lineage, B/Hunan-Lingling/350/2013 exhibited a 7.99-fold elevated IC50 for zanamivir (2.72 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D197N (N2 number), a previously reported antiviral resistant-related amino-acid substitution in the NA gene, was detected in B/Hunan-Lingling/350/2013. These data suggest that recently circulating influenza B viruses in mainland China have retained susceptibility to neuraminidase inhibitors.

  13. Prognosis of hospitalized patients with 2009 H1N1 influenza in Spain: influence of neuraminidase inhibitors

    Science.gov (United States)

    Delgado-Rodríguez, Miguel; Castilla, Jesús; Godoy, Pere; Martín, Vicente; Soldevila, Nuria; Alonso, Jordi; Astray, Jenaro; Baricot, Maretva; Cantón, Rafael; Castro, Ady; Gónzález-Candelas, Fernando; Mayoral, José María; Quintana, José María; Pumarola, Tomás; Tamames, Sonia; Sáez, Marc; Domínguez, Angela

    2012-01-01

    Background The H1N1 influenza pandemic strain has been associated with a poor prognosis in hospitalized patients. The present report evaluates the factors influencing prognosis. Methods A total of 813 patients hospitalized with H1N1 influenza in 36 hospitals (nationwide) in Spain were analysed. Detailed histories of variables preceding hospital admission were obtained by interview, validating data on medications and vaccine with their attending physicians. Data on treatment and complications during hospital stay were recorded. As definition of poor outcome, the endpoints of death and admission to intensive care were combined; and as a further outcome, length of stay was used. Results The mean age was 38.5 years (SD 22.8 years). There were 10 deaths and 79 admissions to intensive care (combined, 88). The use of neuraminidase inhibitors was reported by 495 patients (60.9%). The variables significantly associated with a poor outcome were diabetes (OR = 2.21, 95% CI = 1.21–4.02), corticosteroid therapy (OR = 3.37, 95% CI = 1.39–8.20) and use of histamine-2 receptor antagonists (OR = 2.68, 95% CI = 1.14–6.36), while the use of neuraminidase inhibitors (OR = 0.57, 95% CI = 0.34–0.94) was protective. Neuraminidase inhibitors within the first 2 days after the influenza onset reduced hospital stay by a mean of 1.9 days (95% CI = 4.7–6.6). Conclusions The use of neuraminidase inhibitors decreases the length of hospital stay and admission to intensive care and/or death. PMID:22467633

  14. Adamantane and Neuraminidase resistant influenza A/H3N2 isolated in Iran from 2005 to 2013

    Directory of Open Access Journals (Sweden)

    Jila Yavarian

    2014-04-01

    Conclusion: This study showed circulating A/H3N2 viruses was resistant to adaman-tanes but susceptible to neuraminidase inhibitors. The national data analyzed in this re-search may help increase knowledge about influenza virus antiviral drug resistance, which is a global public health concern. The authors suggested continuing this study and also the investigation of antiviral drug resistance of influenza A/H1N1 and B viruses.

  15. Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

    Science.gov (United States)

    Wible, Ryan S; Sutter, Thomas R

    2017-03-20

    The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

  16. Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport

    International Nuclear Information System (INIS)

    Hempe, J.M.; Cousins, R.J.

    1991-01-01

    The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. The authors have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPCL and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient

  17. Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding

    Science.gov (United States)

    Sherma, Nisha D.

    2014-01-01

    Abstract Significance: Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex biological samples and map disulfide bond structures are crucial to gaining greater insights into how proteins operate in human health and disease. Recent Advances: Many different molecular probes are now commercially available to label and track cysteine residues at great sensitivity. Coupled with mass spectrometry, stable isotope-labeled sulfhydryl-specific reagents can provide previously unprecedented molecular insights into the dynamics of cysteine modification. Likewise, the combined application of modern mass spectrometers with improved sample preparation techniques and novel data mining algorithms is beginning to routinize the analysis of complex protein disulfide structures. Critical Issues: Proper application of these modern tools and techniques, however, still requires fundamental understanding of sulfhydryl chemistry as well as the assumptions that accompany sample preparation and underlie effective data interpretation. Future Directions: The continued development of tools, technical approaches, and corresponding data processing algorithms will, undoubtedly, facilitate site-specific protein sulfhydryl quantification and disulfide structure analysis from within complex biological mixtures with ever-improving accuracy and sensitivity. Fully routinizing disulfide structure analysis will require an equal but balanced focus on sample preparation and corresponding mass spectral dataset reproducibility. Antioxid. Redox Signal. 21, 511–531. PMID:24383618

  18. Evidence for a cysteine-mediated mechanism of excitation energy regulation in a photosynthetic antenna complex

    Science.gov (United States)

    Orf, Gregory S.; Saer, Rafael G.; Niedzwiedzki, Dariusz M.; Zhang, Hao; McIntosh, Chelsea L.; Schultz, Jason W.; Mirica, Liviu M.; Blankenship, Robert E.

    2016-01-01

    Light-harvesting antenna complexes not only aid in the capture of solar energy for photosynthesis, but regulate the quantity of transferred energy as well. Light-harvesting regulation is important for protecting reaction center complexes from overexcitation, generation of reactive oxygen species, and metabolic overload. Usually, this regulation is controlled by the association of light-harvesting antennas with accessory quenchers such as carotenoids. One antenna complex, the Fenna–Matthews–Olson (FMO) antenna protein from green sulfur bacteria, completely lacks carotenoids and other known accessory quenchers. Nonetheless, the FMO protein is able to quench energy transfer in aerobic conditions effectively, indicating a previously unidentified type of regulatory mechanism. Through de novo sequencing MS, chemical modification, and mutagenesis, we have pinpointed the source of the quenching action to cysteine residues (Cys49 and Cys353) situated near two low-energy bacteriochlorophylls in the FMO protein from Chlorobaculum tepidum. Removal of these cysteines (particularly removal of the completely conserved Cys353) through N-ethylmaleimide modification or mutagenesis to alanine abolishes the aerobic quenching effect. Electrochemical analysis and electron paramagnetic resonance spectra suggest that in aerobic conditions the cysteine thiols are converted to thiyl radicals which then are capable of quenching bacteriochlorophyll excited states through electron transfer photochemistry. This simple mechanism has implications for the design of bio-inspired light-harvesting antennas and the redesign of natural photosynthetic systems. PMID:27335466

  19. A structural and mechanistic study of π-clamp-mediated cysteine perfluoroarylation.

    Science.gov (United States)

    Dai, Peng; Williams, Jonathan K; Zhang, Chi; Welborn, Matthew; Shepherd, James J; Zhu, Tianyu; Van Voorhis, Troy; Hong, Mei; Pentelute, Bradley L

    2017-08-11

    Natural enzymes use local environments to tune the reactivity of amino acid side chains. In searching for small peptides with similar properties, we discovered a four-residue π-clamp motif (Phe-Cys-Pro-Phe) for regio- and chemoselective arylation of cysteine in ribosomally produced proteins. Here we report mutational, computational, and structural findings directed toward elucidating the molecular factors that drive π-clamp-mediated arylation. We show the significance of a trans conformation prolyl amide bond for the π-clamp reactivity. The π-clamp cysteine arylation reaction enthalpy of activation (ΔH ‡ ) is significantly lower than a non-π-clamp cysteine. Solid-state NMR chemical shifts indicate the prolyl amide bond in the π-clamp motif adopts a 1:1 ratio of the cis and trans conformation, while in the reaction product Pro3 was exclusively in trans. In two structural models of the perfluoroarylated product, distinct interactions at 4.7 Å between Phe1 side chain and perfluoroaryl electrophile moiety are observed. Further, solution 19 F NMR and isothermal titration calorimetry measurements suggest interactions between hydrophobic side chains in a π-clamp mutant and the perfluoroaryl probe. These studies led us to design a π-clamp mutant with an 85-fold rate enhancement. These findings will guide us toward the discovery of small reactive peptides to facilitate abiotic chemistry in water.

  20. Neuraminidase inhibitor susceptibility and neuraminidase enzyme kinetics of human influenza A and B viruses circulating in Thailand in 2010-2015.

    Science.gov (United States)

    Tewawong, Nipaporn; Marathe, Bindumadhav M; Poovorawan, Yong; Vongpunsawad, Sompong; Webby, Richard J; Govorkova, Elena A

    2018-01-01

    Amino acid substitutions within or near the active site of the viral neuraminidase (NA) may affect influenza virus fitness. In influenza A(H3N2) and B viruses circulating in Thailand between 2010 and 2015, we identified several NA substitutions that were previously reported to be associated with reduced inhibition by NA inhibitors (NAIs). To study the effect of these substitutions on the enzymatic properties of NA and on virus characteristics, we generated recombinant influenza viruses possessing either a wild type (WT) NA or an NA with a single I222V, S331G, or S331R substitution [in influenza A(H3N2) viruses] or a single D342S, A395T, A395V, or A395D NA substitution (in influenza B viruses). We generated recombinant (7:1) influenza A and B viruses on the genetic background of A/Puerto Rico/8/1934 (A/PR/8, H1N1) or B/Yamanashi/166/1998 (B/YAM) viruses, respectively. In contrast to the expected phenotypes, all the recombinant influenza A(H3N2) and B viruses carrying putative NA resistance substitutions were susceptible to NAIs. The Km and Vmax for the NAs of A/PR8-S331G and A/PR8-S331R viruses were higher than for the NA of WT virus, and the corresponding values for the B/YAM-D342S virus were lower than for the NA of WT virus. Although there was initial variation in the kinetics of influenza A and B viruses' replication in MDCK cells, their titers were comparable to each other and to WT viruses at later time points. All introduced substitutions were stable except for B/YAM-D342S and B/YAM-A395V which reverted to WT sequences after three passages. Our data suggest that inferring susceptibility to NAIs based on sequence information alone should be cautioned. The impact of NA substitution on NAI resistance, viral growth, and enzymatic properties is viral context dependent and should be empirically determined.

  1. Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges.

    Science.gov (United States)

    Dhanapala, Pathum; Withanage-Dona, Dulashi; Tang, Mimi L K; Doran, Tim; Suphioglu, Cenk

    2017-02-21

    Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli ( E. coli ) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients' sera. A pool of non-allergic patients' sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis.

  2. Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway.

    Science.gov (United States)

    Liu, Xuehong; Dawson, David C

    2011-11-29

    Cysteine scanning has been widely used to identify pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR). These studies, however, have been typically conducted at room temperature rather than human body temperature. Reports of substantial effects of temperature on gating and anion conduction in CFTR channels as well as an unexpected pattern of cysteine reactivity in the sixth transmembrane segment (TM6) prompted us to investigate the effect of temperature on the reactivity of cysteines engineered into TM6 of CFTR. We compared reaction rates at temperatures ranging from 22 to 37 °C for cysteines placed on either side of an apparent size-selective accessibility barrier previously defined by comparing reactivity toward channel-permeant and channel-impermeant, thiol-directed reagents. The results indicate that the reactivity of cysteines at three positions extracellular to the position of the accessibility barrier, 334, 336, and 337, is highly temperature-dependent. At 37 °C, cysteines at these positions were highly reactive toward MTSES(-), whereas at 22 °C, the reaction rates were 2-6-fold slower to undetectable. An activation energy of 157 kJ/mol for the reaction at position 337 is consistent with the hypothesis that, at physiological temperature, the extracellular portion of the CFTR pore can adopt conformations that differ significantly from those that can be accessed at room temperature. However, the position of the accessibility barrier defined empirically by applying channel-permeant and channel-impermeant reagents to the extracellular aspect of the pore is not altered. The results illuminate previous scanning results and indicate that the assay temperature is a critical variable in studies designed to use chemical modification to test structural models for the CFTR anion conduction pathway.

  3. Cysteine peroxidase activity in rat blood plasma | Razygraev ...

    African Journals Online (AJOL)

    The rat plasma found to be able to accelerate greatly the H2O2-dependent oxidation of cysteine. The activity was a characteristic of a protein fraction precipitated at 30—44% ammonium sulfate saturation, and the specific activity in protein fraction was significantly higher than in plasma. Cysteine:H2O2 oxidoreductase ...

  4. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the...

  5. Reaction mechanism of O-acylhydroxamate with cysteine proteases

    Indian Academy of Sciences (India)

    WINTEC

    such as, cardiovascular, oncology, osteoporosis, and arthritis. Till now, twenty one families of cysteine proteases have been discovered5, almost half of them are found in viruses and others are found in bacteria, fungi, pro- tozoa and plants. In mammals, two main groups of cysteine proteases are present: cytosolic calpains.

  6. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned...

  7. Novel Cysteine Desulfidase CdsB Involved in Releasing Cysteine Repression of Toxin Synthesis in Clostridium difficile

    Directory of Open Access Journals (Sweden)

    Huawei Gu

    2018-01-01

    Full Text Available Clostridium difficile, a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δerm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na2S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ54 (SigL-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR::ermB or sigL::ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation.

  8. Oxidation Resistance of the Sulfur Amino Acids: Methionine and Cysteine

    Directory of Open Access Journals (Sweden)

    Peng Bin

    2017-01-01

    Full Text Available Sulfur amino acids are a kind of amino acids which contain sulfhydryl, and they play a crucial role in protein structure, metabolism, immunity, and oxidation. Our review demonstrates the oxidation resistance effect of methionine and cysteine, two of the most representative sulfur amino acids, and their metabolites. Methionine and cysteine are extremely sensitive to almost all forms of reactive oxygen species, which makes them antioxidative. Moreover, methionine and cysteine are precursors of S-adenosylmethionine, hydrogen sulfide, taurine, and glutathione. These products are reported to alleviate oxidant stress induced by various oxidants and protect the tissue from the damage. However, the deficiency and excess of methionine and cysteine in diet affect the normal growth of animals; thereby a new study about defining adequate levels of methionine and cysteine intake is important.

  9. Expression of Insoluble Influenza Neuraminidase Type 1 (NA1 Protein in Tobacco

    Directory of Open Access Journals (Sweden)

    Teen Lee Pua

    2012-12-01

    Full Text Available The avian influenza virus, particularly H5N1 strain, is highly virulent to poultry and mankind. Several expression systems, like yeast, baculovirus and mammalian cells, have been adopted to produce vaccine candidate for this lethal disease. The present research aimed at developing a recombinant vaccine candidate, neuraminidase type 1 (NA1, for the Malaysia isolate of H5N1 in Nicotiana benthamiana. The NA1 gene was fused directly in-frame in cowpea mosaic virus (CPMV-based pEAQ-HT vector with C-terminal polyhistidine-tag incorporated to ease the subsequent purification step. The expression of the NA1 gene in tobacco was confirmed at RNA and protein levels at 6 days post-infiltration (Dpi. From the insoluble fraction of the protein, a recombinant glycosylated NA1 protein with a molecular weight of ~56 kDa was immunogenically detected by a specific anti-NA polyclonal antibody. We report for the first time the insolubility of the plant-made NA1 protein where a native sequence was used for its expression. This study signifies the necessity of the use of optimised sequences for expression work and provides great opportunity for the exploration of plant-manufactured NA1 protein as vaccine candidate.

  10. Cysteine sulfoxide derivatives in Petiveria alliacea.

    Science.gov (United States)

    Kubec, R; Musah, R A

    2001-11-01

    Two diastereomers of S-benzyl-L-cysteine sulfoxide have been isolated from fresh roots of Petiveria alliacea. Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy and confirmed by comparison with authentic compounds. Both the R(S) and S(S) diastereomers of the sulfoxide are present in all parts of the plant (root, stem, and leaves) with the latter diastereomer being predominant. Their total content greatly varied in different parts of the plant between 0.07 and 2.97 mg g(-1) fr. wt, being by far the highest in the root. S-Benzylcysteine has also been detected in trace amounts (<10 microg g(-1) fr. wt) in all parts of the plant. This represents the first report of the presence of S-benzylcysteine derivatives in nature.

  11. Retaining in-gel zymographic activity of cysteine proteases via a cysteine-supplemented running buffer.

    Science.gov (United States)

    Vootukuri Reddy, Sreekanth; Philpott, Mike P; Trigiante, Giuseppe

    2016-10-01

    Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Residue processing

    Energy Technology Data Exchange (ETDEWEB)

    Gieg, W.; Rank, V.

    1942-10-15

    In the first stage of coal hydrogenation, the liquid phase, light and heavy oils were produced; the latter containing the nonliquefied parts of the coal, the coal ash, and the catalyst substances. It was the problem of residue processing to extract from these so-called let-down oils that which could be used as pasting oils for the coal. The object was to obtain a maximum oil extraction and a complete removal of the solids, because of the latter were returned to the process they would needlessly burden the reaction space. Separation of solids in residue processing could be accomplished by filtration, centrifugation, extraction, distillation, or low-temperature carbonization (L.T.C.). Filtration or centrifugation was most suitable since a maximum oil yield could be expected from it, since only a small portion of the let-down oil contained in the filtration or centrifugation residue had to be thermally treated. The most satisfactory centrifuge at this time was the Laval, which delivered liquid centrifuge residue and centrifuge oil continuously. By comparison, the semi-continuous centrifuges delivered plastic residues which were difficult to handle. Various apparatus such as the spiral screw kiln and the ball kiln were used for low-temperature carbonization of centrifuge residues. Both were based on the idea of carbonization in thin layers. Efforts were also being made to produce electrode carbon and briquette binder as by-products of the liquid coal phase.

  13. Fitness costs for Influenza B viruses carrying neuraminidase inhibitor-resistant substitutions: underscoring the importance of E119A and H274Y.

    Science.gov (United States)

    Burnham, Andrew J; Baranovich, Tatiana; Marathe, Bindumadhav M; Armstrong, Jianling; Webster, Robert G; Govorkova, Elena A

    2014-05-01

    Influenza B viruses cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated pediatric mortality. Neuraminidase (NA) inhibitors (NAIs) are the only available therapy for patients infected with influenza B viruses, and the potential emergence of NAI-resistant viruses is a public health concern. The NA substitutions located within the enzyme active site could not only reduce NAI susceptibility of influenza B virus but also affect virus fitness. In this study, we investigated the effect of single NA substitutions on the fitness of influenza B/Yamanashi/166/1998 viruses (Yamagata lineage). We generated recombinant viruses containing either wild-type (WT) NA or NA with a substitution in the catalytic (R371K) or framework (E119A, D198E, D198Y, I222T, H274Y, and N294S) residues. We assessed NAI susceptibility, NA biochemical properties, NA protein expression, and virus replication in vitro and in differentiated normal human bronchial epithelial (NHBE) cells. Our results showed that four NA substitutions (D198E, I222T, H274Y, and N294S) conferred reduced inhibition by oseltamivir and three (E119A, D198Y, and R371K) conferred highly reduced inhibition by oseltamivir, zanamivir, and peramivir. All NA substitutions, except for D198Y and R371K, were genetically stable after seven passages in MDCK cells. Cell surface NA protein expression was significantly increased by H274Y and N294S substitutions. Viruses with the E119A, I222T, H274Y, or N294S substitution were not attenuated in replication efficiency in vitro or in NHBE cells. Overall, viruses with the E119A or H274Y NA substitution possess fitness comparable to NAI-susceptible virus, and the acquisition of these substitutions by influenza B viruses should be closely monitored.

  14. Evaluation of the Selectivity and Cysteine Dependence of Inhibitors across the Regulator of G Protein-Signaling Family.

    Science.gov (United States)

    Hayes, Michael P; Bodle, Christopher R; Roman, David L

    2018-01-01

    Since their discovery more than 20 years ago, regulators of G protein-signaling (RGS) proteins have received considerable attention as potential drug targets because of their ability to modulate G α activity. Efforts to identify small molecules capable of inhibiting the protein-protein interactions between activated G α subunits and RGS proteins have yielded a substantial number of inhibitors, especially toward the well studied RGS4. These efforts also determined that many of these small molecules inhibit the protein-protein interactions through covalent modification of cysteine residues within the RGS domain that are located distal to the G α -binding interface. As some of these cysteine residues are highly conserved within the RGS family, many of these inhibitors display activity toward multiple RGS family members. In this work, we sought to determine the selectivity of these small-molecule inhibitors against 12 RGS proteins, as well as against the cysteine-null mutants for 10 of these proteins. Using both biochemical and cell-based methods to assess G α -RGS complex formation and G α enzymatic activity, we found that several previously identified RGS4 inhibitors were active against other RGS members, such as RGS14, with comparable or greater potency. Additionally, for every compound tested, activity was dependent on the presence of cysteine residues. This work defines the selectivity of commercially available RGS inhibitors and provides insight into the RGS family members for which drug discovery efforts may be most likely to succeed. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Lysine- and cysteine-based protein adductions derived from toxic metabolites of 8-epidiosbulbin E acetate.

    Science.gov (United States)

    Lin, Dongju; Wang, Kai; Guo, Xiucai; Gao, Huiyuan; Peng, Ying; Zheng, Jiang

    2016-12-15

    Furanoid 8-epidiosbulbin E acetate (EEA) is a major constituent of herbal medicine Dioscorea bulbifera L. (DB), a traditional herbal medicine widely used in Asian nations. Our early studies demonstrated that administration of EEA caused acute hepatotoxicity in mice and the observed toxicity required P450-mediated metabolic activation. Protein modification by reactive metabolites of EEA has been suggested to be an important mechanism of EEA-induced hepatotoxicity. The objectives of the present study were to investigate the interaction of the electrophilic reactive metabolites derived from EEA with lysine and cysteine residues of proteins and to define the correlation of protein adductions of EEA and the hepatotoxicity induced by EEA. EEA-derived cis-enedial was found to modify both lysine and cysteine residues of proteins. The observed modifications increased with the increase in doses administered in the animals. The formation of protein adductions derived from the reactive metabolites of EEA were potentiated by buthionine sulfoximine, but were attenuated by ketoconazole. This work facilitated better understanding of the mechanisms of toxic action of EEA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  16. Overlapping and independent structural roles for human papillomavirus type 16 L2 conserved cysteines.

    Science.gov (United States)

    Conway, Michael J; Alam, Samina; Christensen, Neil D; Meyers, Craig

    2009-10-25

    Cryoelectron microscopy images of HPV16 pseudovirions (PsV) depict that each pentamer of L1 can be occluded with a monomer of L2. Further research suggests that an N-terminal external loop of L2 exists, which is the target of neutralizing and cross-neutralizing antibodies. Here we show that N-terminal L2 cysteine residues, Cys22 and Cys28, have overlapping and independent structural roles, which affect both early- and late-stage assembly events. Substitution of either cysteine residue enhances infectivity markedly in comparison to wild-type HPV16. However, only Cys22Ser 20-day virions become nearly as stable as wild type. In addition, Cys22Ser, and Cys22,28Ser 20-day virions have lost their susceptibility to neutralization by anti-L2 antibodies, whereas Cys28Ser 20-day virions remain partially susceptible. These results suggest that Cys28 is necessary for late-stage stabilization of capsids, while Cys22 is necessary for proper display of L2 neutralizing epitopes.

  17. Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay

    Directory of Open Access Journals (Sweden)

    Stefanie Quosdorf

    2017-11-01

    Full Text Available Neuraminidase is a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2′-(4-methylumbelliferyl-α-d-N-acetylneuraminic acid (MUNANA-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1 and bacterial Vibrio cholerae neuraminidase (VCNA. Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure–activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs® 7630 (root extract of Pelargonium sidoides showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed.

  18. Action of X-rays and neuraminidase on pinocytotic activity and microtubule-microfilament system of Ehrlich ascites tumor cells in monolayer culture

    Energy Technology Data Exchange (ETDEWEB)

    Pfab, R.; Hess, F.; Schachtschabel, D.O.; Paul, N.; Kern, H.F.

    1981-07-01

    Monolayer cultures of Ehrlich ascites tumor cells in exponential growth phase were treated with X-rays and neuraminidase alone or in combination. A radiation dose of 2 Gy (200 rd) and higher effected a significant inhibition of DNA synthesis and cell proliferation. Neuraminidase treatment in addition to irradiation did not modify the growth-inhibitory irradiation effect. Cells pretreated for 0.5 to 1.0 hours with neuraminidase (in Earle's salt solution) and cultured thereafter (4 h) in serum-containing growth medium exhibited an enhanced development of the microtube-microfilament-system. Morphometric analysis of microtubulus on electronmicroscopic sections revealed a nearly 4-fold increased number in neuraminidase treated cells while the average length of microtubules was similar to controls. Pinocytosis as measured by the ultrastructural uptake of ferritin appeared to be increased following neuraminidase treatment. A connection between neuraminic acid containing components (glycoproteins or glycolipids) of the cell membrane or cell surface, on the one hand, and the cytoskeleton and the endocytotic process of these tumor cells, on the other hand, is suggested.

  19. Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

    Directory of Open Access Journals (Sweden)

    Chunxiang Yao

    Full Text Available Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2, react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat, an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

  20. Cysteine peptidases from Phytomonas serpens: biochemical and immunological approaches.

    Science.gov (United States)

    Elias, Camila G R; Aor, Ana Carolina; Valle, Roberta S; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

    2009-12-01

    Phytomonas serpens, a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi. In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens, while a strong 50-kDa cysteine peptidase was observed in T. cruzi. Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens. The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens, including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection.

  1. Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

    Science.gov (United States)

    Yao, Chunxiang; Behring, Jessica B; Shao, Di; Sverdlov, Aaron L; Whelan, Stephen A; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A; Seta, Francesca; Costello, Catherine E; Cohen, Richard A; Matsui, Reiko; Colucci, Wilson S; McComb, Mark E; Bachschmid, Markus M

    2015-01-01

    Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

  2. Subcellular distribution of glutathione and cysteine in cyanobacteria.

    Science.gov (United States)

    Zechmann, Bernd; Tomasić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-10-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.

  3. Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue.

    Science.gov (United States)

    Gupta, Deepak; Varghese Gupta, Sheeba; Dahan, Arik; Tsume, Yasuhiro; Hilfinger, John; Lee, Kyung-Dall; Amidon, Gordon L

    2013-02-04

    Poor oral absorption is one of the limiting factors in utilizing the full potential of polar antiviral agents. The neuraminidase target site requires a polar chemical structure for high affinity binding, thus limiting oral efficacy of many high affinity ligands. The aim of this study was to overcome this poor oral absorption barrier, utilizing prodrug to target the apical brush border peptide transporter 1 (PEPT1). Guanidine oseltamivir carboxylate (GOCarb) is a highly active polar antiviral agent with insufficient oral bioavailability (4%) to be an effective therapeutic agent. In this report we utilize a carrier-mediated targeted prodrug approach to improve the oral absorption of GOCarb. Acyloxy(alkyl) ester based amino acid linked prodrugs were synthesized and evaluated as potential substrates of mucosal transporters, e.g., PEPT1. Prodrugs were also evaluated for their chemical and enzymatic stability. PEPT1 transport studies included [(3)H]Gly-Sar uptake inhibition in Caco-2 cells and cellular uptake experiments using HeLa cells overexpressing PEPT1. The intestinal membrane permeabilities of the selected prodrugs and the parent drug were then evaluated for epithelial cell transport across Caco-2 monolayers, and in the in situ rat intestinal jejunal perfusion model. Prodrugs exhibited a pH dependent stability with higher stability at acidic pHs. Significant inhibition of uptake (IC(50) 30-fold increase in affinity compared to GOCarb. The l-valyl prodrug exhibited significant enhancement of uptake in PEPT1/HeLa cells and compared favorably with the well-absorbed valacyclovir. Transepithelial permeability across Caco-2 monolayers showed that these amino acid prodrugs have a 2-5-fold increase in permeability as compared to the parent drug and showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral

  4. Neuraminidase-1 is required for the normal assembly of elastic fibers.

    Science.gov (United States)

    Starcher, Barry; d'Azzo, Alessandra; Keller, Patrick W; Rao, Gottipati K; Nadarajah, Deepa; Hinek, Alexsander

    2008-10-01

    The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.

  5. Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors.

    Science.gov (United States)

    Li, Ning; Ren, Aihui; Wang, Xiaoshuang; Fan, Xin; Zhao, Yong; Gao, George F; Cleary, Patrick; Wang, Beinan

    2015-01-06

    Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

  6. Effects of vaccination and the new neuraminidase inhibitor, laninamivir, on influenza infection.

    Directory of Open Access Journals (Sweden)

    Takuro Mizuno

    Full Text Available BACKGROUND: Evidence of the effectiveness of influenza vaccination in children and elderly adults is limited, although this population has the highest risk for influenza infection. MATERIALS AND METHODS: We enrolled 4443 participants, aged 3-97 years, who had influenza-kit-positive results during seasons 2007-12, including 2135 with influenza A, 534 with A/H1N1, and 1643 with influenza B. Eligible subjects completed a questionnaire to identify past influenza infection and vaccination history. For the diagnosis of current influenza infection, subjects were examined, and pharyngeal swabs were collected and tested using the Capilia flu rapid diagnosis kit to confirm influenza infection. An interim analysis was performed using clinician-based surveillance data for the entire four seasons of influenza infection in Japan. RESULTS: In 3035 adults aged 14-64 years, administration of the influenza vaccine significantly reduced the frequency of infection (P65 years significantly. Laninamivir, oseltamivir phosphate, zanamivir hydrate, and amantadine hydrochloride were administered to 1381, 2432, 1044, and 100 patients, respectively. They were effective in >97% of patients, with no significant differences being found. Adverse effects were few. However, the recurrence rate of influenza infection after treatment was significantly reduced in patients who received laninamivir compared with that in those who received oseltamivir and zanamivir (P<0.01. The effectiveness of laninamivirdid not decrease. CONCLUSIONS: The vaccines administered had limited efficacy in reducing the frequency of influenza infection in young adults. Laninamivir significantly reduced the recurrence of influenza infection when compared with other neuraminidase inhibitors.

  7. A generic system for the expression and purification of soluble and stable influenza neuraminidase.

    Directory of Open Access Journals (Sweden)

    Peter M Schmidt

    Full Text Available The influenza surface glycoprotein neuraminidase (NA is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir and Relenza (zanamivir that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent 'swine flu' pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1 H(274Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.

  8. Preterm human milk contains a large pool of latent TGF-β, which can be activated by exogenous neuraminidase

    Science.gov (United States)

    Namachivayam, Kopperuncholan; Blanco, Cynthia L.; Frost, Brandy L.; Reeves, Aaron A.; Jagadeeswaran, Ramasamy; MohanKumar, Krishnan; Safarulla, Azif; Mandal, Partha; Garzon, Steven A.; Raj, J. Usha

    2013-01-01

    Human milk contains substantial amounts of transforming growth factor (TGF)-β, particularly the isoform TGF-β2. We previously showed in preclinical models that enterally administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study we hypothesized that premature infants remain at higher risk of NEC than full-term infants, even when they receive their own mother's milk, because preterm human milk contains less bioactive TGF-β than full-term milk. Our objective was to compare TGF-β bioactivity in preterm vs. full-term milk and identify factors that activate milk-borne TGF-β. Mothers who delivered between 23 0/7 and 31 6/7 wk or at ≥37 wk of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling were measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, TGF-β3, and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Preterm milk contains large quantities of TGF-β, but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects. PMID:23558011

  9. Methanolic soluble fractions of lingzhi or reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) extract inhibit neuraminidase activity in Newcastle disease virus (LaSota).

    Science.gov (United States)

    Shamaki, Bala U; Sandabe, Umar K; Ogbe, Adamu O; Abdulrahman, Fanna I; El-Yuguda, Abdul-Dahiru

    2014-01-01

    The antineuraminidase activity of different organic soluble fractions of Ganoderma lucidum extract was investigated using inhibition of hemagglutination and elution of chicken erythrocytes by Newcastle disease virus (NDV). Fractions of methanol, ethylacetate, and normal butanol (n-butanol) of the G. lucidum were tested against neuraminidase producing NDV as antigen. Different dilutions of the organic soluble fractions inhibited elution of 1% red blood cells by neuraminidase of NDV While the methanolic and n-butanol extracts inhibited neuraminidase activity even at a dilution of 1:16 and that of ethylacetate fraction inhibited even at 1:32 respectively. This finding indicates that G. lucidum has some antineuraminidase activity against NDV and may be exploited in the management of NDV infection.

  10. Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin

    International Nuclear Information System (INIS)

    Chu, Y.C.; Wang, R.Y.; Burke, G.T.; Chanley, J.D.; Katsoyannis, P.G.

    1987-01-01

    The authors have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine,2,2,2,-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A 20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A 20 -A 21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A 20 -A 21 amide bond can modulate biological activity independently of receptor binding affinity. The A 20 -A 21 amide bond is thus considered as an important element in the message region of insulin

  11. Organometallic palladium reagents for cysteine bioconjugation

    Science.gov (United States)

    Vinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.

    2015-10-01

    Reactions based on transition metals have found wide use in organic synthesis, in particular for the functionalization of small molecules. However, there are very few reports of using transition-metal-based reactions to modify complex biomolecules, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature and mild pH) and the existence of multiple reactive functional groups found in biomolecules. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants and external thiol nucleophiles. The broad utility of the bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as benchtop reagents for diverse bioconjugation applications.

  12. Residual risk

    African Journals Online (AJOL)

    ing the residual risk of transmission of HIV by blood transfusion. An epidemiological approach assumed that all HIV infections detected serologically in first-time donors were pre-existing or prevalent infections, and that all infections detected in repeat blood donors were new or incident infections. During 1986 - 1987,0,012%.

  13. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

    DEFF Research Database (Denmark)

    Serafimova, Iana M; Pufall, Miles A; Krishnan, Shyam

    2012-01-01

    show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation...

  14. Cysteine String Protein α: A New Role in Vesicle Recycling

    OpenAIRE

    Sheng, Jiansong; Wu, Ling-Gang

    2012-01-01

    Zhang et al. (2012) and Rozas et al. (2012) find that cysteine string protein α, a protein involved in neurodegeneration, regulates vesicle endocytosis via interaction with dynamin 1, which may participate in regulating synaptic transmission and possibly in maintaining synapses.

  15. Computational analysis and determination of a highly conserved surface exposed segment in H5N1 avian flu and H1N1 swine flu neuraminidase

    Directory of Open Access Journals (Sweden)

    Nandy Ashesh

    2010-02-01

    Full Text Available Abstract Background Catalytic activity of influenza neuraminidase (NA facilitates elution of progeny virions from infected cells and prevents their self-aggregation mediated by the catalytic site located in the body region. Research on the active site of the molecule has led to development of effective inhibitors like oseltamivir, zanamivir etc, but the high rate of mutation and interspecies reassortment in viral sequences and the recent reports of oseltamivir resistant strains underlines the importance of determining additional target sites for developing future antiviral compounds. In a recent computational study of 173 H5N1 NA gene sequences we had identified a 50-base highly conserved region in 3'-terminal end of the NA gene. Results We extend the graphical and numerical analyses to a larger number of H5N1 NA sequences (514 and H1N1 swine flu sequences (425 accessed from GenBank. We use a 2D graphical representation model for the gene sequences and a Graphical Sliding Window Method (GSWM for protein sequences scanning the sequences as a block of 16 amino acids at a time. Using a protein sequence descriptor defined in our model, the protein sliding scan method allowed us to compare the different strains for block level variability, which showed significant statistical correlation to average solvent accessibility of the residue blocks; single amino acid position variability results in no correlation, indicating the impact of stretch variability in chemical environment. Close to the C-terminal end the GSWM showed less descriptor-variability with increased average solvent accessibility (ASA that is also supported by conserved predicted secondary structure of 3' terminal RNA and visual evidence from 3D crystallographic structure. Conclusion The identified terminal segment, strongly conserved in both RNA and protein sequences, is especially significant as it is surface exposed and structural chemistry reveals the probable role of this stretch in

  16. Potential role of cysteine and methionine in the protection against hormonal imbalance and mutagenicity induced by furazolidone in female rats.

    Science.gov (United States)

    Ahmed, Hanaa H; El-Aziem, Sekena H Abd; Abdel-Wahhab, Mosaad A

    2008-01-14

    The use of nitrofurans as veterinary drugs has been banned in the EU since 1993 due to doubts on the safety of the protein-bound residues of these drugs in edible products. Furazolidone (FUZ) is a nitrofuran drug, which has been used for many years as an antibacterial drug in veterinary practice. The aim of the current study is to investigate the role of L-cysteine and L-methionine in the protection against hormonal imbalance and the genotoxicity induced by FUZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis in female rats. Forty female Sprague-Dawley rats were divided into four groups included the untreated control group; a group treated with FUZ (300 mg/kg b.w.); a group treated with a mixture of L-cysteine (300 mg/kg b.w.) and L-methionine (42.8 mg/kg b.w.) and a group treated with FUZ plus the mixture of L-cysteine and L-methionine for 10 days. The results indicated that FUZ induced hormonal disturbances involving thyroid, ovarian and adrenal hormones. Moreover, FUZ increased the micronucleus formation and induced changes in polymorphic band patterns. The combined treatment with FUZ and the mixture of L-cysteine and L-methionine succeeded to prevent or diminish the endocrine disturbance and the clastogenic effects of FUZ. The current study is casting new light on the complex mechanisms underlying the ameliorating action of dietary L-cysteine and L-methionine against FUZ toxicity in experimental animals.

  17. MTSET modification of D4S6 cysteines stabilize the fast inactivated state of Nav1.5 sodium channels

    Directory of Open Access Journals (Sweden)

    Michael E O'leary

    2015-06-01

    Full Text Available The transmembrane S6 segments of Na+ sodium channels form the cytoplasmic entrance of the channel and line the internal aspects of the aqueous pore. This region of the channel has been implicated in Na+ channel permeation, gating and pharmacology. In this study we utilized cysteine substitutions and methanethiosulfonate reagent (MTSET to investigate the role of the S6 segment of homologous domain 4 (D4S6 in the gating of the cardiac (Nav1.5 channel. D4S6 cysteine mutants were heterologously expressed in tsA201 cells and currents recorded using whole-cell patch clamp. Internal MTSET reduced the peak Na+ currents, induced hyperpolarizing shifts in steady-state inactivation and slowed the recovery of mutant channels with cysteines inserted near the middle (F1760C, V1763C and C-terminus (Y1767C of the D4S6. These findings suggested a link between the MTSET inhibition and fast inactivation. This was confirmed by expressing the V1763C and Y1767C mutations in non-inactivating Nav1.5 channels. Removing inactivation abolished the MTSET inhibition of the V1763C and Y1767C mutants. The data indicate that the MTSET-induced reduction in current primarily results from slower recovery from inactivation that produces hyperpolarizing shifts in fast inactivation and decreases the steady-state availability of the channels. This contrasted with a cysteine inserted near the C-terminus of the D4S6 (I1770C where MTSET increased the persistent Na+ current at depolarized voltages consistent with impaired fast inactivation. Covalent modification of D4S6 cysteines with MTSET adduct appears to reduce the mobility of the D4S6 segment and stabilize the channels in the fast inactivated state. These findings indicate that residues located near the middle and C-terminus of the D4S6 play an important role in fast inactivation.

  18. Potential role of cysteine and methionine in the protection against hormonal imbalance and mutagenicity induced by furazolidone in female rats

    International Nuclear Information System (INIS)

    Ahmed, Hanaa H.; El-Aziem, Sekena H. Abd; Abdel-Wahhab, Mosaad A.

    2008-01-01

    The use of nitrofurans as veterinary drugs has been banned in the EU since 1993 due to doubts on the safety of the protein-bound residues of these drugs in edible products. Furazolidone (FUZ) is a nitrofuran drug, which has been used for many years as an antibacterial drug in veterinary practice. The aim of the current study is to investigate the role of L-cysteine and L-methionine in the protection against hormonal imbalance and the genotoxicity induced by FUZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis in female rats. Forty female Sprague-Dawley rats were divided into four groups included the untreated control group; a group treated with FUZ (300 mg/kg b.w.); a group treated with a mixture of L-cysteine (300 mg/kg b.w.) and L-methionine (42.8 mg/kg b.w.) and a group treated with FUZ plus the mixture of L-cysteine and L-methionine for 10 days. The results indicated that FUZ induced hormonal disturbances involving thyroid, ovarian and adrenal hormones. Moreover, FUZ increased the micronucleus formation and induced changes in polymorphic band patterns. The combined treatment with FUZ and the mixture of L-cysteine and L-methionine succeeded to prevent or diminish the endocrine disturbance and the clastogenic effects of FUZ. The current study is casting new light on the complex mechanisms underlying the ameliorating action of dietary L-cysteine and L-methionine against FUZ toxicity in experimental animals

  19. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

    OpenAIRE

    Lepelley Maud; Amor Mohamed; Martineau Nelly; Cheminade Gerald; Caillet Victoria; McCarthy James

    2012-01-01

    Abstract Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences ...

  20. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus

    DEFF Research Database (Denmark)

    Willumsen, B M; Norris, K; Papageorge, A G

    1984-01-01

    localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein...... not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue......Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane...

  1. Synthesis, antioxidative and whitening effects of novel cysteine derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Ji Hoon; Kim, Kyoung Mi; Jeong, Yoon Ju; Park, Young Min; Lee, Jae Young; Park, Soo Nam [Dept. of Fine Chemistry, Cosmetic R and D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, Seoul (Korea, Republic of); Park, Jino [Daebong LS. Ltd, Incheon (Korea, Republic of)

    2017-01-15

    Recently, development of biocompatibility functional cosmetic agents as antioxidant or whitening agent has increased. In this study, synthetic cysteine derivatives (DBLS-21, -24, and -33) were developed containing syringic acid and cysteine moieties (l-cysteine ethyl ester, N-acetyl cysteine methyl ester, and N-acetyl cysteine ethyl ester), and their antioxidative and whitening activities were evaluated. The cellular protective effect (τ{sub 50}) of DBLS-21 was 51.1 min at 50 μM on {sup 1}O{sub 2} -induced hemolysis of erythrocytes. This activity was slightly higher than that of α-tocopherol (43.6 min) as a lipophilic antioxidant. In the melanogenesis inhibitory effect, DBLS-21, -24, and -33 was 1.6-, 1.8-, and 2.5-fold higher than arbutin, respectively. In particular, DBLS-21 and -33 was 112.8- and 6.1-fold higher than arbutin, respectively (293.4 μM) on tyrosinase inhibition activity (IC{sub 50} ). But DBLS-24 had no tyrosinase inhibitory activity. These results suggest that cysteine derivatives possess potential for use as an antioxidant agent (DBLS-21) and whitening agents (all derivatives) in cosmetics.

  2. Cysteine homeostasis plays an essential role in plant immunity.

    Science.gov (United States)

    Álvarez, Consolación; Bermúdez, M Ángeles; Romero, Luis C; Gotor, Cecilia; García, Irene

    2012-01-01

    Cysteine is the metabolic precursor of essential biomolecules such as vitamins, cofactors, antioxidants and many defense compounds. The last step of cysteine metabolism is catalysed by O-acetylserine(thiol)lyase (OASTL), which incorporates reduced sulfur into O-acetylserine to produce cysteine. In Arabidopsis thaliana, the main OASTL isoform OAS-A1 and the cytosolic desulfhydrase DES1, which degrades cysteine, contribute to the cytosolic cysteine homeostasis. • Meta-analysis of the transcriptomes of knockout plants for OAS-A1 and for DES1 show a high correlation with the biotic stress series in both cases. • The study of the response of knockout mutants to plant pathogens shows that des1 mutants behave as constitutive systemic acquired resistance mutants, with high resistance to biotrophic and necrotrophic pathogens, salicylic acid accumulation and WRKY54 and PR1 induction, while oas-a1 knockout mutants are more sensitive to biotrophic and necrotrophic pathogens. However, oas-a1 knockout mutants lack the hypersensitive response associated with the effector-triggered immunity elicited by Pseudomonas syringae pv. tomato DC3000 avrRpm1. • Our results highlight the role of cysteine as a crucial metabolite in the plant immune response. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  3. Synthesis, antioxidative and whitening effects of novel cysteine derivatives

    International Nuclear Information System (INIS)

    Ha, Ji Hoon; Kim, Kyoung Mi; Jeong, Yoon Ju; Park, Young Min; Lee, Jae Young; Park, Soo Nam; Park, Jino

    2017-01-01

    Recently, development of biocompatibility functional cosmetic agents as antioxidant or whitening agent has increased. In this study, synthetic cysteine derivatives (DBLS-21, -24, and -33) were developed containing syringic acid and cysteine moieties (l-cysteine ethyl ester, N-acetyl cysteine methyl ester, and N-acetyl cysteine ethyl ester), and their antioxidative and whitening activities were evaluated. The cellular protective effect (τ 50 ) of DBLS-21 was 51.1 min at 50 μM on 1 O 2 -induced hemolysis of erythrocytes. This activity was slightly higher than that of α-tocopherol (43.6 min) as a lipophilic antioxidant. In the melanogenesis inhibitory effect, DBLS-21, -24, and -33 was 1.6-, 1.8-, and 2.5-fold higher than arbutin, respectively. In particular, DBLS-21 and -33 was 112.8- and 6.1-fold higher than arbutin, respectively (293.4 μM) on tyrosinase inhibition activity (IC 50 ). But DBLS-24 had no tyrosinase inhibitory activity. These results suggest that cysteine derivatives possess potential for use as an antioxidant agent (DBLS-21) and whitening agents (all derivatives) in cosmetics

  4. Cysteine transporter SLC3A1 promotes breast cancer tumorigenesis.

    Science.gov (United States)

    Jiang, Yang; Cao, Yuan; Wang, Yongbin; Li, Wei; Liu, Xinyi; Lv, Yixuan; Li, Xiaoling; Mi, Jun

    2017-01-01

    Cysteine is an essential amino acid for infants, aged people as well as patients with metabolic disorders. Although the thiol group of cysteine side chain is active in oxidative reactions, the role of cysteine in cancer remains largely unknown. Here, we report that the expression level of the solute carrier family 3, member 1 (SLC3A1), the cysteine carrier, tightly correlated with clinical stages and patients' survival. Elevated SLC3A1 expression accelerated the cysteine uptake and the accumulation of reductive glutathione (GSH), leading to reduced reactive oxygen species (ROS). ROS increased the stability and activity of PP2Ac, resulting in decreased AKT activity. Hence, SLC3A1 activated the AKT signaling through inhibiting PP2A phosphatase activity. Consistently, overexpression of SLC3A1 enhanced tumorigenesis of breast cancer cells, whereas blocking SLC3A1 either with specific siRNA or SLC3A1 specific inhibitor sulfasalazine suppressed tumor growth and also abolished dietary NAC-promoted tumor growth. Collectively, our data demonstrate that SLC3A1 promotes cysteine uptake and determines cellular response to antioxidant N-acetylcysteine, suggesting SLC3A1 is a potential therapeutic target for breast cancer.

  5. Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

    Science.gov (United States)

    Tseng, Tien-Sheng; Tsai, Keng-Chang; Chen, Wang-Chuan; Wang, Yeng-Tseng; Lee, Yu-Ching; Lu, Chung-Kuang; Don, Ming-Jaw; Chang, Chang-Yu; Lee, Ching-Hsiao; Lin, Hui-Hsiung; Hsu, Hung-Ju; Hsiao, Nai-Wan

    2015-07-15

    Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products.

  6. Residual basins

    International Nuclear Information System (INIS)

    D'Elboux, C.V.; Paiva, I.B.

    1980-01-01

    Exploration for uranium carried out over a major portion of the Rio Grande do Sul Shield has revealed a number of small residual basins developed along glacially eroded channels of pre-Permian age. Mineralization of uranium occurs in two distinct sedimentary units. The lower unit consists of rhythmites overlain by a sequence of black shales, siltstones and coal seams, while the upper one is dominated by sandstones of probable fluvial origin. (Author) [pt

  7. Positive Selection on Hemagglutinin and Neuraminidase Genes of H1N1 Influenza Viruses

    LENUS (Irish Health Repository)

    Li, Wenfu

    2011-04-21

    Abstract Background Since its emergence in March 2009, the pandemic 2009 H1N1 influenza A virus has posed a serious threat to public health. To trace the evolutionary path of these new pathogens, we performed a selection-pressure analysis of a large number of hemagglutinin (HA) and neuraminidase (NA) gene sequences of H1N1 influenza viruses from different hosts. Results Phylogenetic analysis revealed that both HA and NA genes have evolved into five distinct clusters, with further analyses indicating that the pandemic 2009 strains have experienced the strongest positive selection. We also found evidence of strong selection acting on the seasonal human H1N1 isolates. However, swine viruses from North America and Eurasia were under weak positive selection, while there was no significant evidence of positive selection acting on the avian isolates. A site-by-site analysis revealed that the positively selected sites were located in both of the cleaved products of HA (HA1 and HA2), as well as NA. In addition, the pandemic 2009 strains were subject to differential selection pressures compared to seasonal human, North American swine and Eurasian swine H1N1 viruses. Conclusions Most of these positively and\\/or differentially selected sites were situated in the B-cell and\\/or T-cell antigenic regions, suggesting that selection at these sites might be responsible for the antigenic variation of the viruses. Moreover, some sites were also associated with glycosylation and receptor-binding ability. Thus, selection at these positions might have helped the pandemic 2009 H1N1 viruses to adapt to the new hosts after they were introduced from pigs to humans. Positive selection on position 274 of NA protein, associated with drug resistance, might account for the prevalence of drug-resistant variants of seasonal human H1N1 influenza viruses, but there was no evidence that positive selection was responsible for the spread of the drug resistance of the pandemic H1N1 strains.

  8. Partial antiviral activities detection of chicken Mx jointing with neuraminidase gene (NA against Newcastle disease virus.

    Directory of Open Access Journals (Sweden)

    Yani Zhang

    Full Text Available As an attempt to increase the resistance to Newcastle Disease Virus (NDV and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA gene and myxo-virus resistance (Mx and detect the gene expression in transfected mouse fibroblasts (NIH-3T3 cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3 cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA. The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05, indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects.

  9. Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

    Science.gov (United States)

    Parsons, Zachary D; Ruddraraju, Kasi Viswanatharaju; Santo, Nicholas; Gates, Kent S

    2016-06-15

    Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides.

    Science.gov (United States)

    Angell, Y M; Alsina, J; Albericio, F; Barany, G

    2002-11-01

    This study details a series of conditions that may be applied to ensure 'safe' incorporation of cysteine with minimal racemization during automated or manual solid-phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307-4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5-33%) of cysteine racemization. As demonstrated on the tripeptide model H-Gly-Cys-Phe-NH(2), and on the nonapeptide dihydrooxytocin, the following methods are recommended: O-pentafluorophenyl (O-Pfp) ester in DMF; O-Pfp ester/1-hydroxybenzotriazole (HOBt) in DMF; N,N'-diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6-trimethylpyridine (TMP) in DMF (preactivation time 3.5-7.0 min in all of these cases); and HBTU/HOBt/TMP in CH(2)Cl(2)/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58-residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6-dimethylpyridine (lutidine), 2,3,5,6-tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6-di-tert-butyl-4-(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.

  11. Bleogens: Cactus-Derived Anti-Candida Cysteine-Rich Peptides with Three Different Precursor Arrangements

    Directory of Open Access Journals (Sweden)

    Shining Loo

    2017-12-01

    Full Text Available Cysteine-rich peptides (CRPs play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family.

  12. Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

    International Nuclear Information System (INIS)

    Lima, Cassia A.; Sasaki, Sergio D.; Tanaka, Aparecida S.

    2006-01-01

    The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M r of 11kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K i value of 0.1 and 0.6nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis

  13. An environmental pollutant, 9,10-phenanthrenequinone, activates human TRPA1 via critical cysteines 621 and 665.

    Science.gov (United States)

    Muraki, Katsuhiko; Sekine, Takashi; Ando, Yuna; Suzuki, Hiroka; Hatano, Noriyuki; Hirata, Tadashi; Muraki, Yukiko

    2017-08-01

    Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold, mechanical stimulation, and irritant chemicals. In our recent study, 9, 10-phenanthrenequinone (9,10-PQ) is the most potent irritant for activation of NRF2 among 1395 cigarette smoke components and it may be, therefore, important to find its additional targets. Here, we show that 9,10-PQ functions as an activator of TRPA1 in human embryonic kidney (HEK) cells expressing human wild-type TRPA1 (HEK-wTRPA1) and human alveolar A549 (A549) cells. Application of 9,10-PQ at 0.1-10 μmol/L induced a concentration-dependent Ca 2+ response as well as inward currents at -50 mV in HEK-wTRPA1 cells. The current response was blocked by TRPA1 antagonists, HC-030031 (HC) and A-967079. To test whether 9,10-PQ affects the cysteine residues of TRPA1, we expressed mutant TRPA1 channels in HEK cells (HEK-muTRPA1) in which six different cysteine residues were replaced with serine. Among them, a mutation of cysteine 621 (C621S) abolished the 9,10-PQ-induced Ca 2+ and current responses. The channel activity induced by 9,10-PQ was also abolished in excised inside-out patches isolated from HEK-muTRPA1 cells with the C621S substitution. Although a mutation of cysteine 665 (C665S) reduced the 9,10-PQ-induced response, channel sensitization by pretreatment with Cu 2+ plus 1,10-phenanthroline and by internal dialysis of 3 μmol/L Ca 2+ restored the response. However, a double mutant with C621S and C665S substitutions had little response to 9,10-PQ, even when sensitized by Ca 2+ dialysis. In A549 cells, 9,10-PQ induced an HC-sensitive Ca 2+ response. Our findings demonstrate that 9,10-PQ activation of human TRA1 is dependent on cysteine residues 621 and 665. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  14. Functional analysis of C1 family cysteine peptidases in the larval gut of Тenebrio molitor and Tribolium castaneum.

    Science.gov (United States)

    Martynov, Alexander G; Elpidina, Elena N; Perkin, Lindsey; Oppert, Brenda

    2015-02-14

    Larvae of the tenebrionids Tenebrio molitor and Tribolium castaneum have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion. High throughput sequencing was used to quantify and characterize transcripts encoding cysteine peptidases from the C1 papain family in the gut of tenebrionid larvae. For T. castaneum, 25 genes and one questionable pseudogene encoding cysteine peptidases were identified, including 11 cathepsin L or L-like, 11 cathepsin B or B-like, and one each F, K, and O. The majority of transcript expression was from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502). For cathepsin B, the major expression was from genes on chromosome 3 (LOC663145 and LOC663117). Some transcripts were expressed at lower levels or not at all in the larval gut, including cathepsins F, K, and O. For T. molitor, there were 29 predicted cysteine peptidase genes, including 14 cathepsin L or L-like, 13 cathepsin B or B-like, and one each cathepsin O and F. One cathepsin L and one cathepsin B were also highly expressed, orthologous to those in T. castaneum. Peptidases lacking conservation in active site residues were identified in both insects, and sequence analysis of orthologs indicated that changes in these residues occurred prior to evolutionary divergence. Sequences from both insects have a high degree of variability in the substrate binding regions, consistent with the ability of these enzymes to degrade a variety of cereal seed storage proteins and inhibitors. Predicted cathepsin B peptidases from both insects included some with a shortened occluding loop without active site residues in the middle, apparently lacking exopeptidase activity and unique to tenebrionid insects. Docking of specific substrates with models of T. molitor cysteine peptidases indicated that some insect cathepsins B and L bind substrates with affinities similar to human cathepsin L, while

  15. Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay.

    Science.gov (United States)

    Quosdorf, Stefanie; Schuetz, Anja; Kolodziej, Herbert

    2017-11-17

    Neuraminidaseis a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2'-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid (MUNANA)-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure-activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs ® 7630 (root extract of Pelargonium sidoides ) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed.

  16. Two single mutations in the fusion protein of Newcastle disease virus confer hemagglutinin-neuraminidase independent fusion promotion and attenuate the pathogenicity in chickens

    Science.gov (United States)

    The fusion (F) protein of Newcastle disease virus (NDV) plays an important role in viral infection and pathogenicity through mediating membrane fusion between the virion and host cells in the presence of the hemagglutinin-neuraminidase (HN). Previously, we obtained a velogenic NDV genotype VII muta...

  17. Pneumococcal Neuraminidase A (NanA) Promotes Biofilm Formation and Synergizes with Influenza A Virus in Nasal Colonization and Middle Ear Infection.

    Science.gov (United States)

    Wren, John T; Blevins, Lance K; Pang, Bing; Basu Roy, Ankita; Oliver, Melissa B; Reimche, Jennifer L; Wozniak, Jessie E; Alexander-Miller, Martha A; Swords, W Edward

    2017-04-01

    Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae , in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens. Copyright © 2017 American Society for Microbiology.

  18. A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

    Science.gov (United States)

    Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

    2013-01-01

    Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  19. Cysteine Sulfoxidation Increases the Photostability of Red Fluorescent Proteins.

    Science.gov (United States)

    Ren, Haiyan; Yang, Bing; Ma, Cheng; Hu, Ying S; Wang, Peng George; Wang, Lei

    2016-10-21

    Photobleaching of fluorescent proteins (FPs) is a major limitation to their use in advanced microscopy, and improving photostability remains highly challenging due to limited understanding of its molecular mechanism. Here we discovered a new mechanism to increase FP photostability. Cysteine oxidation, implicated in only photobleaching before, was found to drastically enhance FP photostability to the contrary. We generated a far-red FP mStable by introducing a cysteine proximal to the chromophore. Upon illumination, this cysteine was oxidized to sulfinic and sulfonic acids, enabling mStable more photostable than its ancestor mKate2 by 12-fold and surpassing other far-red FPs. mStable outperformed in laser scanning confocal imaging and super-resolution structured illumination microscopy. Moreover, photosensitization to oxidize a cysteine similarly introduced in another FP mPlum also increased its photostability by 23-fold. This postfolding cysteine sulfoxidation cannot be simply substituted by the isosteric aspartic acid, representing a unique mechanism valuable for engineering better photostability into FPs.

  20. Identifying proteins that can form tyrosine-cysteine crosslinks.

    Science.gov (United States)

    Martinie, Ryan J; Godakumbura, Pahan I; Porter, Elizabeth G; Divakaran, Anand; Burkhart, Brandon J; Wertz, John T; Benson, David E

    2012-10-01

    Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.

  1. Detection of cysteine- and lysine-based protein adductions by reactive metabolites of 2,5-dimethylfuran.

    Science.gov (United States)

    Wang, Kai; Li, Weiwei; Chen, Jiaming; Peng, Ying; Zheng, Jiang

    2015-10-08

    Many furan-containing compounds are known to be toxic and/or carcinogenic. Metabolic activation of toxic furans to cis-enediones (cis-enedials or γ-ketoenals) is generally considered as the initial step towards the processes of their toxicities. Sequential modification of key proteins by the electrophilic reactive intermediates is suggested to be an important mechanism of the toxic actions. In the present study, we developed a novel and simple analytical platform to detect protein modification resulting from metabolic activation of model compound 2,5-dimethylfuran (DMF). 4-Bromobenzylamine and 4-bromobenzylmercaptan were employed to trap protein adductions at cysteine and lysine residues, respectively. The resulting protein samples were proteolytically digested by chymotrypsin and Pronase E, followed by LC-MS/MS analysis. Modifications of cysteine and lysine residues of proteins were observed in microsomal incubations and animals after exposure to DMF. In conclusion, the approach established has been proven highly selective and reliable. This advance allows us not only to detect the protein adductions but also to define the structural identities of amino acid residues modified. This technique provides a unique platform to assess protein modifications arising from metabolic activation of potentially harmful furan-containing compounds. Hepatic protein adductions were found to be proportional to the hepatotoxicity of DMF. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Cysteines in the neuropilin-2 MAM domain modulate receptor homooligomerization and signal transduction.

    Science.gov (United States)

    Barton, Rachael; Driscoll, Alyssa; Flores, Samuel; Mudbhari, Durlav; Collins, Theresa; Iovine, M Kathryn; Berger, Bryan W

    2015-07-01

    Neuropilins (NRPs) are transmembrane receptors involved in angiogenesis, lymphangiogenesis, and neuronal development as well as in cancer metastasis. Previous studies suggest that NRPs exist in heteromeric complexes with vascular endothelial growth factors (VEGFs) and VEGF receptors as well as plexins and semaphorins. We determined via site-directed mutagenesis and bioluminescent resonance energy transfer assays that a conserved cysteine (C711) in the Danio rerio NRP2a MAM (meprin, A-5 protein, and protein tyrosine phosphatase μ) domain modulates NRP2a homomeric interactions. Mutation of this residue also disrupts semaphorin-3F binding in NRP2a-transfected COS-7 cells and prevents the NRP2a overexpression effects in a zebrafish vascular model. Collectively, our results indicate the MAM domain plays an important role in defining the NRP2 homodimer structure, which is important for semaphorin-dependent signal transduction via NRP2. © 2015 Wiley Periodicals, Inc.

  3. Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1 influenza virus in ferrets.

    Directory of Open Access Journals (Sweden)

    Natalia A Ilyushina

    2010-05-01

    Full Text Available The acquisition of neuraminidase (NA inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1 influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H. NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50s increased 5- to 940-fold. Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max, K(m and K(i of the avian-like N1 NA glycoproteins were highly consistent with their IC(50 values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6 EID(50 dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01 and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased

  4. Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets.

    Science.gov (United States)

    Ilyushina, Natalia A; Seiler, Jon P; Rehg, Jerold E; Webster, Robert G; Govorkova, Elena A

    2010-05-27

    The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50)s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max), K(m) and K(i)) of the avian-like N1 NA glycoproteins were highly consistent with their IC(50) values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6) EID(50) dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (Pinfluenza drugs that target different virus/host factors and can limit the emergence of resistance.

  5. RESIDUAL RISK ASSESSMENTS - RESIDUAL RISK ...

    Science.gov (United States)

    This source category previously subjected to a technology-based standard will be examined to determine if health or ecological risks are significant enough to warrant further regulation for Coke Ovens. These assesments utilize existing models and data bases to examine the multi-media and multi-pollutant impacts of air toxics emissions on human health and the environment. Details on the assessment process and methodologies can be found in EPA's Residual Risk Report to Congress issued in March of 1999 (see web site). To assess the health risks imposed by air toxics emissions from Coke Ovens to determine if control technology standards previously established are adequately protecting public health.

  6. A computational analysis of S-(2-succino)cysteine sites in proteins.

    Science.gov (United States)

    Miglio, Gianluca; Sabatino, Alessandro Damiano; Veglia, Eleonora; Giraudo, Maria Teresa; Beccuti, Marco; Cordero, Francesca

    2016-02-01

    The adduction of fumaric acid to the sulfhydryl group of certain cysteine (Cys) residues in proteins via a Michael-like reaction leads to the formation of S-(2-succino)cysteine (2SC) sites. Although its role remains to be fully understood, this post-translational Cys modification (protein succination) has been implicated in the pathogenesis of diabetes/obesity and fumarate hydratase-related diseases. In this study, theoretical approaches to address sequence- and 3D-structure-based features possibly underlying the specificity of protein succination have been applied to perform the first analysis of the available data on the succinate proteome. A total of 182 succinated proteins, 205 modifiable, and 1750 non-modifiable sites have been examined. The rate of 2SC sites per protein ranged from 1 to 3, and the overall relative abundance of modifiable sites was 10.8%. Modifiable and non-modifiable sites were not distinguishable when the hydrophobicity of the Cys-flaking peptides, the acid dissociation constant value of the sulfhydryl groups, and the secondary structure of the Cys-containing segments were compared. By contrast, significant differences were determined when the accessibility of the sulphur atoms and the amino acid composition of the Cys-flaking peptides were analysed. Based on these findings, a sequence-based score function has been evaluated as a descriptor for Cys residues. In conclusion, our results indicate that modifiable and non-modifiable sites form heterogeneous subsets when features often discussed to describe Cys reactivity are examined. However, they also suggest that some differences exist, which may constitute the baseline for further investigations aimed at the development of predictive methods for 2SC sites in proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers.

    Science.gov (United States)

    Prahlad, Janani; Hauser, David N; Milkovic, Nicole M; Cookson, Mark R; Wilson, Mark A

    2014-09-01

    The oxidation of a key cysteine residue (Cys106) in the parkinsonism-associated protein DJ-1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106-SO2 (-) (sulfinic acid) form of DJ-1. To study this important post-translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ-1 dimerization, which would severely compromise the protein's function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X-ray crystallography, NMR spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross-linking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol cross-linking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple cross-linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild-type protein. The enhanced flexibility of Glu18 DJ-1 mutants provides a parsimonious explanation for their lower observed cross-linking efficiency in cells. In addition, thiol cross-linkers may have an underappreciated value as qualitative probes of protein conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility. © 2014 International Society for Neurochemistry.

  8. Sequence comparison, molecular modeling, and network analysis predict structural diversity in cysteine proteases from the Cape sundew, Drosera capensis

    Directory of Open Access Journals (Sweden)

    Carter T. Butts

    2016-01-01

    Full Text Available Carnivorous plants represent a so far underexploited reservoir of novel proteases with potentially useful activities. Here we investigate 44 cysteine proteases from the Cape sundew, Drosera capensis, predicted from genomic DNA sequences. D. capensis has a large number of cysteine protease genes; analysis of their sequences reveals homologs of known plant proteases, some of which are predicted to have novel properties. Many functionally significant sequence and structural features are observed, including targeting signals and occluding loops. Several of the proteases contain a new type of granulin domain. Although active site residues are conserved, the sequence identity of these proteases to known proteins is moderate to low; therefore, comparative modeling with all-atom refinement and subsequent atomistic MD-simulation is used to predict their 3D structures. The structure prediction data, as well as analysis of protein structure networks, suggest multifarious variations on the papain-like cysteine protease structural theme. This in silico methodology provides a general framework for investigating a large pool of sequences that are potentially useful for biotechnology applications, enabling informed choices about which proteins to investigate in the laboratory.

  9. Characterization of L-cysteine desulfhydrase from Prevotella intermedia.

    Science.gov (United States)

    Yano, T; Fukamachi, H; Yamamoto, M; Igarashi, T

    2009-12-01

    Hydrogen sulfide is responsible for lysis of red blood cells and is a major compound for oral malodor. To clarify the production mechanism of hydrogen sulfide in Prevotella intermedia, we found an L-cysteine desulfhydrase gene (lcs) homologue on the genome database of P. intermedia ATCC25611 and characterized its gene product. The lcs gene homologue cloned into pGEX6p-1 vector was expressed in Escherichia coli and purified. Lcs activity was assayed by detection of the reaction products (hydrogen sulfide and pyruvate) or its derivatives from L-cysteine. Site-directed mutagenesis was used to convert an amino acid of the Lcs molecule. The purified lcs gene product catalysed the degradation of L-cysteine to pyruvate, ammonia, and hydrogen sulfide, indicating that the protein is L-cysteine desulfhydrase. The enzyme required pyridoxal 5'-phosphate as a cofactor, and it was highly active at pH 7.0 and completely inhibited by ZnCl(2). The K(m) and V(max) of the enzyme were 0.7 mm and 4.2 micromol/min/mg, respectively. Replacement of Tyr-59, Tyr-118, Asp-198, and Lys-233 with any of the amino acids resulted in the complete disappearance of Lcs activity, implying that these amino acids are essential for enzyme activity. In addition, hydrogen sulfide produced by this enzyme lysed sheep red blood cells and modified hemoglobin. These results show the enzymatic properties of L-cysteine desulfhydrase from P. intermedia ATCC25611 and also suggest that the Lcs enzyme, which produces hydrogen sulfide from L-cysteine, is closely associated with the pathogenesis of P. intermedia.

  10. The Role of Cysteine Residues in Catalysis of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Hubálek, Martin; Lepšík, Martin; Bednárová, Lucie; Pazderková, Markéta; Kopecký, V. Jr.; Snášel, Jan; Dostál, Jiří; Pichová, Iva

    2017-01-01

    Roč. 12, č. 1 (2017), č. článku e0170373. E-ISSN 1932-6203 R&D Projects: GA MŠk LO1302; GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : structural insights * metabolism * parameters Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.806, year: 2016 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170373

  11. Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding

    Science.gov (United States)

    Pérez-Sala, Dolores; Oeste, Clara L.; Martínez, Alma E.; Carrasco, M. Jesús; Garzón, Beatriz; Cañada, F. Javier

    2015-01-01

    The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial–mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transport-deficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc. PMID:26031447

  12. Evolution of the neuraminidase gene of seasonal influenza A and B viruses in Thailand between 2010 and 2015.

    Science.gov (United States)

    Tewawong, Nipaporn; Vichiwattana, Preeyaporn; Korkong, Sumeth; Klinfueng, Sirapa; Suntronwong, Nungruthai; Thongmee, Thanunrat; Theamboonlers, Apiradee; Vongpunsawad, Sompong; Poovorawan, Yong

    2017-01-01

    The neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are commonly used for the treatment and control of influenza A and B virus infection. However, the emergence of new influenza virus strains with reduced susceptibility to NAIs may appear with the use of these antivirals or even naturally. We therefore screened the neuraminidase (NA) sequences of seasonal influenza virus A(H1N1), A(H1N1)pdm09, A(H3N2), and influenza B virus strains identified in Thailand for the presence of substitutions previously reported to reduce susceptibility to NAIs. We initially examined oseltamivir resistance (characterized by the H275Y mutation in the NA gene) in 485 A(H1N1)pdm09 strains circulating in Thailand and found that 0.82% (4/485) had this substitution. To further evaluate the evolution of the NA gene, we also randomly selected 98 A(H1N1)pdm09, 158 A(H3N2), and 69 influenza B virus strains for NA gene amplification and sequencing, which revealed various amino acid mutations in the active site of the NA protein previously shown to be associated with reduced susceptibility to NAIs. Phylogenetic analysis of the influenza virus strains from this study and elsewhere around the world, together with the estimations of nucleotide substitution rates and selection pressure, and the predictions of B-cell epitopes and N-linked glycosylation sites all provided evidence for the ongoing evolution of NA. The overall rates of NA evolution for influenza A viruses were higher than for influenza B virus at the nucleotide level, although influenza B virus possessed more genealogical diversity than that of influenza A viruses. The continual surveillance of the antigenic changes associated with the NA protein will not only contribute to the influenza virus database but may also provide a better understanding of selection pressure exerted by antiviral use.

  13. Structure determination of the 1918 H1N1 neuraminidase from a crystal with lattice-translocation defects

    International Nuclear Information System (INIS)

    Zhu, Xueyong; Xu, Xiaojin; Wilson, Ian A.

    2008-01-01

    The structure of the 1918 H1N1 neuraminidase was determined to 1.65 Å from crystals with a lattice-translocation defect using uncorrected, as well as corrected, diffraction data. Few examples of macromolecular crystals containing lattice-translocation defects have been published in the literature. Lattice translocation and twinning are believed to be two common but different crystal-growth anomalies. While the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination of crystals with lattice-translocation defects has not often been reported. To date, only four protein crystal structures containing such a crystal defect have been determined, using corrected, but not uncorrected, intensity data. In this report, the crystallization, structure determination and refinement of N1 neuraminidase derived from the 1918 H1N1 influenza virus (18NA) at 1.65 Å resolution are described. The crystal was indexed in space group C222 1 , with unit-cell parameters a = 117.7, b = 138.5, c = 117.9 Å, and the structure was solved by molecular replacement. The lattice-translocation vector in the 18NA crystal was (0, 1/2, 1/2) or its equivalent vector (1/2, 0, 1/2) owing to the C lattice symmetry. Owing to this special lattice-translocation vector in space group C222 1 , structure refinement could be achieved in two different ways: using corrected or uncorrected diffraction data. In the refinement with uncorrected data, a composite model was built to represent the molecules in the translated and untranslated layers, respectively. This composite structure model provided a unique example to examine how the molecules were arranged in the two lattice domains resulting from lattice-translocation defects

  14. Residual nilpotence and residual solubility of groups

    International Nuclear Information System (INIS)

    Mikhailov, R V

    2005-01-01

    The properties of the residual nilpotence and the residual solubility of groups are studied. The main objects under investigation are the class of residually nilpotent groups such that each central extension of these groups is also residually nilpotent and the class of residually soluble groups such that each Abelian extension of these groups is residually soluble. Various examples of groups not belonging to these classes are constructed by homological methods and methods of the theory of modules over group rings. Several applications of the theory under consideration are presented and problems concerning the residual nilpotence of one-relator groups are considered.

  15. Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex.

    Science.gov (United States)

    Torres, María José; Trejo, Sebastián Alejandro; Natalucci, Claudia Luisa; López, Laura María Isabel

    2013-06-01

    The latex from Vasconcellea quercifolia ("oak leaved papaya"), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s(-1), k cat/K m = 3.46 × 10(3) ± 14 s(-1) M(-1)). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the "papain-like" family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.

  16. Chemoselective PEGylation of cysteine analogs of human basic ...

    African Journals Online (AJOL)

    Purpose: To improve the stability and bioactivity of human basic fibroblast growth factor (hbFGF) by site-specific pegylation. Methods: Four new mutants of hbFGF were designed with substituted Asp68, Lys77, Glu78 and Arg81 with cysteine with the aid of bioinformatics technique, and then cloned into pET21a plasmid, ...

  17. A Serendipitous Formation of a Cysteine-bridged Disaccharide

    African Journals Online (AJOL)

    NICO

    O-acetyl-b-D-glucopyranosylisothiouronium salt and the iodide or tosyl derivatives of L-serine,3 the desulfurization of disulfide- linked glycosyl cysteine derivatives,4 Lewis acid-catalyzed glycosylation,5,6 and solid phase glycosylation.7. Glycosylation of amino acids has previously relied on the use of amino acids protected ...

  18. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes

    Czech Academy of Sciences Publication Activity Database

    Kašperová, A.; Kunert, J.; Horynová, M.; Weigl, E.; Sebela, M.; Lenobel, René; Raška, M.

    2011-01-01

    Roč. 54, č. 5 (2011), E456-E462 ISSN 0933-7407 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cysteine dioxygenase * dermatophytes * recombinant protein * keratinolytic fungi * cDNA Subject RIV: CE - Biochemistry Impact factor: 2.247, year: 2011

  19. Chemoselective PEGylation of Cysteine Analogs of Human Basic ...

    African Journals Online (AJOL)

    crystal structure of FGF2 (PDB code: 1BFG, chain A, 1.6 oA) with high score and lower e- value and maximum sequence identity served as a template. For each cysteine analog, 10,000 molecules were generated and structure quality was validated by PSVS (http:// psvs-1_4- dev.nesg.org/ ). The best structural model was.

  20. Structure and Reactivity of the Cysteine Methyl Ester Radical Cation

    NARCIS (Netherlands)

    Osburn, S.; Steill, J. D.; Oomens, J.; O' Hair, R. A. J.; Van Stipdonk, M.; Ryzhov, V.

    2011-01-01

    The structure and reactivity of the cysteine methyl ester radical cation, CysOMe(center dot+), have been examined in the gas phase using a combination of experiment and density functional theory (DFT) calculations. CysOMe(center dot+) undergoes rapid ion molecule reactions with dimethyl disulfide,

  1. Chemical detection of cysteine-rich circular petides in selected ...

    African Journals Online (AJOL)

    Cysteine-rich circular peptides (CRCs) comprise a large family of gene encoded and low molecular weight polypeptides that has recently engaged the attention of scientists. This class of peptides exhibit a continuous circular configuration and a cystine knot backbone, which defines their resilient nature-directed structural ...

  2. Cloning and characterization of a novel cysteine protease gene ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Latex has been reported to contain a number of proteins such as protease-like enzymes (Arima et al 2000;. Arribere et al 1998), chitinase (Haque and Bradbury 1999;. Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis. SHI-QING PENG. 1,#,*, JIA-HONG ZHU. 1,#, HUI-LIANG LI.

  3. Effect of cysteine supplementation on in vitro maturation of bovine ...

    African Journals Online (AJOL)

    Parham

    2011-11-09

    Nov 9, 2011 ... This study was aimed at determining the effect of cysteine supplement during in vitro maturation of bovine oocytes. Cumulus-oocyte complexes (COCs) from abattoir ovaries were matured in vitro in. Hepes-TCM 199 supplemented with 0.2 mM sodium pyruvate, 1 µg/ml 17-β-estradiol, 10% fetal calf.

  4. Chemoselective PEGylation of Cysteine Analogs of Human Basic ...

    African Journals Online (AJOL)

    Purpose: To improve the stability and bioactivity of human basic fibroblast growth factor (hbFGF) by site-specific pegylation. Methods: Four new mutants of hbFGF were designed with substituted Asp68, Lys77, Glu78 and Arg81 with cysteine with the aid of bioinformatics technique, and then cloned into pET21a plasmid, ...

  5. A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

    Science.gov (United States)

    Söveges, B; Imre, T; Szende, T; Póti, Á L; Cserép, G B; Hegedűs, T; Kele, P; Németh, K

    2016-07-07

    Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

  6. Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

    OpenAIRE

    Himelbloom, B H; Hassan, H M

    1986-01-01

    Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

  7. MHC-peptide binding: dimers of cysteine-containing nonapeptides bind with high affinity to HLA-A2.1 class I molecules.

    Science.gov (United States)

    Di Modugno, F; Mammi, C; Rosanò, L; Rubiu, O; Nisticò, P; Chersi, A

    1997-11-01

    Small peptides, 8-10 amino acids long, derived from degradation of cytoplasmic proteins by a proteasome-proteinase complex, are usually presented and recognized by CD8+ cytolytic T lymphocytes (CTLs) associated with major histocompatibility complex (MHC) class I molecules. Recently synthetic peptides were used for the in vitro induction of tumor-specific CTLs, offering another strategy in the study of the immune-response repertoire and providing a new tool in cancer vaccination and immunotherapy. Peptides derived from otherwise normal proteins, overexpressed in many tumors as products of the protooncogene, may represent a target for an immune response. This is the case of HER-2/neu gene (also known as ErbB-2), encoding a cysteine-rich glycoprotein transmembrane receptor with tyrosine kinase activity (gp185neu). Recent data, demonstrating that HLA-A2.1-related peptides are able to stimulate in vitro CD8+ lymphocytes, Prompted us to study the binding to HLA-A2.1 molecules of several gp185 synthetic peptides containing a cystein residue and to define the relevance of this amino acid residue in the reduced or oxidated form of the sulfhydryl group. We found that monomers and their homodimers, linked by a disulfide bridge, bind to HLA-A2.1 molecules with overlapping affinity. These results suggest that additional amino acids of the nonapeptide do not prevent the binding and the HLA refolding through chemical or sterical interactions. This might be of particular relevance for the in vivo processing of cysteine-rich proteins. Because ErbB-2 molecules, as tumor-differentiation antigens in melanoma, are cysteine-rich molecules, it may be relevant to evaluate the possible role of the cystine residues interacting with the T-cell receptor. The recognition of these heterodimers by CD8+ lymphocytes will require functional in vivo studies.

  8. Vibrio type III effector VPA1380 is related to the cysteine protease domain of large bacterial toxins.

    Directory of Open Access Journals (Sweden)

    Thomas Calder

    Full Text Available Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2, but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.

  9. Vibrio Type III Effector VPA1380 Is Related to the Cysteine Protease Domain of Large Bacterial Toxins

    Science.gov (United States)

    Calder, Thomas; Kinch, Lisa N.; Fernandez, Jessie; Salomon, Dor; Grishin, Nick V.; Orth, Kim

    2014-01-01

    Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator. PMID:25099122

  10. Thermal decomposition of the amino acids glycine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine and histidine.

    Science.gov (United States)

    Weiss, Ingrid M; Muth, Christina; Drumm, Robert; Kirchner, Helmut O K

    2018-01-01

    The pathways of thermal instability of amino acids have been unknown. New mass spectrometric data allow unequivocal quantitative identification of the decomposition products. Calorimetry, thermogravimetry and mass spectrometry were used to follow the thermal decomposition of the eight amino acids G, C, D, N, E, Q, R and H between 185 °C and 280 °C. Endothermic heats of decomposition between 72 and 151 kJ/mol are needed to form 12 to 70% volatile products. This process is neither melting nor sublimation. With exception of cysteine they emit mainly H 2 O, some NH 3 and no CO 2 . Cysteine produces CO 2 and little else. The reactions are described by polynomials, AA→ a NH 3 + b H 2 O+ c CO 2 + d H 2 S+ e residue, with integer or half integer coefficients. The solid monomolecular residues are rich in peptide bonds. Eight of the 20 standard amino acids decompose at well-defined, characteristic temperatures, in contrast to commonly accepted knowledge. Products of decomposition are simple. The novel quantitative results emphasize the impact of water and cyclic condensates with peptide bonds and put constraints on hypotheses of the origin, state and stability of amino acids in the range between 200 °C and 300 °C.

  11. An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins.

    Science.gov (United States)

    Tarasava, Katsiaryna; Freisinger, Eva

    2014-12-01

    Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein species. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site.

    Science.gov (United States)

    Brandt, Erik G; Hellgren, Mikko; Brinck, Tore; Bergman, Tomas; Edholm, Olle

    2009-02-14

    The binding of zinc (Zn) ions to proteins is important for many cellular events. The theoretical and computational description of this binding (as well as that of other transition metals) is a challenging task. In this paper the binding of the Zn ion to four cysteine residues in the structural site of horse liver alcohol dehydrogenase (HLADH) is studied using a synthetic peptide mimic of this site. The study includes experimental measurements of binding constants, classical free energy calculations from molecular dynamics (MD) simulations and quantum mechanical (QM) electron structure calculations. The classical MD results account for interactions at the molecular level and reproduce the absolute binding energy and the hydration free energy of the Zn ion with an accuracy of about 10%. This is insufficient to obtain correct free energy differences. QM correction terms were calculated from density functional theory (DFT) on small clusters of atoms to include electronic polarisation of the closest waters and covalent contributions to the Zn-S coordination bond. This results in reasonably good agreement with the experimentally measured binding constants and Zn ion hydration free energies in agreement with published experimental values. The study also includes the replacement of one cysteine residue to an alanine. Simulations as well as experiments showed only a small effect of this upon the binding free energy. A detailed analysis indicate that the sulfur is replaced by three water molecules, thereby changing the coordination number of Zn from four (as in the original peptide) to six (as in water).

  13. Structural and Immunological Characteristics of a 28-Kilodalton Cruzipain-Like Cysteine Protease of Paragonimus westermani Expressed in the Definitive Host Stage

    Science.gov (United States)

    Yun, Doo-Hee; Chung, Joon-Yong; Chung, Young-Bae; Bahk, Young-Yil; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2000-01-01

    A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis. PMID:11063501

  14. Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping

    Science.gov (United States)

    Dong, Maoqing; Xu, Xiequn; Ball, Alicja M.; Makhoul, Joshua A.; Lam, Polo C.-H.; Pinon, Delia I.; Orry, Andrew; Sexton, Patrick M.; Abagyan, Ruben; Miller, Laurence J.

    2012-01-01

    While it is evident that the carboxyl-terminal region of natural peptide ligands bind to the amino-terminal domain of class B GPCRs, how their biologically critical amino-terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys2 and Cys5 secretin analogues exhibited full activity and retained moderate binding affinity (IC50: 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine-replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand-receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys2 predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser340, 94±7%; Pro341, 84±9%; Phe258, 73±5%; Trp274 62±8%), and Cys5 labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln348, 100%; Ile347, 73±12%; Glu342, 59±10%; Phe351, 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H1-W274, D3-N268, G4-F258) were supported by mutagenesis and residue-residue complementation studies.—Dong, M., Xu, X., Ball, A. M., Makhoul, J. A., Lam, P. C.-H., Pinon, D. I., Orry, A., Sexton, P. M., Abagyan, R., Miller, L. J. Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping. PMID:22964305

  15. Neuraminidase inhibitors in the treatment and post exposure prevention of influenza

    Directory of Open Access Journals (Sweden)

    von der Schulenburg, Johann-Matthias

    2006-01-01

    Full Text Available Introduction: Influenza is a viral respiratory infection which presents itself as an acute febrile disease. It is contracted by virus-laden respiratory secretions from infected individuals. Symptoms usually last three to seven days and are accompanied by severely limited activities during this time. A definite diagnosis, however, can only be made by laboratory analysis. Every year, about 20% of children and 5% of adults develop symptomatic influenza of the serotypes A or B worldwide. Typical complications of influenza include viral or bacterial infections, as well as deterioration of an existing cardio-vascular or respiratory disease which may lead to hospitalization and death. Current policy recommends that individuals, who are at-risk of developing serious complications (patients over sixty years of age or patients with concomitant chronic diseases, as well as people in direct contact with high risk patients (i.e. nursing staff in living and care facilities, should be annually vaccinated with inactivated influenza strains. Various pharmaceutical agents for the treatment and prophylaxis of influenza have been approved. Amantadine, which inhibits the viral M2-ion channel, is only effective in influenza-serotype A. Neuraminidase inhibitors (NI represent a new class of antivirals for prophylaxis and treatment of influenza A and B. NI interrupt various central functions that are vital for the life cycle and spreading of the virus. Two drugs of this substance class, Zanamivir (RelenzaTM and Oseltamivir (Tamiflu®, are licensed for the treatment of influenza. For adults and teenagers over thirteen years of age Oseltamivir is also approved for the prophylaxis of influenza. Zanamivir is a powder which needs to be inhaled, whereas Oseltamivir is licensed as a capsule for oral administration. M2-inhibitors and NI are only effective at an early stage of the influenza infection, i.e. during the first 36 to 48 hours after symptom onset, before replication

  16. Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene.

    Science.gov (United States)

    Wang, C L; Lum, A M; Ozuna, S C; Clark, D S; Keasling, J D

    2001-08-01

    The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

  17. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    Science.gov (United States)

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Dimerization of the Glucan Phosphatase Laforin Requires the Participation of Cysteine 329

    Science.gov (United States)

    Sánchez-Martín, Pablo; Raththagala, Madushi; Bridges, Travis M.; Husodo, Satrio; Gentry, Matthew S.; Sanz, Pascual; Romá-Mateo, Carlos

    2013-01-01

    Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin’s oligomerization. PMID:23922729

  19. A viable recombinant rhabdovirus lacking its glycoprotein gene and expressing influenza virus hemagglutinin and neuraminidase is a potent influenza vaccine.

    Science.gov (United States)

    Ryder, Alex B; Buonocore, Linda; Vogel, Leatrice; Nachbagauer, Raffael; Krammer, Florian; Rose, John K

    2015-03-01

    The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell culture and induced a

  20. Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice.

    Science.gov (United States)

    Wohlbold, Teddy John; Nachbagauer, Raffael; Xu, Haoming; Tan, Gene S; Hirsh, Ariana; Brokstad, Karl A; Cox, Rebecca J; Palese, Peter; Krammer, Florian

    2015-03-10

    In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. Despite the existence of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to cause morbidity and mortality in the human population, emphasizing the continued need for research in the field. While the majority of

  1. iPreny-PseAAC: Identify C-terminal Cysteine Prenylation Sites in Proteins by Incorporating Two Tiers of Sequence Couplings into PseAAC.

    Science.gov (United States)

    Xu, Yan; Wang, Zu; Li, Chunhui; Chou, Kuo-Chen

    2017-01-01

    Occurring at the cysteine residue in the C-terminal of a protein, prenylation is a special kind of post-translational modification (PTM), which may play a key role for statin in altering immune function. Therefore, knowledge of the prenylation sites in proteins is important for drug development as well as for in-depth understanding the biological process concerned. Given a query protein whose C-terminal contains some cysteine residues, which one can be of prenylation or none of them can be prenylated? To address this problem, we have developed a new predictor, called "iPreny-PseAAC", by incorporating two tiers of sequence pair coupling effects into the general form of PseAAC (pseudo amino acid composition). It has been observed by four different cross-validation approaches that all the important indexes in reflecting its prediction quality are quite high and fully consistent to each other. It is anticipated that the iPreny-PseAAC predictor holds very high potential to become a useful high throughput tool in identifying protein C-terminal cysteine prenylation sites and the other relevant areas. To maximize the convenience for most experimental biologists, the webserver for the new predictor has been established at http://app.aporc.org/iPreny-PseAAC/, by which users can easily get their desired results without needing to go through the mathematical details involved in this paper. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

    Science.gov (United States)

    Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

    2000-02-29

    Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.

  3. Fluoresence quenching of riboflavin in aqueous solution by methionin and cystein

    International Nuclear Information System (INIS)

    Droessler, P.; Holzer, W.; Penzkofer, A.; Hegemann, P.

    2003-01-01

    The fluorescence quantum distributions, fluorescence quantum yields, and fluorescence lifetimes of riboflavin in methanol, DMSO, water, and aqueous solutions of the sulphur atom containing amino acids methionin and cystein have been determined. In methanol, DMSO, and water (pH=4-8) only dynamic fluorescence reduction due to intersystem crossing and internal conversion is observed. In aqueous methionin solutions of pH=5.25-9 a pH independent static and dynamic fluorescence quenching occurs probably due to riboflavin anion-methionin cation pair formation. In aqueous cystein solutions (pH range from 4.15 to 9) the fluorescence quenching increases with rising pH due to cystein thiolate formation. The cystein thiol form present at low pH does not react with neutral riboflavin. Cystein thiolate present at high pH seems to react with neutral riboflavin causing riboflavin deprotonation (anion formation) by cystein thiolate reduction to the cystein thiol form

  4. Chemical characterization of complexation behavior of pertechnetate with cysteine

    International Nuclear Information System (INIS)

    Wang A.-Y.; Lin J.-L.; Lo J.-G.

    1992-01-01

    The labeling behavior of cysteine with 99 TcO 4 - ion and/or 99m TcO 4 - ion at different cysteine concentrations reductant and pH values has been studied by chromatography, and the labeling yield was calculated. Three major Tc-complexes, yellow, reddish brown and green can be separated by gel filtration chromatography (GFC). Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and ion-exchange chromatography (IC) were used to separate the complexes collected from GFC. The TLC, HPLC data show the pertechnetate accompanied with a yellow complex; the green and purple complex contain more than two complexes. Electrophoresis and IC data show that the complexes carry a negative charge. The conductivity, UV-VIS, flow beta-detector with HPLC and autoradiography are also applied to analyze complex formation. (author) 20 refs.; 13 figs.; 5 tabs

  5. Thiazolidine prodrugs of cysteamine and cysteine as radioprotective agents

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, J.C.; Koch, K.E.; Detrick, S.R. [Univ. of Utah, Salt Lake City, UT (United States)] [and others

    1995-08-01

    The need for protection against the toxic effects of ionizing radiation comes from many different directions: occupational exposure, nuclear accidents, environmental sources and protection of normal tissue during the therapeutic irradiation of cancer. Sulfhydryl-containing compounds, including cysteamine and L-cysteine, have long been known to possess radioprotective properties, but their therapeutic utility is limited by their side effects at radioprotective doses. To avoid this drawback, thiazolidine prodrugs of cysteamine and L-cysteine were prepared by the condensation of each thiolamine with the aldose monosaccharides, D-ribose and D-glucose, producing RibCyst, GlcCyst, Rib-Cys and GlcCys. The prodrugs were designed to liberate the parent thiolamine nonenzymatically, after ring opening and hydrolysis, which is then available e to function as a radioprotective agent. Cysteamine`s inherent toxicity, measured using Chinese hamster V79 cells growing in culture, was completely eliminated, even at concentrations as high as 25 mM, by providing the thiolamine in the form of a prodrug. Good protection against radiation-induced lethality was demonstrated by the cysteamine prodrugs using a clonogenic assay. Protection against radiation-induced DNA single-strand breaks, as measured by alkaline elution, was also shown by both RibCyst and GlcCyst; this activity was higher than that exhibited by either cysteamine or WR-1065. The L-cysteine prodrugs, RibCys and GlcCys, also possessed radioprotective abilities under most of the conditions studied. Protection against DNA damage was comparable between L-cystein, WR-1065 and RibCys. 42 refs., 7 figs., 2 tabs.

  6. Subcellular distribution of glutathione and cysteine in cyanobacteria

    OpenAIRE

    Zechmann, Bernd; Tomašić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-01-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine,...

  7. Effect of cysteine supplementation on in vitro maturation of bovine ...

    African Journals Online (AJOL)

    B Rahim, S Jalal, N Yosef ... Cumulus-oocyte complexes (COCs) from abattoir ovaries were matured in vitro in Hepes-TCM 199 supplemented with 0.2 mM sodium pyruvate, 1 μg/ml 17-β-estradiol, 10% fetal calf serum (FCS), 0.5 μg/ml bFSH and 0 (control) and 100 or 500 μM/ml of cysteine for 24 h. When COCs matured in ...

  8. Cysteines and N-Glycosylation Sites Conserved among All Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus 1 Infection.

    Science.gov (United States)

    Rider, Paul J F; Naderi, Misagh; Bergeron, Scott; Chouljenko, Vladimir N; Brylinski, Michal; Kousoulas, Konstantin G

    2017-11-01

    Neurotropism is a defining characteristic of alphaherpesvirus pathogenicity. Glycoprotein K (gK) is a conserved virion glycoprotein of all alphaherpesviruses that is not found in other herpesvirus subfamilies. The extracellular amino terminus of gK has been shown to be important to the ability of the prototypic alphaherpesvirus herpes simplex virus 1 (HSV-1) to enter neurons via axonal termini. Here, we determined the role of the two conserved N-linked glycosylation (N48 and N58) sites of gK in virus-induced cell fusion and replication. We found that N-linked glycosylation is important to the regulation of HSV-1-induced membrane fusion since mutating N58 to alanine (N58A) caused extensive virus-induced cell fusion. Due to the known contributions of N-linked glycosylation to protein processing and correct disulfide bond formation, we investigated whether the conserved extracellular cysteine residues within the amino terminus of gK contributed to the regulation of HSV-1-induced membrane fusion. We found that mutation of C37 and C114 residues led to a gK-null phenotype characterized by very small plaque formation and drastic reduction in infectious virus production, while mutation of C82 and C243 caused extensive virus-induced cell fusion. Comparison of N-linked glycosylation and cysteine mutant replication kinetics identified disparate effects on infectious virion egress from infected cells. Specifically, cysteine mutations caused defects in the accumulation of infectious virus in both the cellular and supernatant fractions, while glycosylation site mutants did not adversely affect virion egress from infected cells. These results demonstrate a critical role for the N glycosylation sites and cysteines for the structure and function of the amino terminus of gK. IMPORTANCE We have previously identified important entry and neurotropic determinants in the amino terminus of HSV-1 glycoprotein K (gK). Alphaherpesvirus-mediated membrane fusion is a complex and highly

  9. The effect of cysteine on electrodeposition of gold nanoparticle

    International Nuclear Information System (INIS)

    Dolati, A.; Imanieh, I.; Salehi, F.; Farahani, M.

    2011-01-01

    Highlights: → Cysteine was found as an appropriate additive for electrodeposition of gold nanoparticles. → The deposition mechanism of gold nanoparticle was determined as instantaneous nucleation. → Oxygen reduction on the gold nanoparticle surface was eight times greater than that on the conventional gold deposits. - Abstract: The most applications of gold nanoparticles are in the photo-electronical accessories and bio-chemical sensors. Chloride solution with cysteine additive was used as electrolyte in gold nanoparticles electrodeposition. The nucleation and growing mechanism were studied by electrochemical techniques such as cyclic voltammetry and chronoamperometry, in order to obtain a suitable nano structure. The deposition mechanism was determined as instantaneous nucleation and the dimension of particles was controlled in nanometric particle size range. Atomic Force Microscope was used to evaluate the effect of cysteine on the morphology and topography of gold nanoparticles. Finally the catalytic property of gold nanoparticle electrodeposited was studied in KOH solution, where oxygen reduction on the gold nanoparticle surface was eight times greater than that on the conventional gold deposits.

  10. Detection of influenza B viruses with reduced sensitivity to neuraminidase inhibitor in Morocco during 2014/15 season.

    Science.gov (United States)

    Elfalki, F; Ihazmad, H; Bimouhen, A; Regragui, Z; Benkaroum, S; Bakri, Y; Barakat, A

    2016-10-02

    We monitored phenotypic and genotypic susceptibility of influenza viruses circulating in Morocco during 2014-2015 to oseltamivir and zanamivir. Throat and nasal swab specimens were collected from outpatients (with influenza-like illness) and inpatients (with severe acute respiratory illness) and tested for influenza viruses using real-time reverse transcription polymerase chain reaction. Positive samples were inoculated in MDCK cells and virus phenotypic susceptibility to neuraminidase inhibitors (NAIs) was assessed using fluorescent NA inhibition. Of 440 specimens, 135 were positive for influenza B Yamagata-like virus, 38 were A(H1N1)pdm09 and 25 were A(H3N2). Sixty influenza B viruses isolated from MDCK cells showed no significant resistance to NAIs. However, two of these strains, B/Morocco/176H/2015 and B/Morocco/CP10/2015, showed reduced susceptibility to oseltamivir. The two influenza B viruses with reduced susceptibility to oseltamivir show that ongoing NAI susceptibility surveillance is essential.

  11. Neuraminidase and hemagglutinin matching patterns of a highly pathogenic avian and two pandemic H1N1 influenza A viruses.

    Directory of Open Access Journals (Sweden)

    Yonghui Zhang

    Full Text Available BACKGROUND: Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies. METHODOLOGY/PRINCIPAL FINDINGS: The hemagglutinin (HA and neuraminidase (NA matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains and a highly pathogenic avian influenza A virus (H5N1 were studied using a pseudotyped particle (pp system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005 could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern. CONCLUSIONS/SIGNIFICANCE: Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.

  12. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Guus Koch

    2013-04-01

    Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

  13. Structure and mechanism leading to formation of the cysteine sulfinate product complex of a biomimetic cysteine dioxygenase model.

    Science.gov (United States)

    Sallmann, Madleen; Kumar, Suresh; Chernev, Petko; Nehrkorn, Joscha; Schnegg, Alexander; Kumar, Devesh; Dau, Holger; Limberg, Christian; de Visser, Sam P

    2015-05-11

    Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Long time scale GPU dynamics reveal the mechanism of drug resistance of the dual mutant I223R/H275Y neuraminidase from H1N1-2009 influenza virus.

    Science.gov (United States)

    Woods, Christopher J; Malaisree, Maturos; Pattarapongdilok, Naruwan; Sompornpisut, Pornthep; Hannongbua, Supot; Mulholland, Adrian J

    2012-05-29

    Multidrug resistance of the pandemic H1N1-2009 strain of influenza has been reported due to widespread treatment using the neuraminidase (NA) inhibitors, oseltamivir (Tamiflu), and zanamivir (Relenza). From clinical data, the single I223R (IR(1)) mutant of H1N1-2009 NA reduced efficacy of oseltamivir and zanamivir by 45 and 10 times, (1) respectively. More seriously, the efficacy of these two inhibitors against the double mutant I223R/H275Y (IRHY(2)) was significantly reduced by a factor of 12 374 and 21 times, respectively, compared to the wild-type.(2) This has led to the question of why the efficacy of the NA inhibitors is reduced by the occurrence of these mutations and, specifically, why the efficacy of oseltamivir against the double mutant IRHY was significantly reduced, to the point where oseltamivir has become an ineffective treatment. In this study, 1 μs of molecular dynamics (MD) simulations was performed to answer these questions. The simulations, run using graphical processors (GPUs), were used to investigate the effect of conformational change upon binding of the NA inhibitors oseltamivir and zanamivir in the wild-type and the IR and IRHY mutant strains. These long time scale dynamics simulations demonstrated that the mechanism of resistance of IRHY to oseltamivir was due to the loss of key hydrogen bonds between the inhibitor and residues in the 150-loop. This allowed NA to transition from a closed to an open conformation. Oseltamivir binds weakly with the open conformation of NA due to poor electrostatic interactions between the inhibitor and the active site. The results suggest that the efficacy of oseltamivir is reduced significantly because of conformational changes that lead to the open form of the 150-loop. This suggests that drug resistance could be overcome by increasing hydrogen bond interactions between NA inhibitors and residues in the 150-loop, with the aim of maintaining the closed conformation, or by designing inhibitors that can form

  15. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    International Nuclear Information System (INIS)

    Åmand, Helene L.; Nordén, Bengt; Fant, Kristina

    2012-01-01

    Highlights: ► Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. ► Dimer formation enhances peptiplex stability, resulting in increased transfection. ► By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes (“peptiplexes”) enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the “chelate effect” and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.

  16. The Antioxidant Role of Glutathione and N-Acetyl-Cysteine Supplements and Exercise-Induced Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Willoughby Darryn

    2005-12-01

    Full Text Available Abstract An increase in exercise intensity is one of the many ways in which oxidative stress and free radical production has been shown to increase inside our cells. Effective regulation of the cellular balance between oxidation and antioxidation is important when considering cellular function and DNA integrity as well as the signal transduction of gene expression. Many pathological states, such as cancer, Parkinson's disease, and Alzheimer's disease have been shown to be related to the redox state of cells. In an attempt to minimize the onset of oxidative stress, supplementation with various known antioxidants has been suggested. Glutathione and N-acetyl-cysteine (NAC are antioxidants which are quite popular for their ability to minimize oxidative stress and the downstream negative effects thought to be associated with oxidative stress. Glutathione is largely known to minimize the lipid peroxidation of cellular membranes and other such targets that is known to occur with oxidative stress. N-acetyl-cysteine is a by-product of glutathione and is popular due to its cysteine residues and the role it has on glutathione maintenance and metabolism. The process of oxidative stress is a complicated, inter-twined series of events which quite possibly is related to many other cellular processes. Exercise enthusiasts and researchers have become interested in recent years to identify any means to help minimize the detrimental effects of oxidative stress that are commonly associated with intense and unaccustomed exercise. It is possible that a decrease in the amount of oxidative stress a cell is exposed to could increase health and performance.

  17. Crystal Structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Iain D Kerr

    2010-06-01

    Full Text Available Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.The mature domain of our TbCat*CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat*CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain*K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

  18. Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

    International Nuclear Information System (INIS)

    Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry

    2005-01-01

    Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure

  19. E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient.

    Science.gov (United States)

    L'Huillier, Arnaud G; Abed, Yacine; Petty, Tom J; Cordey, Samuel; Thomas, Yves; Bouhy, Xavier; Schibler, Manuel; Simon, Audrey; Chalandon, Yves; van Delden, Christian; Zdobnov, Evgeny; Boquete-Suter, Patricia; Boivin, Guy; Kaiser, Laurent

    2015-12-01

    An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategies. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella enterica and Escherichia coli.

    Science.gov (United States)

    Loddeke, Melissa; Schneider, Barbara; Oguri, Tamiko; Mehta, Iti; Xuan, Zhenyu; Reitzer, Larry

    2017-08-15

    Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM ) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica Δ cyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica , anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica , and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and

  1. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    International Nuclear Information System (INIS)

    Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M.

    1989-01-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  2. Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

    KAUST Repository

    Liu, Pei

    2014-01-28

    Cellular redox status plays a key role in mediating various physiological and developmental processes often through modulating activities of redox-sensitive proteins. Various stresses trigger over-production of reactive oxygen/nitrogen species which lead to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. Here, we report a high-throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols. The biotin-tagged peptides are affinity purified, labeled with iTRAQ reagents, and analyzed using a paralleled HCD-CID fragmentation mode in an LTQ-Orbitrap. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. This approach allows identification of the specific redox-regulated cysteine residues, and offers an effective tool for elucidation of redox proteomes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. COMPARATIVE ANALYSIS FOR METAL BINDING CAPACITY OF CYSTEINE BY USING UV-VIS SPECTROPHOTOMETER

    OpenAIRE

    Shivendu Ranjan; Nandita Dasgupta; Gyanendra Gour; Rashmi Dubey; Kumari Amrita

    2012-01-01

    The metal binding capacity of cysteine with three different metals Nickel, Copper and Lead was studied using UV-Vis spectrophotometer for which absorbance values were taken after interaction of cysteine with metal salt solutions (10ppm and 100ppm). Before taking above absorbance dilution factor was set using cysteine stock. The increase in peak intensity was observed when metal salt solution and metal saltcysteine solution were compared. Based on peak shift and peak intensity finally it can b...

  4. Cysteine toxicity for oral streptococci and effect of branched-chain amino acids.

    OpenAIRE

    Cowman, R A; Baron, S S; Fitzgerald, R J

    1983-01-01

    Cysteine was bactericidal to strains of Streptococcus mutans and S. salivarius in concentrations that were nontoxic to S. sanguis, S. milleri, or S. mitior when these microorganisms were incubated in a saliva protein-based synthetic medium. Cysteine toxicity for S. mutans also occurred after incubation in synthetic base medium supplemented with amino acids as the nitrogen source for growth. The bactericidal effect of cysteine for S. mutans or S. salivarius in the saliva protein medium was inf...

  5. Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

    OpenAIRE

    Yang, Hyun-Jong; Chung, Young-Bae; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2002-01-01

    The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine protea...

  6. Transforming growth factor-β: activation by neuraminidase and role in highly pathogenic H5N1 influenza pathogenesis.

    Directory of Open Access Journals (Sweden)

    Christina M Carlson

    2010-10-01

    Full Text Available Transforming growth factor-beta (TGF-β, a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β. We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3 except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus-infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis.

  7. Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice.

    Science.gov (United States)

    Yang, Chen; Skiena, Steven; Futcher, Bruce; Mueller, Steffen; Wimmer, Eckard

    2013-06-04

    A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NA(Min), PR8-HA(Min), and PR8-(NA+HA)(Min) (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin-Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD50 values: PR8, 32 plaque-forming units (PFU); HA(Min), 1.7 × 10(3) PFU; NA(Min), 2.4 × 10(5) PFU; (NA+HA)(Min), ≥3.16 × 10(6) PFU. Remarkably, (NA+HA)(Min) was attenuated >100,000-fold, with NA(Min) the major contributor to attenuation. In vaccinated mice (NA+HA)(Min) was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD50) of 2.4 PFU. Moreover, at a PD50 of only 147 or 237, (NA+HA)(Min) conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development.

  8. Vaccination with Adjuvanted Recombinant Neuraminidase Induces Broad Heterologous, but Not Heterosubtypic, Cross-Protection against Influenza Virus Infection in Mice

    Science.gov (United States)

    Wohlbold, Teddy John; Nachbagauer, Raffael; Xu, Haoming; Tan, Gene S.; Hirsh, Ariana; Brokstad, Karl A.; Cox, Rebecca J.; Palese, Peter

    2015-01-01

    ABSTRACT In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. PMID:25759506

  9. The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus.

    Directory of Open Access Journals (Sweden)

    Hongbo Zhou

    Full Text Available The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt, WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49(th to 68(th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.

  10. Virtual screening approach to identifying influenza virus neuraminidase inhibitors using molecular docking combined with machine-learning-based scoring function.

    Science.gov (United States)

    Zhang, Li; Ai, Hai-Xin; Li, Shi-Meng; Qi, Meng-Yuan; Zhao, Jian; Zhao, Qi; Liu, Hong-Sheng

    2017-10-10

    In recent years, an epidemic of the highly pathogenic avian influenza H7N9 virus has persisted in China, with a high mortality rate. To develop novel anti-influenza therapies, we have constructed a machine-learning-based scoring function (RF-NA-Score) for the effective virtual screening of lead compounds targeting the viral neuraminidase (NA) protein. RF-NA-Score is more accurate than RF-Score, with a root-mean-square error of 1.46, Pearson's correlation coefficient of 0.707, and Spearman's rank correlation coefficient of 0.707 in a 5-fold cross-validation study. The performance of RF-NA-Score in a docking-based virtual screening of NA inhibitors was evaluated with a dataset containing 281 NA inhibitors and 322 noninhibitors. Compared with other docking-rescoring virtual screening strategies, rescoring with RF-NA-Score significantly improved the efficiency of virtual screening, and a strategy that averaged the scores given by RF-NA-Score, based on the binding conformations predicted with AutoDock, AutoDock Vina, and LeDock, was shown to be the best strategy. This strategy was then applied to the virtual screening of NA inhibitors in the SPECS database. The 100 selected compounds were tested in an in vitro H7N9 NA inhibition assay, and two compounds with novel scaffolds showed moderate inhibitory activities. These results indicate that RF-NA-Score improves the efficiency of virtual screening for NA inhibitors, and can be used successfully to identify new NA inhibitor scaffolds. Scoring functions specific for other drug targets could also be established with the same method.

  11. Evolutionary history and phylodynamics of influenza A and B neuraminidase (NA genes inferred from large-scale sequence analyses.

    Directory of Open Access Journals (Sweden)

    Jianpeng Xu

    Full Text Available Influenza neuraminidase (NA is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking.Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The d(N/d(S ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding.The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance.

  12. Electrocatalytic Oxidation and Determination of Cysteine at Oxovanadium(IV Salen Coated Electrodes

    Directory of Open Access Journals (Sweden)

    Piyush Kumar Sonkar

    2014-01-01

    Full Text Available A transition metal complex, oxovanadium(IV salen (where salen represents N,N′-bis(salicylideneethylenediamine is immobilized on glassy carbon (GC electrodes and utilized for electrocatalytic oxidation of cysteine. In presence of oxovanadium(IV salen, increased oxidation current is observed due to the effective oxidation of cysteine by the electrogenerated oxovanadium(V salen species. The oxidation current linearly varies with the concentration of cysteine from 0.1 to 1.0 mM. The modified electrode has good sensitivity and low limit of detection. These properties make the oxovanadium(IV salen as an effective electrocatalyst for the determination of cysteine.

  13. Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy.

    Science.gov (United States)

    Liu, Y; Mundt, E; Mundt, A; Sylte, M; Suarez, D L; Swayne, D E; García, M

    2010-03-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus, purified, recombinant N1 protein from A/chicken/Indonesia/PA7/2003 (H5N1) virus. The N1-ELISA showed high selectivity for detection of N1 antibodies, with no cross-reactivity with other neuraminidase subtypes, and broad reactivity with sera to N1 subtype isolates from North American and Eurasian lineages. Sensitivity of the N1-ELISA to detect N1 antibodies in turkey sera, collected 3 wk after H1N1 vaccination, was comparable to detection of avian influenza antibodies by the commercial, indirect ELISAs ProFLOK AIV Plus ELISA Kit (Synbiotics, Kansas City, MO) and Avian Influenza Virus Antibody Test Kit (IDEXX, Westbrook, ME). However, 6 wk after vaccination, the Synbiotics ELISA kit performed better than the N1-ELISA and the IDEXX ELISA kit. An evaluation was made of the ability of the N1-ELISA to discriminate vaccinated chickens from subsequently challenged chickens. Two experiments were conducted, chickens were vaccinated with inactivated H5N2 and H5N9 viruses and challenged with highly pathogenic H5N1 virus, and chickens were vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected at 14 days postchallenge and tested by hemagglutination inhibition (HI), quantitative neuraminidase inhibition (NI), and N1-ELISA. At 2 days postchallenge, oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. The N1-ELISA was in fair agreement with VI and HI results. Although the N1-ELISA showed a lower sensitivity than the NI assay, it was demonstrated that detection of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus in vaccinated chickens. Therefore, N1-ELISA can facilitate a vaccination strategy with differentiation of infected from vaccinated animals using a neuraminidase heterologous approach.

  14. Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments.

    Directory of Open Access Journals (Sweden)

    Eefje J A Schrauwen

    Full Text Available Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1, we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.

  15. Construction and comparison of different source neuraminidase candidate vaccine strains for human infection with Eurasian avian-like influenza H1N1 virus.

    Science.gov (United States)

    Liu, Liqi; Lu, Jian; Zhou, Jianfang; Li, Zi; Zhang, Heng; Wang, Dayan; Shu, Yuelong

    2017-12-01

    Human infections with Eurasian avian-like swine influenza H1N1 viruses have been reported in China in past years. One case resulted in death and others were mild case. In 2016, the World Health Organization recommended the use of A/Hunan/42443/2015(H1N1) virus to construct the first candidate vaccine strain for Eurasian avian-like swine influenza H1N1 viruses. Previous reports showed that the neuraminidase of A/Puerto Rico/8/34(H1N1) might improve the viral yield of reassortant viruses. Therefore, we constructed two reassortant candidate vaccine viruses of A/Hunan/42443/2015(H1N1) by reverse genetic technology, with (6+2) and (7+1) gene constitution, respectively. The (6+2) virus had hemagglutinin and neuraminidase from A/Hunan/42443/2015, and the (7+1) one had hemagglutinin from A/Hunan/42443/2015, while all the other genes were from A/Puerto Rico/8/34. Our data revealed that although the neuraminidase of the (7+1) virus was from high yield A/Puerto Rico/8/34, the hemagglutination titer and the hemagglutinin protein content of the (7+1) virus was not higher than that of the (6+2) virus. Both of the (7+1) and (6+2) viruses reached a similar level to that of A/Puerto Rico/8/34 at the usual harvest time in vitro. Therefore, both reassortant viruses are potential candidate vaccine viruses, which could contribute to pandemic preparedness. Copyright © 2017. Published by Elsevier Masson SAS.

  16. Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis-Menten mechanism.

    Science.gov (United States)

    Sudhamalla, Babu; Kumar, Mahesh; Roy, Karnati R; Kumar, R Sunil; Bhuyan, Abani K

    2013-11-01

    It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease. Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis. The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable. The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.

    Science.gov (United States)

    Smooker, P. M.; Whisstock, J. C.; Irving, J. A.; Siyaguna, S.; Spithill, T. W.; Pike, R. N.

    2000-01-01

    The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases. PMID:11206078

  18. Protein oxidation: an overview of metabolism of sulphur containing amino acid, cysteine.

    Science.gov (United States)

    Ahmad, Saheem; Khan, Hamda; Shahab, Uzma; Rehman, Shahnawaz; Rafi, Zeeshan; Khan, Mohd Yasir; Ansari, Ahsanullah; Siddiqui, Zeba; Ashraf, Jalaluddin Mohammad; Abdullah, Saleh M S; Habib, Safia; Uddin, Moin

    2017-01-01

    The available data suggest that among cellular constituents, proteins are the major target for oxidation primarily because of their quantity and high rate of interactions with ROS. Proteins are susceptible to ROS modifications of amino acid side chains which alter protein structure. Among the amino acids, Cysteine (Cys) is more prone to oxidation by ROS because of its high nucleophilic property. The reactivity of Cys with ROS is due to the presence of thiol group. In the oxidised form, Cys forms disulfide bond, which are primary covalent cross-link found in proteins, and which stabilize the native conformation of a protein. Indirect evidence suggests that thiol modifications by ROS may be involved in neurodegenerative disorders, but the significance and precise extent of the contributions are poorly understood. Here, we review the role of oxidized Cys in different pathological consequences and its biochemistry may increase the research in the discovery of new therapies. The purpose of this review is to re-examine the role and biochemistry of oxidised Cys residues.

  19. Cysteine-Based Protein Adduction by Epoxide-Derived Metabolite(s) of Benzbromarone.

    Science.gov (United States)

    Wang, Hui; Feng, Yukun; Wang, Qian; Guo, Xiucai; Huang, Wenlin; Peng, Ying; Zheng, Jiang

    2016-12-19

    Benzbromarone (BBR) is a therapeutically useful uricosuric agent but can also cause acute liver injury. The hepatotoxicity of BBR is suggested to be associated with its metabolic activation. Our recent metabolic study demonstrated that BBR was metabolized to epoxide intermediate(s) by cytochrome P450 3A, and the intermediate(s) was reactive to N-acetylcysteine. The objectives of the present study were to determine the chemical identity of the interaction of protein with the epoxide intermediate(s) of BBR and to define the association of the protein modification with hepatotoxicity induced by BBR. Microsomal incubation study showed that the reactive intermediate(s) covalently modified microsomal protein at cysteine residues. Such adduction was also observed in hepatic protein obtained from liver of mice given BBR. The protein covalent binding occurred in time- and dose-dependent manners. Pretreatment with ketoconazole attenuated BBR-induced protein modification and hepatotoxicity, while pretreatment with dexamethasone or buthionine sulfoximine potentiated the protein adduction and hepatotoxicity induced by BBR. A good correlation was observed between BBR-induced hepatotoxicity and the epoxide-derived hepatic protein modification in mice. The present study provided in-depth mechanistic insight into BBR-induced hepatotoxicity.

  20. Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization.

    Science.gov (United States)

    Shabelnikov, Sergey; Kiselev, Artem

    2015-01-01

    Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates.

  1. Producing armyworm (spodoptera sp.) Bioinsecticide based on cysteine protease of red ginger (zingiber officinale var. Rubrum)

    Science.gov (United States)

    Afnan, N. T.; Nur, D. F.; Utami, T. S.; Sahlan, M.; Wijanarko, A.; Hermansyah, H.

    2018-03-01

    Armyworm (Spodoptera sp.) is highly polyphagous defoliator on various horticulture and grain plants. Various chemical insecticides have been created to control it. There is a need to create an eco-friendly and specific insecticide which only affect armyworm’s nervous system. This research investigates cysteine-protease’s enzyme activity of red ginger (Zingiber officinale var. Rubrum) which is called zingibain. Its catalytic site matches with residue site in armyworm’s body so it can be used as bioinsecticide raw material which meets the criterias above. Fresh red ginger rhizomes were washed and extracted. The juice was then deposited in low temperature and centrifuged to get rid of its starch content. It was filtrated to remove large contaminants and poured into Potassium Phospate buffer. The liquid was then centrifuged again for 30 minutes before collecting the supernatant. Fresh leaves were then dipped into crude ginger protease extract and fed to fourth instar-armyworms. Leaves dipped into non-diluted extract were barely eaten by armyworm while the 50% and 25% dilution was half eaten and most eaten. The crude red ginger extract was not strong enough to kill them although the research showed its enzymatic activity reaches up to 169 PU. It still needs improvement to be produced as commercial bioinsecticide.

  2. Effect of Neuraminidase Inhibitor–Resistant Mutations on Pathogenicity of Clade 2.2 A/Turkey/15/06 (H5N1) Influenza Virus in Ferrets

    OpenAIRE

    Ilyushina, Natalia A.; Seiler, Jon P.; Rehg, Jerold E.; Webster, Robert G.; Govorkova, Elena A.

    2010-01-01

    The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N...

  3. Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets.

    Directory of Open Access Journals (Sweden)

    Erhard van der Vries

    2011-09-01

    Full Text Available Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223 was compared with a well-characterized reference virus (NL/602. In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R, transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

  4. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    Science.gov (United States)

    Pandey, Chandra Mouli; Sumana, Gajjala; Tiwari, Ida

    2014-09-01

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38 × 10-4 cm s-1. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.

  5. Immunological cross-reactivity of the major allergen from perennial ryegrass (Lolium perenne), Lol p I, and the cysteine proteinase, bromelain.

    Science.gov (United States)

    Pike, R N; Bagarozzi, D; Travis, J

    1997-04-01

    Antibodies prepared in rabbits against the major allergen from ryegrass (Lolium perenne), Lol p I, cross-reacted with the cysteine proteinase bromelain from pineapple and vice versa. Deglycosylation of the proteins showed that the cross-reaction was based on recognition of the carbohydrate moiety of the allergen, but for bromelain the cross-reaction was most likely due to a combination of factors. The results indicate that the carbohydrate residues from these allergens play an important role in cross-reactions found between them and possibly those from other species.

  6. Two cysteine substitutions in the MC1R generate the blue variant of the Arctic fox (Alopex lagopus) and prevent expression of the white winter coat.

    Science.gov (United States)

    Våge, Dag Inge; Fuglei, Eva; Snipstad, Kristin; Beheim, Janne; Landsem, Veslemøy Malm; Klungland, Helge

    2005-10-01

    We have characterized two mutations in the MC1R gene of the blue variant of the arctic fox (Alopex lagopus) that both incorporate a novel cysteine residue into the receptor. A family study in farmed arctic foxes verified that the dominant expression of the blue color phenotype cosegregates completely with the allele harboring these two mutations. Additionally to the altered pigment synthesis, the blue fox allele suppresses the seasonal change in coat color found in the native arctic fox. Consequently, these findings suggest that the MC1R/agouti regulatory system is involved in the seasonal changes of coat color found in arctic fox.

  7. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    Energy Technology Data Exchange (ETDEWEB)

    Bradshaw, William J. [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Kirby, Jonathan M. [Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Thiyagarajan, Nethaji [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Chambers, Christopher J.; Davies, Abigail H. [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom); Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Roberts, April K.; Shone, Clifford C. [Public Health England, Porton Down, Salisbury SP4 0JG (United Kingdom); Acharya, K. Ravi, E-mail: bsskra@bath.ac.uk [University of Bath, Claverton Down, Bath BA2 7AY (United Kingdom)

    2014-07-01

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.

  8. Determination of free and total cyst(e)ine in plasma of dogs and cats.

    Science.gov (United States)

    Tôrres, Cristina L; Miller, Joshua W; Rogers, Quinton R

    2004-01-01

    In human blood, the amino acid cysteine forms disulfide bonds with itself and with other sulfhydryl compounds in their free form and with sulfhydryls in protein. Protein-bound cysteine is lost when plasma proteins are removed before amino acid analysis. The purpose of this study was to assess the time course and extent of cyst(e)ine (cysteine + half-cystine) loss in dog and cat plasma. An equal volume of 6% sulfosalicylic acid was added to plasma aliquots at 0, 2, 4, 10, 16, 24, 36, 48, 60, and 72 hours after separation of blood cells. Tris-2-carboxyethyl-phosphine hydrochloride (TCEP - HCl), a reducing agent, was used to regenerate total plasma cyst(e)ine after 3 months of sample storage (-20 degrees C). Initial free cyst(e)ine concentrations (mean +/- SEM) were higher in canine plasma (77 +/- 4 micromol/L) than in feline plasma (37 +/- 3 micromol/L). Free plasma cyst(e)ine concentrations in dogs and cats decreased after first-order kinetics, with a half-life of 23 and 69 hours, respectively. Total plasma cysteine after TCEP - HCl treatment was similar for dogs (290 micromol/L) and cats (296 micromol/L), but the percentage of free cysteine was higher (P = .02) in dogs (27%) than in cats (13%). Over half of the cyst(e)ine, homocysteine, cysteinylglycine, and glutathione were bound in vivo to plasma proteins. These results emphasize the importance of removing plasma proteins within 1 hour after blood collection for reliable assay of free plasma cyst(e)ine.

  9. Elimination of hydrogen sulphide and β substitution in cystein, catalyzed by the cysteine-lyase of hens yolk-sac and yolk (1961)

    International Nuclear Information System (INIS)

    Chapeville, F.; Fromageot, P.

    1961-01-01

    The yolk of incubated hen's eggs contains a pyridoxal phosphate activated enzyme, free of iron, copper, magnesium and calcium. This enzyme activates the β-carbon atom of cysteine. Its reactivity is demonstrated by the ease with which this β-carbon fixes various sulfur containing substances in which the sulfur has reducing properties: inorganic sulfide, sulfide or cysteine itself. In the absence of substances able to react with the β-carbon atom, the active complex, consisting of the enzyme and the aminated tri-carbon chain, is hydrolysed to pyruvic acid and ammonia. The liberation of hydrogen sulfide thus appears to be the consequence either of the substitution of the β-carbon atom of cysteine or of the decomposition of the complex which this aminoacid forms with the enzyme studied. The latter seems therefore to possess an activity which differs from the activity of the desulfhydrases as yet known. We suggest to call this enzyme cystein-lyase. (authors) [fr

  10. Keap1 cysteine 288 as a potential target for diallyl trisulfide-induced Nrf2 activation.

    Directory of Open Access Journals (Sweden)

    Sanghyun Kim

    Full Text Available Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1 andquinone oxidoreductase-1 (NQO1 in human gastric epithelial (AGS cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2 in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1, a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts

  11. Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron–Sulfur Cluster Biosynthesis

    Science.gov (United States)

    2016-01-01

    Ferredoxins play an important role as an electron donor in iron–sulfur (Fe–S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron–sulfur cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron–sulfur cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe–S cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does. The reduced form of each ferredoxin became oxidized in the presence of the cysteine desulfurase complex when l-cysteine was added, leading to its conversion to l-alanine and the generation of sulfide. In an in vitro reaction, the reduced form of each ferredoxin was found to support Fe–S cluster assembly on ISCU; the rate of cluster assembly was faster with FDX2 than with FDX1. Taken together, these results show that both FDX1 and FDX2 can function in Fe–S cluster assembly in vitro. PMID:28001042

  12. An endogenous inhibitor of cysteine cathepsin B from brain tissues

    Directory of Open Access Journals (Sweden)

    O.L. Lyanna

    2013-11-01

    Full Text Available Lysosomes are the key degradative compartments of the cell in which the processes of protein degradation take place. Lysosomal cathepsins, which are enclosed in the lysosomes, help to maintain the homeostasis of the cell’s metabolism by participating in the degradation of heterophagic and autophagic material. When breaking down the integrity of lysosomal membranes the cathepsins are released into the cytosol and initiate the development of numerous pathological states. Breakdown in the control of protease activity leads to undesired and unregulated proteolysis. This is a cause of many diseases, such as Alzheimer’s disease, cancer, viral infections, cataracts etc. For this reason inhibitors of proteases have the potential to provide successful treatment for a wide range of diseases. Cathepsin B is one of the most abundant and ubiquitously expressed cysteine peptidases of the papain family. It is implicated in a number of pathological states including: inflammatory diseases of the airways, bone and joint disorders, acute pancreatitis, tumour metastasis, Alzheimer’s disease and ischemic neuronal death. The study of specific inhibitors for cathepsin B is considered important for chemotherapy and treatments of other diseases. This article represents part of a complex study of the lysosomal proteolytic-antiproteolytic system and its breakdown in the process of illness. In this article we present a scheme for extraction, purification and characterization of endogenous inhibitors of lysosomal cysteine cathepsin B. The cathepsin inhibitor was purified to homogeneity from the human neocortex. The purification was carried out in several successive stages: ammonium sulfate precipitation, followed by gel-filtration on Sephadex G-150, and ion exchange chromatography using DEAE-Sephadex A-75, followed by gel filtration on Sephadex G-100. Throughout the purification procedure, cathepsin inhibitory activity was controlled against the substrate p

  13. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

    Science.gov (United States)

    Cysteine peptidases are important in many biological processes. In this study, we describe the design, synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-Y, wh...

  14. An iron-oxygen intermediate formed during the catalytic cycle of cysteine dioxygenase.

    Science.gov (United States)

    Tchesnokov, E P; Faponle, A S; Davies, C G; Quesne, M G; Turner, R; Fellner, M; Souness, R J; Wilbanks, S M; de Visser, S P; Jameson, G N L

    2016-07-07

    Cysteine dioxygenase is a key enzyme in the breakdown of cysteine, but its mechanism remains controversial. A combination of spectroscopic and computational studies provides the first evidence of a short-lived intermediate in the catalytic cycle. The intermediate decays within 20 ms and has absorption maxima at 500 and 640 nm.

  15. Cysteine as a non toxic corrosion inhibitor for copper alloys in conservation

    DEFF Research Database (Denmark)

    Gravgaard, Mari; van Lanschot, Jettie

    2012-01-01

    studies of colour changes in the corrosion products. The results obtained in this article demonstrate that cysteine could be a non-toxic alternative to BTA. Cysteine performed as well as BTA on pre-corroded coupons with bronze disease in high humidity and showed acceptable results during testing...

  16. Tuning the carbon nanotube photoluminescence enhancement at addition of cysteine through the change of external conditions

    Energy Technology Data Exchange (ETDEWEB)

    Kurnosov, N.V.; Karachevtsev, M.V.; Leontiev, V.S.; Karachevtsev, V.A., E-mail: karachevtsev@ilt.kharkov.ua

    2017-01-15

    The enhancement of the photoluminescence (PL) from the semiconducting single-walled carbon nanotubes suspended with single-stranded DNA (ssDNA) in water observed after amino acids doping is the largest at cysteine addition. The PL intensity increased through the passivation of p-defects on the carbon nanotube sidewall by the cysteine molecules due to thiol group. The effect of several external factors on the cysteine-induced enhancement of PL from carbon nanotubes covered with ssDNA was studied: UV irradiation, tip or bath sonication treatment of the suspension, the ionic strength and pH of aqueous suspension. It turned out that all these factors have an essential influence on the dependence of the PL enhancement on the cysteine concentration through inducing of additional defects on nanotube as well as a change of the nanotube surface coverage with polymer. The obtained experimental results demonstrated that PL from carbon nanotubes can be exploited successfully for the monitoring of cysteine concentration in aqueous solution. - Highlights: • Cysteine doping enhances carbon nanotube emission more than other amino acids do. • SWNT emission dependence on cysteine concentration is tuned by UV irradiation and pH. • Type of sonication treatment influences SWNT PL dependence on cysteine concentration. • Polymer coverage and defectiveness of nanotubes effect on nanotube emission. • Graphic abstract.

  17. Nitric oxide-generating l-cysteine-grafted graphene film as a blood-contacting biomaterial.

    Science.gov (United States)

    Du, Zhen; Dou, Ruixia; Zu, Mian; Liu, Xueying; Yin, Wenyan; Zhao, Yuliang; Chen, Jingbo; Yan, Liang; Gu, Zhanjun

    2016-06-24

    By using polyethylenimine molecules as the linker, l-cysteine was immobilized onto graphene nanosheets, endowing the biocompatible l-cysteine-functionalized graphene film with the ability for catalytic decomposition of exogenous or endogenous donors to generate nitric oxide, and thus inhibiting the platelet activation and aggregation and reducing platelet adhesion.

  18. Cysteine: a conditionally essential amino acid in low-birth-weight preterm infants?

    NARCIS (Netherlands)

    Riedijk, Maaike A.; van Beek, Ron H. T.; Voortman, Gardi; de Bie, Henrica M. A.; Dassel, Anne C. M.; van Goudoever, Johannes B.

    2007-01-01

    Cyst(e)ine can be synthesized de novo from methionine and serine and is, therefore, a nonessential amino acid in human adults. Several studies have suggested that cyst(e)ine might be a conditionally essential amino acid in preterm infants because of biochemical immaturity. No data are available on

  19. Studies on synthesis and structure-activity relationship (SAR) of derivatives of a new natural product from marine fungi as inhibitors of influenza virus neuraminidase.

    Science.gov (United States)

    Li, Jing; Zhang, Dingmei; Zhu, Xun; He, Zhenjian; Liu, Shu; Li, Mengfeng; Pang, Jiyan; Lin, Yongcheng

    2011-01-01

    Based on the natural isoprenyl phenyl ether from a mangrove-derived fungus, 32 analogues were synthesized and evaluated for inhibitory activity against influenza H1N1 neuraminidase. Compound 15 (3-(allyloxy)-4-hydroxybenzaldehyde) exhibited the most potent inhibitory activity, with IC(50) values of 26.96 μM for A/GuangdongSB/01/2009 (H1N1), 27.73 μM for A/Guangdong/03/2009 (H1N1), and 25.13 μM for A/Guangdong/ 05/2009 (H1N1), respectively, which is stronger than the benzoic acid derivatives (~mM level). These are a new kind of non-nitrogenous aromatic ether Neuraminidase (NA) inhibitors. Their structures are simple and the synthesis routes are not complex. The structure-activity relationship (SAR) analysis revealed that the aryl aldehyde and unsubstituted hydroxyl were important to NA inhibitory activities. Molecular docking studies were carried out to explain the SAR of the compounds, and provided valuable information for further structure modification.

  20. Studies on Synthesis and Structure-Activity Relationship (SAR of Derivatives of a New Natural Product from Marine Fungi as Inhibitors of Influenza Virus Neuraminidase

    Directory of Open Access Journals (Sweden)

    Yongcheng Lin

    2011-10-01

    Full Text Available Based on the natural isoprenyl phenyl ether from a mangrove-derived fungus, 32 analogues were synthesized and evaluated for inhibitory activity against influenza H1N1 neuraminidase. Compound 15 (3-(allyloxy-4-hydroxybenzaldehyde exhibited the most potent inhibitory activity, with IC50 values of 26.96 μM for A/GuangdongSB/01/2009 (H1N1, 27.73 μM for A/Guangdong/03/2009 (H1N1, and 25.13 μM for A/Guangdong/05/2009 (H1N1, respectively, which is stronger than the benzoic acid derivatives (~mM level. These are a new kind of non-nitrogenous aromatic ether Neuraminidase (NA inhibitors. Their structures are simple and the synthesis routes are not complex. The structure-activity relationship (SAR analysis revealed that the aryl aldehyde and unsubstituted hydroxyl were important to NA inhibitory activities. Molecular docking studies were carried out to explain the SAR of the compounds, and provided valuable information for further structure modification.

  1. Surveillance of antiviral resistance markers in Argentina: detection of E119V neuraminidase mutation in a post-treatment immunocompromised patient

    Directory of Open Access Journals (Sweden)

    Andrea Pontoriero

    Full Text Available Although vaccines are the best means of protection against influenza, neuraminidase inhibitors are currently the main antiviral treatment available to control severe influenza cases. One of the most frequent substitutions in the neuraminidase (NA protein of influenza A(H3N2 viruses during or soon after oseltamivir administration is E119V mutation. We describe the emergence of a mixed viral population with the E119E/V mutation in the NA protein sequence in a post-treatment influenza sample collected from an immunocompromised patient in Argentina. This substitution was identified by a real-time reverse transcriptase polymerase chain reaction (RT-PCR protocol and was confirmed by direct Sanger sequencing of the original sample. In 2014, out of 1140 influenza samples received at the National Influenza Centre, 888 samples (78% were A(H3N2 strains, 244 (21.3% were type B strains, and 8 (0.7% were A(H1N1pdm09 strains. Out of 888 A(H3N2 samples, 842 were tested for the E119V substitution by quantitative RT-PCR: 841 A(H3N2 samples had the wild-type E119 genotype and in one sample, a mixture of viral E119/ V119 subpopulations was detected. Influenza virus surveillance and antiviral resistance studies can lead to better decisions in health policies and help in medical treatment planning, especially for severe cases and immunocompromised patients.

  2. Intramolecular synergistic effect of glutamic acid, cysteine and glycine against copper corrosion in hydrochloric acid solution

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Daquan, E-mail: zhdq@sh163.net [Department of Environmental Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Xie Bin; Gao Lixin; Cai Qirui [Department of Environmental Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Joo, Hyung Goun; Lee, Kang Yong [Stress Analysis and Failure Design Laboratory, School of Mechanical Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2011-10-31

    The corrosion protection of copper by glutamic acid, cysteine, glycine and their derivative (glutathione) in 0.5 M hydrochloric acid solution has been studied by the electrochemical impedance spectroscopy and cyclic voltammetry. The inhibition efficiency of the organic inhibitors on copper corrosion increases in the order: glutathione > cysteine > cysteine + glutamic acid + glycine > glutamic acid > glycine. Maximum inhibition efficiency for cysteine reaches about 92.9% at 15 mM concentration level. The glutathione can give 96.4% inhibition efficiency at a concentration of 10 mM. The molecular structure parameters were obtained by PM3 (Parametric Method 3) semi-empirical calculation. The intramolecular synergistic effect of glutamic acid, cysteine and glycine moieties in glutathione is attributed to the lower energy of the lowest unoccupied molecular orbital (E{sub LUMO}) level and to the excess hetero-atom adsorption centers and the bigger coverage on the copper surface.

  3. Intramolecular synergistic effect of glutamic acid, cysteine and glycine against copper corrosion in hydrochloric acid solution

    International Nuclear Information System (INIS)

    Zhang Daquan; Xie Bin; Gao Lixin; Cai Qirui; Joo, Hyung Goun; Lee, Kang Yong

    2011-01-01

    The corrosion protection of copper by glutamic acid, cysteine, glycine and their derivative (glutathione) in 0.5 M hydrochloric acid solution has been studied by the electrochemical impedance spectroscopy and cyclic voltammetry. The inhibition efficiency of the organic inhibitors on copper corrosion increases in the order: glutathione > cysteine > cysteine + glutamic acid + glycine > glutamic acid > glycine. Maximum inhibition efficiency for cysteine reaches about 92.9% at 15 mM concentration level. The glutathione can give 96.4% inhibition efficiency at a concentration of 10 mM. The molecular structure parameters were obtained by PM3 (Parametric Method 3) semi-empirical calculation. The intramolecular synergistic effect of glutamic acid, cysteine and glycine moieties in glutathione is attributed to the lower energy of the lowest unoccupied molecular orbital (E LUMO ) level and to the excess hetero-atom adsorption centers and the bigger coverage on the copper surface.

  4. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  5. The cysteine radical cation: structures and fragmentation pathways.

    Science.gov (United States)

    Zhao, Junfang; Siu, K W Michael; Hopkinson, Alan C

    2008-01-14

    A theoretical study on the structures, relative energies, isomerization reactions and fragmentation pathways of the cysteine radical cation, [NH(2)CH(CH(2)SH)COOH].+, is reported. Hybrid density functional theory (B3LYP) has been used in conjunction with the 6-311++G(d,p) basis set. The isomer at the global minimum, Captodative-1, has the structure NH(2)C.(CH(2)SH)C(OH)(2)+; the stability of this ion is attributed to the captodative effect in which the NH(2) functions as a powerful pi-electron donor and C(OH)(2)+ as a powerful pi-electron acceptor. Ion Distonic-S-1, H(3)N(+)CH(CH(2)S.)COOH, in which the radical is formally situated on the S atom, is higher in enthalpy (DeltaH degrees (0)) than Captodative-1 by 6.1 kcal mol(-1), but is lower in enthalpy than another isomer Distonic-C-1, H(3)N(+)C.(CH(2)SH)COOH, by 8.2 kcal mol(-1). Isomerization of the canonical radical cation of cysteine, [H(2)NCH(CH(2)SH)COOH].+, (Canonical-1), to Captodative-1 has an enthalpy of activation of 25.8 kcal mol(-1), while the barrier against isomerization of Canonical-1 to Distonic-S-1 is only 9.6 kcal mol(-1). Two additional transient tautomers, one with the radical located at C(alpha) and the charge on SH(2), and the other a carboxy radical with the charge on NH(3), are reported. Plausible fragmentation pathways (losses of small molecules, CO(2), CH(2)S, H(2)S and NH(3), and neutral radicals COOH. , HSCH(2). and NH(2).) from Canonical-1 are examined.

  6. Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

    Science.gov (United States)

    Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

    2014-01-01

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

  7. Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad.

    Science.gov (United States)

    Aramini, James M; Rossi, Paolo; Huang, Yuanpeng J; Zhao, Li; Jiang, Mei; Maglaqui, Melissa; Xiao, Rong; Locke, Jessica; Nair, Rajesh; Rost, Burkhard; Acton, Thomas B; Inouye, Masayori; Montelione, Gaetano T

    2008-09-16

    Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.

  8. Residual gas analysis

    International Nuclear Information System (INIS)

    Berecz, I.

    1982-01-01

    Determination of the residual gas composition in vacuum systems by a special mass spectrometric method was presented. The quadrupole mass spectrometer (QMS) and its application in thin film technology was discussed. Results, partial pressure versus time curves as well as the line spectra of the residual gases in case of the vaporization of a Ti-Pd-Au alloy were demonstrated together with the possible construction schemes of QMS residual gas analysers. (Sz.J.)

  9. Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Xue; Kumru, Ozan S.; Blaber, Sachiko I.; Middaugh, C. Russell; Li, Ling; Ornitz, David M.; Sutherland, Mason A.; Tenorio, Connie A.; Blaber, Michael (FSU); (WU-MED); (Kansas)

    2016-07-06

    Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous “second generation” form of FGF-1 for therapeutic application.

  10. The effect of various S-alkylating agents on the chromatographic behavior of cysteine-containing peptides in reversed-phase chromatography.

    Science.gov (United States)

    Jiang, Xuehui; Shamshurin, Dmitry; Spicer, Vic; Krokhin, Oleg V

    2013-02-01

    We investigate the influence of various alkylation chemistries on the reversed phase (RP) HPLC behavior of Cys-containing peptides under the most popular RP-HPLC conditions used in proteomics: C18 phases with trifluoroacetic acid (TFA) or formic acid (FA) as the ion pairing modifiers, and separation at pH 10. Akylating agents studied are iodoacetamide (IAM), iodoacetic acid (IAA), 4-vinylpyridine (4-VP), acrylamide (AA) and methyl methanethiosulfonate (MMTS). These were compared against the retention of identical peptides without alkylation, i.e. free cysteines. The intrinsic hydrophobicity values of the Cys residue under formic acid conditions for these modifications were found to increase in the following order: 4-VPalkylated Cys using TFA eluent. Switching to a basic condition dramatically decreases the retention of free cysteine and IAA-alkylated analytes due to the ionization of side-chains. The opposite effect is observed for 4-VP, which become neutral at basic pHs. The careful measurement of the hydrophobic contributions for these residues is vital to the development of accurate peptide retention prediction models; the incorporation of these modifications into our Sequence Specific Retention Calculator model is presented. Copyright © 2013. Published by Elsevier B.V.

  11. Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

    Science.gov (United States)

    Ueki, Iori; Roman, Heather B.; Valli, Alessandro; Fieselmann, Krista; Lam, Jimmy; Peters, Rachel; Hirschberger, Lawrence L.

    2011-01-01

    Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO+/− mice) were crossed to generate CDO−/−, CDO+/−, and CDO+/+ mice. CDO−/− mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO−/− mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO−/− mice than in CDO+/− or CDO+/+ mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO−/− mice. H2S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S/sulfane sulfur levels and facilitate the use of H2S as a signaling molecule. PMID:21693692

  12. Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages.

    Science.gov (United States)

    Zhu, Zhendong; Ren, Zhanjun; Fan, Xiaoteng; Pan, Yang; Lv, Shan; Pan, Chuanying; Lei, Anmin; Zeng, Wenxian

    2017-01-01

    The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.

  13. Facile and green synthesis of highly stable L-cysteine functionalized copper nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Nikhil, E-mail: nkumar.phd2011.bt@nitrr.ac.in; Upadhyay, Lata Sheo Bachan, E-mail: contactlataupadhyay@gmail.com

    2016-11-01

    Highlights: • A facile and eco-friendly method for the synthesis of L-cysteine functionalized copper nanoparticles is reported. • Synthesis of Highly stable L-cysteine functionalized copper nanoparticles (∼40 nm) was done in an aqueous medium. • FTIR analysis shows that L-cysteine bound to the nanoparticle surface via thiol group. - Abstract: A simple eco-friendly method for L-cysteine capped copper nanoparticles (CCNPs) synthesis in aqueous solution has been developed. Glucose and L-cysteine were used as reducing agent and capping/functionalizing agent, respectively. Different parameters such as capping agent concentration, pH, reaction temperature, and reducing agent concentration were optimized during the synthesis. The L-cysteine capped copper nanoparticle were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, Particle size and zeta potential analyser, and high resolution transmission electron microscopy. Spherical shaped cysteine functionalized/capped copper nanoparticles with an average size of 40 nm were found to be highly stable at room temperature (RT) for a period of 1 month.

  14. A Sensitive Ratiometric Long-Wavelength Fluorescent Probe for Selective Determination of Cysteine/Homocysteine.

    Science.gov (United States)

    Manibalan, Kesavan; Chen, Sin-Ming; Mani, Veerappan; Huang, Tsung-Tao; Huang, Sheng-Tung

    2016-07-01

    The development of sensitive fluorescence probes to detect biothiols such as cysteine and homocysteine has attracted great attention in recent times. Herein, we described the design and synthesis of coumarin based long-wavelength fluorescence probe, Bromo-2-benzothiazolyl-3-cyano-7-hydroxy coumarin (BBCH, 2) for selective detections of cysteine and homocysteine. The probe is rationally designed in such a way that both sulfhydryl and adjacent amino groups of thiols are involved in sensing process. Only cysteine/homocysteine able to react with BBCH to release fluorescence reporter (BCH, 1); while, glutathione and other amino acids unable to react with BBCH due to the absence of adjacent amino groups. In presence of cysteine, the color of BBCH is turns from colorless to red and thus BBCH is a naked eye fluorescence indicator for cysteine. Besides, BBCH can discriminate cysteine and homocysteine based on color changes and different reaction rates. The described sensing platform showed good sensing performances to detect cysteine and homocysteine with detection limits of 0.87 and 0.19 μM, respectively. Practical applicability was verified in biological and pharmaceutical samples.

  15. Proteomic Approaches to Quantify Cysteine Reversible Modifications in Aging and Neurodegenerative Diseases

    Science.gov (United States)

    Gu, Liqing; Robinson, Renã A. S.

    2016-01-01

    Cysteine is a highly reactive amino acid and is subject to a variety of reversible post-translational modifications (PTMs), including nitrosylation, glutathionylation, palmitoylation, as well as formation of sulfenic acid and disulfides. These modifications are not only involved in normal biological activities, such as enzymatic catalysis, redox signaling and cellular homeostasis, but can also be the result of oxidative damage. Especially in aging and neurodegenerative diseases, oxidative stress leads to aberrant cysteine oxidations that affect protein structure and function leading to neurodegeneration as well as other detrimental effects. Methods that can identify cysteine modifications by type, including the site of modification, as well as the relative stoichiometry of the modification can be very helpful for understanding the role of the thiol proteome and redox homeostasis in the context of disease. Cysteine reversible modifications however, are challenging to investigate as they are low abundant, diverse, and labile especially under endogenous conditions. Thanks to the development of redox proteomic approaches, large-scale quantification of cysteine reversible modifications is possible. These approaches cover a range of strategies to enrich, identify, and quantify cysteine reversible modifications from biological samples. This review will focus on nongel-based redox proteomics workflows that give quantitative information about cysteine PTMs and highlight how these strategies have been useful for investigating the redox thiol proteome in aging and neurodegenerative diseases. PMID:27666938

  16. Facile and green synthesis of highly stable L-cysteine functionalized copper nanoparticles

    International Nuclear Information System (INIS)

    Kumar, Nikhil; Upadhyay, Lata Sheo Bachan

    2016-01-01

    Highlights: • A facile and eco-friendly method for the synthesis of L-cysteine functionalized copper nanoparticles is reported. • Synthesis of Highly stable L-cysteine functionalized copper nanoparticles (∼40 nm) was done in an aqueous medium. • FTIR analysis shows that L-cysteine bound to the nanoparticle surface via thiol group. - Abstract: A simple eco-friendly method for L-cysteine capped copper nanoparticles (CCNPs) synthesis in aqueous solution has been developed. Glucose and L-cysteine were used as reducing agent and capping/functionalizing agent, respectively. Different parameters such as capping agent concentration, pH, reaction temperature, and reducing agent concentration were optimized during the synthesis. The L-cysteine capped copper nanoparticle were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, Particle size and zeta potential analyser, and high resolution transmission electron microscopy. Spherical shaped cysteine functionalized/capped copper nanoparticles with an average size of 40 nm were found to be highly stable at room temperature (RT) for a period of 1 month

  17. The mechanism of cysteine detection in biological media by means of vanadium oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Bezerra, A. G. [Universidade Tecnologica Federal do Parana, Departamento Academico de Fisica (Brazil); Barison, A. [Universidade Federal do Parana, Departamento de Quimica (Brazil); Oliveira, V. S. [Universidade Federal do Parana, Departamento de Fisica (Brazil); Foti, L.; Krieger, M. A. [Fundacao Oswaldo Cruz, Instituto de Biologia Molecular do Parana (Brazil); Dhalia, R.; Viana, I. F. T. [Fundacao Oswaldo Cruz, Centro de Pesquisas Aggeu Magalhaes (Brazil); Schreiner, W. H., E-mail: wido@fisica.ufpr.br [Universidade Federal do Parana, Departamento de Fisica (Brazil)

    2012-09-15

    We report on the interaction of vanadate nanoparticles, produced using the laser ablation in liquids synthesis, with cysteine in biological molecules. Cysteine is a very important amino acid present in most proteins, but also because cysteine and the tripeptide glutathione are the main antioxidant molecules in our body system. Detailed UV-Vis absorption spectra and dynamic light scattering measurements were done to investigate the detection of cysteine in large biological molecules. The intervalence band of the optical absorption spectra shows capability for quantitative cysteine sensing in the {mu}M range in biological macromolecules. Tests included cytoplasmic repetitive antigen and flagellar repetitive antigen proteins of the Trypanosoma cruzi protozoa, as well as the capsid p24 proteins from Human Immunodeficiency Virus type 1 and type 2. Detailed NMR measurements for hydrogen, carbon, and vanadium nuclei show that cysteine in contact with the vanadate looses hydrogen of the sulphydryl side chain, while the vanadate is reduced. The subsequent detachment of two deprotonated molecules to form cystine and the slow return to the vanadate complete the oxidation-reduction cycle. Therefore, the vanadate acts as a charge exchanging catalyst on cysteine to form cystine. The NMR results also indicate that the nanoparticles are not formed by the common orthorhombic V{sub 2}O{sub 5} form.

  18. Transcriptome Analysis of Maize Immature Embryos Reveals the Roles of Cysteine in Improving Agrobacterium Infection Efficiency

    Science.gov (United States)

    Liu, Yan; Zhang, Zhiqiang; Fu, Junjie; Wang, Guoying; Wang, Jianhua; Liu, Yunjun

    2017-01-01

    Maize Agrobacterium-mediated transformation efficiency has been greatly improved in recent years. Antioxidants, such as, cysteine, can significantly improve maize transformation frequency through improving the Agrobacterium infection efficiency. However, the mechanism underlying the transformation improvement after cysteine exposure has not been elucidated. In this study, we showed that the addition of cysteine to the co-cultivation medium significantly increased the Agrobacterium infection efficiency of hybrid HiII and inbred line Z31 maize embryos. Reactive oxygen species contents were higher in embryos treated with cysteine than that without cysteine. We further investigated the mechanism behind cysteine-related infection efficiency increase using transcriptome analysis. The results showed that the cysteine treatment up-regulated 939 genes and down-regulated 549 genes in both Z31 and HiII. Additionally, more differentially expressed genes were found in HiII embryos than those in Z31 embryos, suggesting that HiII was more sensitive to the cysteine treatment than Z31. GO analysis showed that the up-regulated genes were mainly involved in the oxidation reduction process. The up-regulation of these genes could help maize embryos to cope with the oxidative stress stimulated by Agrobacterium infection. The down-regulated genes were mainly involved in the cell wall and membrane metabolism, such as, aquaporin and expansin genes. Decreased expression of these cell wall integrity genes could loosen the cell wall, thereby improving the entry of Agrobacterium into plant cells. This study offers insight into the role of cysteine in improving Agrobacterium-mediated transformation of maize immature embryos. PMID:29089955

  19. Homocysteine, cysteine, and risk of incident colorectal cancer in the Women's Health Initiative observational cohort123

    Science.gov (United States)

    Beresford, Shirley AA; Neuhouser, Marian L; Cheng, Ting-Yuan David; Song, Xiaoling; Brown, Elissa C; Zheng, Yingye; Rodriguez, Beatriz; Green, Ralph; Ulrich, Cornelia M

    2013-01-01

    Background: Inflammation underlies the etiology of colorectal cancer (CRC). Hyperhomocysteinemia is associated with inflammation and may be a risk marker for CRC. Cysteine is a metabolic product of homocysteine and a precursor of the antioxidant glutathione. It is unknown whether cysteine is associated with CRC. Objective: The objective was to assess the associations between homocysteine and cysteine and CRC incidence in postmenopausal women. Design: Associations between homocysteine and cysteine and incident CRC in the Women's Health Initiative observational cohort were assessed by using a nested case-control design. Cases and controls (n = 988/group) were matched for age (mean ± SD age: 67 ± 7 y), ethnicity (85.2% white, 8.9% black, 2.2% Hispanic/Latina, and 3.6% other), hysterectomy status, and date of blood draw. Homocysteine and cysteine were measured by HPLC with postcolumn fluorimetric detection. Results: Multivariate-adjusted ORs (95% CIs) for CRC were 1.46 (1.05, 2.04) for the highest quartile of homocysteine (>9.85 μmol/L) compared with the lowest quartile (≤6.74 μmol/L) (P = 0.02) and 0.57 (0.40, 0.82) for the highest quartile of cysteine (>309 μmol/L) compared with the lowest quartile (≤260 μmol/L) (P = 0.01). The association with homocysteine was significant for proximal colon tumors (P = 0.008) but not for distal or rectal tumors, whereas the association with cysteine was significant for rectal tumors (P = 0.02), borderline for proximal tumors (P = 0.06), and not significant for distal tumors. The associations with both homocysteine and cysteine were significant for localized tumors (P ≤ 0.01) but not for metastases. Conclusion: High plasma homocysteine is associated with increased risk of CRC, whereas high cysteine is associated with decreased risk. This trial was registered at clinicaltrials.gov as NCT 00000611. PMID:23426034

  20. l-cysteine suppresses ghrelin and reduces appetite in rodents and humans

    Science.gov (United States)

    McGavigan, A K; O'Hara, H C; Amin, A; Kinsey-Jones, J; Spreckley, E; Alamshah, A; Agahi, A; Banks, K; France, R; Hyberg, G; Wong, C; Bewick, G A; Gardiner, J V; Lehmann, A; Martin, N M; Ghatei, M A; Bloom, S R; Murphy, K G

    2015-01-01

    Background: High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. Methods: We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. Results: l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. Conclusions: Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety. PMID:25219528

  1. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    International Nuclear Information System (INIS)

    Suzuki, Nobuhiro; Yamazaki, Yasuo; Brown, R. Lane; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2008-01-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn 2+ -bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn 2+ ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn 2+ binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels

  2. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Nobuhiro [Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Yamazaki, Yasuo [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Brown, R. Lane [Neurological Science Institute, Oregon Health and Science University, Beaverton, Oregon 97006 (United States); Fujimoto, Zui [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Morita, Takashi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Mizuno, Hiroshi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); VALWAY Technology Center, NEC Soft Ltd, Koto-ku, Tokyo 136-8627 (Japan); Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, Tsukuba, Ibaraki 305-8566 (Japan); Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan)

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

  3. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    Science.gov (United States)

    Jung, Woongsic; Campbell, Robert L; Gwak, Yunho; Kim, Jong Im; Davies, Peter L; Jin, EonSeon

    2016-01-01

    Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

  4. Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.

    Directory of Open Access Journals (Sweden)

    Martin R Challand

    Full Text Available The 'radical S-adenosyl-L-methionine (AdoMet' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA, from numerous antibiotics, including agents (e.g. linezolid, retapamulin that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2 carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA. In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.

  5. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, Chad R. [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States); Hao, Quan [MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University, Ithaca, NY 14853-8001 (United States); Stipanuk, Martha H., E-mail: mhs6@cornell.edu [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States)

    2005-11-01

    Recombinant rat cysteine dioxygenase (CDO) has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.5 Å resolution. Cysteine dioxygenase (CDO; EC 1.13.11.20) is an ∼23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O{sub 2}, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Å resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Å, α = β = γ = 90°. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

  6. Agricultural pesticide residues

    International Nuclear Information System (INIS)

    Fuehr, F.

    1984-01-01

    The utilization of tracer techniques in the study of agricultural pesticide residues is reviewed under the following headings: lysimeter experiments, micro-ecosystems, translocation in soil, degradation of pesticides in soil, biological availability of soil-applied substances, bound residues in the soil, use of macro- and microautography, double and triple labelling, use of tracer labelling in animal experiments. (U.K.)

  7. Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803: formation of the persulfide intermediate.

    Science.gov (United States)

    Behshad, Elham; Bollinger, J Martin

    2009-12-22

    Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with lambda(max) approximately 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5'-phosphate (PLP) cofactor. The second intermediate, with absorption features at approximately 417 and approximately 340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate alpha-deuterium equilibrium isotope effect ((2)H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional approximately 506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect ((2)H-KIE) on its formation, which is similar in magnitude to the (2)H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is

  8. A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

    Science.gov (United States)

    Godahewa, G I; Perera, N C N; Lee, Sukkyoung; Kim, Myoung-Jin; Lee, Jehee

    2017-09-05

    Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone. Copyright © 2017. Published by Elsevier

  9. Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

    Science.gov (United States)

    Wang, Chun-Hsiung; Hsu, Chi-Hsin; Wu, Yi-Min; Luo, Yu-Chun; Tu, Mei-Hui; Chang, Wei-hau; Cheng, R. Holland

    2010-01-01

    The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles. PMID:20446029

  10. Distinct Mechanism of Cysteine Oxidation-Dependent Activation and Cold Sensitization of Human Transient Receptor Potential Ankyrin 1 Channel by High and Low Oxaliplatin

    Directory of Open Access Journals (Sweden)

    Takahito Miyake

    2017-11-01

    Full Text Available Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1 is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD, which causes sensitization of TRPA1 to reactive oxygen species (ROS. Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1 with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1 mM induced outward-rectifier whole-cell currents and increased the intracellular Ca2+ concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca2+ influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala-expressing cells showed similar whole-cell currents and Ca2+ influx. By contrast, a lower concentration of oxaliplatin (100 μM did not increase the intracellular Ca2+ concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower

  11. Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

    Science.gov (United States)

    Firouzamandi, Masoumeh; Moeini, Hassan; Hosseini, Davood; Bejo, Mohd Hair; Omar, Abdul Rahman; Mehrbod, Parvaneh; Ideris, Aini

    2016-03-01

    The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

  12. Functional properties and genetic relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat virus.

    Science.gov (United States)

    Krüger, Nadine; Hoffmann, Markus; Drexler, Jan Felix; Müller, Marcel Alexander; Corman, Victor Max; Sauder, Christian; Rubin, Steven; He, Biao; Örvell, Claes; Drosten, Christian; Herrler, Georg

    2015-04-01

    A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests

  13. Antiviral resistance due to deletion in the neuraminidase gene and defective interfering-like viral polymerase basic 2 RNA of influenza A virus subtype H3N2

    DEFF Research Database (Denmark)

    Trebbien, Ramona; Christiansen, Claus Bohn; Fischer, Thea Kølsen

    2018-01-01

    Background and objective: Antiviral treatment of influenza virus infections can lead to drug resistance of virus. This study investigates a selection of mutations in the full genome of H3N2 influenza A virus isolated from a patient in treatment with oseltamivir. Study design: Respiratory samples...... from a patient were collected before, during, and after antiviral treatment. Whole genome sequencing of the influenza virus by next generation sequencing, and low-frequency-variant analysis was performed. Neuraminidase-inhibition tests were performed with oseltamivir and zanamivir, and viruses were......, indicating a potential risk for transmission of the deleted virus. Full genome deep sequencing was useful to reveal variant mutations that might be selected due to antiviral treatment, and defective interfering-like viral PB2 RNA in the respiratory samples was detected....

  14. The unique cysteine knot regulates the pleotropic hormone leptin.

    Directory of Open Access Journals (Sweden)

    Ellinor Haglund

    Full Text Available Leptin plays a key role in regulating energy intake/expenditure, metabolism and hypertension. It folds into a four-helix bundle that binds to the extracellular receptor to initiate signaling. Our work on leptin revealed a hidden complexity in the formation of a previously un-described, cysteine-knotted topology in leptin. We hypothesized that this unique topology could offer new mechanisms in regulating the protein activity. A combination of in silico simulation and in vitro experiments was used to probe the role of the knotted topology introduced by the disulphide-bridge on leptin folding and function. Our results surprisingly show that the free energy landscape is conserved between knotted and unknotted protein, however the additional complexity added by the knot formation is structurally important. Native state analyses led to the discovery that the disulphide-bond plays an important role in receptor binding and thus mediate biological activity by local motions on distal receptor-binding sites, far removed from the disulphide-bridge. Thus, the disulphide-bridge appears to function as a point of tension that allows dissipation of stress at a distance in leptin.

  15. In situ molecular identification of the influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City.

    Science.gov (United States)

    Ocadiz-Delgado, Rodolfo; Albino-Sanchez, Martha Estela; Garcia-Villa, Enrique; Aguilar-Gonzalez, Maria Guadalupe; Cabello, Carlos; Rosete, Dora; Mejia, Fidencio; Manjarrez-Zavala, Maria Eugenia; Ondarza-Aguilera, Carmen; Rivera-Rosales, Rosa Ma; Gariglio, Patricio

    2013-01-18

    In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City's hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1) triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. In order to detect A (H1N1) virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1) Neuraminidase (NA) RNA using in situ RT-PCR. We detected NA transcripts in 100% of the previously diagnosed A (H1N1)-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1)-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1)-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. We have successfully amplified specific influenza A (H1N1) NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1) outbreak in Mexico City.

  16. In situ molecular identification of the Influenza A (H1N1 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City

    Directory of Open Access Journals (Sweden)

    Ocadiz-Delgado Rodolfo

    2013-01-01

    Full Text Available Abstract Background In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City’s hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1 triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1 2009 strains is possible in archival material such as paraffin-embedded lung samples. Methods In order to detect A (H1N1 virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1 Neuraminidase (NA RNA using in situ RT-PCR. Results We detected NA transcripts in 100% of the previously diagnosed A (H1N1-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. Conclusions We have successfully amplified specific influenza A (H1N1 NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1 outbreak in Mexico City.

  17. Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens

    International Nuclear Information System (INIS)

    Pushko, Peter; Tretyakova, Irina; Hidajat, Rachmat; Zsak, Aniko; Chrzastek, Klaudia; Tumpey, Terrence M.; Kapczynski, Darrell R.

    2017-01-01

    Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine. - Highlights: •VLPs were prepared that co-localized H5, H7 and H9 subtypes in a VLP envelope. •VLPs were characterized including electron microscopy, HA assay and NA enzyme activity. •Experimental VLP vaccine was evaluated in an avian influenza challenge model. •VLPs induced immune responses against heterologous H5, H7 and H9 virus challenges.

  18. Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1 virus have higher neuraminidase activities than the spherical wild-type.

    Directory of Open Access Journals (Sweden)

    Jill Seladi-Schulman

    Full Text Available Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1 [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.

  19. Handling of Solid Residues

    International Nuclear Information System (INIS)

    Medina Bermudez, Clara Ines

    1999-01-01

    The topic of solid residues is specifically of great interest and concern for the authorities, institutions and community that identify in them a true threat against the human health and the atmosphere in the related with the aesthetic deterioration of the urban centers and of the natural landscape; in the proliferation of vectorial transmitters of illnesses and the effect on the biodiversity. Inside the wide spectrum of topics that they keep relationship with the environmental protection, the inadequate handling of solid residues and residues dangerous squatter an important line in the definition of political and practical environmentally sustainable. The industrial development and the population's growth have originated a continuous increase in the production of solid residues; of equal it forms, their composition day after day is more heterogeneous. The base for the good handling includes the appropriate intervention of the different stages of an integral administration of residues, which include the separation in the source, the gathering, the handling, the use, treatment, final disposition and the institutional organization of the administration. The topic of the dangerous residues generates more expectation. These residues understand from those of pathogen type that are generated in the establishments of health that of hospital attention, until those of combustible, inflammable type, explosive, radio-active, volatile, corrosive, reagent or toxic, associated to numerous industrial processes, common in our countries in development

  20. Cysteine-free peptides in scorpion venom: geographical distribution ...

    African Journals Online (AJOL)

    GRACE

    2006-12-29

    Dec 29, 2006 ... The most remarkable characteristic is the high lysine content (11) and the amount of charged residues (17 in total). The positive (11 lysine and 1 arginine) and negative (1 aspartate and 4 glutamate) charges are located at opposite ends of the molecule. (Verdonck et al., 2000). The peptide conforms to an α-.

  1. High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity : application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

    NARCIS (Netherlands)

    Stijntjes, G.J.; te Koppele, J.M.; Vermeulen, N P

    1992-01-01

    An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid,

  2. Facile and green synthesis of highly stable L-cysteine functionalized copper nanoparticles

    Science.gov (United States)

    Kumar, Nikhil; Upadhyay, Lata Sheo Bachan

    2016-11-01

    A simple eco-friendly method for L-cysteine capped copper nanoparticles (CCNPs) synthesis in aqueous solution has been developed. Glucose and L-cysteine were used as reducing agent and capping/functionalizing agent, respectively. Different parameters such as capping agent concentration, pH, reaction temperature, and reducing agent concentration were optimized during the synthesis. The L-cysteine capped copper nanoparticle were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, Particle size and zeta potential analyser, and high resolution transmission electron microscopy. Spherical shaped cysteine functionalized/capped copper nanoparticles with an average size of 40 nm were found to be highly stable at room temperature (RT) for a period of 1 month

  3. A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione.

    Science.gov (United States)

    Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng

    2013-02-14

    We report a turn-on fluorescent sensor based on nitrothiophenolate boron dipyrromethene (BODIPY) derivatives for the discrimination of cystein (Cys) from homocystein (Hcy) and glutathione (GSH). The sensor was applied for detection of Cys in living cells.

  4. Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

    Science.gov (United States)

    Yang, Hyun-Jong; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2002-01-01

    The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products. PMID:12073734

  5. 7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

    Science.gov (United States)

    He, Xiaobo; Xia, Qingsu; Ma, Liang; Fu, Peter P

    2016-01-01

    Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

  6. Controllable synthesis of TiO2 nanomaterials by assisting with l-cysteine and ethylenediamine

    KAUST Repository

    Tao, Yugui

    2013-11-21

    This paper reports a facile l-cysteine-assisted solvothermal synthesis of TiO2 nanomaterials using ethylenediamine (En) and distilled water as solvent. The influence of reaction time, temperature, l-cysteine and solvent was initially investigated. Results demonstrated the reaction temperature, l-cysteine and En significantly imposed impact on the phase and morphology of the particles. Amorphous nanosheets, mixed-crystal nanorods and pure anatase nanoparticles were controllably synthesized by varying reaction temperature. The formation of the amorphous nanosheets and mixed-crystal nanorods were directly affected by the presence of l-cysteine and En. And the presence of En distinctly affected the crystal phase of the products, which was rarely mentioned in other studies. Moreover, the photocatalytic activities of three typical samples were excellent. The possible formation mechanism of the sample was also discussed. © 2013 Springer Science+Business Media New York.

  7. Study of the histochemical detection of cysteine desulfhydrase in the vitellin sac of birds (1961)

    International Nuclear Information System (INIS)

    Chapeville, F.; Khau Van Kien, L.

    1961-01-01

    We have developed a method for the histochemical detection of cysteine desulfhydrase in the vitellin sac of the chicken embryo. The enzyme is localized in the presence of a lead salt by lead sulphide formed in situ from hydrogen sulphide liberated from the cysteine. The micrographs obtained are histological and show the presence of the enzyme in the different types of endoderm cell. (authors) [fr

  8. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur

    OpenAIRE

    Stipanuk, Martha H.; Ueki, Iori

    2010-01-01

    Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine β-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when meth...

  9. Structural influence in the interaction of cysteine with five coordinated copper complexes: Theoretical and experimental studies

    Science.gov (United States)

    Huerta-Aguilar, Carlos Alberto; Thangarasu, Pandiyan; Mora, Jesús Gracia

    2018-04-01

    Copper complexes of N,N,N‧,N‧-tetrakis(pyridyl-2-ylmethyl)-1,2-diaminoethane (L1) and N,N,N‧,N‧-tetrakis(pyridyl-2-ylmethyl)-1,3-diaminopropane (L2) prepared were characterized completely by different analytical methods. The X-structure of the complexes shows that Cu(II) presents in trigonal bi-pyramidal (TBP) geometry, consisting with the electronic spectra where two visible bands corresponding to five coordinated structure were observed. Thus TD-DFT was used to analyze the orbital contribution to the electronic transitions for the visible bands. Furthermore, the interaction of cysteine with the complexes was spectrally studied, and the results were explained through DFT analysis, observing that the geometrical parameters and oxidation state of metal ions play a vital role in the binding of cysteine with copper ion. It appears that the TBP structure is being changed into octahedral geometry during the addition of cysteine to the complexes as two bands (from complex) is turned to a broad band in visible region, signifying the occupation of cysteine molecule at sixth position of octahedral geometry. In the molecular orbital analysis, the existence of a strong overlapping of HOMOs (from cysteine) with LUMOs of Cu ion was observed. The total energy of the systems calculated by DFT shows that cysteine binds favorably with copper (I) than that with Cu(II).

  10. Cysteinome: The first comprehensive database for proteins with targetable cysteine and their covalent inhibitors.

    Science.gov (United States)

    Wu, Sijin; Luo Howard, Huizhe; Wang, Haina; Zhao, Weijie; Hu, Qiwan; Yang, Yongliang

    2016-09-23

    The covalent modification of intrinsically nucleophilic cysteine in proteins is crucial for diverse biochemical events. Bioinformatics approaches may prove useful in the design and discovery of covalent molecules targeting the cysteine in proteins to tune their functions and activities. Herein, we describe the Cysteinome, the first online database that provides a rich resource for the display, search and analysis of structure, function and related annotation for proteins with targetable cysteine as well as their covalent modulators. To this end, Cysteinome compiles 462 proteins with targetable cysteine from 122 different species along with 1217 covalent modulators curated from existing literatures. Proteins are annotated with a detailed description of protein families, biological process and related diseases. In addition, covalent modulators are carefully annotated with chemical name, chemical structure, binding affinity, physicochemical properties, molecule type and related diseases etc. The Cysteinome database may serve as a useful platform for the identification of crucial proteins with targetable cysteine in certain cellular context. Furthermore, it may help biologists and chemists for the design and discovery of covalent chemical probes or inhibitors homing at functional cysteine of critical protein targets implicated in various physiological or disease process. The Cysteinome database is freely available to public at http://www.cysteinome.org/. Copyright © 2016. Published by Elsevier Inc.

  11. Depletion of circulating cyst(e)ine by oral and intravenous mesna.

    Science.gov (United States)

    Stofer-Vogel, B.; Cerny, T.; Küpfer, A.; Junker, E.; Lauterburg, B. H.

    1993-01-01

    The sulfhydryl status of normal and tumour cells is critically important in determining their susceptibility to various cytostatic agents. As a sulfhydryl compound, mesna (sodium 2-mercaptoethane-sulfonate) which is used in large doses to prevent haemorrhagic cystitis associated with certain chemotherapeutic regimens might derange cellular thiol homeostasis. In order to investigate the effects of mesna on the concentrations of thiols in plasma, cysteine, glutathione and their disulfides were measured by HPLC following the oral and intravenous administration of mesna to healthy volunteers. After 7.3 mmol mesna i.v. free cysteine rose from 8.2 (95% CI 7.0-9.4) nmol ml-1 to 53.6 (47.4-59.8) nmol ml-1 at 5 min, most likely due to reduction of circulating cystine by the sulfhydryl drug. This initial rise was followed by a marked decrease of total cyst(e)ine in plasma from 276 (215-337) nmol ml-1 to a nadir of 102 (89-115) nmol ml-1 between 30-120 min after infusion, most likely due to an increased uptake of cysteine into cells and an increased urinary excretion of cyst(e)ine. Qualitatively similar changes were seen after oral mesna. The present data indicate that mesna depletes circulating cyst(e)ine and may thereby markedly alter the sulfhydryl status of cells in vivo although the drug itself is not taken up by most cells. PMID:8353049

  12. [Construction of RNA-containing virus-like nanoparticles expression vector with cysteine residues on surface and fluorescent decoration].

    Science.gov (United States)

    Cheng, Yang-Jian; Liang, Ji-Xuan; Li, Qing-Ge

    2005-08-01

    Site-directed mutagenesis was performed at the codon 15 of the MS2 bacteriophage coat protein gene,which had been cloned to the virus-like particles expression vector containing non-self RNA fragment. The produced expression vector,termed pARSC, was transformed to E. coli DH5alpha. The positive clones were selected and proliferated. The harvested cells were treated with sonication and the supernatant was then subjected to linear sucrose density gradients centrifugation (15% to 60%) at 32000 r/min for 4 h at 4 degrees C. The virus-like particles, VLP-Cy, were collected at 35% sucrose density. The particles were examined by transmission electron microscopy and the spherical viral particles of approximately 27 nm in diameter were found. The thiolated VLP-Cy was then chemically modified with fluorescein -5'-maleimide. The covalent fluorescent labeling was confirmed by absorption analysis, SDS-PAGE and MALDI-TOF mass spectroscopy. This is the first report of preparation of RNA-containing natural fluorescent nanoparticles. The study highlight the versatility of MS2 bacteriophage capsids as building blocks for functional nanomaterials construction for a variety of application purposes.

  13. Use of Engineered Unique Cysteine Residues to Facilitate Oriented Coupling of Proteins Directly to a Gold Substrate

    NARCIS (Netherlands)

    Magis, G.J; Olsen, J.D.; Reynolds, N.P.; Leggett, G.J.; Hunter, C.N.; Aartsma, T.J.; Frese, R.N.

    2011-01-01

    A prerequisite for any "lab on a chip" device that utilizes an electrical signal from the sensor protein is the ability to attach the protein in a specific orientation onto a conducting substrate. Here, we demonstrate the covalent attachment to a gold surface of light-harvesting membrane proteins,

  14. [Residual neuromuscular blockade].

    Science.gov (United States)

    Fuchs-Buder, T; Schmartz, D

    2017-06-01

    Even small degrees of residual neuromuscular blockade, i. e. a train-of-four (TOF) ratio >0.6, may lead to clinically relevant consequences for the patient. Especially upper airway integrity and the ability to swallow may still be markedly impaired. Moreover, increasing evidence suggests that residual neuromuscular blockade may affect postoperative outcome of patients. The incidence of these small degrees of residual blockade is relatively high and may persist for more than 90 min after a single intubating dose of an intermediately acting neuromuscular blocking agent, such as rocuronium and atracurium. Both neuromuscular monitoring and pharmacological reversal are key elements for the prevention of postoperative residual blockade.

  15. TENORM: Wastewater Treatment Residuals

    Science.gov (United States)

    Water and wastes which have been discharged into municipal sewers are treated at wastewater treatment plants. These may contain trace amounts of both man-made and naturally occurring radionuclides which can accumulate in the treatment plant and residuals.

  16. Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin.

    Science.gov (United States)

    Kranawetvogl, Andreas; Küppers, Jim; Gütschow, Michael; Worek, Franz; Thiermann, Horst; Elsinghorst, Paul W; John, Harald

    2017-08-01

    Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Residuation in orthomodular lattices

    Directory of Open Access Journals (Sweden)

    Chajda Ivan

    2017-04-01

    Full Text Available We show that every idempotent weakly divisible residuated lattice satisfying the double negation law can be transformed into an orthomodular lattice. The converse holds if adjointness is replaced by conditional adjointness. Moreover, we show that every positive right residuated lattice satisfying the double negation law and two further simple identities can be converted into an orthomodular lattice. In this case, also the converse statement is true and the corresponence is nearly one-to-one.

  18. Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

    Science.gov (United States)

    Kassmann, Mario; Hansel, Alfred; Leipold, Enrico; Birkenbeil, Jan; Lu, Song-Qing; Hoshi, Toshinori; Heinemann, Stefan H.

    2010-01-01

    Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion. PMID:18369661

  19. Characterization of Hospital Residuals

    International Nuclear Information System (INIS)

    Blanco Meza, A.; Bonilla Jimenez, S.

    1997-01-01

    The main objective of this investigation is the characterization of the solid residuals. A description of the handling of the liquid and gassy waste generated in hospitals is also given, identifying the source where they originate. To achieve the proposed objective the work was divided in three stages: The first one was the planning and the coordination with each hospital center, in this way, to determine the schedule of gathering of the waste can be possible. In the second stage a fieldwork was made; it consisted in gathering the quantitative and qualitative information of the general state of the handling of residuals. In the third and last stage, the information previously obtained was organized to express the results as the production rate per day by bed, generation of solid residuals for sampled services, type of solid residuals and density of the same ones. With the obtained results, approaches are settled down to either determine design parameters for final disposition whether for incineration, trituration, sanitary filler or recycling of some materials, and storage politics of the solid residuals that allow to determine the gathering frequency. The study concludes that it is necessary to improve the conditions of the residuals handling in some aspects, to provide the cleaning personnel of the equipment for gathering disposition and of security, minimum to carry out this work efficiently, and to maintain a control of all the dangerous waste, like sharp or polluted materials. In this way, an appreciable reduction is guaranteed in the impact on the atmosphere. (Author) [es

  20. Metabolism of cysteine and cysteinesulfinate in rat kidney tubules

    International Nuclear Information System (INIS)

    De La Rosa, J.; Stipanuk, M.H.

    1986-01-01

    In studies with rat hepatocytes, hypotaurine plus taurine production accounted for less than 5% of the total amount of cysteine (CYS) catabolized, whereas more than 90% of the metabolized cysteinesulfinate (CSA) was converted to taurine plus hypotaurine. Similar studies have been carried out with kidney tubules isolated from fed rats and incubated with 2 mM [1- 14 C]CYS or 25 mM [1- 14 C]CSA at 37 0 C for up to 40 min. The production of 14 CO 2 from CSA (3.1 +/- 1.3 nmol/sup ./ min -1 /sup ./ mg dry wt -1 ) was equivalent to the accumulation of N in NH 4 + plus glutamate. Substantial oxidation of CYS was observed (16 +/- 11 nmol CO 2 x min -1 x mg dry wt -1 ), but only 12% of the expected amount of N was recovered as NH 4 + plus glutamate. Accumulation of hypotaurine plus taurine was equivalent to 20% of the observed rate of 14 CO 2 production from CSA but accounted for only 2% of the observed rate of 14 CO 2 production from CYS. Addition of unlabeled CSA to incubations with varying levels of CYS had no effect on production of 14 CO 2 . Addition of 2 mM α-ketoglutarate to the incubation mixtures resulted in an increased in 14 CO 2 production from CSA to 290% of the control level but had no effect on CYS oxidation. In agreement with the authors findings for rat hepatocytes, these data suggest that most metabolism of CYS by the rat kidney tubule occurs by a CSA-independent pathway. However, in contrast to the metabolism of CSA almost entirely to taurine in the hepatocyte, kidney tubules appeared to metabolize CSA primarily by the transamination pathway

  1. Kininogens: More than cysteine protease inhibitors and kinin precursors.

    Science.gov (United States)

    Lalmanach, Gilles; Naudin, Clément; Lecaille, Fabien; Fritz, Hans

    2010-11-01

    Two kininogens are found in mammalian sera: HK (high molecular weight kininogen) and LK (low molecular weight kininogen) with the exception of the rat which encompasses a third kininogen, T-Kininogen (TK). Kininogens are multifunctional glycosylated molecules related to cystatins (clan IH, family I25). They harbor three cystatin domains but only two of them are tight-binding inhibitors of cysteine cathepsins. HK and LK, but not TK, are precursors of potent peptide hormones, the kinins, which are released proteolytically by tissue and plasma kallikreins. Besides these classical features novel functions of kininogens have been recently discovered; they are described in the second part of this review. HKa, which corresponds to the kinin-free two-chain HK and its isolated domain D5 (kininostatin), possesses angiostatic and pro-apoptotic properties, inhibits the proliferation of endothelial cells and participates in the regulation of angiogenesis. Moreover, some HK-derived peptides display potent and broad-spectrum microbicidal properties against both Gram-positive and Gram-negative bacteria, and thus may offer a promising alternative to conventional antibiotic therapy. Of seminal interest, a kininogen-derived peptide inhibits activation of the contact phase system of coagulation and protects mice with invasive Streptococcus pyogenes infection from pulmonary lesions. On the other hand, TK is a biomarker of aging at the end of lifespan of elderly rats. However, although TK has been initially identified as an acute phase reactant, and earlier known as alpha-l-acute phase globulin, the increase of TK in liver and plasma is not known to relate to any inflammatory event during the senescence process. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  2. Oxidation of hypotaurine and cysteine sulphinic acid by peroxynitrite

    Science.gov (United States)

    2005-01-01

    Peroxynitrite mediates the oxidation of the sulphinic group of both HTAU (hypotaurine) and CSA (cysteine sulphinic acid), producing the respective sulphonates, TAU (taurine) and CA (cysteic acid). The reaction is associated with extensive oxygen uptake, suggesting that HTAU and CSA are oxidized by the one-electron transfer mechanism to sulphonyl radicals, which may initiate an oxygen-dependent radical chain reaction with the sulphonates as final products. Besides the one-electron mechanism, HTAU and CSA can be oxidized by the two-electron pathway, leading directly to sulphonate formation without oxygen consumption. The apparent second-order rate constants for the direct reaction of peroxynitrite with HTAU and CSA at pH 7.4 and 25 °C are 77.4±5 and 76.4±9 M−1·s−1 respectively. For both sulphinates, the apparent second-order rate constants increase sharply with decrease in pH, and the sigmoidal curves obtained are consistent with peroxynitrous acid as the species responsible for sulphinate oxidation. The kinetic data, together with changes in oxygen uptake, sulphinate depletion, sulphonate production, and product distribution of nitrite and nitrate, suggest that oxidation of sulphinates by peroxynitrite may take place by the two reaction pathways whose relative importance depends on reagent concentrations and pH value. In the presence of bicarbonate, the direct reaction of sulphinates with peroxynitrite is inhibited and the oxidative reaction probably involves only the radicals •NO2 and CO3•−, generated by decomposition of the peroxynitrite-CO2 adduct. PMID:15740460

  3. Preparation, Crystallization and X-ray Diffraction Analysis to 1.5 A Resolution of Rat Cysteine Dioxygenase, a Mononuclear Iron Enzyme Responsible for Cysteine Thiol Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons,C.; Hao, Q.; Stipanuk, M.

    2005-01-01

    Cysteine dioxygenase (CDO; EC 1.13.11.20) is an {approx}23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O2, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Angstroms resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Angstrom, {alpha} = {beta} = {gamma} = 90. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

  4. Oxidative stress in mammalian cells impinges on the cysteines redox state of human XRCC3 protein and on its cellular localization.

    Directory of Open Access Journals (Sweden)

    Pierre-Marie Girard

    Full Text Available In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR, a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3 in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT, which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.

  5. Structure-based mutational studies ofO-acetylserine sulfhydrylase reveal the reason for the loss of cysteine synthase complex formation inBrucella abortus.

    Science.gov (United States)

    Dharavath, Sudhaker; Raj, Isha; Gourinath, Samudrala

    2017-03-23

    Cysteine biosynthesis takes place via a two-step pathway in bacteria, fungi, plants and protozoan parasites, but not in humans, and hence, the machinery of cysteine biosynthesis is an opportune target for therapeutics. The decameric cysteine synthase complex (CSC) is formed when the C-terminal tail of serine acetyltransferase (SAT) binds in the active site of O -acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS from Brucella abortus (BaOASS) does not interact with its cognate SAT C-terminal tail. Crystal structures of native BaOASS showed that residues Gln96 and Tyr125 occupy the active-site pocket and interfere with the entry of the SAT C-terminal tail. The BaOASS (Q96A-Y125A) mutant showed relatively strong binding ( K d  = 32.4 μM) to BaSAT C-terminal peptides in comparison with native BaOASS. The mutant structure looks similar except that the active-site pocket has enough space to bind the SAT C-terminal end. Surface plasmon resonance results showed a relatively strong (7.3 μM K d ) interaction between BaSAT and the BaOASS (Q96A-Y125A), but no interaction with native BaOASS. Taken together, our observations suggest that the CSC does not form in B. abortus . © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  6. Management of NORM Residues

    International Nuclear Information System (INIS)

    2013-06-01

    The IAEA attaches great importance to the dissemination of information that can assist Member States in the development, implementation, maintenance and continuous improvement of systems, programmes and activities that support the nuclear fuel cycle and nuclear applications, and that address the legacy of past practices and accidents. However, radioactive residues are found not only in nuclear fuel cycle activities, but also in a range of other industrial activities, including: - Mining and milling of metalliferous and non-metallic ores; - Production of non-nuclear fuels, including coal, oil and gas; - Extraction and purification of water (e.g. in the generation of geothermal energy, as drinking and industrial process water; in paper and pulp manufacturing processes); - Production of industrial minerals, including phosphate, clay and building materials; - Use of radionuclides, such as thorium, for properties other than their radioactivity. Naturally occurring radioactive material (NORM) may lead to exposures at some stage of these processes and in the use or reuse of products, residues or wastes. Several IAEA publications address NORM issues with a special focus on some of the more relevant industrial operations. This publication attempts to provide guidance on managing residues arising from different NORM type industries, and on pertinent residue management strategies and technologies, to help Member States gain perspectives on the management of NORM residues

  7. Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

    Directory of Open Access Journals (Sweden)

    Morten K. Grøftehauge

    2015-07-01

    Full Text Available As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

  8. Acetaminophen-cysteine adducts during therapeutic dosing and following overdose

    Directory of Open Access Journals (Sweden)

    Judge Bryan S

    2011-03-01

    Full Text Available Abstract Background Acetaminophen-cysteine adducts (APAP-CYS are a specific biomarker of acetaminophen exposure. APAP-CYS concentrations have been described in the setting of acute overdose, and a concentration >1.1 nmol/ml has been suggested as a marker of hepatic injury from acetaminophen overdose in patients with an ALT >1000 IU/L. However, the concentrations of APAP-CYS during therapeutic dosing, in cases of acetaminophen toxicity from repeated dosing and in cases of hepatic injury from non-acetaminophen hepatotoxins have not been well characterized. The objective of this study is to describe APAP-CYS concentrations in these clinical settings as well as to further characterize the concentrations observed following acetaminophen overdose. Methods Samples were collected during three clinical trials in which subjects received 4 g/day of acetaminophen and during an observational study of acetaminophen overdose patients. Trial 1 consisted of non-drinkers who received APAP for 10 days, Trial 2 consisted of moderate drinkers dosed for 10 days and Trial 3 included subjects who chronically abuse alcohol dosed for 5 days. Patients in the observational study were categorized by type of acetaminophen exposure (single or repeated. Serum APAP-CYS was measured using high pressure liquid chromatography with electrochemical detection. Results Trial 1 included 144 samples from 24 subjects; Trial 2 included 182 samples from 91 subjects and Trial 3 included 200 samples from 40 subjects. In addition, we collected samples from 19 subjects with acute acetaminophen ingestion, 7 subjects with repeated acetaminophen exposure and 4 subjects who ingested another hepatotoxin. The mean (SD peak APAP-CYS concentrations for the Trials were: Trial 1- 0.4 (0.20 nmol/ml, Trial 2- 0.1 (0.09 nmol/ml and Trial 3- 0.3 (0.12 nmol/ml. APAP-CYS concentrations varied substantially among the patients with acetaminophen toxicity (0.10 to 27.3 nmol/ml. No subject had detectable APAP

  9. The effect of L-cysteine on the portion-selective uptake of cadmium in the renal proximal tubule

    International Nuclear Information System (INIS)

    Murakami, Masataka; Sano, Kenichi; Webb, M.

    1987-01-01

    Cadmium (Cd), co-administered with an excess of L-cysteine, accumulates rapidly in the kidneys of the rat. After subcutaneous (s.c.) injection of 3 μmol CdCl 2 /kg body wt the concentrations of Cd in the blood and kidneys increase with the dose of cysteine over the range 0.06-5.0 mmol/kg body wt. At cysteine doses of less than 1.5 mmol/kg body wt the ratio of the concentrations of Cd in the outer medulla and cortex of the kidney remains the same as that after the injection of Cd alone. This ratio, however, is more than doubled at dose levels of 5-10 mmol cysteine/kg body wt. Hepatic uptake of Cd is unaffected by doses of cysteine below 1.5 mmol/kg body wt but decreases markedly at higher doses. In animals that are dosed simultaneously with 5 mmol cysteine/kg body wt, renal uptake of 109 Cd is known to occur in the straight segments of the proximal tubules. At a dose level of less than 1.5 mmol cysteine/kg body wt the present autoradiographical studies show that 109 Cd is taken up predominantly by the proximal convoluted tubules of the kidney cortex. At the critical dose level (1.5 mmol/kg body wt), cysteine decreases the retention of Cd at the s.c. injection site, but probably has little effect on the distribution of Cd between protein and other carrier molecules in the blood. This distribution, however, is altered at higher cysteine dose levels. It is suggested that, under the latter conditions, stable Cd-cysteine complexes are formed in the blood and are filtered readily through the glomeruli. These complexes are taken up in the kidney at the sites of cysteine reabsorption which, by studies with L-[ 35 S]-cysteine, are identified as the straight segments of the proximal tubules. (orig.)

  10. Residual-stress measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ezeilo, A.N.; Webster, G.A. [Imperial College, London (United Kingdom); Webster, P.J. [Salford Univ. (United Kingdom)

    1997-04-01

    Because neutrons can penetrate distances of up to 50 mm in most engineering materials, this makes them unique for establishing residual-stress distributions non-destructively. D1A is particularly suited for through-surface measurements as it does not suffer from instrumental surface aberrations commonly found on multidetector instruments, while D20 is best for fast internal-strain scanning. Two examples for residual-stress measurements in a shot-peened material, and in a weld are presented to demonstrate the attractive features of both instruments. (author).

  11. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination.

    Science.gov (United States)

    Lepelley, Maud; Amor, Mohamed Ben; Martineau, Nelly; Cheminade, Gerald; Caillet, Victoria; McCarthy, James

    2012-03-01

    Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death

  12. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

    Directory of Open Access Journals (Sweden)

    Lepelley Maud

    2012-03-01

    Full Text Available Abstract Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP and four cysteine proteinase inhibitor (CPI gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is

  13. Mechanistic Studies of the Spore Photoproduct Lyase (SPL) via a Single Cysteine Mutation

    Science.gov (United States)

    Yang, Linlin; Lin, Gengjie; Nelson, Renae S.; Jian, Yajun; Telser, Joshua; Li, Lei

    2012-01-01

    5-thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photo-damage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5′-dA• generated by SAM cleavage reaction to abstract the H6proR atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely the cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO2− and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for the TpTSO2− formation. Should any other protein residue serve as the H donor, its presence would result in TpT as the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, due to the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton coupled electron transfer) process, contrasting to the H atom transfer mechanism in the WT SPL reaction. The C141A mutant repairs SP at a rate which is ~3-fold slower than the WT enzyme. Formation of TpTSO2− and TpT exhibit a Vmax deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2 respectively, which is smaller than the DVmax KIE of 2.8 ± 0.3 determined in the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process in the enzyme catalysis. As expected, the pre-reduced C141A mutant only supports ~ 0.4 turnover, which is in sharp contrast to the > 5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is

  14. Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Dharmendra; Subramanian, Kartik; Bisaria, Virendra S.; Sreekrishnan, T.R.; Gomes, James [Indian Inst. of Technology, Dept. of Biochemical Engineering and Biotechnology, New Delhi (India)

    2005-02-01

    The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l{sup -1} of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l{sup -1} h{sup -1}) every 2 h to the production medium increased the production of methionine to 3.39 g l{sup -1}. A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers. (Author)

  15. Observation of Cysteine Thiolate and -S...H-O Intermolecular Hydrogen Bond

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Hin-koon; Lau, Kai Chung; Wang, Xue B.; Wang, Lai S.

    2006-11-23

    The cysteine anion was produced in the gas phase by electrospray ionization and investigated by photoelectron spectroscopy at low-temperature (70K). The cysteine anion was found to exhibit the thiolate form [-SCH2CH(NH2)CO2H], rather than the expected carboxylate form [HSCH2CH(NH2)CO2 -]. This observation was confirmed by two control experiments i.e. methyl cysteine [CH3SCH2CH(NH2)CO2-] and cysteine methyl ester [-SCH2CH(NH2)CO2CH3]. The electron binding energy of [-CH2CH(NH2)CO2H] was measured to be about 0.7 eV blue-shifted relative to [-SCH2CH(NH2)CO2CH3] due to the formation of an intramolecular –S-…HO2C– hydrogen bond in the cysteine hiolate. Theoretical calculations at the CCSD(T)/6-311++G(2df,p) and B3LYP/6-311++G(2df,p) levels were carried out to estimate the strength of this intramolecular –S-…HO2C– hydrogen bond. Combining experimental measurements and theoretical calculations yielded an estimated value of 16.4 ± 2.0 kcal/mol for the –S-…HO2C– intramolecular hydrogen bond strength.

  16. Cysteines 208 and 241 in Ero1α are required for maximal catalytic turnover

    Directory of Open Access Journals (Sweden)

    Thomas Ramming

    2016-04-01

    Full Text Available Endoplasmic reticulum (ER oxidoreductin 1α (Ero1α is a disulfide producer in the ER of mammalian cells. Besides four catalytic cysteines (Cys94, Cys99, Cys394, Cys397, Ero1α harbors four regulatory cysteines (Cys104, Cys131, Cys208, Cys241. These cysteines mediate the formation of inhibitory intramolecular disulfide bonds, which adapt the activation state of the enzyme to the redox environment in the ER through feedback signaling. Accordingly, disulfide production by Ero1α is accelerated by reducing conditions, which minimize the formation of inhibitory disulfides, or by mutations of regulatory cysteines. Here we report that reductive stimulation enhances Ero1α activity more potently than the mutation of cysteines. Specifically, mutation of Cys208/Cys241 does not mechanistically mimic reductive stimulation, as it lowers the turnover rate of Ero1α in presence of a reducing agent. The Cys208/Cys241 pair therefore fulfills a function during catalysis that reaches beyond negative regulation. In agreement, we identify a reciprocal crosstalk between the stabilities of the Cys208–Cys241 disulfide and the inhibitory disulfide bonds involving Cys104 and Cys131, which also controls the recruitment of the H2O2 scavenger GPx8 to Ero1α. Two possible mechanisms by which thiol–disulfide exchange at the Cys208/Cys241 pair stimulates the catalytic turnover under reducing conditions are discussed.

  17. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

    Science.gov (United States)

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir

    2015-02-01

    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions. Copyright © 2014 Elsevier GmbH. All rights reserved.

  18. Electronic structures of the L-cysteine film on dental alloys

    Energy Technology Data Exchange (ETDEWEB)

    Ogawa, K., E-mail: e7141@cc.saga-u.ac.jp [Synchrotron Light Application Center, Saga University, Saga 840-8502 (Japan); Tsujibayashi, T. [Department of Physics, Osaka Dental University, Osaka 573-1121 (Japan); Takahashi, K.; Azuma, J. [Synchrotron Light Application Center, Saga University, Saga 840-8502 (Japan); Kakimoto, K. [Department of Geriatric Dentistry, Osaka Dental University, Osaka 573-1121 (Japan); Kamada, M. [Synchrotron Light Application Center, Saga University, Saga 840-8502 (Japan)

    2011-04-15

    Research highlights: {yields} The electronic structures of dental alloys and L-cysteine film were studied by PES. {yields} The density of states in the dental alloy originates from Au and Cu as constituents. {yields} The Cu-3d states contribute dominantly to the occupied states near the Fermi level. {yields} The electronic structure of L-cysteine thin film is different from the thick film. {yields} The bonding between Cu-3d and S-3sp states are formed at the interface. - Abstract: Metal-organic interfaces have been attracting continuous attention in many fields including basic biosciences. The surface of dental alloys could be one of such interfaces since they are used in a circumstance full of organic compounds such as proteins and bacteria. In this work, electronic structures of Au-dominant dental alloys, which have Ag and Cu besides Au, and those of L-cysteine on the dental alloys have been studied by photoelectron spectroscopy with synchrotron radiation. It was found that the density of states in the dental alloy originate from gold and copper as constituents, and the Cu-3d states contribute dominantly to the occupied states near the Fermi level. It was also found that the electronic structure of the L-cysteine thin film on the dental alloy is different from that of the L-cysteine thick film. The result indicates the formation of the orbital bonding between Cu-3d and S-3sp states in the thin film on the dental alloy.

  19. Cysteine proteases: Modes of activation and future prospects as pharmacological targets

    Directory of Open Access Journals (Sweden)

    Sonia eVerma

    2016-04-01

    Full Text Available Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria and parasite to the higher organisms (mammals. Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases and metallo-proteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a pro-domain (regulatory and a mature domain (catalytic. The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.

  20. Experimental and theoretical investigation on corrosion inhibition of AA5052 aluminium alloy by L-cysteine in alkaline solution

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Dapeng; Gao, Lixin [School of Environmental and Chemical Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Zhang, Daquan, E-mail: zhangdaquan@shiep.edu.cn [School of Environmental and Chemical Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Yang, Dong [School of Environmental and Chemical Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Wang, Hongxia; Lin, Tong [Institute for Frontier Materials, Deakin University, Geelong, VIC 3216 (Australia)

    2016-02-01

    The corrosion inhibition of L-cysteine on AA5052 aluminium alloy in 4 mol/L NaOH solution was investigated by hydrogen gas evolution experiment, polarisation curve, galvanostatic discharge, electrochemical impedance spectroscopy measurements and quantum chemical calculations. The adsorption of L-cysteine on aluminium alloy surface obeyed the amended Langmuir's adsorption isotherm. The polarisation curves indicated that L-cysteine acted as a cathodic inhibitor to inhibit cathodic reaction. The inhibition mechanism was dominated by the geometric covering effect. The galvanostatic discharge shows that the additives restrain the hydrogen evolution and increase the anodic utilization rate. Quantum chemical calculations indicated that L-cysteine molecules mainly interacted with on the carboxyl groups on the aluminium alloy surface. A strong hybridization occurred between the s-orbital and p-orbital of reactive sites in the L-cysteine molecule and the sp-orbital of Aluminium. - Highlights: • L-cysteine was used as corrosion inhibitor for Al alloy in alkaline solution. • Adsorption of L-cysteine on Al alloy surface obeyed the amended Langmuir's isotherm. • L-cysteine molecules interacted with the carboxyl groups on the Al alloy surface. • A strong orbital hybridization occurred between the reactive sites in L-cysteine and Al.

  1. Spectroscopic investigations on the effect of N-Acetyl-L-cysteine-Capped CdTe Quantum Dots on catalase

    Science.gov (United States)

    Sun, Haoyu; Yang, Bingjun; Cui, Erqian; Liu, Rutao

    2014-11-01

    Quantum dots (QDs) are recognized as some of the most promising semiconductor nanocrystals in biomedical applications. However, the potential toxicity of QDs has aroused wide public concern. Catalase (CAT) is a common enzyme in animal and plant tissues. For the potential application of QDs in vivo, it is important to investigate the interaction of QDs with CAT. In this work, the effect of N-Acetyl-L-cysteine-Capped CdTe Quantum Dots with fluorescence emission peak at 612 nm (QDs-612) on CAT was investigated by fluorescence, synchronous fluorescence, fluorescence lifetime, ultraviolet-visible (UV-vis) absorption and circular dichroism (CD) techniques. Binding of QDs-612 to CAT caused static quenching of the fluorescence, the change of the secondary structure of CAT and the alteration of the microenvironment of tryptophan residues. The association constants K were determined to be K288K = 7.98 × 105 L mol-1 and K298K = 7.21 × 105 L mol-1. The interaction between QDs-612 and CAT was spontaneous with 1:1 stoichiometry approximately. The CAT activity was also inhibited for the bound QDs-612. This work provides direct evidence about enzyme toxicity of QDs-612 to CAT in vitro and establishes a new strategy to investigate the interaction between enzyme and QDs at a molecular level, which is helpful for clarifying the bioactivities of QDs in vivo.

  2. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    Energy Technology Data Exchange (ETDEWEB)

    Stiegler, A.; Burden, S; Hubbard, S

    2009-01-01

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

  3. Cy-preds: An algorithm and a web service for the analysis and prediction of cysteine reactivity.

    Science.gov (United States)

    Soylu, İnanç; Marino, Stefano M

    2016-02-01

    Cysteine (Cys) is a critically important amino acid, serving a variety of functions within proteins including structural roles, catalysis, and regulation of function through post-translational modifications. Predicting which Cys residues are likely to be reactive is a very sought after feature. Few methods are currently available for the task, either based on evaluation of physicochemical features (e.g., pKa and exposure) or based on similarity with known instances. In this study, we developed an algorithm (named HAL-Cy) which blends previous work with novel implementations to identify reactive Cys from nonreactive. HAL-Cy present two major components: (i) an energy based part, rooted on the evaluation of H-bond network contributions and (ii) a knowledge based part, composed of different profiling approaches (including a newly developed weighting matrix for sequence profiling). In our evaluations, HAL-Cy provided significantly improved performances, as tested in comparisons with existing approaches. We implemented our algorithm in a web service (Cy-preds), the ultimate product of our work; we provided it with a variety of additional features, tools, and options: Cy-preds is capable of performing fully automated calculations for a thorough analysis of Cys reactivity in proteins, ranging from reactivity predictions (e.g., with HAL-Cy) to functional characterization. We believe it represents an original, effective, and very useful addition to the current array of tools available to scientists involved in redox biology, Cys biochemistry, and structural bioinformatics. © 2015 Wiley Periodicals, Inc.

  4. A free cysteine prolongs the half-life of a homing peptide and improves its tumor-penetrating activity

    Science.gov (United States)

    Pang, Hong-Bo; Braun, Gary B.; She, Zhi-Gang; Kotamraju, Venkata R.; Sugahara, Kazuki N.; Teesalu, Tambet; Ruoslahti, Erkki

    2014-01-01

    The accessibility of extravascular tumor tissue to drugs is critical for therapeutic efficacy. We previously described a tumor-targeting peptide (iRGD) that elicits active transport of drugs and macromolecules (covalently coupled or co-administered) across the vascular wall into tumor tissue. Short peptides (iRGD is a 9-amino acid cyclic peptide) generally have a plasma half-life measured in minutes. Since short half-life limits the window of activity obtained with a bolus injection of iRGD, we explored to extend the half-life of the peptide. We show here that addition of a cysteine residue prolongs the plasma half-life of iRGD and increases the accumulation of the peptide in tumors. This modification prolongs the activity of iRGD in inducing macromolecular extravasation and leads to greater drug accumulation in tumors than is obtained with the unmodified peptide. This effect is mediated by covalent binding of iRGD to plasma albumin through a disulfide bond. Our study provides a simple strategy to improve peptide pharmacokinetics and activity. Applied to RGD, it provides a means to increase the entry of therapeutic agents into tumors. PMID:24345789

  5. Cysteine Specific Targeting of the Functionally Distinct Peroxiredoxin and Glutaredoxin Proteins by the Investigational Disulfide BNP7787

    Directory of Open Access Journals (Sweden)

    Aulma R. Parker

    2015-03-01

    Full Text Available Glutaredoxin (Grx, peroxiredoxin (Prx, and thioredoxin (Trx are redoxin family proteins that catalyze different types of chemical reactions that impact cell growth and survival through functionally distinct intracellular pathways. Much research is focused on understanding the roles of these redoxin proteins in the development and/or progression of human diseases. Grx and Prx are overexpressed in human cancers, including human lung cancers. BNP7787 is a novel investigational agent that has been evaluated in previous clinical studies, including non-small cell lung cancer (NSCLC studies. Herein, data from activity assays, mass spectrometry analyses, and X-ray crystallographic studies indicate that BNP7787 forms mixed disulfides with select cysteine residues on Grx and Prx and modulates their function. Studies of interactions between BNP7787 and Trx have been conducted and reported separately. Despite the fact that Trx, Grx, and Prx are functionally distinct proteins that impact oxidative stress, cell proliferation and disease processes through different intracellular pathways, BNP7787 can modify each protein and appears to modulate function through mechanisms that are unique to each target protein. Tumor cells are often genomically heterogeneous containing subpopulations of cancer cells that often express different tumor-promoting proteins or that have multiple dysregulated signaling pathways modulating cell proliferation and drug resistance. A multi-targeted agent that simultaneously modulates activity of proteins important in mediating cell proliferation by functionally distinct intracellular pathways could have many potentially useful therapeutic applications.

  6. TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2.

    Science.gov (United States)

    Findling, Simone; Stotz, Henrik U; Zoeller, Maria; Krischke, Markus; Zander, Mark; Gatz, Christiane; Berger, Susanne; Mueller, Martin J

    2018-01-01

    Reactive electrophile species (RES), including prostaglandins, phytoprostanes and 12-oxo phytodienoic acid (OPDA), activate detoxification responses in plants and animals. However, the pathways leading to the activation of defense reactions related to abiotic or biotic stress as a function of RES formation, accumulation or treatment are poorly understood in plants. Here, the thiol-modification of proteins, including the RES-activated basic region/leucine zipper transcription factor TGA2, was studied. TGA2 contains a single cysteine residue (Cys186) that was covalently modified by reactive cyclopentenones but not required for induction of detoxification genes in response to OPDA or prostaglandin A1. Activation of the glutathione-S-transferase 6 (GST6) promoter was responsive to cyclopentenones but not to unreactive cyclopentanones, including jasmonic acid suggesting that thiol reactivity of RES is important to activate the TGA2-dependent signaling pathway resulting in GST6 activation We show that RES modify thiols in numerous proteins in vivo, however, thiol reactivity alone appears not to be sufficient for biological activity as demonstrated by the failure of several membrane permeable thiol reactive reagents to activate the GST6 promoter.

  7. Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

    Directory of Open Access Journals (Sweden)

    Lee Dae-Hee

    2009-03-01

    Full Text Available Abstract Background Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A and cyclohexanone monooxygenase (CHMO. Results We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively. Conclusion The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.

  8. Composition of carbonization residues

    Energy Technology Data Exchange (ETDEWEB)

    Hupfer; Leonhardt

    1943-11-27

    This report compared the composition of samples from Wesseling and Leuna. In each case the sample was a residue from carbonization of the residues from hydrogenation of the brown coal processed at the plant. The composition was given in terms of volatile components, fixed carbon, ash, water, carbon, hydrogen, oxygen, nitrogen, volatile sulfur, and total sulfur. The result of carbonization was given in terms of (ash and) coke, tar, water, gas and losses, and bitumen. The composition of the ash was given in terms of silicon dioxide, ferric oxide, aluminum oxide, calcium oxide, magnesium oxide, potassium and sodium oxides, sulfur trioxide, phosphorus pentoxide, chlorine, and titanium oxide. The most important difference between the properties of the two samples was that the residue from Wesseling only contained 4% oil, whereas that from Leuna had about 26% oil. Taking into account the total amount of residue processed yearly, the report noted that better carbonization at Leuna could save 20,000 metric tons/year of oil. Some other comparisons of data included about 33% volatiles at Leuna vs. about 22% at Wesseling, about 5 1/2% sulfur at Leuna vs. about 6 1/2% at Leuna, but about 57% ash for both. Composition of the ash differed quite a bit between the two. 1 table.

  9. Designing with residual materials

    NARCIS (Netherlands)

    Walhout, W.; Wever, R.; Blom, E.; Addink-Dölle, L.; Tempelman, E.

    2013-01-01

    Many entrepreneurial businesses have attempted to create value based on the residual material streams of third parties. Based on ‘waste’ materials they designed products, around which they built their company. Such activities have the potential to yield sustainable products. Many of such companies

  10. Thermo-Kinetic Investigation of Comparative Ligand Effect on Cysteine Iron Redox Reaction

    Directory of Open Access Journals (Sweden)

    Masood Ahmad Rizvi

    2015-03-01

    Full Text Available Transition metal ions in their free state bring unwanted biological oxidations generating oxidative stress. The ligand modulated redox potential can be indispensable in prevention of such oxidative stress by blocking the redundant bio-redox reactions. In this study we investigated the comparative ligand effect on the thermo-kinetic aspects of biologically important cysteine iron (III redox reaction using spectrophotometric and potentiometric methods. The results were corroborated with the complexation effect on redox potential of iron(III-iron(II redox couple. The selected ligands were found to increase the rate of cysteine iron (III redox reaction in proportion to their stability of iron (II complex (EDTA < terpy < bipy < phen. A kinetic profile and the catalytic role of copper (II ions by means of redox shuttle mechanism for the cysteine iron (III redox reaction in presence of 1,10-phenanthroline (phen ligand is also reported.

  11. S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II.

    Science.gov (United States)

    Kubec, Roman; Kim, Seokwon; Musah, Rabi A

    2002-11-01

    Three cysteine derivatives, (R)-S-(2-hydroxyethyl)cysteine, together with (R(S)R(C))- and (S(S)R(C))-S-(2-hydroxyethyl)cysteine sulfoxides, have been isolated from the roots of Petiveria alliacea. Furthermore, three additional amino acids, S-methyl-, S-ethyl-, and S-propylcysteine derivatives, were detected. They were present only in trace amounts (<3 microg g(-1) fr. wt), precluding determination of their absolute configurations and oxidation states. In addition, four thiosulfinates, S-(2-hydroxyethyl) (2-hydroxyethane)-, S-(2-hydroxyethyl) phenylmethane-, S-benzyl (2-hydroxyethane)- and S-benzyl phenylmethanethiosulfinates, have been found in a homogenate of the roots. The formation pathways of various benzyl/phenyl-containing compounds previously found in the plant were also discussed.

  12. A Critical Role for Cysteine 57 in the Biological Functions of Selenium Binding Protein-1.

    Science.gov (United States)

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M; Yang, Wancai

    2015-11-18

    The concentration of selenium-binding protein1 (SBP1) is often lower in tumors than in the corresponding tissue and lower levels have been associated with poor clinical outcomes. SBP1 binds tightly selenium although what role selenium plays in its biological functions remains unknown. Previous studies indicated that cysteine 57 is the most likely candidate amino acid for selenium binding. In order to investigate the role of cysteine 57 in SBP1, this amino acid was altered to a glycine and the mutated protein was expressed in human cancer cells. The SBP1 half-life, as well as the cellular response to selenite cytotoxicity, was altered by this change. The ectopic expression of SBP1(GLY) also caused mitochondrial damage in HCT116 cells. Taken together, these results indicated that cysteine 57 is a critical determinant of SBP1 function and may play a significant role in mitochondrial function.

  13. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

    Science.gov (United States)

    Bernardim, Barbara; Cal, Pedro M. S. D.; Matos, Maria J.; Oliveira, Bruno L.; Martínez-Sáez, Nuria; Albuquerque, Inês S.; Perkins, Elizabeth; Corzana, Francisco; Burtoloso, Antonio C. B.; Jiménez-Osés, Gonzalo; Bernardes, Gonçalo J. L.

    2016-10-01

    Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications.

  14. L-Cysteine Capped CdSe Quantum Dots Synthesized by Photochemical Route.

    Science.gov (United States)

    Singh, Avinash; Kunwar, Amit; Rath, M C

    2018-05-01

    L-cysteine capped CdSe quantum dots were synthesized via photochemical route in aqueous solution under UV photo-irradiation. The as grown CdSe quantum dots exhibit broad fluorescence at room temperature. The CdSe quantum dots were found to be formed only through the reactions of the precursors, i.e., Cd(NH3)2+4 and SeSO2-3 with the photochemically generated 1-hydroxy-2-propyl radicals, (CH3)2COH radicals, which are formed through the process of H atom abstraction by the photoexcited acetone from 2-propanol. L-Cysteine was found to act as a suitable capping agent for the CdSe quantum dots and increases their biocompatability. Cytotoxicty effects of these quantum dots were evaluated in Chinese Hamster Ovary (CHO) epithelial cells, indicated a significant lower level for the L-cysteine capped CdSe quantum dots as compare to the bare ones.

  15. Hieronymain I, a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae).

    Science.gov (United States)

    Bruno, Mariela A; Pardo, Marcelo F; Caffini, Néstor O; López, Laura M I

    2003-02-01

    A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5-9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.

  16. Single cysteine substitution in Bacillus thuringiensis Cry7Ba1 improves the crystal solubility and produces toxicity to Plutella xylostella larvae.

    Science.gov (United States)

    Peng, Donghai; Wang, Fenshan; Li, Nisha; Zhang, Zhenyu; Song, Rong; Zhu, Zimin; Ruan, Lifang; Sun, Ming

    2011-10-01

    Many Bacillus thuringiensis isolates have no demonstrated toxicity against insects. In this study, a novel holotype crystal protein gene cry7Ba1 was isolated from a 'non-insecticidal'B. thuringiensis strain YBT-978. The Cry7Ba1 protein showed high toxicity against Plutella xylostella larvae after the crystals were dissolved at pH 12.5, suggesting that the 'non-insecticidal' properties of this protein were due to insolubility in the normal insect midgut pH environment. After the C-terminal half of Cry7Ba1 was replaced by that of Cry1Ac or Cry1C proteins, the recombinant protein inclusions could be dissolved at pH 9.5, and exhibited high toxicity against P. xylostella larvae. This result proved the insolubility of Cry7Ba1 crystal was determined by the structure of its C-terminal half. Further, six mutations were constructed by substituting cysteine residues with serine. Solubility studies showed that the crystals from mutants C697S, C834S and C854S could be dissolved at lower pH (10.5, 9.5 and 11.5 respectively). Bioassays showed that crystals from mutant C834S were toxic to P. xylostella larvae. Our discoveries suggest that a single cysteine residue located in the C-terminal half of the protein determines the solubility and toxicity of some nontoxic crystal proteins. This study provides a strategy to isolate novel insecticidal crystal protein genes from 'non-insecticidal'B. thuringiensis strains. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  17. YbdK is a carboxylate-amine ligase with a gamma-glutamyl:Cysteine ligase activity: crystal structure and enzymatic assays.

    Science.gov (United States)

    Lehmann, Christopher; Doseeva, Victoria; Pullalarevu, Sadhana; Krajewski, Wojciech; Howard, Andrew; Herzberg, Osnat

    2004-08-01

    The Escherichia coli open reading frame YbdK encodes a member of a large bacterial protein family of unknown biological function. The sequences within this family are remotely related to the sequence of gamma-glutamate-cysteine ligase (gamma-GCS), an enzyme in the glutathione biosynthetic pathway. A gene encoding gamma-GCS in E. coli is already known. The 2.15 A resolution crystal structure of YbdK reveals an overall fold similar to that of glutamine synthetase (GS), a nitrogen metabolism enzyme that ligates glutamate and ammonia to yield glutamine. GS and gamma-GCS perform related chemical reactions and require ATP and Mg2+ for their activity. The Mg2+-dependent binding of ATP to YbdK was confirmed by fluorescence spectroscopy employing 2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate, and yielding a dissociation constant of 3 +/- 0.5 microM. The structure of YbdK contains a crevice that corresponds to the binding sites of ATP, Mg2+ and glutamate in GS. Many of the GS residues that coordinate the metal ions and interact with glutamic acid and the phosphoryl and ribosyl groups of ATP are also present in YbdK. GS amino acids that have been associated with ammonia binding have no obvious counterparts in YbdK, consistent with a substrate specificity that is different from that of GS. Ligase activity between glutamic acid and each of the twenty amino acid residues was tested on high performance liquid chromatography (HPLC) by following the hydrolysis of ATP to ADP. Catalysis was observed only with cysteine. A pyruvate kinase/lactic acid dehydrogenase coupled assay was used to rule out GS activity and to determine that YbdK exhibits gamma-GCS activity. The catalytic rate was found to be approximately 500-fold slower than that reported for authentic gamma-GCS. Copyright 2004 Wiley-Liss, Inc.

  18. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

    Science.gov (United States)

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

    2016-09-01

    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functio