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Sample records for additional cysteine residues

  1. TRPA1 is activated by direct addition of cysteine residues to the N-hydroxysuccinyl esters of acrylic and cinnamic acids.

    PubMed

    Sadofsky, Laura R; Boa, Andrew N; Maher, Sarah A; Birrell, Mark A; Belvisi, Maria G; Morice, Alyn H

    2011-01-01

    The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.

  2. The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

    PubMed

    Husain, S S; Lowe, G

    1970-04-01

    Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.

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

    EPA Science Inventory

    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 ...

  4. Conservation of cysteine residues in fungal histidine acid phytases.

    PubMed

    Mullaney, Edward J; Ullah, Abul H J

    2005-03-11

    Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

  5. Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid peroxides.

    PubMed

    Dremina, Elena S; Sharov, Victor S; Davies, Michael J; Schöneich, Christian

    2007-10-01

    The oxidative modification of proteins plays an important role in a wide range of pathological processes and aging. Proteins are modified by numerous biologic oxidants including hydrogen peroxide, peroxynitrite, singlet oxygen, and oxygen- and nitrogen-centered radicals. More recently, an additional class of physiologically important oxidants has been identified, peptide and protein peroxides. The latter react quite rapidly and selectively with protein cysteine residues. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is reversibly regulated through NO-dependent S-glutathiolation of specific cysteine residues. The irreversible oxidation of these cysteine residues could, therefore, impair NO-dependent muscle relaxation. Here, we show that specific protein-derived (amino acid) peroxides react selectively with a subset of the 22 reduced cysteine residues of SERCA1, including a peptide-containing Cys674 and Cys675, where Cys674 (in SERCA2) represents one of the targets for NO-dependent S-glutathiolation. Out of 11 tested amino acid, peptide, and protein peroxides, those derived from free tryptophan and free tyrosine showed the highest reactivity towards SERCA, while no oxidation under similar experimental conditions was detected through hydrogen peroxide. Among the peroxides from tryptophan, those of free tryptophan showed a significantly higher reactivity as compared to those from N- and C-terminally blocked tryptophan. Quantitative HPLC-MS/MS analysis demonstrated that the highest reactivity of the tryptophan-derived peroxides was observed for Cys774 and Cys938, cysteine residues, which are embedded within the transmembrane domains of SERCA1. This unusual reactivity of transmembrane domains cannot be solely rationalized by the hydrophobicity of the oxidant, as the peroxide from dl-tryptophan shows considerable higher reactivity as compared to the one derived from N-acetyl-tryptophan methyl ester. Our data demonstrate a potential role of peptide- and protein

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

    PubMed

    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

    2015-06-30

    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.

  7. Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity.

    PubMed

    Müller, Sandra; Schöttler, Manuela; Schön, Sylvia; Prante, Christian; Brinkmann, Thomas; Kuhn, Joachim; Götting, Christian; Kleesiek, Knut

    2005-03-01

    XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

  8. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery

    PubMed Central

    Hallenbeck, Kenneth K.; Turner, David M.; Renslo, Adam R.; Arkin, Michelle R.

    2017-01-01

    The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an ‘allele specific’ manner. For the medicinal chemist, the structure-guided design of cysteine-reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteine-modifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues. PMID:27449257

  9. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery.

    PubMed

    Hallenbeck, Kenneth K; Turner, David M; Renslo, Adam R; Arkin, Michelle R

    2017-01-01

    The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an 'allele specific' manner. For the medicinal chemist, the structure-guided design of cysteine- reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteinemodifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues.

  10. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry

    PubMed Central

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A.; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization - tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT. PMID:23880742

  11. Identification of an active site cysteine residue in Escherichia coli pyruvate oxidase.

    PubMed

    Koland, J G; Gennis, R B

    1982-06-10

    The cysteine-directed reagent N-ethylmaleimide rapidly and completely inactivates pyruvate oxidase. This inactivation is correlated with the reaction of one cysteine residue per enzyme monomer. In the presence of the cofactor, thiamin pyrophosphate, the enzyme is not inhibited by N-ethylmaleimide. Furthermore, the N-ethylmaleimide-inactivated enzyme exhibits a very low affinity for the cofactor as determined by a fluorescence quenching technique. The presence of a reactive cysteine residue at the thiamin pyrophosphate binding site is therefore indicated. Although N-ethylmaleimide completely inactivates the enzyme, a second sulfhydryl reagent methylmethanethiosulfonate is only partially inhibitory. It is shown that methylmethanethiosulfonate and N-ethylmaleimide react with the same cysteine residue. Thus, the N-ethylmaleimide-sensitive residue is probably not directly involved in catalysis.

  12. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    PubMed

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  13. Selective Loss of Cysteine Residues and Disulphide Bonds in a Potato Proteinase Inhibitor II Family

    PubMed Central

    Li, Xiu-Qing; Zhang, Tieling; Donnelly, Danielle

    2011-01-01

    Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution. PMID:21494600

  14. Characterization of a novel y-type HMW-GS with eight cysteine residues from Triticum monococcum ssp. monococcum.

    PubMed

    Li, Zenglin; Li, Hongyu; Chen, Gang; Kou, Chunlan; Ning, Shunzong; Yuan, Zhongwei; Jiang, Qi; Zheng, Youliang; Liu, Dengcai; Zhang, Lianquan

    2015-11-15

    The composition and number of high-molecular-weight glutenin subunits (HMW-GSs) play important roles in determining the grain-processing quality of common wheat. The Glu-1Ay allele is silent in common wheat. In this study, an active y-type HMW-GS allele termed 1Ay8.2 (GenBank No. KP137569) was identified from Triticum monococcum L. ssp. monococcum (AmAm, 2n=2x=14), a species with a genome related to the A-genome of common wheat. Compared with previously reported active 1Ay subunits, this novel subunit contained an extra cysteine residue at position 103 of the amino acid sequence in the N-terminal region, in addition to the six cysteines in the N- and C-terminal regions found in most active 1Ay subunits and the one in the repetitive region that appears in only a few 1Ay alleles. This subunit was expressed in an amphiploid (AAAmAmBB, 2n=6x=42) between Triticum turgidum L. ssp. dicoccon and T. monococcum ssp. monococcum. This amphiploid could be used as a bridge to transfer 1Ay8.2 into common wheat cultivars. Replacing the silenced 1Ay in common wheat with the active 1Ay8.2 allele harboring an extra cysteine residue is expected to improve the quality by increasing the number of HMW-GSs and promoting the formation of covalent interactions through disulfide bonds with the extra cysteine residue.

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

    PubMed

    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.

  16. Do cysteine residues regulate transient receptor potential canonical type 6 channel protein expression?

    PubMed

    Thilo, Florian; Liu, Ying; Krueger, Katharina; Förste, Nora; Wittstock, Antje; Scholze, Alexandra; Tepel, Martin

    2012-03-01

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

  17. Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue.

    PubMed

    Belenghi, Beatrice; Romero-Puertas, Maria C; Vercammen, Dominique; Brackenier, Anouk; Inzé, Dirk; Delledonne, Massimo; Van Breusegem, Frank

    2007-01-12

    Nitric oxide (NO) regulates a number of signaling functions in both animals and plants under several physiological and pathophysiological conditions. S-Nitrosylation linking a nitrosothiol on cysteine residues mediates NO signaling functions of a broad spectrum of mammalian proteins, including caspases, the main effectors of apoptosis. Metacaspases are suggested to be the ancestors of metazoan caspases, and plant metacaspases have previously been shown to be genuine cysteine proteases that autoprocess in a manner similar to that of caspases. We show that S-nitrosylation plays a central role in the regulation of the proteolytic activity of Arabidopsis thaliana metacaspase 9 (AtMC9) and hypothesize that this S-nitrosylation affects the cellular processes in which metacaspases are involved. We found that AtMC9 zymogens are S-nitrosylated at their active site cysteines in vivo and that this posttranslational modification suppresses both AtMC9 autoprocessing and proteolytic activity. However, the mature processed form is not prone to NO inhibition due to the presence of a second S-nitrosylation-insensitive cysteine that can replace the S-nitrosylated cysteine residue within the catalytic center of the processed AtMC9. This cysteine is absent in caspases and paracaspases but is conserved in all reported metacaspases.

  18. The identification of free cysteine residues within antibodies and a potential role for free cysteine residues in covalent aggregation because of agitation stress.

    PubMed

    Huh, Joon H; White, April J; Brych, Stephen R; Franey, Heather; Matsumura, Masazumi

    2013-06-01

    Human immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) antibodies contain multiple disulfide bonds, which are an integral part of the structure and stability of the protein. Open disulfide bonds have been detected in a number of therapeutic and serum derived antibodies. This report details a method that fluorescently labels free cysteine residues, quantifies, and identifies the proteolytic fragments by liquid chromatography coupled to online mass spectrometry. The majority of the open disulfide bonds in recombinant and serum derived IgG1 and IgG2 antibodies were in the constant domains. This method was applied to the identification of cysteines in an IgG2 antibody that are involved in the formation of covalent intermolecular bonds because of the application of a severe agitation stress. The free cysteine in the CH 1 domain of the IgG2 decreased upon application of the stress and implicates open disulfide bonds in this domain as the likely source of free cysteines involved in the formation of intermolecular disulfide bonds. The presence of comparable levels of open disulfide bonds in recombinant and endogenous antibodies suggests that open disulfide bonds are an inherent feature of antibodies and that the susceptibility of intermolecular disulfide bond formation is similar for recombinant and serum-derived IgG antibodies.

  19. HIGH-THROUGHPUT IDENTIFICATION OF CATALYTIC REDOX-ACTIVE CYSTEINE RESIDUES

    EPA Science Inventory

    Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by se...

  20. Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.

    PubMed

    Fatehi, Mohammad; Linsdell, Paul

    2009-04-01

    Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.

  1. Conformational preferences and pKa value of cysteine residue.

    PubMed

    Lee, Joo Yun; Byun, Byung Jin; Kang, Young Kee

    2008-09-11

    The conformational preferences of the Cys dipeptides with thiol and thiolate groups (Ac-Cys-NHMe and Ac-Cys (-)-NHMe, respectively) and the apparent (i.e., macroscopic) p K a value of the Cys dipeptide have been studied at the hybrid density functional B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level with the conductor-like polarizable continuum model in the gas phase and in water. The hydrogen bonds and/or favorable interactions between the backbone and the thiol group of the side chain resulted in the different conformational preferences of the Cys and Cys (-) dipeptides from those of the Ala dipeptide in the gas phase and in water, although the preferred conformations of the Cys dipeptide are in part similar to those of the Ala dipeptide. In particular, the interactions between the thiolate group and the backbone amide groups appear to play a role in stabilizing the alpha- or 3 10-helical conformations for the Cys (-) dipeptide in the gas phase and in water. The p K a value of the Cys residue is estimated to be 8.58 at 25 degrees C using the statistically weighted free energies of all feasible conformations for the Cys and Cys (-) dipeptides in the gas phase and solvation free energies, which is consistent with the observed values of 8.3 and 8.22 +/- 0.16.

  2. Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

    SciTech Connect

    Elliott, J.I.; Ljungdahl, L.G.

    1982-04-01

    The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4'-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K/sub m/ values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4'-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20 to 30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20 to 30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.

  3. Comprehensive cysteine-scanning mutagenesis reveals Claudin-2 pore-lining residues with different intrapore locations.

    PubMed

    Li, Jiahua; Zhuo, Min; Pei, Lei; Rajagopal, Madhumitha; Yu, Alan S L

    2014-03-07

    The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. We aimed to map the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutations within the ECL1 by expression in polyclonal Madin-Darby canine kidney II Tet-Off cells and found nine mutants that displayed a significant decrease of conductance after treatment with the thiol-reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate, indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal Madin-Darby canine kidney I Tet-Off cells and exposed them to thiol-reactive reagents. The maximum degree of inhibition of conductance, size selectivity of degree of inhibition, and size dependence of the kinetics of reaction were used to deduce the location of residues within the pore. Our data support the following sequence of pore-lining residues located from the narrowest to the widest part of the pore: Ser(68), Ser(47), Thr(62)/Ile(66), Thr(56), Thr(32)/Gly(45), and Met(52). The paracellular pore appears to primarily be lined by polar side chains, as expected for a predominantly aqueous environment. Furthermore, our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp(65)-Ser(68) that form a major part of the lining of the pore.

  4. Solution oxygen-17 NMR application for observing a peroxidized cysteine residue in oxidized human SOD1

    NASA Astrophysics Data System (ADS)

    Fujiwara, Noriko; Yoshihara, Daisaku; Sakiyama, Haruhiko; Eguchi, Hironobu; Suzuki, Keiichiro

    2016-12-01

    NMR active nuclei, 1H, 13C and 15N, are usually used for determination of protein structure. However, solution 17O-NMR application to proteins is extremely limited although oxygen is an essential element in biomolecules. Proteins are oxidized through cysteine residues by two types of oxidation. One is reversible oxidation such as disulphide bonding (Cys-S-S-Cys) and the other is irreversible oxidation to cysteine sulfinic acid (Cys-SO 2H) and cysteine sulfonic acid (Cys-SO 3H). Copper,Zinc-superoxide dismutase (SOD1) is a key enzyme in the protection of cells from the superoxide anion radical. The SH group at Cys 111 residue in human SOD1 is selectively oxidized to -SO 2H and -SO 3H with atmospheric oxygen, and this oxidized human SOD1 is also suggested to play an important role in the pathophysiology of various neurodegenerative diseases, probably mainly via protein aggregation. Therefore, information on the structural and the dynamics of the oxidized cysteine residue would be crucial for the understanding of protein aggregation mechanism. Although the -SO 3H group on proteins cannot be directly detected by conventional NMR techniques, we successfully performed the site-specific 17O-labeling of Cys 111 in SOD1 using ^{17}it {O}2 gas and the 17O-NMR analysis for the first time. We observed clear 17O signal derived from a protein molecule and show that 17O-NMR is a sensitive probe for studying the structure and dynamics of the 17O-labeled protein molecule. This novel and unique strategy can have great impact on many research fields in biology and chemistry.

  5. Expression and mutational analysis of Autographa californica nucleopolyhedrovirus HCF-1: functional requirements for cysteine residues.

    PubMed

    Wilson, Joyce A; Forney, Scott D; Ricci, Alessondra M; Allen, Emily G; Hefferon, Kathleen L; Miller, Lois K

    2005-11-01

    The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.

  6. Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue.

    PubMed

    Yee, Estella F; Diensthuber, Ralph P; Vaidya, Anand T; Borbat, Peter P; Engelhard, Christopher; Freed, Jack H; Bittl, Robert; Möglich, Andreas; Crane, Brian R

    2015-12-09

    Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

  7. Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

    PubMed Central

    Yee, Estella F.; Diensthuber, Ralph P.; Vaidya, Anand T.; Borbat, Peter P.; Engelhard, Christopher; Freed, Jack H.; Bittl, Robert; Möglich, Andreas; Crane, Brian R.

    2015-01-01

    Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications. PMID:26648256

  8. Characterization of high-molecular-weight glutenin subunits from Eremopyrum bonaepartis and identification of a novel variant with unusual high molecular weight and altered cysteine residues.

    PubMed

    Jiang, Qian-Tao; Zhang, Xiao-Wei; Ma, Jian; Wei, Long; Zhao, Shan; Zhao, Quan-Zhi; Qi, Peng-Fei; Lu, Zhen-Xiang; Zheng, You-Liang; Wei, Yu-Ming

    2014-04-01

    We characterized two high-molecular-weight glutenin subunit (HMW-GS) variants from Eremopyrum bonaepartis, determined their complete open reading frames, and further expressed them in a bacterial system. The variants have many novel structural features compared with typical subunits encoded by Glu-1 loci: 1Fx3.7 and 1Fy1.5 exhibit hybrid properties of x- and y-type subunits. In addition, unusual molecular mass and altered number and distribution of cysteine residues were unique features of HMW-GSs encoded by Glu-F1 from E. bonaepartis. The mature 1Fx3.7 subunit has a full length of 1,223 amino acid residues, making it the largest subunit found thus far, while 1Fy1.5 is just 496 residues. In addition, the mutated PGQQ repeat motif was found in the repetitive region of 1Fx3.7. Although it has a similar molecular mass to that previously reported for 1Dx2.2, 1Dx2.2* and 1S(sh)x2.9 subunits, 1Fx3.7 appears to have had a different evolutionary history. The N-terminal and repetitive regions have a total of four additional cysteine residues, giving 1Fx3.7 a total of eight cysteines, while 1Fy1.5 has only six cysteines because the GHCPTSPQQ nonapeptide at the end of the repetitive region is deleted. With its extra cysteine residues and the longest repetitive region, features that are relevant to good wheat quality, the 1Fx3.7 subunit gene could be an excellent candidate for applications in wheat quality improvement.

  9. Effect of cysteine and cystine addition on sensory profile and potent odorants of extruded potato snacks.

    PubMed

    Majcher, Małgorzata A; Jeleń, Henryk H

    2007-07-11

    Aromas generated in extruded potato snacks without and with addition of 0.25, 0.5, and 1% (w/w) of flavor precursors, cysteine and cystine, were compared and evaluated by descriptive sensory profiling. The results showed that high addition of cysteine (0.5 and 1%) resulted in the formation of undesirable odor and taste described as mercaptanic/sulfur, onion-like, and bitter; on the contrary, addition of cystine even at high concentration gave product with pleasant odor and taste, slightly changed into breadlike notes. GC/O analysis showed cysteine to be a much more reactive flavor precursor than cystine, stimulating formation of 12 compounds with garlic, sulfury, burnt, pungent/beer, cabbage/mold, meatlike, roasted, and popcorn odor notes. Further analysis performed by the AEDA technique identified 2-methyl-3-furanthiol (FD 2048) as a most potent odorant of extruded potato snacks with 1% addition of cysteine. Other identified compounds with high FD were butanal, 3-methyl-2-butenethiol, 2-methylthiazole, methional, 2-acetyl-1-pyrroline, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone. In the case of cystine addition (1%) the highest FD factors were calculated for butanal, 2-acetyl-1-pyrroline, benzenemethanethiol, methional, phenylacetaldehyde, dimethyltrisulfide, 1-octen-3-ol, 1,5-octadien-3-one, and 2-acetylpyrazine.

  10. Redox Biology: Computational Approaches to the Investigation of Functional Cysteine Residues

    PubMed Central

    Marino, Stefano M.

    2011-01-01

    Abstract Cysteine (Cys) residues serve many functions, such as catalysis, stabilization of protein structure through disulfides, metal binding, and regulation of protein function. Cys residues are also subject to numerous post-translational modifications. In recent years, various computational tools aiming at classifying and predicting different functional categories of Cys have been developed, particularly for structural and catalytic Cys. On the other hand, given complexity of the subject, bioinformatics approaches have been less successful for the investigation of regulatory Cys sites. In this review, we introduce different functional categories of Cys residues. For each category, an overview of state-of-the-art bioinformatics methods and tools is provided, along with examples of successful applications and potential limitations associated with each approach. Finally, we discuss Cys-based redox switches, which modify the view of distinct functional categories of Cys in proteins. Antioxid. Redox Signal. 15, 135–146. PMID:20812876

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

    PubMed Central

    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

  12. Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide.

    PubMed

    Haag, Andreas F; Kerscher, Bernhard; Dall'Angelo, Sergio; Sani, Monica; Longhi, Renato; Baloban, Mikhail; Wilson, Heather M; Mergaert, Peter; Zanda, Matteo; Ferguson, Gail P

    2012-03-30

    The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

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

    PubMed

    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.

  14. Importance of cysteine residues in the thyroid hormone transporter MCT8.

    PubMed

    Lima de Souza, Elaine C; Groeneweg, Stefan; Visser, W Edward; Peeters, Robin P; Visser, Theo J

    2013-05-01

    The thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) is crucial for brain development as demonstrated by the severe psychomotor retardation in patients with MCT8 mutations. MCT8 contains 10 residues of the reactive amino acid cysteine (Cys) whose functional roles were studied using the Cys-specific reagent p-chloromercurybenzenesulfonate (pCMBS) and by site-directed mutagenesis. Pretreatment of JEG3 cells with pCMBS resulted in a dose- and time-dependent decrease of subsequent T3 uptake. Pretreatment with dithiothreitol did not affect TH transport or its inhibition by pCMBS. However, pCMBS inhibition of MCT8 was reversed by dithiothreitol. Inhibition of MCT8 by pCMBS was prevented in the presence of T3. The single and double mutation of C481A and C497A did not affect T3 transport, but the single mutants were less sensitive and the double mutant was completely insensitive to pCMBS. Similar effects on MCT8 were obtained using HgCl2 instead of pCMBS. In conclusion, we have identified Cys481 and Cys497 in MCT8 as the residues modified by pCMBS or HgCl2. These residues are probably located at or near the substrate-recognition site in MCT8. It remains to be investigated whether MCT8 function is regulated by modification of these Cys residues under pathophysiological conditions.

  15. Dipeptide-derived nitriles containing additional electrophilic sites: potentially irreversible inhibitors of cysteine proteases.

    PubMed

    Löser, Reik; Gütschow, Michael

    2009-12-01

    Heterocyclic and open-chain dipeptide-derived nitriles have been synthesized, containing an additional electrophilic center enabling the subsequent covalent modification of the thioimidate nitrogen formed in situ at the active site of the enzyme. The inhibitory potential of these nitriles against the cysteine proteases papain and cathepsins L, S, and K was determined. The open-chain dipeptide nitriles 8 and 10 acted as moderate reversible inhibitors, but no evidence for an irreversible inhibition of these enzymes was discernable.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28135343

  17. Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein.

    PubMed

    Katsuma, Susumu; Sugano, Yudai; Kiuchi, Takashi; Shimada, Toru

    2015-10-23

    We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.

  18. Alanine substitutions of noncysteine residues in the cysteine-stabilized αβ motif

    PubMed Central

    Yang, Ying-Fang; Cheng, Kuo-Chang; Tsai, Ping-Hsing; Liu, Chung-Cheng; Lee, Tian-Ren; Ping-Chiang Lyu

    2009-01-01

    The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized αβ motif (CSαβ) consists of an α-helix and an antiparallel triple-stranded β-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. PMID:19533758

  19. Mutational analysis of extracellular cysteine residues of rat secretin receptor shows that disulfide bridges are essential for receptor function.

    PubMed

    Vilardaga, J P; Di Paolo, E; Bialek, C; De Neef, P; Waelbroeck, M; Bollen, A; Robberecht, P

    1997-05-15

    We attempted to express point-mutant secretin receptors where each of the 10 extracellular Cys residues was replaced by a Ser residue, in Chinese hamster ovary (CHO) cells. Six of the point-mutant receptors (C24-->S, C44-->S, C53-->S, C67-->S, C85-->S and C101-->S) could not be detected by binding or functional studies: the mutations resulted in functional inactivation of the receptor. In contrast, the four other point-mutant receptors (C11-->S, C186-->S, C193-->S and C263-->S) were able to bind poorly 125I-secretin, and to activate adenylate cyclase with high secretin EC50 values. These results suggest that cysteine residues 24, 44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263-->S), the four triple mutants, and through double mutants C186,193-->S and C186,263-->S with a very high (microM) EC50 value, suggesting that, in the wild-type receptor, disulfide bridges are formed between C11-C186, and between C193-C263. Prior treatment with dithiothreitol resulted in a marked EC50 increase of the wild-type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the C11-C186 (but not the C193-C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild-type receptor.

  20. Role of a cysteine residue in the active site of ERK and the MAPKK family

    SciTech Connect

    Ohori, Makoto; Kinoshita, Takayoshi; Yoshimura, Seiji; Warizaya, Masaichi; Nakajima, Hidenori . E-mail: hidenori.nakajima@jp.astellas.com; Miyake, Hiroshi

    2007-02-16

    Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGF{beta}-induced AP-1-dependent luciferase expression with respective IC{sub 50} values of 0.08 and 0.05 {mu}M. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the {alpha},{beta}-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to S{gamma} of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, N{zeta} of Lys114, backbone C=O of Ser153, N{delta}2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPK{alpha}/{beta}/{gamma}/{delta} which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.

  1. Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.

    PubMed

    Ang, Swee Kim; Zhang, Mengqi; Lodi, Tiziana; Lu, Hui

    2014-06-01

    Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.

  2. Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.

    PubMed Central

    Oteiza, P I; Adamo, A M; Aloise, P A; Paladini, A C; Paladini, A A; Soto, E F

    1987-01-01

    Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr. PMID:3663175

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

    SciTech Connect

    Hara, Satoshi; Tatenaka, Yuki; Ohuchi, Yuya; Hisabori, Toru

    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.

  4. Proximity of reactive cysteine residue and flavin in Escherichia coli pyruvate oxidase as estimated by fluorescence energy transfer.

    PubMed

    Koland, J G; Gennis, R B

    1982-08-31

    Pyruvate oxidase of Escherichia coli possesses a reactive cysteine residue believed to be associated with the thiamin pyrophosphate (TPP) binding site. This residue is not reactive in the presence of TPP. Exposure of the enzyme to cysteine-directed fluorescent reagents results in the formation of fluorescent protein conjugates. Although these reagents do not react solely with the TPP-protectable cysteine residue, the fluorescence emission spectrum of a probe attached to this residue can be obtained by a difference technique. It was determined that the fluorescence emission of probes at the TPP-protectable site is very low due to energy transfer to the FAD coenzyme and that this fluorescence is greatly enhanced upon reduction or extraction of the flavin. Application of fluorescence energy transfer theory enabled the determination of an upper limit for the distance between the probes at the TPP-protectable site and the flavin adenine dinucleotide (FAD) (roughly 20 A). Thus, the TPP binding site and the FAD coenzyme are likely in close proximity.

  5. Predicting the redox state and secondary structure of cysteine residues using multi-dimensional classification analysis of NMR chemical shifts.

    PubMed

    Wang, Ching-Cheng; Lai, Wen-Chung; Chuang, Woei-Jer

    2016-09-01

    A tool for predicting the redox state and secondary structure of cysteine residues using multi-dimensional analyses of different combinations of nuclear magnetic resonance (NMR) chemical shifts has been developed. A data set of cysteine [Formula: see text], (13)C(α), (13)C(β), (1)H(α), (1)H(N), and (15)N(H) chemical shifts was created, classified according to redox state and secondary structure, using a library of 540 re-referenced BioMagResBank (BMRB) entries. Multi-dimensional analyses of three, four, five, and six chemical shifts were used to derive rules for predicting the structural states of cysteine residues. The results from 60 BMRB entries containing 122 cysteines showed that four-dimensional analysis of the C(α), C(β), H(α), and N(H) chemical shifts had the highest prediction accuracy of 100 and 95.9 % for the redox state and secondary structure, respectively. The prediction of secondary structure using 3D, 5D, and 6D analyses had the accuracy of ~90 %, suggesting that H(N) and [Formula: see text] chemical shifts may be noisy and made the discrimination worse. A web server (6DCSi) was established to enable users to submit NMR chemical shifts, either in BMRB or key-in formats, for prediction. 6DCSi displays predictions using sets of 3, 4, 5, and 6 chemical shifts, which shows their consistency and allows users to draw their own conclusions. This web-based tool can be used to rapidly obtain structural information regarding cysteine residues directly from experimental NMR data.

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

    PubMed

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

    2011-01-01

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

  7. Antioxidant activity of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin in oil-in-water emulsions.

    PubMed

    Elias, Ryan J; McClements, D Julian; Decker, Eric A

    2005-12-28

    Proteins dispersed in the continuous phase of oil-in-water emulsions are capable of inhibiting lipid oxidation reactions. The antioxidant activity of these proteins is thought to encompass both free radical scavenging by amino acid residues and chelation of prooxidative transition metals; however, the precise mechanism by which this occurs remains unclear. In this study, the oxidative stability of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin (beta-Lg) in a Brij-stabilized menhaden oil-in-water emulsion was determined. The presence of low concentrations of continuous phase beta-Lg (250 and 750 microg/mL) significantly inhibited lipid oxidation as determined by lipid hydroperoxides and thiobarbituric acid reactive substances analysis. It was observed that cysteine oxidized before tryptophan in beta-Lg, and both residues oxidized before lipid oxidation could be detected. No oxidation of the methionine residues of beta-Lg was observed despite its reported high oxidative susceptibility. It is conceivable that surface exposure of amino acid residues greatly affects their oxidation kinetics, which may explain why some residues are preferentially oxidized relative to others. Further elucidation of the mechanisms governing free radical scavenging of amino acids could lead to more effective applications of proteins as antioxidants within oil-in-water food emulsions.

  8. Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Scotcher, Jenna; Clarke, David J.; Weidt, Stefan K.; Mackay, C. Logan; Hupp, Ted R.; Sadler, Peter J.; Langridge-Smith, Pat R. R.

    2011-05-01

    The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

  9. Crystal structure of a papain-fold protein without the catalytic residue: a novel member in the cysteine proteinase family.

    PubMed

    Zhang, Min; Wei, Zhiyi; Chang, Shaojie; Teng, Maikun; Gong, Weimin

    2006-04-21

    A 31kDa cysteine protease, SPE31, was isolated from the seeds of a legume plant, Pachyrizhus erosus. The protein was purified, crystallized and the 3D structure solved using molecular replacement. The cDNA was obtained by RT PCR followed by amplification using mRNA isolated from the seeds of the legume plant as a template. Analysis of the cDNA sequence and the 3D structure indicated the protein to belong to the papain family. Detailed analysis of the structure revealed an unusual replacement of the conserved catalytic Cys with Gly. Replacement of another conserved residue Ala/Gly by a Phe sterically blocks the access of the substrate to the active site. A polyethyleneglycol molecule and a natural peptide fragment were bound to the surface of the active site. Asn159 was found to be glycosylated. The SPE31 cDNA sequence shares several features with P34, a protein found in soybeans, that is implicated in plant defense mechanisms as an elicitor receptor binding to syringolide. P34 has also been shown to interact with vegetative storage proteins and NADH-dependent hydroxypyruvate reductase. These roles suggest that SPE31 and P34 form a unique subfamily within the papain family. The crystal structure of SPE31 complexed with a natural peptide ligand reveals a unique active site architecture. In addition, the clear evidence of glycosylated Asn159 provides useful information towards understanding the functional mechanism of SPE31/P34.

  10. Positioning of cysteine residues within the N-terminal portion of the BST-2/tetherin ectodomain is important for functional dimerization of BST-2.

    PubMed

    Welbourn, Sarah; Kao, Sandra; Du Pont, Kelly E; Andrew, Amy J; Berndsen, Christopher E; Strebel, Klaus

    2015-02-06

    BST-2/tetherin is a cellular host factor capable of restricting the release of a variety of enveloped viruses, including HIV-1. Structurally, BST-2 consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ectodomain, and a C-terminal membrane anchor. The BST-2 ectodomain encodes three cysteine residues in its N-terminal half, each of which can contribute to the formation of cysteine-linked dimers. We previously reported that any one of the three cysteine residues is sufficient to produce functional BST-2 dimers. Here we investigated the importance of cysteine positioning on the ectodomain for functional dimerization of BST-2. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. The resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. We found significant flexibility in the positioning of cysteine residues, although the propensity to form cysteine-linked dimers generally decreased with increasing distance from the N terminus. Interestingly, all BST-2 variants, including the one lacking all three ectodomain cysteines, retained the ability to form non-covalent dimers, and all of the BST-2 variants were efficiently expressed at the cell surface. Importantly, not all BST-2 variants capable of forming cysteine-linked dimers were functional, suggesting that cysteine-linked dimerization of BST-2 is necessary but not sufficient for inhibiting virus release. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell.

  11. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    SciTech Connect

    Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  12. Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition.

    PubMed

    Van Soom, A; Yuan, Y Q; Peelman, L J; de Matos, D G; Dewulf, J; Laevens, H; de Kruif, A

    2002-03-15

    Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.

  13. Cinchona Alkaloid Catalyzed Sulfa-Michael Addition Reactions Leading to Enantiopure β-Functionalized Cysteines.

    PubMed

    Breman, Arjen C; Telderman, Suze E M; van Santen, Roy P M; Scott, Jamie I; van Maarseveen, Jan H; Ingemann, Steen; Hiemstra, Henk

    2015-11-06

    Sulfa-Michael additions to α,β-unsaturated N-acylated oxazolidin-2-ones and related α,β-unsaturated α-amino acid derivatives have been enantioselectively catalyzed by Cinchona alkaloids functionalized with a hydrogen bond donating group at the C6' position. The series of Cinchona alkaloids includes known C6' (thio)urea and sulfonamide derivatives and several novel species with a benzimidazole, squaramide or a benzamide group at the C6' position. The sulfonamides were especially suited as bifunctional organocatalysts as they gave the products in very good diastereoselectivity and high enantioselectivity. In particular, the C6' sulfonamides catalyzed the reaction with the α,β-unsaturated α-amino acid derivatives to afford the products in a diastereomeric ratio as good as 93:7, with the major isomer being formed in an ee of up to 99%. The products of the organocatalytic sulfa-Michael addition to α,β-unsaturated α-amino acid derivatives were subsequently converted in high yields to enantiopure β-functionalized cysteines suitable for native chemical ligation.

  14. An Additive Effect of Oral N-Acetyl Cysteine on Eradication of Helicobacter pylori

    PubMed Central

    Hamidian, Seyed Mohammad-Taghi; Aletaha, Najmeh-sadat; Taslimi, Reza; Montazeri, Mohammad

    2015-01-01

    Background. Helicobacter pylori is highly adapted to the gastric environment where it lives within or beneath the gastric mucous layer. The aim of this study was to evaluate whether the addition of N-acetyl cysteine to the treatment regimen of H. pylori infection would affect eradication rates of the disease. Methods. A total of 79 H. pylori positive patients were randomized to two therapeutic groups. Both groups received a 14-day course of three-drug regimen including amoxicillin/clarithromycin/omeprazole. Experimental group (38 subjects) received NAC, and control group (41 subjects) received placebo, besides three-drug regimen. H. pylori eradication was evaluated by urea breath test at least 4 weeks after the cessation of therapy. Results. The rate of H. pylori eradication was 72.9% and 60.9% in experimental and control groups, respectively (P = 0.005). By logistic regression modeling, female gender (OR 3.68, 95% CI: 1.06–5.79; P = 0.040) and treatment including NAC (OR 1.88, 95% CI: 0.68–3.15; P = 0.021) were independent factors associated with H. pylori eradication. Conclusion. The results of the present study show that NAC has an additive effect on the eradication rates of H. pylori obtained with three-drug regimen and appears to be a promising means of eradicating H. pylori infection. PMID:26421191

  15. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    SciTech Connect

    Kurooka, Hisanori; Sugai, Manabu; Mori, Kentaro; Yokota, Yoshifumi

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

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

    PubMed

    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.

  17. A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death1[OPEN

    PubMed Central

    Elmore, James M.; Creer, Athena Y.; Feng, Baomin; Franco, Jessica Y.; He, Ping; Phinney, Brett

    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. PMID:27852951

  18. Cysteine residues in the major capsid protein, Vp1, of the JC virus are important for protein stability and oligomer formation.

    PubMed

    Kobayashi, Shintaro; Suzuki, Tadaki; Igarashi, Manabu; Orba, Yasuko; Ohtake, Noriko; Nagakawa, Keita; Niikura, Kenichi; Kimura, Takashi; Kasamatsu, Harumi; Sawa, Hirofumi

    2013-01-01

    The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.

  19. Contribution of cysteine residues to the structure and function of herpes simplex virus gH/gL

    SciTech Connect

    Cairns, Tina M. . E-mail: tmcairns@biochem.dental.upenn.edu; Landsburg, Daniel J. . E-mail: dlandsbu@temple.edu; Charles Whitbeck, J. . E-mail: whitbeck@biochem.dental.upenn.edu; Eisenberg, Roselyn J. . E-mail: roselyn@biochem.dental.upenn.edu; Cohen, Gary H. . E-mail: cohen@biochem.dental.upenn.edu

    2005-02-20

    In HSV types 1 and 2, gH forms a noncovalent heterodimer with gL. Previous studies demonstrated that the first 323 amino acids of gH1 and the first 161 amino acids of gL1 are sufficient for gH/gL binding. For gL1, substitution of any of its four cysteine (C) residues (all located within the gH/gL binding region) destroyed gH binding and function. Although gH1 contains 8 cysteines in its ectodomain, gH 2 contains 7 (C3 of gH1 is replaced by arginine in gH2). We found that mutation of any of the four C-terminal cysteines led to a reduction or loss of gH/gL function. Mutation of C5 or C6 in gH1 or gH2 rendered the proteins non-functional. However, substitution of C7 and/or C8 in gH1 has a definite negative impact on cell-cell fusion, although these mutations had less effect on complementation. Remarkably, all four gH1 N-terminal cysteines could be mutated simultaneously with little effect on fusion or complementation. As gH2 already lacks C3, we constructed a triple mutant (gH2-C1/2/4) which exhibited a similar phenotype. Since gH1 is known to bind gL2 and vice versa, we wondered whether binding of gH2 to the heterologous gL1 would enhance the fusion defect seen with the gH2-C2 mutant. The combination of mutant gH2-C2 with wild-type gL1 was nonfunctional in a cell-cell fusion assay. Interestingly, the reciprocal was not true, as gH1-C2 could utilize both gL1 and gL2. These findings suggest that there is a structural difference in the gH2 N-terminus as compared to gH1. We also present genetic evidence for at least one disulfide bond within gH2, between cysteines 2 and 4.

  20. The Presence of Cysteine in the Cytoplasmic Domain of the Vesicular Stomatitis Virus Glycoprotein is Required for Palmitate Addition

    NASA Astrophysics Data System (ADS)

    Rose, John K.; Adams, Gregg A.; Gallione, Carol J.

    1984-04-01

    The transmembrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is known to contain 1-2 mol of covalently linked fatty acid (palmitate) per mol of protein. G protein is oriented in cellular membranes such that the carboxyl-terminal 29 amino acids protrude into the cytoplasm. We have obtained expression in eukaryotic cells of mutagenized cDNA clones that encode VSV G proteins lacking portions of this cytoplasmic domain. Labeling of these truncated proteins with [3H]palmitate indicated that the palmitate might be linked to an amino acid residue within the first 14 residues on the carboxyl-terminal side of the transmembrane domain. Using oligonucleotide directed mutagenesis, we changed the single codon specifying cysteine in this domain to a codon specifying serine. Expression of this mutant gene results in synthesis of a G protein lacking palmitate. We suggest that linkage of palmitate to G protein is through the cysteine in the cytoplasmic domain and that such a linkage may occur in many viral and cellular glycoproteins. The G protein lacking palmitate is glycosylated and is transported normally to the cell surface.

  1. Kinetic and site-directed mutagenesis studies of the cysteine residues of bovine low molecular weight phosphotyrosyl protein phosphatase.

    PubMed

    Davis, J P; Zhou, M M; Van Etten, R L

    1994-03-25

    The roles of the 8 conserved cysteines and 1 arginine in the low molecular weight phosphotyrosyl protein phosphatases were investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for Cys-12, Cys-17, and Arg-18. The CD spectra of the purified proteins were effectively superimposable, consistent with the conclusion that no major structural alterations had occurred, but 1H NMR spectroscopy did reveal some spectral shifts in the aromatic region. Kinetic analysis of the mutant proteins demonstrated that only Cys-12, Cys-17, and Arg-18 had significantly altered catalytic activity toward the substrate p-nitrophenyl phosphate at pH 5. The Cys-12 and Arg-18 mutants were effectively inactive. Thus, it is concluded that Cys-12 is the catalytic nucleophile, and Arg-18 presumably serves an essential function in substrate binding. The C17S mutant had 6% residual activity compared with wild type protein, whereas the C17A mutant had 37% activity. Consistent with the observed activity of the Cys-17 mutant, a covalent phosphocysteine intermediate was trapped and identified by 31P NMR. Further kinetic analysis of C17A using several aryl phosphate monoester substrates with different leaving group pK alpha values indicated that no change in the rate-determining step of the catalytic mechanism had occurred, that is, dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. The C17A mutant had a 4-fold higher phosphate Ki and slightly higher Km values for p-nitrophenyl phosphate suggesting that Cys-17 may be important for optimal positioning of the substrate phosphate moiety.

  2. The role of individual cysteine residues in the processing, structure, and function of human macrophage colony-stimulating factor.

    PubMed

    Deng, P; Wang, Y L; Pattengale, P K; Rettenmier, C W

    1996-11-12

    The shortest form of human macrophage colony-stimulating factor (M-CSF alpha, CSF-1(256) is expressed on the cell surface as a homodimeric type I transmembrane glycoprotein. The seven cysteine residues in CSF-1(256) form three intrachain disulfide bonds (Cys7-Cys90, Cys48-Cys139, and Cys 102-Cys146), and one interchain disulfide bond (Cys31-Cys31). To examine the role of the seven cysteine residues in CSF-1(256), we replaced each half-cystine by a serine using site-directed mutagenesis, and stably expressed the mutated genes in mouse NIH 3T3 cells. We showed that each of the seven cysteines of CSF-1(256) is essential for its biological activity. Our data further show that substitution of Cys48 or Cys139 totally blocked dimer formation and cell surface expression of CSF-1(256), and that substitution of Cys102 and Cys146 severely impaired CSF-1 dimer formation and cell surface expression. In contrast, substitution of Cys7 or Cys90 affected CSF-1 dimer formation to a lesser degree but did not significantly affect cell surface expression of CSF-1. Furthermore, disruption of the interchain disulfide bond led to efficient cell surface expression of monomeric CSF-1. All of the cell surface expressed mutant CSF-1 proteins, either dimeric or monomeric, still underwent efficient ectodomain cleavage. The electrophoretic mobilities of the cleaved dimeric ectodomains of these mutant CSF-1 proteins on SDS-PAGE exhibited distinctly different patterns as compared with the wild type. Substitution of either Cys7 or Cys90 produced the same shift, while substitution of either Cys102 or Cys146 resulted in a shift distinct from that caused by substitution of Cys7 or Cys90. These data suggest that replacement of either of a pair of intrachain half-cystine residues results in similar conformational changes, and may provide a novel method for mapping intrachain disulfide bonds in dimeric proteins.

  3. Spectral Envelopes and Additive + Residual Analysis/Synthesis

    NASA Astrophysics Data System (ADS)

    Rodet, Xavier; Schwarz, Diemo

    The subject of this chapter is the estimation, representation, modification, and use of spectral envelopes in the context of sinusoidal-additive-plus-residual analysis/synthesis. A spectral envelope is an amplitude-vs-frequency function, which may be obtained from the envelope of a short-time spectrum (Rodet et al., 1987; Schwarz, 1998). [Precise definitions of such an envelope and short-time spectrum (STS) are given in Section 2.] The additive-plus-residual analysis/synthesis method is based on a representation of signals in terms of a sum of time-varying sinusoids and of a non-sinusoidal residual signal [e.g., see Serra (1989), Laroche et al. (1993), McAulay and Quatieri (1995), and Ding and Qian (1997)]. Many musical sound signals may be described as a combination of a nearly periodic waveform and colored noise. The nearly periodic part of the signal can be viewed as a sum of sinusoidal components, called partials, with time-varying frequency and amplitude. Such sinusoidal components are easily observed on a spectral analysis display (Fig. 5.1) as obtained, for instance, from a discrete Fourier transform.

  4. Selective Fluorescence Detection of Cysteine over Homocysteine and Glutathione Based on a Cysteine-Triggered Dual Michael Addition/Retro-aza-aldol Cascade Reaction.

    PubMed

    Liu, Yawei; Lv, Xin; Hou, Min; Shi, Yawei; Guo, Wei

    2015-11-17

    In this work, a cysteine (Cys)-triggered dual Michael addition/retro-aza-aldol cascade reaction has been exploited and utilized to construct a fluorescent probe for Cys for the first time. The resulting fluorescent probe 8-alkynylBodipy 1 contains an activated alkynyl unit as Michael receptor and a Bodipy dye as fluorescence reporter and can highly selectively detect Cys over homocysteine (Hcy)/glutathione (GSH) as well as other amino acids with a significant fluorescence off-on response (∼4500-fold) and an ultralow detection limit (0.38 nM). The high selectivity of 1 for Cys could be attributed to a kinetically favored five-membered cyclic intermediate produced by the dual Michael addition of Cys with the activated alkynyl unit of 1. The big fluorescence off-on response is due to the subsequent retro-aza-aldol reaction of the five-membered cyclic intermediate that results in the release of a highly fluorescent 8-methylBodipy dye 2. The probe has been successfully used to detect and image Cys in serum and cells, respectively.

  5. Influence of Protein – Micelle Ratios and Cysteine Residues on the Kinetic Stability and Unfolding Rates of Human Mitochondrial VDAC-2

    PubMed Central

    Maurya, Svetlana Rajkumar; Mahalakshmi, Radhakrishnan

    2014-01-01

    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 – 13000∶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. PMID:24494036

  6. Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Yutthanasirikul, Rayakorn; Nagano, Takanori; Jimbo, Haruhiko; Hihara, Yukako; Kanamori, Takashi; Ueda, Takuya; Haruyama, Takamitsu; Konno, Hiroki; Yoshida, Keisuke; Hisabori, Toru; Nishiyama, Yoshitaka

    2016-03-11

    Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

  7. Delayed Lysis with Salmonella Bacteriophage P22: Induction of Lysis by Addition of Cysteine or Histidine to the Growth Medium

    PubMed Central

    Cohen, Larry W.

    1969-01-01

    A mutant (Lys−) of Salmonella bacteriophage P22 showed a delay in lysis of more than 3 hr in infections in unsupplemented M9 medium. The infected cells were induced to lyse during that interval by addition of histidine or sulfhydryl compounds cysteine, mercaptoethanol, glutathione, or ergothioneine. Urocanic acid, the first intermediate in the catabolic histidine pathway, did not induce lysis, nor did histamine, imidazolelactate, or carnosine. None of the other amino acids common to protein had any inductive effect. Both the d and l forms of histidine were effective in inducing lysis, suggesting that the incorporation of the histidine into protein is not involved. Chloramphenicol inhibited lysis when added at 60 min with or without histidine, but did not inhibit the induction of lysis when added with cysteine. Bacterial cells infected with Lys+ phage were induced to lyse prematurely when cysteine was added at 30 min but not at 20 min of infection. Iodoacetate inhibited lysis of Lys+-infected cells when added at 20 min but not at 30 min. PMID:16789095

  8. Substitution of conserved cysteine residues in Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Substitutions in the amino-terminal region of Wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution a...

  9. Covalent targeting of remote cysteine residues to develop CDK12 and 13 inhibitors

    PubMed Central

    Zhang, Tinghu; Kwiatkowski, Nicholas; Olson, Calla M; Dixon-Clarke, Sarah E; Abraham, Brian J; Greifenberg, Ann K; Ficarro, Scott B; Elkins, Jonathan M; Liang, Yanke; Hannett, Nancy M; Manz, Theresa; Hao, Mingfeng; Bartkowiak, Bartlomiej; Greenleaf, Arno L; Marto, Jarrod A; Geyer, Matthias; Bullock, Alex N; Young, Richard A; Gray, Nathanael S

    2016-01-01

    Cyclin-dependent kinases 12 and 13 (CDK12 and 13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and 13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and 13 covalent inhibitor, THZ531. Co-crystallization with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super–enhancer–associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and 13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities. PMID:27571479

  10. Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

    PubMed

    Bastin, Guillaume; Singh, Kevin; Dissanayake, Kaveesh; Mighiu, Alexandra S; Nurmohamed, Aliya; Heximer, Scott P

    2012-08-17

    Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

  11. Influences of Proline and Cysteine Residues on Fragment Yield in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Smargiasso, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2014-06-01

    Matrix-assisted laser desorption/ionization in-source decay produces highly informative fragments for the sequencing of peptides/proteins. Among amino acids, cysteine and proline residues were found to specifically influence the fragment yield. As they are both frequently found in small peptide structures for which de novo sequencing is mandatory, the understanding of their specific behaviors would allow useful fragmentation rules to be established. In the case of cysteine, a c•/ w fragment pair originating from Xxx-Cys is formed by side-chain loss from the cysteine residue. The presence of a proline residue contributes to an increased yield of ISD fragments originating from N-Cα bond cleavage at Xxx1-Xxx2Pro, which is attributable to the cyclic structure of the proline residue. Our results suggest that the aminoketyl radical formed by MALDI-ISD generally induces the homolytic N-Cα bond cleavage located on the C-terminal side of the radical site. In contrast, N-Cα bond cleavage at Xxx-Pro produces no fragments and the N-Cα bond at the Xxx1-Xxx2Pro bond is alternatively cleaved via a heterolytic cleavage pathway.

  12. An exposed cysteine residue of human angiostatic mini tryptophanyl-tRNA synthetase.

    PubMed

    Wakasugi, Keisuke

    2010-04-13

    Human tryptophanyl-tRNA synthetase (TrpRS) catalyzes the aminoacylation of tRNA(Trp). Human TrpRS exists in two forms: a major form that is the full-length protein and a truncated form (mini TrpRS) in which most of the N-terminal extension is absent. Human mini, but not full-length, TrpRS has angiostatic activity. Because the full-length protein, which lacks angiostatic activity, has all of the amino acid determinants of the mini form, which has activity, I searched for conformational differences between the two proteins. Using a disulfide cross-linking assay, I showed that the molecular environment around Cys62 is significantly different between the two proteins. This difference can be explained by inspection of the three-dimensional structure of the full-length protein. These results give a clear demonstration of a significant difference, around a specific residue (Cys62), between a potent angiostatic and nonangiostatic version of human TrpRS.

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

    PubMed Central

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

    2010-01-01

    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 identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  14. The number of cysteine residues per mole in apolipoprotein E affects systematically synchronous neural interactions in women's healthy brains.

    PubMed

    Leuthold, Arthur C; Mahan, Margaret Y; Stanwyck, John J; Georgopoulos, Angeliki; Georgopoulos, Apostolos P

    2013-05-01

    Apolipoprotein E (apoE) is involved in lipid metabolism in the brain, but its effects on brain function are not understood. Three apoE isoforms (E4, E3, and E2) are the result of cysteine-arginine interchanges at two sites: there are zero interchanges in E4, one interchange in E3, and two interchanges in E2. The resulting six apoE genotypes (E4/4, E4/3, E4/2, E3/3, E3/2, E2/2) yield five groups with respect to the number of cysteine residues per mole (CysR/mole), as follows. ApoE4/4 has zero cysteine residues per mole (0-CysR/mole), E4/3 has one (1-CysR/mole), E4/2 and E3/3 each has two (2-CysR/mole), E3/2 has three (3-CysR/mole), and E2/2 has four (4-CysR/mole). The use of the number of CysR/mole to characterize the apoE molecule converts the categorical apoE genotype scale, consisting of 6 distinct genotypes above, to a 5-point continuous scale (0-4 CysR/mole). This allows the use of statistical analyses suitable for continuous variables (e.g. regression) to quantify the relations between various variables and apoE. Using such analyses, here, we show for the first time that apoE affects in a graded and orderly manner neural communication, as assessed by analyzing the relation between the number of CysR/mole and synchronous neural interactions (SNI) measured by magnetoencephalography (MEG) in 130 cognitively healthy women. At the one end of the CysR/mole range, the 4-CysR/mole (E2/2) SNI distribution had the highest mean, lowest variance, lowest range, and lowest coefficient of variation, whereas at the other end, 0-CysR/mole (E4/4) SNI distribution had the lowest mean, highest variance, highest range, and highest coefficient of variation. The special status of the 4-CysR/mole distribution was reinforced by the results of a hierarchical tree analysis where the 4-CysR/mole (E2/2) SNI distribution occupied a separate branch by itself and the remaining CysR/mole SNI distributions were placed at increasing distances from the 4-CysR/mole distribution, according to

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

    PubMed Central

    Zhang, Yuanwei; Zheng, Qingqing; Sun, Congcong; Song, Jinxing; Gao, Lina; Zhang, Shizhu; Muñoz, Alberto; Read, Nick D.; Lu, Ling

    2016-01-01

    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. PMID:27058039

  16. Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.

    PubMed

    Telezhkin, Vsevolod; Brazier, Stephen P; Mears, Ruth; Müller, Carsten T; Riccardi, Daniela; Kemp, Paul J

    2011-06-01

    The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

  17. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  18. Substitution of cysteine for glycine at residue 415 of one allele of the alpha 1(I) chain of type I procollagen in type III/IV osteogenesis imperfecta.

    PubMed Central

    Nicholls, A C; Oliver, J; Renouf, D V; Keston, M; Pope, F M

    1991-01-01

    We have examined the type I collagen in a patient with type III/IV osteogenesis imperfecta. Two forms of alpha 1(I) chain were produced, one normal and the other containing a cysteine residue within the triple helical domain of the molecule. Cysteine is not normally present in this domain of type I collagen. Peptide mapping experiments localised the mutation to peptide alpha 1(I)CB3 which spans residues 403 to 551 of the triple helix. Subsequent PCR amplification of cDNA covering this region followed by sequencing showed a G to T single base change in the GGC codon for glycine 415 generating TGC, the codon for cysteine. The effect of the mutation on the protein is to delay secretion from the cell, reduce the thermal stability of the molecule by 2 degrees C, and cause excessive post-translational modification of all chains in molecules containing one or more mutant alpha 1(I) chains. The clinical phenotype observed in this patient and the position of the mutation conform to the recent prediction of Starman et al that Gly----Cys mutations in the alpha 1(I) chain have a gradient of severity decreasing from the C-terminus to the N-terminus. Images PMID:1770532

  19. Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

    PubMed

    Khan, Samir A; Rossi, Ana M; Riley, Andrew M; Potter, Barry V L; Taylor, Colin W

    2013-04-15

    IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

  20. Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing

    PubMed Central

    Melo-Nava, Brenda; Casas-González, Patricia; Pérez-Solís, Marco A.; Castillo-Badillo, Jean; Maravillas-Montero, José L.; Jardón-Valadez, Eduardo; Zariñán, Teresa; Aguilar-Rojas, Arturo; Gallay, Nathalie; Reiter, Eric; Ulloa-Aguirre, Alfredo

    2016-01-01

    Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these

  1. Effect of organic residues addition on the technological properties of clay bricks.

    PubMed

    Demir, Ismail

    2008-01-01

    The objective of this study is to investigate the utilization potential of several organic residues in clay bricks. Sawdust, tobacco residues, and grass are widespread by-products of industrial and agricultural processes in Turkey. These residue materials have long cellulose fibres. Sawdust and tobacco residues generally are used as fuel, and the grass is utilized for agricultural purposes. The insulation capacity of brick increases with the increasing porosity of the clay body. Combustible, organic types of pore-forming additives are most frequently used for this purpose. For this reason, increasing amounts of organic residues (0%, 2.5%, 5% and 10% in wt.) were mixed with raw brick-clay. All samples were fired at 900 degrees C. Effects on shaping, plasticity, density, and mechanical properties were investigated. The organic residue additions were found to be effective for pore-forming in the clay body with the clay maintaining acceptable mechanical properties. It was observed that the fibrous nature of the residues did not create extrusion problems. However, higher residue addition required a higher water content to ensure the right plasticity. As a result, sawdust, tobacco residues, and grass can be utilized in an environmentally safe way as organic pore-forming agents in brick-clay.

  2. Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins.

    PubMed

    LeMaster, David M; Hernández, Griselda

    2007-02-01

    The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.

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

    PubMed

    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.

  4. Mutagenesis of varicella-zoster virus glycoprotein I (gI) identifies a cysteine residue critical for gE/gI heterodimer formation, gI structure, and virulence in skin cells.

    PubMed

    Oliver, Stefan L; Sommer, Marvin H; Reichelt, Mike; Rajamani, Jaya; Vlaycheva-Beisheim, Leonssia; Stamatis, Shaye; Cheng, Jason; Jones, Carol; Zehnder, James; Arvin, Ann M

    2011-05-01

    Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.

  5. Analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in Nicotiana benthamiana plants and cross-linking of peptides to VLPs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs...

  6. Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to Cysteines.

    PubMed

    Krenske, Elizabeth H; Petter, Russell C; Houk, K N

    2016-12-02

    Additions of cysteine thiols to Michael acceptors underpin the mechanism of action of several covalent drugs (e.g., afatinib, osimertinib, ibrutinib, neratinib, and CC-292). Reversible Michael acceptors have been reported in which an additional electron-withdrawing group was added at the α-carbon of a Michael acceptor. We have performed density functional theory calculations to determine why thiol additions to these Michael acceptors are reversible. The α-EWG group stabilizes the anionic transition state and intermediate of the Michael addition, but less intuitively, it destabilizes the neutral adduct. This makes the reverse reaction (elimination) both faster and more thermodynamically favorable. For thiol addition to be reversible, the Michael acceptor must also contain a suitable substituent on the β-carbon, such as an aryl or branched alkyl group. Computations explain how these structural elements contribute to reversibility and the ability to tune the binding affinities and the residence times of covalent inhibitors.

  7. Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.

    PubMed

    Sigal, I S; Harwood, B G; Arentzen, R

    1982-12-01

    We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.

  8. Determination of ceftiofur metabolite desfuroylceftiofur cysteine disulfide in bovine tissues using liquid chromatography-tandem mass spectrometry as a surrogate marker residue for ceftiofur.

    PubMed

    Feng, Shixia; Chiesa, Oscar A; Kijak, Philip; Chattopadhaya, Chaitali; Lancaster, Vicki; Smith, Elizabeth A; Girard, Lauren; Sklenka, Sara; Li, Hui

    2014-06-04

    Ceftiofur is a widely used cephalosporin β-lactam antibiotic with frequently reported residue violations. This paper reports a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining a ceftiofur metabolite, desfuroylceftiofur cysteine disulfide (DCCD), in bovine kidney, liver, and muscle tissues. Incurred tissue samples were obtained from dosed animals and analyzed to evaluate the utility of the method. For kidney, the target tissue, the method utilized a simple extraction with phosphate buffer followed by solid phase extraction (SPE) cleanup. For liver and muscle, acetonitrile and hexane were used to remove most proteins and fat from the initial buffer extract before the SPE cleanup. Method accuracy was between 97 and 107%, and the coefficient of variation was between 3.4 and 11.0% for all three types of tissues. The relationship between the new and regulatory methods for bovine kidney was established. It was concluded that DCCD is a suitable surrogate marker residue for ceftiofur in bovine kidney.

  9. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue.

    PubMed

    Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S; Žusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres

    2016-11-15

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.

  10. Determination of Bulk Residual Stresses in Electron Beam Additive-Manufactured Aluminum

    NASA Astrophysics Data System (ADS)

    Brice, Craig A.; Hofmeister, William H.

    2013-11-01

    Additive-manufactured aluminum alloy deposits were analyzed using neutron diffraction to characterize the effect of intermediate stress relief anneal heat treatment on bulk residual stresses in the final part. Based on measured interplanar spacing, stresses were calculated at various locations along a single bead, stacked wall deposit. A comparison between an uninterrupted deposited wall and an interrupted, stress-relieved, and annealed deposited wall showed a measureable reduction in residual stress magnitude at the interface with a corresponding shift in stress character into the deposit. This shift changes the interface stresses from purely compressive to partially tensile. The residual stress profile varied along the length of the deposit, and the heat-treatment procedure reduced the overall magnitude of the stress at the interface by 10 through 25 MPa. These results are interpreted in terms of thermal gradients inherent to the process and compared with prior residual stress-characterization studies in additive-manufactured metallic structures.

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

    SciTech Connect

    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.

  12. Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

    PubMed

    Eldefrawi, M E; Eldefrawi, A T; Wilson, D B

    1975-09-23

    Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.

  13. Role of Cysteine Residues in the Structure, Stability, and Alkane Producing Activity of Cyanobacterial Aldehyde Deformylating Oxygenase

    PubMed Central

    Hayashi, Yuuki; Yasugi, Fumitaka; Arai, Munehito

    2015-01-01

    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. PMID:25837679

  14. Effects of Ca/sup 2 +/ and subunit interactions on surface accessibility of cysteine residues in cardiac troponin

    SciTech Connect

    Ingraham, R.H.; Hodges, R.S.

    1988-08-09

    Rabbit and bovine cardiac troponin (Tn) subunits and complexes were labeled with iodo(/sup 14/C)acetamide in the presence and absence of Ca/sup 2 +/ to determine the effects of tertiary and quaternary structure on exposure of Cys SH groups. This procedure serves both to map region of subunit interaction and the effects of Ca/sup 2 +/-induced conformational change and to indicate which Cys residues should be useful attachment sites for spectroscopic or cross-linking probes. After being labeled, Tn subunits were purified by using reversed-phase HPLC and subjected to tryptic cleavage with or without prior citraconylation. Cys-containing fragments were isolated by RP-HPLC, and the percent labeling was determined. Cys-75 and -92 of TnI were completely accessible to iodoacetamide both when TnI was labeled alone or when in the TnC-TnI complex. Both residues were largely inaccessible when Tn or the TnI-TnT complex was labeled, suggesting burial in the TnI-TnT interface. In contrast, the Cys from the N-terminal region of bovine TnT was stoichiometrically labeled when TnT was labeled alone, in native Tn or in a troponin-tropomyosin complex. Cys-35 and -84 of TnC are located in the nonfunctional Ca/sup 2 +/ binding loop I of cardiac TnC and helix D, respectively. For TnC alone, the percent labelings of Cys-35 and -84 were 11% and 26%, respectively (minus Ca/sup 2 +/), and 16% and 63%, respectively (plus Ca/sup 2 +/). For TnC labeled within Tn, the percent labelings of Cys-35 and -84 were 20% and 52%, respectively (minus Ca/sup 2 +/), and 20% and 78%, respectively (plus Ca/sup 2 +/). The Ca/sup 2 +/-induced exposure of these residues, especially Cys-84, supports the Ca/sup 2 +/-activated model of turkey skeletal TnC derived from crystallographic data.

  15. Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis.

    PubMed

    Williamson, Heather R; Sehanobish, Esha; Shiller, Alan M; Sanchez-Amat, Antonio; Davidson, Victor L

    2017-02-21

    The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

  16. Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

    PubMed Central

    Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S.; Žusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres

    2016-01-01

    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease. PMID:27845418

  17. Improving the Stability of the EC1 Domain of E-cadherin by Thiol Alkylation of the Cysteine Residue

    PubMed Central

    Trivedi, Maulik; Laurence, Jennifer S.; Williams, Todd D.; Middaugh, C. Russell; Siahaan, Teruna J.

    2012-01-01

    The objective of this work was to improve chemical and physical stability of the EC1 protein derived from the extracellular domain of E-cadherin. In solution, the EC1 protein has been shown to form a covalent dimer via a disulfide bond formation followed by physical aggregation and precipitation. To improve solution stability of the EC1 protein, the thiol group of the Cys13 residue in EC1 was alkylated with iodoacetate, iodoacetamide, and maleimide-PEG-5000 to produce thioether derivatives called EC1-IA, EC1-IN, and EC1-PEG. The physical and chemical stabilities of the EC1 derivatives and the parent EC1 were evaluated at various pHs (3.0, 7.0, and 9.0) and temperatures (0, 3, 70 °C). The structural characteristics of each molecule were analyzed by circular dichroism (CD) and fluorescence spectroscopy and the derivatives have similar secondary structure as the parent EC1 protein at pH 7.0. Both EC1-IN and EC1-PEG derivatives showed better chemical and physical stability profiles than did the parent EC1 at pH 7.0. EC1-PEG had the best stability profile compared to EC1-IN and EC1 in solution under various conditions. PMID:22531851

  18. "Keep a low profile": pesticide residue, additives, and freon use in Australian tobacco manufacturing

    PubMed Central

    Chapman, S

    2003-01-01

    Objectives: To review the Australian tobacco industry's knowledge of pesticide residue on Australian tobacco and its policies and practices on resisting calls by tobacco control advocates that consumers should be informed about pesticide residue as well as additives. Methods: Review of previously internal industry documents relevant to pesticides and additives in Australian tobacco located from the Master Settlement Agreement websites. Results: Between 1972 and 1994 Philip Morris Australia was aware that its leaf samples were often contaminated with pesticide residue, sometimes including organochlorine levels described by PM's European laboratories as being "extremely high". Consumers were not advised of the contamination nor products withdrawn. From 1981, the industry also resisted calls to declare fully the extent of use and long term safety data on all additives used in their products. They developed standard public responses that were evasive and misleading and, in 2000, implemented voluntary additive disclosure which allowed the companies to continue to avoid disclosure of any ingredient they deemed to be a trade secret. There was extensive use of ozone depleting freon in Australian tobacco manufacturing. Again, the industry kept this information away from consumers. Conclusions: Australian smokers are unable to make informed decisions about smoking because pesticide and additive disclosure remains voluntary. The Australian government should regulate tobacco to require full disclosure including information on the likely health consequences of inhaling pesticide and additive pyrolysis products. PMID:14645948

  19. Monitoring Residual Solvent Additives and Their Effects in Solution Processed Solar Cells

    NASA Astrophysics Data System (ADS)

    Fogel, Derek M.; Basham, James I.; Engmann, Sebastian; Pookpanratana, Sujitra J.; Bittle, Emily G.; Jurchescu, Oana D.; Gundlach, David J.

    2015-03-01

    High boiling point solvent additives are a widely adopted approach for increasing bulk heterojunction (BHJ) solar cell efficiency. However, experiments show residual solvent can persist for hours after film deposition, and certain common additives are unstable or reactive. We report here on the effects of residual 1,8-diiodooctane on the electrical performance of poly(3-hexylthiophene-2,5-diyl) (P3HT): phenyl-C71-butyric acid methyl ester (PC[71]BM) BHJ photovoltaic cells. We optimized our fabrication process for efficiency at an active layer thickness of 220 nm, and all devices were processed in parallel to minimize unintentional variations between test structures. The one variable in this study is the active layer post spin drying time. Immediately following the cathode deposition, we measured the current-voltage characteristics at one sun equivalent illumination intensity, and performed impedance spectroscopy to quantify charge density, lifetime, and recombination process. Spectroscopic ellipsometry, FTIR, and XPS are also used to monitor residual solvent and correlated with electrical performance. We find that residual additive degrades performance by increasing the series resistance and lowering efficiency, fill factor, and free carrier lifetime.

  20. L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus.

    PubMed

    Shiau, Chia-Yang; Liu, Yu-Tien

    2002-04-12

    L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.

  1. Functional consequences of sulfhydryl modification of the γ-aminobutyric acid transporter 1 at a single solvent-exposed cysteine residue.

    PubMed

    Omoto, Jaison J; Maestas, Matthew J; Rahnama-Vaghef, Ali; Choi, Ye E; Salto, Gerardo; Sanchez, Rachel V; Anderson, Cynthia M; Eskandari, Sepehr

    2012-12-01

    The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of γ-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1-2.5 mM for 5-20 min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.

  2. The secondary structure of apolipoproteins in human HDL3 particles after chemical modification of their tyrosine, lysine, cysteine or arginine residues. A Fourier transform infrared spectroscopy study.

    PubMed

    Herzyk, E; Owen, J S; Chapman, D

    1988-09-02

    Fourier transform infrared spectra of apolipoprotein E-depleted human HDL3 have been obtained in H2O and 2H2O buffers. The absorption bands in the protein amide I and amide II regions (1700-1500 cm-1) were assigned to alpha-helical, disordered and beta-strand/beta-turn structures of apolipoproteins A-I and A-II (apoA-I and apoA-II), the apolipoprotein constituents of HDL3. Modification of HDL3 by tetranitromethane (TNM) treatment, acetylation, reduction plus alkylation and 1,2-cyclohexanedione treatment derivatised tyrosine, lysine, cysteine and arginine residues, respectively, and caused alteration of the secondary structure of the HDL3 apolipoproteins to different extents. Each of the chemical modifications caused changes in the frequency of bands associated with beta-strands/beta-turns, but only TNM treatment of HDL3, as judged by the second- and fourth-derivative spectra, resulted in a shift of the band assigned to the alpha-helical structure of the proteins. In agreement with other workers, only TNM treatment of HDL3 particles was found to inhibit their binding by high-affinity cell membrane receptors. It is proposed, therefore, that receptor recognition of HDL3 particles is dependent on conservation of the alpha-helix structures within apoA-I and apoA-II, and that beta-strand/beta-turn structures are not involved. This conclusion is consistent with the predominance of amphipathic alpha-helical structures in both apolipoproteins and with the relaxed specificity of the receptors which are thought to recognise both apoA-I and apoA-II.

  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.

    PubMed

    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 (VH+CH1) 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.

  4. Nitro-fatty acid reaction with glutathione and cysteine. Kinetic analysis of thiol alkylation by a Michael addition reaction.

    PubMed

    Baker, Laura M S; Baker, Paul R S; Golin-Bisello, Franca; Schopfer, Francisco J; Fink, Mitchell; Woodcock, Steven R; Branchaud, Bruce P; Radi, Rafael; Freeman, Bruce A

    2007-10-19

    Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M(-1)S(-1) and 355 M(-1)S(-1), respectively, at pH 7.4 and 37 degrees C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A2 and 15-deoxy-Delta(12,14)-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-beta-d-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

  5. Evaluation of an eastern shale oil residue as an asphalt additive

    SciTech Connect

    Thomas, K.P.; Harnsberger, P.M.

    1995-09-01

    An evaluation of eastern shale oil (ESO) residue as an asphalt additive to reduce oxidative age hardening and moisture susceptibility was conducted by Western Research Institute (WRI). The ESO residue, have a viscosity of 23.9 Pa{lg_bullet}s at 60{degree}C (140{degree}F), was blended with three different petroleum-derived asphalts, ASD-1, AAK-1, and AAM-1, which are known to be very susceptible to oxidative aging. Rheological and infrared analyses of the unaged and aged asphalts and the blends were then conducted to evaluate oxidative age hardening. In addition, the petroleum-derived asphalts and the blends were coated onto three different aggregates, Lithonia granite (RA), a low-absorption limestone (RD), and a siliceous Gulf Coast gravel (RL), and compacted into briquettes. Successive freeze-thaw cycling was then conducted to evaluate the moisture susceptibility of the prepared briquettes. The rheological analyses of the unaged petroleum-derived asphalts and their respective blends indicate that the samples satisfy the rutting requirement. However, the aging indexes for the rolling thin film oven (RTFO)-aged and RTFO/pressure aging vessel (PAV)-aged samples indicate that the blends are stiffer than the petroleum-derived asphalts. This means that when in service the blends will be more prone to pavement embrittlement and fatigue cracking than the petroleum-derived asphalts. Infrared analyses were also conducted on the three petroleum-derived asphalts and the blends before and after RTFO/PAV aging. In general, upon RTFO/PAV aging, the amounts of carbonyls and sulfoxides in the samples increase, indicating that the addition of the ESO residue does not mitigate the chemical aging (oxidation) of the petroleum-derived asphalts. This information correlates with the rheological data and the aging indexes that were calculated for the petroleum-derived asphalts and the blends.

  6. Effect of additives on the tensile performance and protein solubility of industrial oilseed residual based plastics.

    PubMed

    Newson, William R; Kuktaite, Ramune; Hedenqvist, Mikael S; Gällstedt, Mikael; Johansson, Eva

    2014-07-16

    Ten chemical additives were selected from the literature for their proposed modifying activity in protein-protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals.

  7. Dynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox active cysteine residues

    PubMed Central

    Zhang, Yan; Romero, Hector; Salinas, Gustavo; Gladyshev, Vadim N

    2006-01-01

    Background Selenocysteine (Sec) is co-translationally inserted into protein in response to UGA codons. It occurs in oxidoreductase active sites and often is catalytically superior to cysteine (Cys). However, Sec is used very selectively in proteins and organisms. The wide distribution of Sec and its restricted use have not been explained. Results We conducted comparative genomics and phylogenetic analyses to examine dynamics of Sec decoding in bacteria at both selenium utilization trait and selenoproteome levels. These searches revealed that 21.5% of sequenced bacteria utilize Sec, their selenoproteomes have 1 to 31 selenoproteins, and selenoprotein-rich organisms are mostly Deltaproteobacteria or Firmicutes/Clostridia. Evolutionary histories of selenoproteins suggest that Cys-to-Sec replacement is a general trend for most selenoproteins. In contrast, only a small number of Sec-to-Cys replacements were detected, and these were mostly restricted to formate dehydrogenase and selenophosphate synthetase families. In addition, specific selenoprotein gene losses were observed in many sister genomes. Thus, the Sec/Cys replacements were mostly unidirectional, and increased utilization of Sec by existing protein families was counterbalanced by loss of selenoprotein genes or entire selenoproteomes. Lateral transfers of the Sec trait were an additional factor, and we describe the first example of selenoprotein gene transfer between archaea and bacteria. Finally, oxygen requirement and optimal growth temperature were identified as environmental factors that correlate with changes in Sec utilization. Conclusion Our data reveal a dynamic balance between selenoprotein origin and loss, and may account for the discrepancy between catalytic advantages provided by Sec and the observed low number of selenoprotein families and Sec-utilizing organisms. PMID:17054778

  8. Quantum mechanics/molecular mechanics modeling of covalent addition between EGFR-cysteine 797 and N-(4-anilinoquinazolin-6-yl) acrylamide.

    PubMed

    Capoferri, Luigi; Lodola, Alessio; Rivara, Silvia; Mor, Marco

    2015-03-23

    Irreversible epidermal growth factor receptor (EGFR) inhibitors can circumvent resistance to first-generation ATP-competitive inhibitors in the treatment of nonsmall-cell lung cancer. They covalently bind a noncatalytic cysteine (Cys797) at the surface of EGFR active site by an acrylamide warhead. Herein, we used a hybrid quantum mechanics/molecular mechanics (QM/MM) potential in combination with umbrella sampling in the path-collective variable space to investigate the mechanism of alkylation of Cys797 by the prototypical covalent inhibitor N-(4-anilinoquinazolin-6-yl) acrylamide. Calculations show that Cys797 reacts with the acrylamide group of the inhibitor through a direct addition mechanism, with Asp800 acting as a general base/general acid in distinct steps of the reaction. The obtained reaction free energy is negative (ΔA = -12 kcal/mol) consistent with the spontaneous and irreversible alkylation of Cys797 by N-(4-anilinoquinazolin-6-yl) acrylamide. Our calculations identify desolvation of Cys797 thiolate anion as a key step of the alkylation process, indicating that changes in the intrinsic reactivity of the acrylamide would have only a minor impact on the inhibitor potency.

  9. Interaction of 5'-P-sulfonylbenzoyl adenosine with cysteine residues of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

    SciTech Connect

    El-Maghrabi, M.R.; Lively, M.O.; Pilkis, S.J.

    1987-05-01

    The kinase and bisphosphatase reactions of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase appear to be catalyzed at separate active sites. The kinase site contains 3 cysteinyl residues that are important for sugar phosphate binding but not for ATP binding. These groups are readily alkylated with iodoacetamide which decreases by 15-fold the affinity for Fru 6-P but also increases the maximal velocity of the reaction by the same extent. Incubation of the enzyme with 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP analog, has no effect on the bisphosphatase activity but inactivates the kinase. The addition of dithiothreitol completely reactivates the kinase, suggesting that the reagent affected sulfhydryl groups critical for sugar phosphate binding and not the ATP site of the enzyme. Similarly, 8-Azido-ATP/UV-photoinactivated enzyme is also reactivated by dithiothreitol and involves the same sulfhydryl groups, since alkylation of the latter with iodoacetamide protects the enzyme from inactivation by FSBA and from 8-azido ATP. Cyanogen bromide cleavage of enzyme that had been alkylated with iodo(I-/sup 14/C)acetamide yielded a 20,000 dalton peptide which contained the three cysteinyl residues. It is concluded that the site of action of ATP analogs to inactivate the kinase are these cysteinyl residues rather than the ATP binding site per se.

  10. Evaluation of an eastern shale oil residue as an asphalt additive

    SciTech Connect

    Thomas, K.P.; Harnsberger, P.M.

    1995-12-19

    An evaluation of eastern shale oil (ESO) residue as an asphalt additive to reduce oxidative age-hardening and moisture susceptibility was conducted. The ESO residue, having a viscosity of 23.9 Pa{sm_bullet}s at 60{degrees}C (140{degrees}F), was blended with three different petroleum-derived asphalts, AAD-1, AAK-1, and AAM-1, that are known to be very susceptible to oxidative aging. Rheological and infrared analyses of the unaged and aged asphalts and the blends were then conducted to evaluate oxidative age-hardening. In addition, the petroleum-derived asphalts and the blends were coated onto three different aggregates, Lithonia granite (RA), a low-absorption limestone (RD), and a silicious Gulf Coast gravel (RL), and compacted into briquets. Successive freeze-thaw cycling was then conducted to evaluate the moisture susceptibility of the prepared briquets. The abbreviations used above for the asphalts and the aggregates are part of the Strategic Highway Research Program nomenclature.

  11. Hybrid Residual Flexibility/Mass-Additive Method for Structural Dynamic Testing

    NASA Technical Reports Server (NTRS)

    Tinker, M. L.

    2003-01-01

    A large fixture was designed and constructed for modal vibration testing of International Space Station elements. This fixed-base test fixture, which weighs thousands of pounds and is anchored to a massive concrete floor, initially utilized spherical bearings and pendulum mechanisms to simulate Shuttle orbiter boundary constraints for launch of the hardware. Many difficulties were encountered during a checkout test of the common module prototype structure, mainly due to undesirable friction and excessive clearances in the test-article-to-fixture interface bearings. Measured mode shapes and frequencies were not representative of orbiter-constrained modes due to the friction and clearance effects in the bearings. As a result, a major redesign effort for the interface mechanisms was undertaken. The total cost of the fixture design, construction and checkout, and redesign was over $2 million. Because of the problems experienced with fixed-base testing, alternative free-suspension methods were studied, including the residual flexibility and mass-additive approaches. Free-suspension structural dynamics test methods utilize soft elastic bungee cords and overhead frame suspension systems that are less complex and much less expensive than fixed-base systems. The cost of free-suspension fixturing is on the order of tens of thousands of dollars as opposed to millions, for large fixed-base fixturing. In addition, free-suspension test configurations are portable, allowing modal tests to be done at sites without modal test facilities. For example, a mass-additive modal test of the ASTRO-1 Shuttle payload was done at the Kennedy Space Center launch site. In this Technical Memorandum, the mass-additive and residual flexibility test methods are described in detail. A discussion of a hybrid approach that combines the best characteristics of each method follows and is the focus of the study.

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

    PubMed Central

    2017-01-01

    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. PMID:28299326

  13. Titration of the bacteriorhodopsin Schiff base involves titration of an additional protein residue.

    PubMed

    Zadok, Uri; Asato, Alfred E; Sheves, Mordechai

    2005-06-14

    The retinal protein protonated Schiff base linkage plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment, the Schiff base (SB) is titrated with a pK(a) of approximately 13, but following light absorption, it experiences a decrease in the pK(a) and undergoes several alterations, including a deprotonation process. We have studied the SB titration using retinal analogues which have intrinsically lower pK(a)'s which allow for SB titrations over a much lower pH range. We found that above pH 9 the channel for the SB titration is perturbed, and the titration rate is considerably reduced. On the basis of studies with several mutants, it is suggested that the protonation state of residue Glu204 is responsible for the channel perturbation. We suggest that above pH 12 a channel for the SB titration is restored probably due to titration of an additional protein residue. The observations may imply that during the bR photocycle and M photointermediate formation the rate of Schiff base protonation from the bulk is decreased. This rate decrease may be due to the deprotonation process of the "proton-releasing complex" which includes Glu204. In contrast, during the lifetime of the O intermediate, the protonated SB is exposed to the bulk. Possible implications for the switch mechanism, and the directionality of the proton movement, are discussed.

  14. Survey of residue levels of organic solvents in "existing food additives" and health food materials by head-space GC.

    PubMed

    Uematsu, Yoko; Ogimoto, Mami; Suzuki, Kumi; Kabashima, Junichirou; Ito, Koichi; Nakazato, Mitsuo

    2008-01-01

    Organic solvent residue levels in "Existing Food Additives" (n=145), health food materials (n=23), and commercial health food products (n=19) were surveyed. Ethanol was the dominant solvent found in the samples, suggesting its use in the manufacturing process. Methanol, acetone, 2-propanol and ethyl acetate was also found. No residual solvent exceeded the limits set by the Food Sanitation Law.

  15. Nitrification in lake sediment with addition of drinking water treatment residuals.

    PubMed

    Wang, Changhui; Liu, Juanfeng; Wang, Zhixin; Pei, Yuansheng

    2014-06-01

    Drinking water treatment residuals (WTRs), non-hazardous by-products generated during potable water production, can effectively reduce the lake internal phosphorus (P) loading and improve water quality in lakes. It stands to reason that special attention regarding the beneficial reuse of WTRs should be given not only to the effectiveness of P pollution control, but also to the effects on the migration and transformation of other nutrients (e.g., nitrogen (N)). In this work, based on laboratory enrichment tests, the effects of WTRs addition on nitrification in lake sediment were investigated using batch tests, fluorescence in situ hybridization, quantitative polymerase chain reaction and phylogenetic analysis techniques. The results indicated that WTRs addition had minor effects on the morphologies of AOB and NOB; however, the addition slightly enhanced the sediment nitrification potential from 12.8 to 13.2 μg-N g(-1)-dry sample h(-1) and also increased the ammonia oxidation bacteria (AOB) and nitrite oxidizing bacteria (NOB) abundances, particularly the AOB abundances (P < 0.05), which increased from 1.11 × 10(8) to 1.31 × 10(8) copies g(-1)-dry sample. Moreover, WTRs addition was beneficial to the enrichment of Nitrosomonas and Nitrosospira multiformis and promoted the emergence of a new Nitrospira cluster, causing the increase in AOB and NOB diversities. Further analysis showed that the variations of nitrification in lake sediment after WTRs addition were primarily due to the decrease of bioavailable P, the introduction of new nitrifiers and the increase of favorable carriers for microorganism attachment in sediments. Overall, these results suggested that WTRs reuse for the control of lake internal P loading would also lead to conditions that are beneficial to nitrification.

  16. 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.

    PubMed

    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.

  17. Determining the Volume of Additive Solution and Residual Plasma in Whole Blood Filtered and Buffy Coat Processed Red Cell Concentrates

    PubMed Central

    Jordan, Andrew; Acker, Jason P.

    2016-01-01

    Summary Background Residual plasma in transfused red cell concentrates (RCCs) has been associated with adverse transfusion outcomes. Despite this, there is no consensus on the standard procedure for measuring residual plasma volume. Methods The volumes of residual plasma and additive solution were measured in RCCs processed using two separation methods: whole blood filtration (WBF) and buffy coat (BC)/RCC filtration. The concentration of mannitol and albumin in RCC components was measured using colorimetric assays. Mannitol concentration was used to calculate additive solution volume. Residual plasma volume was calculated using two methods. Results Calculated RCC supernatant volumes were much lower in BC-processed components compared to WBF-processed components (BC = 97 ± 6 ml, WBF = 109 ± 4 ml; p < 0.05). Calculated additive solution volumes were greater in WBF- than in BC-processed components (BC = 81 ± 4 ml, WBF = 105 ± 2 ml; p < 0.05). Absolute residual plasma volume varied significantly based on the calculation method used. Conclusion Disparity between plasma volume calculation methods was observed. Efforts should be made to standardize residual plasma volume measurement methods in order to accurately assess the impact of residual plasma on transfusion outcomes. PMID:27330533

  18. A Quantitative Mass-Spectrometry Platform to Monitor Changes in Cysteine Reactivity

    PubMed Central

    Qian, Yu; Weerapana, Eranthie

    2017-01-01

    Summary Cysteine residues on proteins serve diverse functional roles in catalysis and regulation and are susceptible to numerous posttranslational modifications. Methods to monitor the reactivity of cysteines within the context of a complex proteome have facilitated the identification and functional characterization of cysteine residues on disparate proteins. Here, we describe the use of a cysteine-reactive iodoacetamide probe coupled to isotopically labeled, cleavable linkers to identify and quantify cysteine-reactivity changes from two biological samples. PMID:27778278

  19. Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs.

    PubMed

    Danaher, Martin; Campbell, Katrina; O'Keeffe, Michael; Capurro, Emiliana; Kennedy, Glenn; Elliott, Christopher T

    2008-01-01

    A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002-2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 microg kg(-1), respectively. DNC residues were found at >200 microg kg(-1) in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 microg kg(-1). Analysis of poultry, carried out as part of official food inspection in the period 2004-2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 microg kg(-1), to approximately 7%. Low levels of DNC residues continue to be found in <2% of egg samples.

  20. Reaction of Cysteine(s) with Phenyldichloroarsine

    DTIC Science & Technology

    1990-01-01

    acetyl -L- cysteine reacted like the two Cys-l 3 residucs are spatially not In close L-Cys teine- a nd Iformed a 1:1 adduct when the ratio proximity...were obtained when L- cysteine methyl ester and N- acetyl -L- cysteine 0.0 were used in our studies. For the N- acetyl -L- cysteine , the sample decomposed...the N- acetyl derivatives of L- cysteine .... ... .. , also formed 1:1 adducts, Another possibility is that solvent plays a role in the adducts fornmd

  1. Associating cooking additives with sodium hydroxide to pretreat bamboo residues for improving the enzymatic saccharification and monosaccharides production.

    PubMed

    Huang, Caoxing; He, Juan; Wang, Yan; Min, Douyong; Yong, Qiang

    2015-10-01

    Cooking additive pulping technique is used in kraft mill to increase delignification degree and pulp yield. In this work, cooking additives were firstly applied in the sodium hydroxide pretreatment for improving the bioconversion of bamboo residues to monosaccharides. Meanwhile, steam explosion and sulfuric acid pretreatments were also carried out on the sample to compare their impacts on monosaccharides production. Results indicated that associating anthraquinone with sodium hydroxide pretreatment showed the best performance in improving the original carbohydrates recovery, delignification, enzymatic saccharification, and monosaccharides production. After consecutive pretreatment and enzymatic saccharification process, 347.49 g, 307.48 g, 142.93 g, and 87.15 g of monosaccharides were released from 1000 g dry bamboo residues pretreated by sodium hydroxide associating with anthraquinone, sodium hydroxide, steam explosion and sulfuric acid, respectively. The results suggested that associating cooking additive with sodium hydroxide is an effective pretreatment for bamboo residues to enhance enzymatic saccharification for monosaccharides production.

  2. Chemical Protein Modification through Cysteine.

    PubMed

    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.

  3. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1999-01-01

    Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Glyn-Cys-Glu10, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25-65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu10 in the presence of Arg5 leads to the formation of Cys-Arg5 in 50% yield. The significance of these results for prebiotic chemistry is discussed.

  4. The effect of cysteine oxidation on isolated hepatocytes.

    PubMed Central

    Viña, J; Saez, G T; Wiggins, D; Roberts, A F; Hems, R; Krebs, H A

    1983-01-01

    Isolated hepatocytes incubated with 4mM-cysteine lose reduced glutathione, adenine nucleotides and intracellular enzymes, thus showing extensive membrane damage. The toxic effects of cysteine are enhanced by NH4Cl. Lactate, ethanol and unsaturated fatty acids afford significant protection against cysteine-induced cytoxicity. Addition of catalase to the incubation medium also protected against cysteine toxicity, indicating that H2O2 formed during the oxidation of cysteine is involved in the toxic effects observed. Under anaerobic conditions cysteine did not cause leakage of lactate dehydrogenase from cells, confirming that rapid autoxidation is an essential condition for development of the toxic effects of cysteine. PMID:6870855

  5. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

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

    2015-01-01

    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. PMID:25416785

  6. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.

    PubMed

    Salsbury, Freddie R; Knutson, Stacy T; Poole, Leslie B; Fetrow, Jacquelyn S

    2008-02-01

    Cysteine sulfenic acid (Cys-SOH), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. This post-translational modification is not random: specific features near the cysteine control its reactivity. To identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known Cys-SOH sites) was compiled. Modifiable cysteines are found in proteins from most structural classes and many functional classes, but have no propensity for any one type of protein secondary structure. To identify features affecting cysteine reactivity, these sites were analyzed using both functional site profiling and electrostatic analysis. Overall, the solvent exposure of modifiable cysteines is not different from the average cysteine. The combined sequence, structure, and electrostatic approaches reveal mechanistic determinants not obvious from overall sequence comparison, including: (1) pKaS of some modifiable cysteines are affected by backbone features only; (2) charged residues are underrepresented in the structure near modifiable sites; (3) threonine and other polar residues can exert a large influence on the cysteine pKa; and (4) hydrogen bonding patterns are suggested to be important. This compilation of Cys-SOH modification sites and their features provides a quantitative assessment of previous observations and a basis for further analysis and prediction of these sites. Agreement with known experimental data indicates the utility of this combined approach for identifying mechanistic determinants at protein functional sites.

  7. Characterization and comparative study of coal combustion residues from a primary and additional flue gas secondary desulfurization process

    SciTech Connect

    Gomes, S.; Francois, M.; Evrard, O.; Pellissier, C.

    1998-11-01

    An extensive characterization and comparative study was done on two flue gas desulfurization (FGD) residues derived from the same coal. LR residues (originated from Loire/Rhone in the south of Lyon, France) are obtained after a primary desulfurization process (SO{sub 2} is trapped by reaction with CaO at a temperature of about 1100 C), and LM residues (originating from La Maxe, near Metz in the east of France) are obtained after an additional secondary desulfurization process (SO{sub 2} is removed further by reaction with Ca(OH){sub 2} at a temperature of about 120 C). Various and complementary investigation methods were used to determine their chemical, physical, and mineralogical properties: x-ray fluorescence and diffraction, scanning electron microscopy, differential scanning calorimetry, thermogravimetry analysis, granulometric distribution, pycnometric density, BET specific surface area and pH, conductivity measurements, and chemical analysis of their insoluble fraction. The FGD residues contain basically two main components: a silico-aluminous fly ash part and calcic FGD phases. In the LR residues the two components can be considered as independent, whereas they are linked in the LM residues because chemical reactions have occurred, leading to the formation of silico-calcic gel CSH, hydrated aluminate AFm, and AFt phases.

  8. Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

    PubMed

    Tiwari, Pratibha; Singh, Noopur; Dixit, Aparna; Choudhury, Devapriya

    2014-10-01

    The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes.

  9. Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues.

    PubMed Central

    Amara, Amro A; Rehm, Bernd H A

    2003-01-01

    The class II PHA (polyhydroxyalkanoate) synthases [PHA(MCL) synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHA(MCL)s using CoA thioesters of medium-chain-length 3-hydroxy fatty acids (C6-C14) as a substrate. Only recently PHA(MCL) synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHA(MCL) synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the alpha/beta-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the alpha/beta-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved gamma-turns of the alpha/beta-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved

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

    PubMed

    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.

  11. 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.

    PubMed

    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).

  12. Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.

    PubMed

    Urzica, Eugen; Pierik, Antonio J; Mühlenhoff, Ulrich; Lill, Roland

    2009-06-09

    Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N- and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in Escherichia coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a (55)Fe radiolabeling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modeling using a structurally but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.

  13. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env.

    PubMed

    Heydarchi, Behnaz; Center, Rob J; Bebbington, Jonathan; Cuthbertson, Jack; Gonelli, Christopher; Khoury, Georges; Mackenzie, Charlene; Lichtfuss, Marit; Rawlin, Grant; Muller, Brian; Purcell, Damian

    2017-04-01

    We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.

  14. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env

    PubMed Central

    Center, Rob J.; Bebbington, Jonathan; Cuthbertson, Jack; Khoury, Georges; Lichtfuss, Marit; Rawlin, Grant; Purcell, Damian

    2017-01-01

    ABSTRACT We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection. PMID:27996375

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

    PubMed

    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.

  16. The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

    PubMed Central

    Calvete, J. J.; Moreno-Murciano, M. P.; Sanz, L.; Jürgens, M.; Schrader, M.; Raida, M.; Benjamin, D. C.; Fox, J. W.

    2000-01-01

    The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed. PMID:10933502

  17. Additive effect of waste tire on the hydrogenolysis reaction of coal liquefaction residue

    SciTech Connect

    Motoyuki Sugano; Daigorou Onda; Kiyoshi Mashimo

    2006-12-15

    A numerous amount of waste tire is landfilled or dumped all over the world, which causes environmental problems, such as destruction of natural places and the risk of fires. On the other hand, the coal liquefaction residue (CLR) is produced in 30% yield through the process supporting unit (PSU) of the NEDOL coal liquefaction process. Therefore, the investigation on an effective method for utilization of waste tire and CLR is required. In this study, the simultaneous hydrogenolysis of CLR and pulverized waste tire was carried out by using tetralin. The yields in the simultaneous hydrogenolysis were compared with algebraic sum of the yields of the individual hydrogenolyses of waste tire alone and coal alone. In the simultaneous hydrogenolysis, the synergistic effects to upgrading, such as an increase in the yield of the oil constituent and a decrease in the yield of the asphaltene constituent, occurred because of the stabilization of asphaltenic radicals from CLR with aliphatic radicals from tire. The decrease in asphaltene yield in the simultaneous hydrogenolysis was pronounced with the increase in the tire:CLR ratio because the solvent effects of liquefied tire, such as stabilization of radicals, hydrogen shuttling, and heat transfer, were enhanced. Accordingly, it is estimated that the simultaneous hydrogenolysis of CLR and waste tire is an effective method for processing both materials. 15 refs., 3 figs., 2 tabs.

  18. Impact of addition of amendments on the degradation of DDT and its residues partitioned on soil.

    PubMed

    Singh, Swatantra P; Bose, Purnendu; Guha, Saumyen; Gurjar, Suresh K; Bhalekar, Santosh

    2013-08-01

    Market-grade DDT used for mosquito control and other purposes is a mixture of 4,4-DDT, 2,4-DDT and smaller amounts of 4,4-DDD, 2,4-DDD, 4,4-DDE and 4,4-DDMU. All above components (together known as DDTr) are strongly hydrophobic and hence are present in the environment predominantly in the soil/sediment phases. The persistence of DDTr and the feasibility of attenuation of DDTr concentration in soil matrix through addition of amendments is a subject of ongoing interest. The objective of this study was to compare the decline of soil-partitioned DDTr concentration through, (1) the natural attenuation process, (2) enhanced aerobic and anaerobic biodegradation processes involving addition of acclimatized seed and co-metabolites and (3) Nanoscale Zero Valent Iron (NZVI) addition. The extent of decline in soil DDTr concentration in control experiments, where biodegradation and photolysis were excluded, was around 10-15% in ∼100d. Extent of DDTr decline in natural attenuation experiments was 25-30% and 15-20% under aerobic and anaerobic conditions respectively. In enhanced biodegradation experiments, addition of acclimatized seed and/or co-metabolites did not enhance the extent of DDTr attenuation over and above the natural attenuation rates both in aerobic and anaerobic conditions. It thus appeared that biodegradation of DDTr adsorbed on soil was severely limited and controlled by desorption and consequent bioavailability of DDTr in the aqueous phase. In case of NZVI addition, the rate of DDTr degradation was much faster, with 40% decrease in DDTr concentration within 28h of NZVI addition. Here, the faster DDTr degradation may be through direct electron transfer between NZVI particles and DDTr molecules adsorbed on soil. Increase in the concentration of 4,4-DDD and 2,4-DDD during NZVI addition suggest that these compounds are either intermediate or end products of DDT degradation process.

  19. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  20. Investigation of the causes for the occurrence of residues of the anticoccidial feed additive nicarbazin in commercial poultry.

    PubMed

    O'Keeffe, M; Capurro, E; Danaher, M; Campbell, K; Elliott, C T

    2007-09-01

    Investigations were undertaken to identify causes for the occurrence of high levels of the zootechnical feed additive nicarbazin in broiler liver at slaughter. The first investigation on 32 commercial broiler flocks involved sampling and analysis for nicarbazin (as dinitrocarbanilide, DNC) in liver from birds during a 3-10-day period after withdrawal of nicarbazin from their feed and before commercial slaughter. DNC residues in liver samples of broilers scheduled as being withdrawn from nicarbazin for > or =6 days ranged from 20 to >1600 microg kg(-1) (the specified withdrawal period for nicarbazin is 5 days and the Joint Expert Committee on Food Additives (JECFA) maximum residue limit (MRL) is 200 microg kg(-1) liver). Further on-farm investigations on 12 of these flocks, selected on the basis of the feeding system in use and the levels of DNC residues determined in liver, identified issues in feed management contributing to elevated residues in broiler liver. A significant correlation (0.81, p < 0.01, n = 10) between DNC residues in liver samples and in feed samples from the feeding pans was observed. The second investigation on 12 commercial broiler flocks involved sampling and analysis for DNC in liver samples and feed samples from feeding pans and from the feed mill at the three thinnings of birds for commercial slaughter. In the case of one flock, a clear relationship between nicarbazin in feed from the feed mill (10.5 mg kg(-1) DNC), in feed from the feeding pans (6.6 mg kg(-1) DNC) and in liver (583 microg kg(-1) DNC) at first thinning (9 days scheduled withdrawal from nicarbazin) was observed. Such a clear relationship was not observed in other cases, particularly at second and third thinnings, pointing to re-exposure of birds to nicarbazin late in the flock production cycle, probably from the litter. Guidelines outlining best farm practice to eliminate nicarbazin residues in poultry have been published in booklet and poster format for broiler producers

  1. An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

    PubMed Central

    Makino, Yuki; Sato, Takaaki; Kawamura, Hiroki; Hachisuka, Shin-ichi; Takeno, Ryo; Imanaka, Tadayuki; Atomi, Haruyuki

    2016-01-01

    Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization. PMID:27857065

  2. Cysteines in the stalk of the nipah virus G glycoprotein are located in a distinct subdomain critical for fusion activation.

    PubMed

    Maar, Dianna; Harmon, Brooke; Chu, David; Schulz, Belinda; Aguilar, Hector C; Lee, Benhur; Negrete, Oscar A

    2012-06-01

    Paramyxoviruses initiate entry through the concerted action of the tetrameric attachment glycoprotein (HN, H, or G) and the trimeric fusion glycoprotein (F). The ectodomains of HN/H/G contain a stalk region important for oligomeric stability and for the F triggering resulting in membrane fusion. Paramyxovirus HN, H, and G form a dimer-of-dimers consisting of disulfide-linked dimers through their stalk domain cysteines. The G attachment protein stalk domain of the highly pathogenic Nipah virus (NiV) contains a distinct but uncharacterized cluster of three cysteine residues (C146, C158, C162). On the basis of a panoply of assays, we report that C158 and C162 of NiV-G likely mediate covalent subunit dimerization, while C146 mediates the stability of higher-order oligomers. For HN or H, mutation of stalk cysteines attenuates but does not abrogate the ability to trigger fusion. In contrast, the NiV-G stalk cysteine mutants were completely deficient in triggering fusion, even though they could still bind the ephrinB2 receptor and associate with F. Interestingly, all cysteine stalk mutants exhibited constitutive exposure of the Mab45 receptor binding-enhanced epitope, previously implicated in F triggering. The enhanced binding of Mab45 to the cysteine mutants relative to wild-type NiV-G, without the addition of the receptor, implicates the stalk cysteines in the stabilization of a pre-receptor-bound conformation and the regulation of F triggering. Sequence alignments revealed that the stalk cysteines were adjacent to a proline-rich microdomain unique to the Henipavirus genus. Our data propose that the cysteine cluster in the NiV-G stalk functions to maintain oligomeric stability but is more importantly involved in stabilizing a unique microdomain critical for triggering fusion.

  3. An iron–oxygen intermediate formed during the catalytic cycle of cysteine dioxygenase† †Electronic supplementary information (ESI) available: Experimental and computational details. See DOI: 10.1039/c6cc03904a Click here for additional data file.

    PubMed Central

    Tchesnokov, E. P.; Faponle, A. S.; Davies, C. G.; Quesne, M. G.; Turner, R.; Fellner, M.; Souness, R. J.; Wilbanks, S. M.

    2016-01-01

    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. PMID:27297454

  4. Influence of Residue and Nitrogen Fertilizer Additions on Carbon Mineralization in Soils with Different Texture and Cropping Histories

    PubMed Central

    Chen, Xianni; Wang, Xudong; Liebman, Matt; Cavigelli, Michel; Wander, Michelle

    2014-01-01

    To improve our ability to predict SOC mineralization response to residue and N additions in soils with different inherent and dynamic organic matter properties, a 330-day incubation was conducted using samples from two long-term experiments (clay loam Mollisols in Iowa [IAsoil] and silt loam Ultisols in Maryland [MDsoil]) comparing conventional grain systems (Conv) amended with inorganic fertilizers with 3 yr (Med) and longer (Long), more diverse cropping systems amended with manure. A double exponential model was used to estimate the size (Ca, Cs) and decay rates (ka, ks) of active and slow C pools which we compared with total particulate organic matter (POM) and occluded-POM (OPOM). The high-SOC IAsoil containing highly active smectite clays maintained smaller labile pools and higher decay rates than the low-SOC MDsoil containing semi-active kaolinitic clays. Net SOC loss was greater (2.6 g kg−1; 8.6%) from the IAsoil than the MDsoil (0.9 g kg−1, 6.3%); fractions and coefficients suggest losses were principally from IAsoil’s resistant pool. Cropping history did not alter SOC pool size or decay rates in IAsoil where rotation-based differences in OPOM-C were small. In MDsoil, use of diversified rotations and manure increased ka by 32% and ks by 46% compared to Conv; differences mirrored in POM- and OPOM-C contents. Residue addition prompted greater increases in Ca (340% vs 230%) and Cs (38% vs 21%) and decreases in ka (58% vs 9%) in IAsoil than MDsoil. Reduced losses of SOC from residue-amended MDsoil were associated with increased OPOM-C. Nitrogen addition dampened CO2-C release. Clay type and C saturation dominated the IAsoil’s response to external inputs and made labile and stable fractions more vulnerable to decay. Trends in OPOM suggest aggregate protection influences C turnover in the low active MDsoil. Clay charge and OPOM-C contents were better predictors of soil C dynamics than clay or POM-C contents. PMID:25078458

  5. Modeling and simulation of cooling-induced residual stresses in heated particulate mixture depositions in additive manufacturing

    NASA Astrophysics Data System (ADS)

    Zohdi, T. I.

    2015-10-01

    One key aspect of many additive manufacturing processes is the deposition of heated mixtures of particulate materials onto surfaces, which then bond and cool, leading to complex microstructures and possible residual stresses. The overall objective of this work is to construct a straightforward computational approach that researchers in the field can easily implement and use as a numerically-efficient simulation and design tool. Specifically because multifield coupling is present, a recursive, staggered, temporally-adaptive, finite difference time domain scheme is developed to resolve the internal microstructural thermal and mechanical fields, accounting for the simultaneous elasto-plasticity and damage. The time step adaptation allows the numerical scheme to iteratively resolve the changing physical fields by refining the time-steps during phases of the process when the system is undergoing large changes on a relatively small time-scale and can also enlarge the time-steps when the processes are relatively slow. The spatial discretization grids are uniform and dense. The deposited microstructure is embedded into spatial discretization. The regular grid allows one to generate a matrix-free iterative formulation which is amenable to rapid computation and minimal memory requirements, making it ideal for laptop computation. Numerical examples are provided to illustrate the approach. This formulation is useful for material scientists who seek ways to deposit such materials while simultaneously avoiding inadvertent excessive residual stresses.

  6. Mechanistic study for immobilization of cysteine-labeled oligopeptides on UV-activated surfaces.

    PubMed

    Ong, Lian Hao; Ding, Xiaokang; Yang, Kun-Lin

    2014-10-01

    In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules.

  7. Evaluation of a zirconium additive for the mitigation of molten ash formation during combustion of residual fuel oil

    SciTech Connect

    1996-12-01

    Florida Power & Light Company (FP&L) currently fires a residual fuel oil (RFO) containing catalyst fines, which results in a troublesome black aluminosilicate liquid phase that forms on heat-transfer surfaces, remains molten, and flows to the bottom of the boiler. When the unit is shut down for a scheduled outage, this liquid phase freezes to a hard black glass that damages the contracting waterwalls of the boiler. Cleaning the boiler bottom and repairing damaged surfaces increase the boiler downtime, at a significant cost to FP&L. The Energy & Environmental Research Center (EERC) proposed to perform a series of tests for FP&L to evaluate the effectiveness of a zirconium additive to modify the mechanism that forms this liquid phase, resulting in the formation of a dry refractory phase that may be easily handled during cleanup of the boiler.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Modifications of substituted seryl and threonyl residues in phosphopeptides and a polysialoglycoprotein by beta-elimination and nucleophile additions.

    PubMed

    Mega, T; Nakamura, N; Ikenaka, T

    1990-01-01

    The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.

  10. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  11. Reclamation of acidic mine residues by creation of technosoils with the addition of biochar and marble waste

    NASA Astrophysics Data System (ADS)

    Moreno-Barriga, Fabián; Díaz, Vicente; Acosta, José; Faz, Ángel; Zornoza, Raul

    2016-04-01

    This study reports the short-term effect of biochar and marble waste addition for the reclamation of acidic mine residues. A lab incubation was carried out for 90 days. Biochars derived from pig manure (PM), crop residues (CR) and municipal solid waste (MSW) were added to the soil at a rate of 20 g kg-1. The marble waste (MW) was added at a rate of 200 g kg-1. Bochars and MW were applied independently and combined. A control soil was used without application of amendments. The evolution of different physical, chemical and biochemical properties and availability of heavy metals was periodically monitored. Results showed that original pH (2.8) was increased with all amendments, those samples containing MW being the ones with the highest pH (~8.0). The electrical conductivity (EC) decreased from 6.6 to 3.0-4.5 mS cm-1 in all the treatments receiving MW. Soil organic C (SOC) increased in all samples receiving biochar up to 18-20 g kg-1, with no shifts during the 90 d incubation, indicating the high stability of the C supplied. Recalcitrant organic C accounted for ~90-98% of the SOC. No significant effect of amendment addition was observed for carbohydrates, soluble C, microbial biomass C and β-glucosidase activity. However, arylesterase activity increased with amendments, highly related to pH. The availability of heavy metals decreased up to 90-95% owing to the addition of amendments, mainly in samples containing MW. The MW provided conditions to increase pH and decrease EC and metals mobility. Biochar was an effective strategy to increase SOC, recalcitrant C and AS, essential to create soil structure. However, a labile source of organic matter should be added together with the proposed amendments to promote the activation of microbial communities. Acknowledgement : This work has been funded by Fundación Séneca (Agency of Science and Technology of the Region of Murcia, Spain) by the project 18920/JLI/13

  12. Effects of particle size and acid addition on the remediation of chromite ore processing residue using ferrous sulfate.

    PubMed

    Jagupilla, Santhi Chandra; Moon, Deok Hyun; Wazne, Mahmoud; Christodoulatos, Christos; Kim, Min Gyu

    2009-08-30

    A bench-scale treatability study was conducted to assess the effects of particle size and acid addition on the remediation of chromite ore processing residue (COPR) using ferrous sulfate. The remediation scheme entailed the chemical reduction of hexavalent chromium [Cr(VI)] and the mitigation of swell potential. Leaching tests and the EQ3/6 geochemical model were used to estimate the acid dosage required to destabilize Cr(VI)-bearing and swell-causing minerals. The model predicted greater acid dosage than that estimated from the batch leaching tests. This indicated that mass transfer limitation may be playing a significant role in impeding the dissolution of COPR minerals following acid addition and hence hindering the remediation of COPR. Cr(VI) concentrations determined by alkaline digestion for the treated samples were less than the current NJDEP standard. However, Cr(VI) concentrations measured by X-ray absorption near edge structure (XANES) were greater than those measured by alkaline digestion. Greater Cr(VI) percentages were reduced for acid pretreated and also for smaller particle size COPR samples. Upon treatment, brownmillerite content was greatly reduced for the acid pretreated samples. Conversely, ettringite, a swell-causing mineral, was not observed in the treated COPR.

  13. Review stapling peptides using cysteine crosslinking.

    PubMed

    Fairlie, David P; Dantas de Araujo, Aline

    2016-11-01

    Stapled peptides are an emerging class of cyclic peptide molecules with enhanced biophysical properties such as conformational and proteolytic stability, cellular uptake and elevated binding affinity and specificity for their biological targets. Among the limited number of chemistries available for their synthesis, the cysteine-based stapling strategy has received considerable development in the last few years driven by facile access from cysteine-functionalized peptide precursors. Here we present some recent advances in peptide and protein stapling where the side-chains of cysteine residues are covalently connected with a range of different crosslinkers affording bisthioether macrocyclic peptides of varying topology and biophysical properties. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 843-852, 2016.

  14. Effect of water treatment additives on lime softening residual trace chemical composition--implications for disposal and reuse.

    PubMed

    Cheng, Weizhi; Roessler, Justin; Blaisi, Nawaf I; Townsend, Timothy G

    2014-12-01

    Drinking water treatment residues (WTR) offer potential benefits when recycled through land application. The current guidance in Florida, US allows for unrestricted land application of lime softening WTR; alum and ferric WTR require additional evaluation of total and leachable concentrations of select trace metals prior to land application. In some cases a mixed WTR is produced when lime softening is accompanied by the addition of a coagulant or other treatment chemical; applicability of the current guidance is unclear. The objective of this research was to characterize the total and leachable chemical content of WTR from Florida facilities that utilize multiple treatment chemicals. Lime and mixed lime WTR samples were collected from 18 water treatment facilities in Florida. Total and leachable concentrations of the WTR were measured. To assess the potential for disposal of mixed WTR as clean fill below the water table, leaching tests were conducted at multiple liquid to solid ratios and under reducing conditions. The results were compared to risk-based soil and groundwater contamination thresholds. Total metal concentrations of WTR were found to be below Florida soil contaminant thresholds with Fe found in the highest abundance at a concentration of 3600 mg/kg-dry. Aluminum was the only element that exceeded the Florida groundwater contaminant thresholds using SPLP (95% UCL = 0.23 mg/L; risk threshold = 0.2 mg/L). Tests under reducing conditions showed elevated concentrations of Fe and Mn, ranging from 1 to 3 orders of magnitude higher than SPLP leachates. Mixed lime WTR concentrations (total and leachable) were lower than the ferric and alum WTR concentrations, supporting that mixed WTR are appropriately represented as lime WTR. Testing of WTR under reducing conditions demonstrated the potential for release of certain trace metals (Fe, Al, Mn) above applicable regulatory thresholds; additional evaluation is needed to assess management options where

  15. Π-Clamp-mediated cysteine conjugation.

    PubMed

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J; Santos, Michael S; Van Voorhis, Troy; Pentelute, Bradley L

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  16. π-Clamp Mediated Cysteine Conjugation

    PubMed Central

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.

    2016-01-01

    Site-selective functionalization of complex molecules is a grand challenge in chemistry. Protecting groups or catalysts must be used to selectively modify one site among many that are similarly reactive. General strategies are rare such the local chemical environment around the target site is tuned for selective transformation. Here we show a four amino acid sequence (Phe-Cys-Pro-Phe), which we call the “π-clamp”, tunes the reactivity of its cysteine thiol for the site-selective conjugation with perfluoroaromatic reagents. We used the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues (e.g. antibodies and cysteine-based enzymes), which was impossible with prior cysteine modification methods. The modified π-clamp antibodies retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates (ADCs) for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach for site-selective chemistry and provides opportunities to modify biomolecules for research and therapeutics. PMID:26791894

  17. π-Clamp-mediated cysteine conjugation

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  18. Investigation on the eco-toxicity of lake sediments with the addition of drinking water treatment residuals.

    PubMed

    Yuan, Nannan; Wang, Changhui; Pei, Yuansheng

    2016-08-01

    Drinking water treatment residuals (WTRs) have a potential to realize eutrophication control objectives by reducing the internal phosphorus (P) load of lake sediments. Information regarding the ecological risk of dewatered WTR reuse in aquatic environments is generally lacking, however. In this study, we analyzed the eco-toxicity of leachates from sediments with or without dewatered WTRs toward algae Chlorella vulgaris via algal growth inhibition testing with algal cell density, chlorophyll content, malondialdehyde content, antioxidant enzyme superoxide dismutase activity, and subcellular structure indices. The results suggested that leachates from sediments unanimously inhibited algal growth, with or without the addition of different WTR doses (10% or 50% of the sediment in dry weight) at different pH values (8-9), as well as from sediments treated for different durations (10 or 180days). The inhibition was primarily the result of P deficiency in the leachates owing to WTR P adsorption, however, our results suggest that the dewatered WTRs were considered as a favorable potential material for internal P loading control in lake restoration projects, as it shows acceptably low risk toward aquatic plants.

  19. Standard addition method for the determination of pharmaceutical residues in drinking water by SPE-LC-MS/MS.

    PubMed

    Cimetiere, Nicolas; Soutrel, Isabelle; Lemasle, Marguerite; Laplanche, Alain; Crocq, André

    2013-01-01

    The study of the occurrence and fate of pharmaceutical compounds in drinking or waste water processes has become very popular in recent years. Liquid chromatography with tandem mass spectrometry is a powerful analytical tool often used to determine pharmaceutical residues at trace level in water. However, many steps may disrupt the analytical procedure and bias the results. A list of 27 environmentally relevant molecules, including various therapeutic classes and (cardiovascular, veterinary and human antibiotics, neuroleptics, non-steroidal anti-inflammatory drugs, hormones and other miscellaneous pharmaceutical compounds), was selected. In this work, a method was developed using ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) and solid-phase extraction to determine the concentration of the 27 targeted pharmaceutical compounds at the nanogram per litre level. The matrix effect was evaluated from water sampled at different treatment stages. Conventional methods with external calibration and internal standard correction were compared with the standard addition method (SAM). An accurate determination of pharmaceutical compounds in drinking water was obtained by the SAM associated with UPLC-MS/MS. The developed method was used to evaluate the occurrence and fate of pharmaceutical compounds in some drinking water treatment plants in the west of France.

  20. Cysteine Modifications in the Pathogenesis of ALS

    PubMed Central

    Valle, Cristiana; Carrì, Maria Teresa

    2017-01-01

    Several proteins are found misfolded and aggregated in sporadic and genetic forms of amyotrophic lateral sclerosis (ALS). These include superoxide dismutase (SOD1), transactive response DNA-binding protein (TDP-43), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), p62, vasolin-containing protein (VCP), Ubiquilin-2 and dipeptide repeats produced by unconventional RAN-translation of the GGGGCC expansion in C9ORF72. Up to date, functional studies have not yet revealed a common mechanism for the formation of such diverse protein inclusions. Consolidated studies have demonstrated a fundamental role of cysteine residues in the aggregation process of SOD1 and TDP43, but disturbance of protein thiols homeostatic factors such as protein disulfide isomerases (PDI), glutathione, cysteine oxidation or palmitoylation might contribute to a general aberration of cysteine residues proteostasis in ALS. In this article we review the evidence that cysteine modifications may have a central role in many, if not all, forms of this disease. PMID:28167899

  1. S-carboxymethyl-L-cysteine.

    PubMed

    Mitchell, Steve C; Steventon, Glyn B

    2012-05-01

    S-carboxymethyl-L-cysteine, the side-chain carboxymethyl derivative of the sulfur-containing amino acid, cysteine, has been known and available for almost 80 years. During this time, it has been put to a variety of uses, but it is within the field of respiratory medicine that, presently, it has found a clinical niche. Early studies indicated that this compound underwent a rather simplistic, predictable pattern of metabolism, whereas later investigations alluded to more subtle interactions with the pathways of intermediary metabolism, as may be expected for an amino acid derivative. In addition, suggestions of polymorphic influences and circadian rhythms within metabolic profiles have emerged. These latter factors may underlie the conflicting reports regarding the therapeutic efficacy of this compound: that it appears to work well in some patients, but has no measurable effects in others. The relevant literature pertaining to the fate of this compound within living systems has been reviewed and a comprehensive précis advanced. Hopefully, this article will serve as a vade mecum for those interested in S-carboxymethyl-L-cysteine and as a catalyst for future research.

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

    PubMed

    Yoon, Aerin; Shin, Jung Won; Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    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.

  3. Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation.

    PubMed

    Srikanth, R; Wilson, Jonathan; Bridgewater, Juma D; Numbers, Jason R; Lim, Jihyeon; Olbris, Mark R; Kettani, Ali; Vachet, Richard W

    2007-08-01

    Oxidative modifications to the side chains of sulfur-containing amino acids often limit the number of product ions formed during collision-induced dissociation (CID) and thus make it difficult to obtain sequence information for oxidized peptides. In this work, we demonstrate that electron-transfer dissociation (ETD) can be used to improve the sequence information obtained from peptides with oxidized cysteine and methionine residues. In contrast to CID, ETD is found to be much less sensitive to the side-chain chemistry, enabling extensive sequence information to be obtained in cases where CID fails to provide this information. These results indicate that ETD is a valuable technique for studying oxidatively modified peptides and proteins. In addition, we report a unique and very abundant product ion that is formed in the CID spectra of peptides having N-terminal cysteine sulfinic acid residues. The mechanism for this unique dissociation pathway involves a six-membered cyclic intermediate and leads to the facile loss of NH(3) and SO(2), which corresponds to a mass loss of 81 Da. While the facile nature of this dissociation pathway limits the sequence information present in CID spectra of peptides with N-terminal cysteine sulfinic acid residues, extensive sequence information for these peptides can be obtained with ETD.

  4. Remediation of hexavalent chromium contamination in chromite ore processing residue by sodium dithionite and sodium phosphate addition and its mechanism.

    PubMed

    Li, Yunyi; Cundy, Andrew B; Feng, Jingxuan; Fu, Hang; Wang, Xiaojing; Liu, Yangsheng

    2017-05-01

    Large amounts of chromite ore processing residue (COPR) wastes have been deposited in many countries worldwide, generating significant contamination issues from the highly mobile and toxic hexavalent chromium species (Cr(VI)). In this study, sodium dithionite (Na2S2O4) was used to reduce Cr(VI) to Cr(III) in COPR containing high available Fe, and then sodium phosphate (Na3PO4) was utilized to further immobilize Cr(III), via a two-step procedure (TSP). Remediation and immobilization processes and mechanisms were systematically investigated using batch experiments, sequential extraction studies, X-ray diffraction (XRD) and X-ray Photoelectron Spectroscopy (XPS). Results showed that Na2S2O4 effectively reduced Cr(VI) to Cr(III), catalyzed by Fe(III). The subsequent addition of Na3PO4 further immobilized Cr(III) by the formation of crystalline CrPO4·6H2O. However, addition of Na3PO4 simultaneously with Na2S2O4 (via a one-step procedure, OSP) impeded Cr(VI) reduction due to the competitive reaction of Na3PO4 and Na2S2O4 with Fe(III). Thus, the remediation efficiency of the TSP was much higher than the corresponding OSP. Using an optimal dosage in the two-step procedure (Na2S2O4 at a dosage of 12× the stoichiometric requirement for 15 days, and then Na3PO4 in a molar ratio (i.e. Na3PO4: initial Cr(VI)) of 4:1 for another 15 days), the total dissolved Cr in the leachate determined via Toxicity Characteristic Leaching Procedure (TCLP Cr) testing of our samples was reduced to 3.8 mg/L (from an initial TCLP Cr of 112.2 mg/L, i.e. at >96% efficiency).

  5. The intrinsic cysteine and histidine residues of the anti-Salmonella antibody Se155-4: a model for the introduction of new functions into antibody-binding sites.

    PubMed

    Young, N Martin; Watson, David C; Cunningham, Anna M; MacKenzie, C Roger

    2014-10-01

    New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a 'reagentless' fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity.

  6. Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition.

    PubMed Central

    Simonovic, M.; Gettins PGW; Volz, K.

    2000-01-01

    CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA. PMID:10975564

  7. Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins

    PubMed Central

    Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph

    2015-01-01

    ABSTRACT Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. IMPORTANCE Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional

  8. Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

    PubMed Central

    Dubois, Thomas; Dancer-Thibonnier, Marie; Monot, Marc; Hamiot, Audrey; Bouillaut, Laurent; Soutourina, Olga; Martin-Verstraete, Isabelle

    2016-01-01

    The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601. PMID:27297391

  9. Evaluation of certain veterinary drug residues in food. Seventy-eighth report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2014-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues of food. The first part of the report considers general principles regarding the evaluation of residues of veterinary drugs within the terms of reference of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), including extrapolation of maximum residue limits (MRLs) to minor species, MRLs for veterinary drug residues in honey, MRLs relating to fish and fish species, dietary exposure assessment methodologies, the decision-tree approach to the evaluation of residues of veterinary drugs and guidance for JECFA experts. Summaries follow of the Committee's evaluations of toxicology and residue data on a variety of veterinary drugs: two anthelminthic agents (derquantel, monepantel), three antiparasitic agents (emanectin benzoate, ivermectin, lasalocid sodium), one antibacterial, antifungal and anthelminthic agent (gentian violet), a production aid (recombinant bovine somatotropins) and an adrenoceptor agonist and growth promoter (zilpaterol hydorchloride). Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes (ADIs)) and proposed MRLs.

  10. Effect of second coagulant addition on coagulation efficiency, floc properties and residual Al for humic acid treatment by Al13 polymer and polyaluminum chloride (PACl).

    PubMed

    Xu, Weiying; Gao, Baoyu; Wang, Yan; Yue, Qinyan; Ren, Haijing

    2012-05-15

    Influence of second dose on coagulation efficiency, floc re-growth, fractal structure and residual Al of the effluent in humic acid (HA) coagulation with Al(13) polymer ([Al(13)O(4)(OH)(24)(H(2)O)(12)](7+)) and PACl were comparatively investigated in this study. Effects of breakage shear on the floc properties generated in the coagulation with and without additional dose were also investigated. The results indicated that additional dose during breakage could essentially improve the HA removal efficiency and floc re-growth in both Al(13) and PACl coagulations. Second doses of Al(13) at 0.5 and 1.0mg/L resulted in better turbidity and UV(254) removal as well as floc re-growth rather than higher additional dose of 1.5 and 2.0mg/L; while in PACl coagulation, more efficient HA removal and better floc re-growth were obtained at higher additional doses (1.0, 1.5 and 2.0mg/L). Small additional Al(13) could apparently increase the D(f) of re-formed flocs while the additional PACl displayed inconspicuous effect on floc D(f). The additional coagulant dose could alleviate the further decrease of re-grown floc size with increased breakage shear for both coagulants. The residual Al analysis implied that two-stage addition contributed to lower residual Al in effluent than one-time addition mode with the same total coagulant concentration.

  11. A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes

    PubMed Central

    Liu, Ying; Lei, Xiao-Yu; Chen, Lian-Fu; Bian, Yin-Bing; Yang, Hong; Ibrahim, Salam A.; Huang, Wen

    2015-01-01

    Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes. PMID:26054293

  12. Synthesis and antimicrobial activity of cysteine-free coprisin nonapeptides.

    PubMed

    Lee, Jaeho; Lee, Daeun; Choi, Hyemin; Kim, Ha Hyung; Kim, Ho; Hwang, Jae Sam; Lee, Dong Gun; Kim, Jae Il

    2014-01-10

    Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. CopA3 (LLCIALRKK-NH₂), a 9-mer peptide containing a single free cysteine residue at position 3 of its sequence, was derived from the α-helical region of coprisin and exhibits potent antibacterial and anti-inflammatory activities. The single cysteine implies a tendency for dimerization; however, it remains unknown whether this cysteine residue is indispensible for CopA3's antimicrobial activity. To address this issue, in the present study we synthesized eight cysteine-substituted monomeric CopA3 analogs and two dimeric analogs, CopA3 (Dimer) and CopIK (Dimer), and evaluated their antimicrobial effects against bacteria and fungi, as well as their hemolytic activity toward human erythrocytes. Under physiological conditions, CopA3 (Mono) exhibits a 6/4 (monomer/dimer) molar ratio in HPLC area percent, indicating that its effects on bacterial strains likely reflect a CopA3 (Mono)/CopA3 (Dimer) mixture. We also report the identification of CopW, a new cysteine-free nonapeptide derived from CopA3 that has potent antimicrobial activity with virtually no hemolytic activity. Apparently, the cysteine residue in CopA3 is not essential for its antimicrobial function. Notably, CopW also exhibited significant synergistic activity with ampicillin and showed more potent antifungal activity than either wild-type coprisin or melittin.

  13. Misincorporation proton-alkyl exchange (MPAX): engineering cysteine probes into proteins.

    PubMed

    Burguete, Alondra Schweizer; Harbury, Pehr B; Pfeffer, Suzanne R

    2005-12-01

    This unit describes a rapid and efficient method to screen a polypeptide for amino acid residues that contribute to protein-protein interaction interfaces. Cysteine residues are introduced as positional probes in a protein at random by co-expression in bacteria with specific cysteine misincorporator tRNAs. The protein is then purified as an ensemble of polypeptides containing cysteine at low frequency, at different positions in each molecule. The ability of the native protein structure to protect different cysteine residues from chemical modification by iodoacetamide is determined to obtain a protein surface map that reveals candidate surface residues that are likely to be important for protein-protein interaction. Cysteine mutants with altered ligand binding can also be selected simultaneously by affinity chromatography.

  14. Evaluation of certain veterinary drug residues in food. Sixty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2006-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues in food. The first part of the report considers general principles regarding the evaluation of veterinary drugs within the terms of reference of JECFA, including compounds without an ADI or MRL; recommendations on principles and methods in derivation of MRLs, including a new procedure for estimating chronic dietary intakes; the use of a spreadsheet-based procedure for the statistical evaluation of residue depletion data; a revised approach for the derivation of microbiological ADIs; and the Committee's review of and comments on documents provided by the Codex Committee on Residues of Veterinary Drugs. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: three antimicrobial agents (colistin, erythromycin, flumequine), two production aids (melengestrol acetate, ractopamine hydrochloride), an insecticide (trichlorfon (metrifonate)) and an anthelminthic (triclabendazole). In addition, the attempt by the Committee to use tylosin as an example to investigate if evaluations are possible based on published data in the absence of data submissions from sponsors is described. Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes and proposed maximum residue limits.

  15. FORMATION OF FINE PARTICLES FROM RESIDUAL OIL COMBUSTION: REDUCING ULTRAFINE NUCLEI THROUGH THE ADDITION OF INORGANIC SORBENT

    EPA Science Inventory

    The paper gives results of an investigation, using an 82-kW-rated laboratory-scale refractory-lined combustor, of the characteristics of particulate matter emitted from residual oil combustion and the reduction of ultrafine nuclei by postflame sorbent injection. Without sorbent a...

  16. Fluorescent nitrile-based inhibitors of cysteine cathepsins.

    PubMed

    Frizler, Maxim; Mertens, Matthias D; Gütschow, Michael

    2012-12-15

    Cysteine cathepsins play an important role in many (patho)physiological conditions. Among them, cathepsins L, S, K and B are subjects of several drug discovery programs. Besides their role as drug targets, cysteine cathepsins are additionally considered to be possible biomarkers for inflammation and cancer. Herein, we describe the design, synthesis, biological evaluation and spectral properties of fluorescently labeled dipeptide- and azadipeptide nitriles.

  17. Evaluation of certain veterinary drug residues in food. Eighty-first report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2016-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in food and to recommend maximum levels for such residues in food. The first part of the report considers general principles regarding the evaluation of residues of veterinary drugs within the terms of reference of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), including MRLs for generic fish species, acute reference doses (ARfDs) for veterinary drugs, an approach for dietary exposure assessment of compounds used for multiple purposes (i.e veterinary drugs and pesticides), dietary exposure assessment for less-than-lifetime exposure, and the assessment of short-term (90-day and 12-month) studies in dogs. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: two insecticides (diflubenzuron and teflubenzuron), an antiparasitic agent (ivermectin), an ectoparasiticide (sisapronil) and a β2-adrenoceptor agonist (zilpaterol hydrochloride). In addition, the Committee considered issues raised in concern forms from the Codex Committee on Residues of Veterinary Drugs in Foods on lasalocid sodium, an antiparasitic agent. Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes (ADIs), ARfDs and proposed MRLs.

  18. The cardiac L-type calcium channel alpha subunit is a target for direct redox modification during oxidative stress - the role of cysteine residues in the alpha interacting domain.

    PubMed

    Muralidharan, Padmapriya; Cserne Szappanos, Henrietta; Ingley, Evan; Hool, Livia C

    2017-03-17

    Cardiovascular disease is the leading cause of death in the Western world. The incidence of cardiovascular disease is predicted to further rise with the increase in obesity and diabetes and with the ageing population. Even though the survival rate from ischemic heart disease has improved over the past 30 years, many patients progress to a chronic pathological condition, known as cardiac hypertrophy that is associated with an increase in morbidity and mortality. Reactive oxygen species (ROS) and calcium play an essential role in mediating cardiac hypertrophy. The L-type calcium channel is the main route for calcium influx into cardiac myocytes. There is now good evidence for a direct role for the L-type calcium channel in the development of cardiac hypertrophy. Cysteines on the channel are targets for redox modification and glutathionylation of the channel can modulate the function of the channel protein leading to the onset of pathology. The cysteine responsible for modification of L-type calcium channel function has now been identified. Detailed understanding of the role of cysteines as possible targets during oxidative stress may assist in designing therapy to prevent the development of hypertrophy and heart failure. This article is protected by copyright. All rights reserved.

  19. Development of an Eastern Shale Oil Residue as an Asphalt Additive - Subtask 2.5: Topical report, February 1, 1994-February 1, 1995

    SciTech Connect

    1997-12-31

    An evaluation of eastern shale oil as an asphalt additive to reduce oxidative age hardening and moisture susceptibility is being conducted. An eastern shale oil residue having a viscosity of 1.30 Pa`s at 60{degrees}C (140{degrees}F) was blended with three different petroleum-derived asphalts that are known to be very susceptible to oxidative aging. In addition, blends of the eastern shale oil residue and the petroleum-derived asphalts are being coated onto three different aggregates that are known to be susceptible to water stripping. The oxidative age hardening portion of this study is not complete at this time. To date, information has been obtained on the unaged samples and two of the aged petroleum-derived asphalts (AAD-1 and AAK-1). When complete, this data will include rheological data on the unaged, RTFO-aged, and the RTFO/PAV-aged samples and infrared data on the unaged and RTFO/PAV-aged samples. With respect to the rheological data, asphalt AAD-1 meets the specifications of a PG 58 asphalt while asphalt AAK-1 does not. In the latter case this indicates that AAK-1 is more appropriately evaluated at a higher temperature range. The infrared spectroscopic data obtained for the eastern shale oil residue show that it contains appreciable amounts of carbonyl and sulfoxide compound types, 0.22 absorbance units and 0. 27 moles/L, respectively. Thus, upon the addition of this residue to the three petroleum-derived asphalts the blends contain increased amounts of these functional groups relative to the petroleum-derived asphalts. This has been observed with other additives and is not considered detrimental. In addition, the data that has been collected to date indicate that the moisture susceptibility of blends of eastern shale oil residue and asphalt AAK-1 are somewhat improved when coated onto Lithonia granite.

  20. Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

    PubMed Central

    Zhang, Xinqun; Hunter, Joshua H.; Emmerton, Kim K.; Miyamoto, Jamie B.; Lewis, Timothy S.; Senter, Peter D.

    2016-01-01

    Abstract A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification. PMID:27966822

  1. The cysteine proteinases of the pineapple plant.

    PubMed

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

  2. The cysteine proteinases of the pineapple plant.

    PubMed Central

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-01-01

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. Images Fig. 4. Fig. 5. PMID:2327970

  3. Effect of lactic acid bacteria inoculant and beet pulp addition on fermentation characteristics and in vitro ruminal digestion of vegetable residue silage.

    PubMed

    Cao, Y; Cai, Y; Takahashi, T; Yoshida, N; Tohno, M; Uegaki, R; Nonaka, K; Terada, F

    2011-08-01

    The objective of this study was to determine the effect of beet pulp (BP) and lactic acid bacteria (LAB) on silage fermentation quality and in vitro ruminal dry matter (DM) digestion of vegetable residues, including white cabbage, Chinese cabbage, red cabbage, and lettuce. Silage was prepared using a small-scale fermentation system, and treatments were designed as control silage without additive or with BP (30% fresh matter basis), LAB inoculant Chikuso-1 (Lactobacillus plantarum, 5mg/kg, fresh matter basis), and BP+LAB. In vitro incubation was performed using rumen fluid mixed with McDougall's artificial saliva (at a ratio of 1:4, vol/vol) at 39°C for 6h to determine the ruminal fermentability of the vegetable residue silages. These vegetable residues contained high levels of crude protein (20.6-22.8% of DM) and moderate levels of neutral detergent fiber (22.7-33.6% of DM). In all silages, the pH sharply decreased and lactic acid increased, and the growth of bacilli, coliform bacteria, molds, and yeasts was inhibited by the low pH at the early stage of ensiling. The silage treated with BP or LAB had a lower pH and a higher lactic acid content than the control silage. After 6h of incubation, all silages had relatively high DM digestibility (38.6-44.9%); in particular, the LAB-inoculated silage had the highest DM digestibility and the lowest methane production. The vegetable residues had high nutritional content and high in vitro DM digestibility. Also, both the addition of a LAB inoculant and moisture adjustment with BP improved the fermentation quality of the vegetable residue silages. In addition, LAB increased DM digestibility and decreased ruminal methane production.

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

    SciTech Connect

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

    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.

  5. Cysteines under ROS attack in plants: a proteomics view.

    PubMed

    Akter, Salma; Huang, Jingjing; Waszczak, Cezary; Jacques, Silke; Gevaert, Kris; Van Breusegem, Frank; Messens, Joris

    2015-05-01

    Plants generate reactive oxygen species (ROS) as part of their metabolism and in response to various external stress factors, potentially causing significant damage to biomolecules and cell structures. During the course of evolution, plants have adapted to ROS toxicity, and use ROS as signalling messengers that activate defence responses. Cysteine (Cys) residues in proteins are one of the most sensitive targets for ROS-mediated post-translational modifications, and they have become key residues for ROS signalling studies. The reactivity of Cys residues towards ROS, and their ability to react to different oxidation states, allow them to appear at the crossroads of highly dynamic oxidative events. As such, a redox-active cysteine can be present as S-glutathionylated (-SSG), disulfide bonded (S-S), sulfenylated (-SOH), sulfinylated (-SO2H), and sulfonylated (-SO3H). The sulfenic acid (-SOH) form has been considered as part of ROS-sensing pathways, as it leads to further modifications which affect protein structure and function. Redox proteomic studies are required to understand how and why cysteines undergo oxidative post-translational modifications and to identify the ROS-sensor proteins. Here, we update current knowledge of cysteine reactivity with ROS. Further, we give an overview of proteomic techniques that have been applied to identify different redox-modified cysteines in plants. There is a particular focus on the identification of sulfenylated proteins, which have the potential to be involved in plant signal transduction.

  6. Increase in the carbohydrate content of the microalgae Spirulina in culture by nutrient starvation and the addition of residues of whey protein concentrate.

    PubMed

    Vieira Salla, Ana Cláudia; Margarites, Ana Cláudia; Seibel, Fábio Ivan; Holz, Luiz Carlos; Brião, Vandré Barbosa; Bertolin, Telma Elita; Colla, Luciane Maria; Costa, Jorge Alberto Vieira

    2016-06-01

    Non-renewable sources that will end with time are the largest part of world energy consumption, which emphasizes the necessity to develop renewable sources of energy. This necessity has created opportunities for the use of microalgae as a biofuel. The use of microalgae as a feedstock source for bioethanol production requires high yields of both biomass and carbohydrates. With mixotrophic cultures, wastewater can be used to culture algae. The aim of the study was to increase the carbohydrate content in the microalgae Spirulina with the additions of residues from the ultra and nanofiltration of whey protein. The nutrient deficit in the Zarrouk medium diluted to 20% and the addition of 2.5% of both residue types led to high carbohydrate productivity (60 mg L(-1) d(-1)). With these culture conditions, the increase in carbohydrate production in Spirulina indicated that the conditions were appropriate for use with microalgae as a feedstock in the production of bioethanol.

  7. Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase.

    PubMed

    Sigman, J A; Sharky, M L; Walsh, S T; Pabon, A; Glucksman, M J; Wolfson, A J

    2003-08-01

    Thimet oligopeptidase is a metalloenzyme involved in regulating neuropeptide processing. Three cysteine residues (246, 248, 253) are known to be involved in thiol activation of the enzyme. In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S) displays increased activity in the absence of dithiothreitol. Dimers, purportedly formed through cysteines 246, 248 and 253, have been thought to be inactive. However, analysis of the triple mutant by native gel electrophoresis reveals the existence of dimers and multimers, implying that oligomer formation is mediated by other cysteines, probably on the surface, and that some of these forms are enzymatically active. Isolation and characterization of iodoacetate-modified monomers and dimers of the triple mutant revealed that, indeed, certain dimeric forms of the enzyme are still fully active, whereas others show reduced activity. Cysteine residues potentially involved in dimerization were identified by modeling of thimet oliogopeptidase to its homolog, neurolysin. Five mutants were constructed; all contained the triple mutation C246S/C248S/C253S and additional substitutions. Substitutions at C46 or C682 and C687 prevented multimer formation and inhibited dimer formation. The C46S mutant had enzymatic activity comparable to the parent triple mutant, whereas that of C682S/C687S was reduced. Thus, the location of intermolecular disulfide bonds, rather than their existence per se, is relevant to activity. Dimerization close to the N-terminus is detrimental to activity, whereas dimerization near the C-terminus has little effect. Altering disulfide bond formation is a potential regulatory factor in the cell owing to the varying oxidation states in subcellular compartments and the different compartmental locations and functions of the enzyme.

  8. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.

    PubMed

    Kuravsky, Mikhail; Barinova, Kseniya; Marakhovskaya, Aleksandra; Eldarov, Mikhail; Semenyuk, Pavel; Muronetz, Vladimir; Schmalhausen, Elena

    2014-10-01

    Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.

  9. A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML).

    PubMed

    Gu, Liqing; Robinson, Renã A S

    2016-04-01

    Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall

  10. Chikungunya nsP2 protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine.

    PubMed

    Saisawang, Chonticha; Saitornuang, Sawanan; Sillapee, Pornpan; Ubol, Sukathida; Smith, Duncan R; Ketterman, Albert J

    2015-11-24

    Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target.

  11. Chikungunya nsP2 protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine

    PubMed Central

    Saisawang, Chonticha; Saitornuang, Sawanan; Sillapee, Pornpan; Ubol, Sukathida; Smith, Duncan R.; Ketterman, Albert J.

    2015-01-01

    Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target. PMID:26597768

  12. Immediate postoperative radiotherapy in residual nonfunctioning pituitary adenoma: Beneficial effect on local control without additional negative impact on pituitary function and life expectancy

    SciTech Connect

    Bergh, Alfons C.M. van den . E-mail: a.c.m.van.den.bergh@rt.umcg.nl; Berg, Gerrit van den; Schoorl, Michiel A.; Sluiter, Wim J.; Vliet, Anton M. van der; Hoving, Eelco W.; Szabo, Ben G.; Langendijk, Johannes A.; Wolffenbuttel, Bruce H.R.; Dullaart, Robin P.F.

    2007-03-01

    Purpose: To demonstrate the benefit of immediate postoperative radiotherapy in residual nonfunctioning pituitary adenoma (NFA) in perspective to the need for hormonal substitution and life expectancy. Methods and Materials: Retrospective cohort analysis of 122 patients, operated for NFA between 1979 and 1998. Recurrence was defined as regrowth on computed tomography or magnetic resonance imaging. The occurrence of hormonal deficiencies was defined as the starting date of hormonal substitution therapy. Results: Seventy-six patients had residual NFA after surgery and received immediate postoperative radiotherapy (Group 1); three patients developed a recurrence, resulting in a 95% local control rate at 10 years. Twenty-eight patients had residual NFA after surgery, but were followed by a wait-and-see policy (Group 2). Sixteen developed a recurrence, resulting in a local control rate of 49% at 5 years and 22% at 10 years (p < 0.001 compared with Group 1). There were no differences between Group 1 and 2 regarding the need for substitution with thyroid hormone, glucocorticoids, and sex hormones before first surgery, directly after surgery and at end of follow-up. There were no differences in hormone substitution free survival between Group 1 and Group 2 during the study period after first surgery. Life expectancy was similar in Group 1 and 2, and their median life expectancy did not differ from median life expectancy in the general population. Conclusions: Immediate postoperative radiotherapy provides a marked improvement of local control among patients with residual NFA compared with surgery alone, without an additional deleterious effect on pituitary function and life expectancy.

  13. The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

    PubMed

    Rahman, Mohammad Aminur; Senga, Takeshi; Oo, Myat Lin; Hasegawa, Hitoki; Biswas, Md Helal Uddin; Mon, Naing Naing; Huang, Pengyu; Ito, Satoko; Yamamoto, Tadashi; Hamaguchi, Michinari

    2008-04-01

    The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

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

    PubMed

    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.

  15. Evaluation of western shale-oil residue as an additive to petroleum asphalt for use as a pavement crack and joint sealant material

    SciTech Connect

    Harnsberger, P.M.; Wolf, J.M.; Robertson, R.E.

    1992-11-01

    The objective of this study was to perform a preliminary evaluation of using a distillation residue from Green River Formation (western) shale oil as an additive to a petroleum asphalt for use as a crack and joint filler material in portland cement concrete and asphaltic pavements. A commercially available rubberized asphalt crack and joint filler material was also tested for comparison. ASTM specification tests for sealant materials used in concrete and asphalt pavements were performed on the sealant materials. Portland cement concrete briquets prepared with an asphalt material sandwiched between two concrete wafers were tested in a stress-relaxation experiment to evaluate the relaxation and recovery properties of the sealant materials. The results show that the shale-oil modified petroleum asphalts and the neat petroleum asphalt do not pass the extension portion of the ASTM test; however, there is indication of improvement in the adhesive properties of the shale-oil modified asphalts. There is also evidence that the addition of shale-oil residue to the petroleum asphalt, especially at the 20% level, improves the relaxation and recovery properties compared with the petroleum asphalt.

  16. Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.

    PubMed

    Saito, Ryota; Suzuki, Takafumi; Hiramoto, Keiichiro; Asami, Soichiro; Naganuma, Eriko; Suda, Hiromi; Iso, Tatsuro; Yamamoto, Hirotaka; Morita, Masanobu; Baird, Liam; Furusawa, Yuki; Negishi, Takaaki; Ichinose, Masakazu; Yamamoto, Masayuki

    2015-11-02

    The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

  17. The cold and menthol receptor TRPM8 contains a functionally important double cysteine motif.

    PubMed

    Dragoni, Ilaria; Guida, Elizabeth; McIntyre, Peter

    2006-12-08

    We have investigated the glycosylation, disulfide bonding, and subunit structure of mouse TRPM8. To do this, amino-terminal c-myc or hemagglutinin epitope-tagged proteins were incorporated and expressed in Chinese hamster ovary cells. These modifications had no obvious effects on channel function in intracellular calcium imaging assays upon application of agonists, icilin or menthol, and cold temperatures. Unmodified TRPM8 migrates with an apparent mass of 129 kDa and can be glycosylated in Chinese hamster ovary cells to give glycoproteins with apparent masses of 136 and 147 kDa. We identified two potential N-linked glycosylation sites in TRPM8 (Asn-821 and Asn-934) and mutated them to show that only the site in the putative pore region at position 934 is modified and that glycosylation of this site is not absolutely necessary for cell surface expression or responsiveness to icilin, menthol, and cool temperatures. Enzymatic cleavage of the carbohydrate chains indicated that they are complex carbohydrate. The glycosylation site is flanked in the pore by two cysteine residues that we mutated, to prove that they are involved in a conserved double cysteine motif, which is essential for channel function. Mutation of either of these cysteines abolishes function and forces the formation of a non-functional complex of the size of a homodimer. The double cysteine mutant is also non-functional. Finally, we showed in Perfluoro-octanoic acid-polyacrylamide gels that TRPM8 can form a tetramer (in addition to dimer and trimer forms), consistent with current thinking that functional TRP ion channels are tetrameric.

  18. The spectrum character of photoreaction of Hypocrellin A and cysteine

    NASA Astrophysics Data System (ADS)

    Zhang, Jucheng; Liu, Wei; Li, Ying; Zhang, Pei; Yi, Zhongzhou; Min, Yong; Huang, Zhaolong; Yao, Lihua; Lu, Haiju

    2008-12-01

    In the current work, Hypocrellin A (HA) is one of the nature photosensitizer was recognized by researchers, and it used as a probe to research the molecular recognition and interaction with protein, the work suggested the HA can as the medicine to treat some disease. This paper study the spectrum character of photoreaction of Hypocrellin A and cysteine in different pH value, the spectrum show an isosbestic point at 495nm, and the absorption peak at 478nm was red-shifted to about 500nm. The result suggested the HA can react with cysteine in this condition, and farther illuminated the cysteine residue may is one of the target of the interaction of HA or HB with protein.

  19. The Cysteine Proteome

    PubMed Central

    Go, Young-Mi; Chandler, Joshua D.; Jones, Dean P.

    2015-01-01

    The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metallation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to discriminate network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome. PMID:25843657

  20. Influence of commercial and residual sorbents and silicates as additives on the stabilisation/solidification of organic and inorganic industrial waste.

    PubMed

    Coz, A; Andrés, A; Soriano, S; Viguri, J R; Ruiz, M C; Irabien, J A

    2009-05-30

    An environmental problem of the foundry activities is the management of industrial waste generated in different processes. The foundry sludge from gas wet cleaning treatment that contains organic and inorganic compounds and a high content of water is an interesting example. Due to their characteristics, they can be managed using different stabilisation/solidification (S/S) technologies prior to land disposal. The purpose of this work is to study S/S formulations in order to improve the control of the mobility of the pollutants and the ecotoxicity of the samples. Different mixtures of cement or lime as binders and additives (foundry sand, silica fume, sodium silicate, silicic acid, activated carbon and black carbon) have been used in order to reduce the mobility of the chemical and ecotoxicological regulated parameters and to compare the results for commercial and residual additives. The best results have been obtained with sorbents (activated carbon and black carbon) or sodium silicate. The results of the foundry sand ash as additive can conclude that it can be used as replacement in the cement products. However, silica fume in the samples with lime and siliceous resin sand as additives gives products that do not fulfil the regulated limits. Finally, some linear expressions between the chemical parameters and the quantity of material used in the samples have been obtained.

  1. Artemisolide is a typical inhibitor of I{kappa}B kinase {beta} targeting cysteine-179 residue and down-regulates NF-{kappa}B-dependent TNF-{alpha} expression in LPS-activated macrophages

    SciTech Connect

    Kim, Byung Hak; Lee, Jun-Young; Seo, Jee Hee; Lee, Hwa Young; Ryu, Shi Yong; Ahn, Byung Woo; Lee, Chong-Kil; Hwang, Bang Yeon; Han, Sang-Bae; Kim, Youngsoo

    2007-09-28

    Nuclear factor (NF)-{kappa}B regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-{kappa}B inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-{kappa}B activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of I{kappa}B kinase {beta} (IKK{beta}), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-{kappa}B activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKK{beta} and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKK{beta} activity was completely abolished by substitution of Cys-179 residue of IKK{beta} to Ala residue, indicating direct targeting site of ATM. ATM could inhibit I{kappa}B{alpha} phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-{kappa}B activation. Further, we demonstrate that ATM down-regulates NF-{kappa}B-dependent TNF-{alpha} expression. Taken together, this study provides a pharmacological potential of ATM in NF-{kappa}B-dependent inflammatory disorders.

  2. Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

    PubMed Central

    Cheng, H; Tchaikovskaya, T; Tu, Y S; Chapman, J; Qian, B; Ching, W M; Tien, M; Rowe, J D; Patskovsky, Y V; Listowsky, I; Tu, C P

    2001-01-01

    Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress. PMID:11368767

  3. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-01-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake Ac

  4. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-09-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.

  5. Backbone 1H, 13C and 15N assignments of YibK and avariant containing a unique cysteine residue at C-terminus in 8 M urea-denatured states [corrected].

    PubMed

    Hsieh, Shu-Ju Micky; Mallam, Anna L; Jackson, Sophie E; Hsu, Shang-Te Danny

    2014-10-01

    YibK is a tRNA methyltransferase from Haemophilus influenzae, which forms a stable homodimer in solution and contains a deep trefoil 31 knot encompassing the C-terminal helix that threads through a long loop. It has been a model system for investigating knotted protein folding pathways. Recent data have shown that the polypeptide chain of YibK remains loosely knotted under highly denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for YibK and its variant in the presence of 8 M urea. This work forms the basis for further analysis using NMR techniques such as paramagnetic relaxation enhancement, residual dipolar couplings and spin-relaxation dynamics analysis.

  6. Methionine-to-Cysteine Recycling in Klebsiella aerogenes

    PubMed Central

    Seiflein, Thomas A.; Lawrence, Jeffrey G.

    2001-01-01

    In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5′-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella. PMID:11114934

  7. A New Class of Serine and Cysteine Protease Inhibitor with Chemotherapeutic Potential

    DTIC Science & Technology

    1999-06-01

    also be used to produce a serine protease inhibitor. Similar to the cysteine inhibitors, a dipeptide side chain is attached to the ring which is...which relieves the 7 strain (Figure 3). Serine and cysteine proteases use a mechanism to cleave peptide bonds which involves addition of a catalytic...serine and cysteine proteases share a similar mechanism for hydrolyzing amide bonds , we expect that 4-heterocyclohexanones should be good inhibitors

  8. Effects on aflatoxin M1 residues in milk by addition of hydrated sodium calcium aluminosilicate to aflatoxin-contaminated diets of dairy cows.

    PubMed

    Harvey, R B; Phillips, T D; Ellis, J A; Kubena, L F; Huff, W E; Petersen, H D

    1991-09-01

    Hydrated sodium calcium aluminosilicate (HSCAS), an anticaking agent for agricultural feeds, was added to aflatoxin (AF)-contaminated diets of 3 lactating dairy cows and evaluated for its potential to reduce aflatoxin M1 (AFM1) residues in milk. During phase I, cows were fed alternating diets that consisted of 200 micrograms of AF/kg of feed for 7 days, 0.5% HSCAS plus 200 micrograms of AF/kg of feed for 7 days, and feed with the HSCAS removed for a final 7 days. The AFM1 milk concentrations from the intervals with HSCAS added to diets were compared with those times when HSCAS was absent. The presence of 0.5% HSCAS in feed containing 200 micrograms of AF/kg reduced AFM1 secretion into the milk by an average of 0.44 micrograms/L (from pretreatment of 1.85 micrograms/L to 1.41 micrograms/L with HSCAS, a 24% reduction). Following a 10-day period of noncontaminated feed consumption and no AFM1 residues in the milk, phase II of the study was begun. The same experimental design as phase I was used, but the dosages of HSCAS and AF were changed to 1.0% and 100 micrograms/kg of feed, respectively. The addition of 1.0% HSCAS in feed containing 100 micrograms of AF/kg decreased AFM1 content in the milk by an average of 0.40 micrograms/L (from a pretreatment of 0.91 micrograms/L to 0.51 micrograms/L when HSCAS was present, a 44% reduction). These findings suggest that HSCAS, a high-affinity sorbent compound for AF in vitro, is capable of reducing the secretion of AFM1 into milk.

  9. Prediction of the disulfide-bonding state of cysteines in proteins at 88% accuracy

    PubMed Central

    Martelli, Pier Luigi; Fariselli, Piero; Malaguti, Luca; Casadio, Rita

    2002-01-01

    The task of predicting the cysteine-bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine-containing segments extracted from 969 nonhomologous proteins of well-resolved three-dimensional structure. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task. PMID:12381855

  10. Differential responses of needle and branch order-based root decay to nitrogen addition: dominant effects of acid-unhydrolyzable residue and microbial enzymes

    NASA Astrophysics Data System (ADS)

    Kou, Liang; Chen, Weiwei; Zhang, Xinyu; Gao, Wenlong; Yang, Hao; Li, Dandan; Li, Shenggong

    2016-04-01

    Both chemical differences between foliage and different orders of fine roots and their contrasting decomposing microenvironments may affect their decomposition. However, little is known about how foliage and branch order-based root decomposition responds to increased N availability and the response mechanisms behind. The effects of different doses of N addition on the decomposition of needles and order-based roots of Pinus elliottii (slash pine) were monitored using the litterbag method for 524 days in a subtropical slash pine plantation in south China. The acid-unhydrolyzable residue (AUR) concentration and microbial extracellular enzymatic activities (EEA) in decomposing needles and roots were also determined. Our results indicate that the responses of needle and order-based root decomposition were N-dose-specific. The decomposition of both needles and lower-order roots was inhibited under the high N dose rate. The retarded decomposition of lower-order roots could be explained more by the increased binding of AUR to inorganic N ions, while the retarded decomposition of needles could be explained more by the reduced microbial EEA. Further, in contrast to lower-order roots, N addition had no effect on the decomposition of higher-order roots. We conclude that the decomposition of foliage and fine roots may fail to mirror each other at ambient conditions or in response to N deposition due to their contrasting decomposition microenvironments and tissue chemistry. Given the differential effects of N addition on order-based roots, our findings highlight the need to consider the tissue chemistry heterogeneity within branching fine root systems when predicting the responses of root decomposition to N loading.

  11. Cysteine-containing peptides having antioxidant properties

    DOEpatents

    Bielicki, John K.

    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.

  12. Cysteine-containing peptides having antioxidant properties

    DOEpatents

    Bielicki, John K.

    2009-10-13

    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.

  13. Chromium remediation or release? Effect of iron(II) sulfate addition on chromium(VI) leaching from columns of chromite ore processing residue.

    PubMed

    Geelhoed, Jeanine S; Meeussen, Johannes C L; Roe, Martin J; Hillier, Stephen; Thomas, Rhodri P; Farmer, John G; Paterson, Edward

    2003-07-15

    Chromite ore processing residue (COPR), derived from the so-called high lime processing of chromite ore, contains high levels of Cr(III) and Cr(VI) and has a pH between 11 and 12. Ferrous sulfate, which is used for remediation of Cr(VI) contamination in wastewater and soils via reduction to Cr(III) and subsequent precipitation of iron(III)/chromium(III) hydroxide, has also been proposed for remediation of Cr(VI) in COPR. Instead, however, addition of FeSO4 to the infiltrating solution in column experiments with COPR greatly increased leaching of Cr(VI). Leached Cr(VI) increased from 3.8 to 12.3 mmol kg(-1) COPR in 25 pore volumes with 20 mM FeSO4, reaching solution concentrations as high as 1.6 mM. Fe(II) was ineffective in reducing Cr(VI) to Cr(III) because it precipitated when it entered the column due to the high pH of COPR, while Cr(VI) in solution was transported away with the infiltrating solution. The large increase in leaching of Cr(VI) upon infiltration of sulfate, either as FeSO4 or Na2SO4, was caused by anion exchange of sulfate for chromate in the layered double hydroxide mineral hydrocalumite, a process for which scanning electron microscopy with energy-dispersive X-ray microanalysis provided direct evidence.

  14. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.

    PubMed

    Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

    2017-01-01

    Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance.

  15. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant

    PubMed Central

    Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

    2017-01-01

    Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance. PMID:27282255

  16. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  17. Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pKa values by MALDI-TOF mass spectrometry

    PubMed Central

    Nelson, Kimberly J.; Day, Amanda E.; Zeng, Bubing B.; King, S. Bruce; Poole, Leslie B.

    2008-01-01

    Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e. the thiolate) within the active site. While this is likely to be an important chemical attribute of many thiol-based enzymes including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pKa values have been determined experimentally. Presented here is a new technique for determining the pKa value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d5) and protiated (d0) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pKa values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and > 10, in good agreement with previous reports using chemical modification approaches. This technique allows the pKa of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest. PMID:18162165

  18. Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

    PubMed Central

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

    2015-01-01

    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. PMID:25741692

  19. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

    NASA Astrophysics Data System (ADS)

    Yang, Jianhong; Wang, Yuxi; Wang, Taijing; Jiang, Jian; Botting, Catherine H.; Liu, Huanting; Chen, Qiang; Yang, Jinliang; Naismith, James H.; Zhu, Xiaofeng; Chen, Lijuan

    2016-06-01

    Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.

  20. Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

    PubMed

    Miniaci, Maria Concetta; Irace, Carlo; Capuozzo, Antonella; Piccolo, Marialuisa; Di Pascale, Antonio; Russo, Annapina; Lippiello, Pellegrino; Lepre, Fabio; Russo, Giulia; Santamaria, Rita

    2016-02-01

    L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

  1. Electrochemical cysteine determination in serum samples by Hg thin film sensor.

    PubMed

    Sezginturk, Mustafa Kemal; Dinckaya, Erhan

    2011-01-01

    Cysteine is a nonessential aminoacid, meaning that cysteine can be made in the human body. It is one of the few amino acids that contain sulfur. This allows cysteine to bond in a special way and maintain the structures of proteins in the body. Cysteine strengthens the protective lining of the stomach and intestines, which may help prevent damage caused by aspirin and similar drugs. In addition, cysteine may play an important role in the communication between immune system cells. In this study, glassy carbon electrodes modified with mercury (Hg) were used as working electrode. Mercury thin film on glassy carbon electrode was deposited by holding the electrode potential at -0.7 V; the measurement period for the coating process was 2 minutes. pH and temperature effects on the electrode response were carried out by working at different pHs and temperatures. The calibration graph for cysteine was drawn in the range of 5-120 μM cysteine. Repeatability and interferences studies were investigated. GSH had an interference effect of about 13% of cysteine response. Finally, the sensor was applied to real samples for cysteine determination and the method was validated by Ellman's reagent.

  2. Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

    PubMed

    Andreeßen, Christina; Gerlt, Vanessa; Steinbüchel, Alexander

    2017-04-01

    3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.

  3. In silico designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation

    PubMed Central

    Sajjadi, Seyed Mehdi; Rahimi, Hamzeh; Mohammadi, Saeed; Faranoush, Mohammad; Mirzahoseini, Hasan; Toogeh, Gholamreza

    2017-01-01

    Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the in silico approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results. PMID:28255315

  4. Gluten gel and film properties in the presence of cysteine and sodium alginate.

    PubMed

    Yuno-Ohta, Naoko; Yamada, Mariko; Inomata, Masako; Konagai, Hiromi; Kataoka, Tomomi

    2009-08-01

    Wheat flour has an ability of forming dough by mixing with water, which exhibits a rheological property required for making bread. The major protein is gluten, which is a valuable protein material for food industry. In this study, gluten protein gels and films were formed with cysteine and sodium alginate. Adding cysteine improved gel and film properties (stress relaxation behavior, bending strength). The gel containing 0.01 M cysteine had a longer relaxation time and was more rigid than the gel without cysteine. Although adding sodium alginate to the gluten suspension containing cysteine improved the water-holding ability and homogeneity of the gel network, the film from this gel was more brittle than the gluten film with cysteine alone. Microstructural observations of the gels and films with scanning electron microscopy suggested that water evaporation was more heterogeneous from the gel containing sodium alginate than from the gel with cysteine alone. Fourier transform-infrared (FT-IR) analysis during film formation suggested that the presence of cysteine encourages interaction between gluten molecules and results in intermolecular beta-sheet formation in earlier stages than in the no additive condition. FT-IR results also suggested that the combined effect of sodium alginate and cysteine on the protein secondary structure was remarkably different from that of cysteine alone. Our results suggest that addition of a suitable amount of cysteine (0.01 M) and heat treatment to 80 degrees C during gluten gel and film formation induces a homogenous network in the gel and film by regulating disulfide-sulfide interactions.

  5. Functional characterization of enzymes involved in cysteine biosynthesis and H(2)S production in Trypanosoma cruzi.

    PubMed

    Marciano, Daniela; Santana, Marianela; Nowicki, Cristina

    2012-10-01

    Trypanosoma cruzi is expected to synthetize de novo cysteine by different routes, among which the two-step pathway involving serine acetyltransferase and cysteine synthase (CS) is comprised. Also, cystathionine β synthase (CBS) might contribute to the de novo generation of cysteine in addition to catalyze the first step of the reverse transsulfuration route producing cystathionine. However, neither the functionality of CS nor that of cystathionine γ lyase (CGL) has been assessed. Our results show that T. cruzi CS could participate notably more actively than CBS in the de novo synthesis of cysteine. Interestingly, at the protein level T. cruzi CS is more abundant in amastigotes than in epimastigotes. Unlike the mammalian homologues, T. cruzi CGL specifically cleaves cystathionine into cysteine and is unable to produce H(2)S. The expression pattern of T. cruzi CGL parallels that of CBS, which unexpectedly suggests that in addition to the de novo synthesis of cysteine, the reverse transsulfuration pathway could be operative in the mammalian and insect stages. Besides, T. cruzi CBS produces H(2)S by decomposing cysteine or via condensation of cysteine with homocysteine. The latter reaction leads to cystathionine production, and is catalyzed remarkably more efficiently than the breakdown of cysteine. In T. cruzi like in other organisms, H(2)S could exert regulatory effects on varied metabolic processes. Notably, T. cruzi seems to count on stage-specific routes involved in cysteine production, the multiple cysteine-processing alternatives could presumably reflect this parasite's high needs of reducing power for detoxification of reactive oxygen species.

  6. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs.

    PubMed

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ("Arg-type" enzymes) and some having a Gln substituted for this Arg ("Gln-type" enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis "Arg-type" enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha "Gln-type" CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among "Gln-type" CDO enzymes, we conclude that the "Gln-type" CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  7. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs

    PubMed Central

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases. PMID:25307852

  8. Expression and purification of cysteine introduced recombinant saporin.

    PubMed

    Günhan, Emine; Swe, Mimi; Palazoglu, Mine; Voss, John C; Chalupa, Leo M

    2008-04-01

    Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC(50) values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity.

  9. Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

    SciTech Connect

    Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry . E-mail: thierry.giardina@univ.u-3mrs.fr

    2005-05-06

    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.

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

    PubMed

    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.

  11. Enhanced incorporation yield of cysteine for glutathione overproduction by fed-batch fermentation of Saccharomyces cerevisiae.

    PubMed

    Lorenz, Eric; Schmacht, Maximilian; Stahl, Ulf; Senz, Martin

    2015-12-20

    In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54±0.01mol/mol) and GSH content (1650.7±42.8mg/l; 1.76±0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40±0.1mol/mol), biomass (84.2±1.2g/l) and GSH content (1304.7±61.4mg/l).

  12. Impact of the addition of different plant residues on nitrogen mineralization-immobilization turnover and carbon content of a soil incubated under laboratory conditions

    NASA Astrophysics Data System (ADS)

    Kaleeem Abbasi, M.; Tahir, M. Mahmood; Sabir, N.; Khurshid, M.

    2015-02-01

    Application of plant residues as soil amendment may represent a valuable recycling strategy that affects carbon (C) and nitrogen (N) cycling in soil-plant systems. The amount and rate of nutrient release from plant residues depend on their quality characteristics and biochemical composition. A laboratory incubation experiment was conducted for 120 days under controlled conditions (25 °C and 58% water-filled pore space) to quantify initial biochemical composition and N mineralization of leguminous and non-leguminous plant residues, i.e., the roots, shoots and leaves of Glycine max, Trifolium repens, Zea mays, Populus euramericana, Robinia pseudoacacia and Elaeagnus umbellata, incorporated into the soil at the rate of 200 mg residue N kg-1 soil. The diverse plant residues showed a wide variation in total N, C, lignin, polyphenols and C / N ratio with higher polyphenol content in the leaves and higher lignin content in the roots. The shoot of Glycine max and the shoot and root of Trifolium repens displayed continuous mineralization by releasing a maximum of 109.8, 74.8 and 72.5 mg N kg-1 and representing a 55, 37 and 36% recovery of N that had been released from these added resources. The roots of Glycine max and Zea mays and the shoot of Zea mays showed continuous negative values throughout the incubation. After an initial immobilization, leaves of Populus euramericana, Robinia pseudoacacia and Elaeagnus umbellata exhibited net mineralization by releasing a maximum of 31.8, 63.1 and 65.1 mg N kg-1, respectively, and representing a 16, 32 and 33% N recovery, respectively. Nitrogen mineralization from all the treatments was positively correlated with the initial residue N contents (r = 0.89; p ≤ 0.01) and negatively correlated with lignin content (r = -0.84; p ≤ 0.01), C / N ratio (r = -0.69; p ≤ 0.05), lignin / N ratio (r = -0.68; p ≤ 0.05), polyphenol / N ratio (r = -0.73; p ≤ 0.05) and (lignin + polyphenol) : N ratio (r = -0.70; p ≤ 0.05) indicating a

  13. Impact of the addition of different plant residues on carbon-nitrogen content and nitrogen mineralization-immobilization turnover in a soil incubated under laboratory conditions

    NASA Astrophysics Data System (ADS)

    Abbasi, M. K.; Tahir, M. M.; Sabir, N.; Khurshid, M.

    2014-10-01

    Application of plant residues as soil amendment may represent a valuable recycling strategy that affects on carbon (C) and nitrogen (N) cycling, soil properties improvement and plant growth promotion. The amount and rate of nutrient release from plant residues depend on their quality characteristics and biochemical composition. A laboratory incubation experiment was conducted for 120 days under controlled conditions (25 °C and 58% water filled pore space (WFPS)) to quantify initial biochemical composition and N mineralization of leguminous and non-leguminous plant residues i.e. the roots, shoots and leaves of Glycine max, Trifolium repens, Zea mays, Poplus euramericana, Rubinia pseudoacacia and Elagnus umbellate incorporated into the soil at the rate of 200 mg residue N kg-1 soil. The diverse plant residues showed wide variation in total N, carbon, lignin, polyphenols and C/N ratio with higher polyphenol content in the leaves and higher lignin content in the roots. The shoot of G. max and the shoot and root of T. repens displayed continuous mineralization by releasing a maximum of 109.8, 74.8 and 72.5 mg N kg-1 and representing a 55, 37 and 36% of added N being released from these resources. The roots of G. max and Z. mays and the shoot of Z. mays showed continuous negative values throughout the incubation showing net immobilization. After an initial immobilization, leaves of P. euramericana, R. pseudoacacia and E. umbellate exhibited net mineralization by releasing a maximum of 31.8, 63.1 and 65.1 mg N kg-1, respectively and representing a 16, 32 and 33% of added N being released. Nitrogen mineralization from all the treatments was positively correlated with the initial residue N contents (r = 0.89; p ≤ 0.01), and negatively correlated with lignin content (r = -0.84; p ≤ 0.01), C/N ratio (r = -0.69; p ≤ 0.05), lignin/N ratio (r = -0.68; p ≤ 0.05), polyphenol/N ratio (r = -0.73; p ≤ 0.05) and ligin + polyphenol/N ratio (r = -0.70; p ≤ 0.05) indicating

  14. Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

    SciTech Connect

    Jurgenson, Christopher T.; Burns, Kristin E.; Begley, Tadhg P.; Ealick, Steven E.

    2008-10-02

    The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 {angstrom} resolution. CysM (Rv1336) is a PLP-containing {beta}-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 {angstrom} resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

  15. Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus).

    PubMed

    Whang, Ilson; De Zoysa, Mahanama; Nikapitiya, Chamilani; Lee, Youngdeuk; Kim, Yucheol; Lee, Sukkyoung; Oh, Chulhong; Jung, Sung-Ju; Oh, Myung-Joo; Choi, Cheol Young; Yeo, Sang-Yeob; Kim, Bong-Seok; Kim, Se-Jae; Lee, Jehee

    2011-03-01

    Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

  16. Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

    PubMed Central

    Kreisberg, R.; Buchner, V.; Arad, D.

    1995-01-01

    This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur. PMID:8563638

  17. Delivery of a foreign epitope by sharing amino acid residues with the carrier matrix.

    PubMed

    Cheong, Wan-Shoo; Drummer, Heidi Edelgard; Netter, Hans-Jürgen

    2009-06-01

    A broad range of structural viral proteins has the ability to assemble into virus-like particles (VLPs). Under the condition that modified subunits are still competent to assemble into VLPs, they are epitope delivery platforms suitable for vaccination purposes. The insertion of foreign sequences can be detrimental for the formation of chimeric VLPs as a result of misfolded subunit proteins. Hence, a strategy was adopted to screen for locations allowing the use of shared residues between the wildtype subunit sequence and the foreign insert. The insertion of a cysteine-containing sequence of hepatitis C virus (HCV) envelope protein 2 (E2) without adding an additional cysteine residue retained the ability of recombinant small hepatitis B surface antigen (HBsAg-S) to form secretion competent VLPs. A cysteine residue shared by the insert and the template protein avoided the formation of non-native disulfide bonds, and allowed the formation of VLPs. The chimeric HBsAg-S VLPs were similar to wildtype VLPs in density exposing the inserted foreign epitope and being immunogenic. Overall, the use of shared sequences between the insert and the subunit will facilitate the design of chimeric VLPs carrying multiple epitopes.

  18. Benzoquinone reveals a cysteine-dependent desensitization mechanism of TRPA1.

    PubMed

    Ibarra, Yessenia; Blair, Nathaniel T

    2013-05-01

    The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium)ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel.

  19. Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

    PubMed

    Vallés, Diego; Bruno, Mariela; López, Laura M I; Caffini, Néstor O; Cantera, Ana María B

    2008-08-01

    A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

  20. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors.

    PubMed

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; Roush, William R

    2008-11-15

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

  1. Chemical modification of glycerinated stalks shows tyrosine residues essential for spasmoneme contraction of Vorticella sp.

    PubMed

    Fang, Jie; Zhang, Bei; Chen, Ning; Asai, Hiroshi

    2004-05-01

    Chemical modification of glycerinated stalks of Vorticella with TNM is used to investigate the role of tyrosine residues in the Ca(2+)-induced contraction of the spasmoneme. Tetranitromethane (TNM) is often employed as a specific reagent for the nitration of tyrosine residues in a protein at neutral and slightly alkaline pHs although TNM can also oxidize cysteine residues in the acidic and neutral pH range. Prior incubation with Ca(2+) of stalks to be treated with TNM can protect the spasmoneme from irreversible denaturation. On the other hand, TNM treatment in the absence of free Ca(2+) causes an irreversible denaturation of the spasmoneme. It was revealed by us that an isolated Ca(2+)-binding protein called spasmin could not bind with Ca(2+) after TNM treatment, even if the treatment was performed in the presence of Ca(2+). In an additional experiment, we confirmed that the chemical modification of cysteine residues in the spasmoneme with N-7-dimethyl-amino-4methyl- coumarinyl- maleimide (DACM) has no effect on the contractibility. These results suggest that tyrosine residues in spasmin are essential for spasmoneme contraction and are protected from TNM in the presence of Ca(2+) when spasmin binds with its receptor protein in the spasmoneme.

  2. Mechanical and chemical properties of cysteine-modified kinesin molecules.

    PubMed

    Iwatani, S; Iwane, A H; Higuchi, H; Ishii, Y; Yanagida, T

    1999-08-10

    To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.

  3. Use of cysteine-reactive crosslinkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers

    PubMed Central

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

    2014-01-01

    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, CD spectroscopy, sedimentation equilibrium ultracentrifugation, and crosslinking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol crosslinking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple crosslinked 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 crosslinking efficiency in cells. In addition, thiol crosslinkers may have an underappreciated value as qualitative probes of protein conformational flexibility. PMID:24832775

  4. Risk-based approach to developing a national residue sampling plan for testing under European Union regulation for veterinary medicinal products and coccidiostat feed additives in domestic animal production.

    PubMed

    Danaher, Martin; Shanahan, Conor; Butler, Francis; Evans, Rhodri; O'Sullivan, Dan; Glynn, Denise; Camon, Tim; Lawlor, Peadar; O'Keeffe, Michael

    2016-07-01

    A ranking system for veterinary medicinal products and coccidiostat feed additives has been developed as a tool to be applied in a risk-based approach to the residue testing programme for foods of animal origin in the Irish National Residue Control Plan (NRCP). Three characteristics of substances that may occur as residues in food are included in the developed risk ranking system: Potency, as measured by the acceptable daily intake assigned by the European Medicines Agency Committee for Medicinal Products for Veterinary Use, to each substance; Usage, as measured by the three factors of Number of Doses, use on Individual animals or for Group treatment, and Withdrawal Period; and Residue Occurrence, as measured by the number of Non-Compliant Samples in the NRCP. For both Number of Doses and Non-Compliant Samples, data for the 5-year period 2008-12 have been used. The risk ranking system for substances was developed for beef cattle, sheep and goats, pigs, chickens and dairy cattle using a scoring system applied to the various parameters described above to give an overall score based on the following equation: Potency × Usage (Number of Doses + Individual/Group Use + Withdrawal Period) × Residue Occurrence. Applying this risk ranking system, the following substances are ranked very highly: antimicrobials such as amoxicillin (for all species except pigs), marbofloxacillin (for beef cattle), oxytetracycline (for all species except chickens), sulfadiazine with trimethoprim (for pigs and chickens) and tilmicosin (for chickens); antiparasitic drugs, such as the benzimidazoles triclabendazole (for beef and dairy cattle), fenbendazole/oxfendazole (for sheep/goats and dairy cattle) and albendazole (for dairy cattle), the avermectin ivermectin (for beef cattle), and anti-fluke drugs closantel and rafoxanide (for sheep/goats); the anticoccidials monensin, narasin, nicarbazin and toltrazuril (for chickens). The risk ranking system described is a relatively simple system

  5. Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: an integrated approach to explore the cysteine oxidation.

    PubMed

    Chang, Yuan-Chang; Huang, Chien-Ning; Lin, Chia-Hung; Chang, Huan-Cheng; Wu, Chih-Che

    2010-08-01

    Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI-MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in tryptic digests of proteins based on ionic affinity capture using polyarginine-coated nanodiamonds as high-affinity probes. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a complex peptide mixture in which the abundance of the sulfonated analyte is as low as 0.02%. The polyarginine-coated probes exhibit a higher affinity for peptides containing multiple sulfonic acids than peptides containing single sulfonic acid. The limit of the detection is in the femtomole range, with the MALDI-TOF mass spectrometer operating in the negative ion mode. The results show that the new approach has good specificity even in the presence of phosphopeptides. An application of this method for selective enrichment and structural identification of sulfopeptides is demonstrated with the tryptic digests of performic-acid-oxidized BSA.

  6. Cysteine sensing by plasmons of silver nanocubes

    NASA Astrophysics Data System (ADS)

    Elfassy, Eitan; Mastai, Yitzhak; Salomon, Adi

    2016-09-01

    Noble metal nanoparticles are considered to be valuable nanostructures in the field of sensors due to their spectral response sensitivity to small changes in the surrounding refractive index which enables them to detect a small amount of molecules. In this research, we use silver nanocubes of about 50 nm length to detect low concentrations of cysteine, a semi-essential amino acid. Following cysteine adsorption onto the nanocubes, a redshift in the plasmonic modes was observed, enabling the detection of cysteine down to 10 μM and high sensitivity of about 125 nm/RIU (refractive index units). Furthermore, we found that multilayer adsorption of cysteine leads to the stabilization of the silver nanocubes. The cysteine growth onto the nanocubes was also characterized by high-resolution transmission electron microscopy (HR-TEM).

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

    NASA Astrophysics Data System (ADS)

    Adams, Eri; Miyazaki, Takae; Hayaishi-Satoh, Aya; Han, Minwoo; Kusano, Miyako; Khandelia, Himanshu; Saito, Kazuki; Shin, Ryoung

    2017-02-01

    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 that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation.

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

    PubMed Central

    Adams, Eri; Miyazaki, Takae; Hayaishi-Satoh, Aya; Han, Minwoo; Kusano, Miyako; Khandelia, Himanshu; Saito, Kazuki; Shin, Ryoung

    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 that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation. PMID:28230101

  9. Supported oligomethionine sulfoxide and Ellman's reagent for cysteine bridges formation.

    PubMed

    Ronga, Luisa; Verdié, Pascal; Sanchez, Pierre; Enjabal, Christine; Maurras, Amélie; Jullian, Magalie; Puget, Karine; Martinez, Jean; Subra, Gilles

    2013-02-01

    A large number of bioactive peptides are cyclized through a disulfide bridge. This structural feature is very important for both bioactivity and stability. The oxidation of cysteine side chains is challenging not only to avoid intermolecular reaction leading to oligomers and oxidation of other residues but also to remove solvents and oxidant such as dimethyl sulfoxide. Supported reagents advantageously simplify the work-up of such disulfide bond formation, but may lead to a significant decrease in yield of the oxidized product. In this study, two resins working through different mechanisms were evaluated: Clear-Ox, a supported version of Ellman's reagent and Oxyfold, consisting in a series of oxidized methionine residues. The choice of the supported reagent is discussed on the light of reaction speed, side-products formation and yield considerations.

  10. Construction, purification, and immunogenicity of recombinant cystein-cystein type chemokine receptor 5 vaccine.

    PubMed

    Wu, Kongtian; Xue, Xiaochang; Wang, Zenglu; Yan, Zhen; Shi, Jihong; Han, Wei; Zhang, Yingqi

    2006-09-01

    Cystein-Cystein type chemokine receptor 5 (CCR5) is a seven-transmembrane, G-protein coupled receptor. It is a major coreceptor with CD4 glycoprotein mediating cellular entry of CCR5 strains of HIV-1. A lack of cell-surface expression of CCR5 found in the homozygous Delta32 CCR5 mutation, upregulation of CC chemokines and antibodies to CCR5 are associated with resistance to HIV infection. In addition, CCR5 can be blocked by three CC chemokines and antibodies to three extracellular domains of CCR5. Consequently, CCR5 is considered an attractive therapeutic target against HIV infection. In the current study, we constructed a recombinant vaccine by coupling a T helper epitope AKFVAAWTLKAA (PADRE) to the N terminus of CCR5 extracellular domains (PADRE-CCR5) and expressed this protein in Escherichia coli. We have developed an inexpensive and scalable purification process for the fusion protein from inclusion bodies and the final yields of 6mg purified fusion protein per gram of cell paste was obtained. The immunogenicity of the recombinant vaccine generated was examined in BALB/c mice. Sera from the vaccinated mice demonstrated high-titer specific antibodies to the recombinant vaccine, suggesting that PADRE-rCCR5 may be used as a candidate of active CCR5 vaccine.

  11. Salt Effect Accelerates Site-Selective Cysteine Bioconjugation

    PubMed Central

    2016-01-01

    Highly efficient and selective chemical reactions are desired. For small molecule chemistry, the reaction rate can be varied by changing the concentration, temperature, and solvent used. In contrast for large biomolecules, the reaction rate is difficult to modify by adjusting these variables because stringent biocompatible reaction conditions are required. Here we show that adding salts can change the rate constant over 4 orders of magnitude for an arylation bioconjugation reaction between a cysteine residue within a four-residue sequence (π-clamp) and a perfluoroaryl electrophile. Biocompatible ammonium sulfate significantly enhances the reaction rate without influencing the site-specificity of π-clamp mediated arylation, enabling the fast synthesis of two site-specific antibody–drug conjugates that selectively kill HER2-positive breast cancer cells. Computational and structure–reactivity studies indicate that salts may tune the reaction rate through modulating the interactions between the π-clamp hydrophobic side chains and the electrophile. On the basis of this understanding, the salt effect is extended to other bioconjugation chemistry, and a new regioselective alkylation reaction at π-clamp cysteine is developed. PMID:27725962

  12. S-sulfhydration: a cysteine posttranslational modification in plant systems.

    PubMed

    Aroca, Ángeles; Serna, Antonio; Gotor, Cecilia; Romero, Luis C

    2015-05-01

    Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems.

  13. Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine 1

    PubMed Central

    Sekiya, Jiro; Schmidt, Ahlert; Wilson, Lloyd G.; Filner, Philip

    1982-01-01

    Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H2S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H2S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H2S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H2S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H2S formation and its release occurred in response to l-cysteine. Feeding experiments with [35S]l-cysteine showed that most of the sulfur in H2S was derived from sulfur in the l-cysteine supplied and that the H2S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. 35S-labeled SO32− and SO42− were found in the tissue extract in addition to internal soluble S2−. These findings

  14. Structural Basis of Conserved Cysteine in the Fibroblast Growth Factor Family: Evidence for a Vestigial Half-Cystine

    SciTech Connect

    Lee, Jihun; Blaber, Michael

    2010-11-09

    The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in {approx} 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.

  15. Structural basis of conserved cysteine in the fibroblast growth factor family: evidence for a vestigial half-cystine.

    PubMed

    Lee, Jihun; Blaber, Michael

    2009-10-16

    The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in approximately 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.

  16. Spectroscopic studies on the interaction of cysteine capped CuS nanoparticles with tyrosine

    SciTech Connect

    Prasanth, S.; Raj, D. Rithesh; Kumar, T. V. Vineesh; Sudarsanakumar, C.

    2015-06-24

    Biocompatible cysteine coated CuS nanoparticles were synthesized by a simple aqueous solution method. Hexagonal phase of the samples were confirmed from X-ray diffraction and particle size found to be 9 nm. The possible interaction between the bioactive cysteine capped CuS nanoparticles and tyrosine were investigated using spectroscopic techniques such as UV-Visible absorption and fluorescence spectroscopy. It is observed that the luminescence intensity of tyrosine molecule enhanced by the addition CuS nanoparticles.

  17. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer 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

  18. L-Cysteine and L-AP4 microinjections in the rat caudal ventrolateral medulla decrease arterial blood pressure.

    PubMed

    Takemoto, Yumi

    2014-12-01

    The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM.

  19. Blends of cysteine-containing proteins

    NASA Astrophysics Data System (ADS)

    Barone, Justin

    2005-03-01

    Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

  20. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  1. Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

    PubMed Central

    2014-01-01

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054. PMID:25181601

  2. Differential cysteine labeling and global label-free proteomics reveals an altered metabolic state in skeletal muscle aging.

    PubMed

    McDonagh, Brian; Sakellariou, Giorgos K; Smith, Neil T; Brownridge, Philip; Jackson, Malcolm J

    2014-11-07

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054.

  3. IscS from Archaeoglobus fulgidus has no desulfurase activity but may provide a cysteine ligand for [Fe2S2] cluster assembly.

    PubMed

    Pagnier, Adrien; Nicolet, Yvain; Fontecilla-Camps, Juan C

    2015-06-01

    Iron sulfur ([Fe-S]) clusters are essential prosthetic groups involved in fundamental cell processes such as gene expression regulation, electron transfer and Lewis acid base chemistry. Central components of their biogenesis are pyridoxal-5'-phosphate (PLP) dependent l-cysteine desulfurases, which provide the necessary S atoms for [Fe-S] cluster assembly. The archaeon Archaeoglobus fulgidus (Af) has two ORFs, which although annotated as l-cysteine desulfurases of the ISC type (IscS), lack the essential Lys residue (K199 in Af) that forms a Schiff base with PLP. We have previously determined the structure of an Af(IscU-D35A-IscS)2 complex heterologously expressed in Escherichia coli and found it to contain a [Fe2S2] cluster. In order to understand the origin of sulfide in that structure we have performed a series of functional tests using wild type and mutated forms of AfIscS. In addition, we have determined the crystal structure of an AfIscS-D199K mutant. From these studies we conclude that: i) AfIscS has no desulfurase activity; ii) in our in vitro [Fe2S2] cluster assembly experiments, sulfide ions are non-enzymatically generated by a mixture of iron, l-cysteine and PLP and iii) the physiological role of AfIscS may be to provide a cysteine ligand to the nascent cluster as observed in the [Fe2S2]-Af(IscU-D35A-IscS)2 complex. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  4. Profiling the Reactivity of Cyclic C-Nucleophiles towards Electrophilic Sulfur in Cysteine Sulfenic Acid

    PubMed Central

    Gupta, Vinayak; Carroll, Kate S.

    2015-01-01

    Oxidation of a protein cysteine thiol to sulfenic acid, termed S-sulfenylation, is a reversible post-translational modification that plays a crucial role in regulating protein function and is correlated with disease states. The majority of reaction-based small molecule and immunochemical probes used for detecting sulfenic acids are based on the 5,5-dimethyl-1,3-cyclohexanedione (dimedone) scaffold, which is selective, but suffers from low reactivity. In addition, mechanistic details and features that diminish or enhance nucleophile reactivity remain largely unknown. A significant hurdle to resolving the aforementioned issues has been the chemically unstable nature of small-molecule sulfenic acid models. Herein, we report a facile mass spectrometry-based assay and repurposed dipeptide-based model to screen a library of cyclic C-nucleophiles for reactivity with sulfenic acid under aqueous conditions. Observed rate constants for ~100 cyclic C-nucleophiles were obtained and, from this collection, we have identified novel compounds with more than 200-fold enhanced reactivity, as compared to dimedone. The increase in reactivity and retention of selectivity of these C-nucleophiles were validated in secondary assays, including a protein model for sulfenic acid. Together, this work represents a significant step toward developing new chemical reporters for detecting protein S-sulfenylation with superior kinetic resolution. The enhanced rates and varied composition of the C-nucleophiles should enable more comprehensive analyses of the sulfenome and serve as the foundation for reversible or irreversible nucleophilic covalent inhibitors that target oxidized cysteine residues in therapeutically important proteins. PMID:26819701

  5. Glutamyl cysteine dipeptide suppresses ferritin expression and alleviates liver injury in iron-overload rat model.

    PubMed

    Salama, Samir A; Al-Harbi, Mohammad S; Abdel-Bakky, Mohamed S; Omar, Hany A

    2015-08-01

    Despite its biological importance, iron is a pro-oxidant element and its accumulation results in tissue injury. Iron overload diseases such as thalassemia and hereditary hemochromatosis are commonly associated with liver tissue injury. Glutamyl cysteine (GC) is a dipeptide with antioxidant properties owing to its cysteine residue. The aim of the current work was to investigate the hepatoprotective effect of GC against iron overload-induced liver injury. Rats were distributed into five groups; normal control, GC control, iron-treated (150 mg/kg ip injection) and both iron and GC-treated (total iron: 150 mg/kg ip and GC: 50 mg or 100 mg/kg/day ip for 30 days). Our results showed that treatment with GC at the two-dose levels attenuated iron-induced liver tissue injury as evidenced by significant reduction in serum activity of liver enzymes ALT and AST, amelioration of iron-induced histopathological alteration, suppression of iron-induced oxidative stress as demonstrated by significant reduction of malondialdehyde and protein carbonyl content beside elevation of total antioxidant capacity, reduced glutathione and the antioxidant enzymes GPx and SOD in liver tissue. In addition, GC significantly reduced levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β and activity of the apoptotic marker caspase-3 in liver tissues. To our surprise, GC reduced liver iron content and ferritin expression, denoting the possible iron chelation competency. Collectively our results highlight evidence for the hepatoprotective effect of GC against iron overload-induced liver injury that is potentially mediated through suppression of oxidative tissue injury, attenuation of inflammatory response, amelioration of hepatocellular apoptosis and possibly through iron chelation.

  6. A Review on Various Uses of N-Acetyl Cysteine

    PubMed Central

    Mokhtari, Vida; Afsharian, Parvaneh; Shahhoseini, Maryam; Kalantar, Seyed Mehdi; Moini, Ashraf

    2017-01-01

    N-acetyl cysteine (NAC), as a nutritional supplement, is a greatly applied antioxidant in vivo and in vitro. NAC is a precursor of L-cysteine that results in glutathione elevation biosynthesis. It acts directly as a scavenger of free radicals, especially oxygen radicals. NAC is a powerful antioxidant. It is also recommended as a potential treatment option for different disorders resulted from generation of free oxygen radicals. Additionally, it is a protected and endured mucolytic drug that mellows tenacious mucous discharges. It has been used for treatment of various diseases in a direct action or in a combination with some other medications. This paper presents a review on various applications of NAC in treatment of several diseases. PMID:28367412

  7. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  8. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGES

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  9. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  10. Formation of three N-acetyl-L-cysteine monoadducts and one diadduct by the reaction of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide with N-acetyl-L-cysteine at physiological conditions: chemical mechanisms and toxicological implications.

    PubMed

    Barshteyn, Nella; Elfarra, Adnan A

    2007-10-01

    Previously, our laboratory has shown that S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by a flavin-containing monooxygenase 3 (FMO3)-mediated oxidation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. In the present study, we characterized reactions of DCVCS with nucleophilic amino acids. DCVCS incubations with N-acetyl-L-cysteine (NAC) at pH 7.4 and 37 degrees C for 1 h resulted in the formation of three monoadducts and one diadduct characterized by LC/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The formation of all adducts (with relative ratios of 29, 31, 24, and 12%, respectively) was rapid and time-dependent; the half-lives of the two DCVCS diastereomers in the presence of NAC were 13.8 (diastereomer I) and 9.4 min (diastereomer II). Adducts 1 and 2 were determined to be diastereomers of S-[1-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed by Michael addition of NAC to the terminal vinylic carbon of DCVCS followed by loss of HCl. Adduct 4 was determined to be S-[2-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed from the initial Michael addition product followed by a less favorable loss of HCl and/or by a rearrangement of adduct 2 through the formation of a cyclic chloronium ion. The addition of another molecule of NAC to monoadducts 1, 2, or 4 resulted in the formation of the novel diadduct, S-[2,2-( N-acetyl-L-cystein-S-yl)vinyl]-L-cysteine sulfoxide (adduct 3), whose detection in relatively large amount suggests that DCVCS could act as a cross-linking agent. DCVCS was not reactive with N-acetyl-L-lysine or L-valinamide at similar incubation conditions. Collectively, the results suggest selective reactivity of DCVCS toward protein sulfhydryl groups. Furthermore, the cross-linking properties of DCVCS may in part explain its high nephrotoxic potency.

  11. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGES

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  12. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  13. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    PubMed Central

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  14. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    PubMed

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  15. Cysteine S-conjugate β-lyases

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Pinto, John T.; Bruschi, Sam A.

    2010-01-01

    Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH2CH(NH3+)CO2−] and selenium Se-conjugates [RSeCH2CH(NH3+)CO2−] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a β-lyase reaction, but catalyze a non-physiological β-lyase side reaction that depends on the electron-withdrawing properties of the –SR or –SeR moiety. In the case of the cysteine S-conjugates, if the eliminated RSH is stable the compound may be S-thiomethylated and excreted (thiomethyl shunt) or S-glucuronidated and harmlessly excreted [the possibility that RSeH compounds may be similarly metabolized has not been extensively studied]. If, however, RSH is chemically reactive the cysteine S-conjugate may be toxic as a result of the β-lyase reaction. The cysteine S-conjugate β-lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. PMID:20949433

  16. The effects of fuel composition and ammonium sulfate addition on PCDD, PCDF, PCN and PCB concentrations during the combustion of biomass and paper production residuals.

    PubMed

    Lundin, Lisa; Jansson, Stina

    2014-01-01

    The use of waste wood as an energy carrier has increased during the last decade. However, the higher levels of alkali metals and chlorine in waste wood compared to virgin biomass can promote the formation of deposits and organic pollutants. Here, the effect of fuel composition and the inhibitory effects of ammonium sulfate, (NH4)2SO4, on the concentrations of persistent organic pollutants (POPs) in the flue gas of a lab-scale combustor was investigated. Ammonium sulfate is often used as a corrosion-preventing additive and may also inhibit formation of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). In addition to PCDDs and PCDFs, polychlorinated naphthalenes (PCN) and biphenyls (PCB) were also analyzed. It was found that the flue gas composition changed dramatically when (NH4)2SO4 was added: CO, SO2, and NH3 levels increased, while those of HCl decreased to almost zero. However, the additive's effects on POP formation were less pronounced. When (NH4)2SO4 was added to give an S:Cl ratio of 3, only the PCDF concentration was reduced, indicating that this ratio was not sufficient to achieve a general reduction in POP emissions. Conversely, at an S:Cl ratio of 6, significant reductions in the WHO-TEQ value and the PCDD and PCDF contents of the flue gas were observed. The effect on the PCDF concentration was especially pronounced. PCN formation seemed to be promoted by the elevated CO concentrations caused by adding (NH4)2SO4.

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

    PubMed Central

    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

    2009-01-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defences and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and its self-processed mature form. 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 20Å 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. PMID:17993455

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

    PubMed

    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.

  19. Direct observation of an anisotropic in-plane residual stress induced by B addition as an origin of high magnetic anisotropy field of Ru/FeCoB film

    SciTech Connect

    Hirata, Ken-ichiro; Gomi, Shunsuke; Mashiko, Yasuhiro; Nakagawa, Shigeki

    2010-05-15

    Although boron-free FeCo films prepared on a Ru underlayer exhibits isotropic in-plane magnetic property, boron added FeCoB films prepared on Ru underlayer revealed large in-plane magnetic anisotropy with a high anisotropy field of 500 Oe. The effect of boron addition on the in-plane anisotropic residual stress in FeCoB film was investigated using sin{sup 2} {psi} method of x-ray diffraction analysis. Large isotropic compressive stress was observed in Ru/FeCo film. In contrast, anisotropic in-plane residual stress was observed in Ru/FeCoB film. The compressive stress along the easy axis of Ru/FeCoB film is released more than that along the hard axis. Such anisotropic residual stress is regarded as an origin of the in-plane magnetic anisotropy through inverse magnetostriction effect. Owing to the configuration of the facing targets sputtering system, boron atoms are sputtered and deposited anisotropically, and so they penetrate FeCo crystals and release the compressive stress along the incidence direction.

  20. Induction of mitochondrial fusion by cysteine-alkylators ethacrynic acid and N-ethylmaleimide.

    PubMed

    Bowes, Timothy J; Gupta, Radhey S

    2005-03-01

    Mitochondrial fusion and fission are important aspects of eukaryotic cell function that permit the adoption of varied mitochondrial morphologies depending upon cellular physiology. We previously observed that ethacrynic acid (EA) induced mitochondrial fusion in cultured BSC-1 and CHO/wt cells. However, the mechanism responsible for it was not clear since EA has a number of known cellular effects including glutathione (GSH) depletion and alkylation of cysteine residues. To gain insight, we have tested the effects of a variety of compounds on EA induced cellular toxicity and mitochondrial fusion. N-acetyl cysteine (NAC), a GSH precursor, was found to abrogate both the toxic and fusion-inductive effects, whereas diethylmaleate (dEM), a GSH depletor, potentiated both these effects in a dose-dependent manner. However, treatment with dEM alone, which depleted GSH to the same degree as EA, did not induce mitochondrial fusion. These results indicate that although detoxification of EA via formation of GSH conjugates is dependant upon GSH levels, the depletion of GSH by EA is not responsible for its effect on mitochondrial fusion. Dihydro-EA (DH-EA), a saturated EA analogue, lacked EA's toxicity and effect on fusion, indicating that the alpha,beta-unsaturated ketone is central to its observed effects. N-ethylmaleimide (NEM), another well-known cysteine-alkylator, also induced mitochondrial fusion at near toxic concentrations. These data suggests that cysteine-alkylation is the causative factor for fusion and toxicity. In live BSC-1 cells, EA induced fusion of mitochondria occurred very rapidly (<20 min), which suggests that it is inducing fusion by modifying certain critical cysteine residue(s) in proteins involved in the process.

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

    PubMed

    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.

  2. Structural Determinants of the Closed KCa3.1 Channel Pore in Relation to Channel Gating: Results from a Substituted Cysteine Accessibility Analysis

    PubMed Central

    Klein, Hélène; Garneau, Line; Banderali, Umberto; Simoes, Manuel; Parent, Lucie; Sauvé, Rémy

    2007-01-01

    In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca2+-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 Å) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 Å) resulted in modification rates 103–104 faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg+, and Ag+ appeared poorly state dependent, whereas modification rates by MTSET were 103 faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg+, and Ag+, arguing

  3. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.

    PubMed

    Uprety, Rajendra; Luo, Ji; Liu, Jihe; Naro, Yuta; Samanta, Subhas; Deiters, Alexander

    2014-08-18

    We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

  4. Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines.

    PubMed

    Eastland, Adrienne; Hornick, Jessica; Kawamura, Ryo; Nanavati, Dhaval; Marko, John F

    2016-09-01

    We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.

  5. Cysteine as a Biological Probe for Comparing Plasma Sources

    NASA Astrophysics Data System (ADS)

    Lackmann, Jan-Wilm; Golda, Judith; Kogelheide, Friederike; Held, Julian; Schulz-von-der-Gathen, Volker; Stapelmann, Katharina

    2016-09-01

    A large variety of plasma sources are available in the plasma medicine community. While enabling to choose the most promising source for a certain biomedical application, comparison of the different sources with a focus on their effect on biological targets is rather challenging. To allow for better comparison of various sources, the recent European COST action MP1101 was used to design the COST reference microplasma jet. Cysteine is a promising candidate investigate the impact of plasma from various sources on a standardized biological molecule, which is especially relevant for the investigations on a molecular level after plasma treatment. The simple structure of cysteine allows for a more in-depth analysis of each chemical group after plasma treatment and enables a comparison between different plasma sources and treatment parameters on each chemical group. The model itself has already been successfully established using a dielectric barrier discharge. Here, additional plasma sources are compared by the means of their impact on cysteine samples, showing e.g. the influence of feed-gas variations by adding oxygen or nitrogen admixture This work was supported by the German Research Foundation (DFG) with the packet grant PAK816 (PlaCID).

  6. Addition of microbially-treated sugar beet residue and a native bacterium increases structural stability in heavy metal-contaminated Mediterranean soils.

    PubMed

    Carrasco, L; Caravaca, F; Azcón, R; Kohler, J; Roldán, A

    2009-10-15

    A mesocosm experiment was conducted to investigate the effect of the addition of Aspergillus niger-treated sugar beet waste, in the presence of rock phosphate, and inoculation with a native, metal-tolerant bacterium, Bacillus thuringiensis, on the stabilisation of soil aggregates of two mine tailings, with differing pH values, from a semiarid Mediterranean area and on the stimulation of growth of Piptatherum miliaceum. Bacterium combined with organic amendment enhanced structural stability (38% in acidic soil and 106% in neutral soil compared with their corresponding controls). Only the organic amendment increased pH, electrical conductivity, water-soluble C, water-soluble carbohydrates and plant growth, in both soils. While in neutral soil both organic amendment and bacterium increased dehydrogenase activity, only organic amendment had a significant effect in acidic soil. This study demonstrates that the use of P. miliaceum in combination with organic amendment and bacterium is a suitable tool for the stabilisation of the soil structure of degraded mine tailings, although its effectiveness is dependent on soil pH.

  7. Cysteine Transport into Cultured Tobacco Cells

    PubMed Central

    Harrington, H. Michael; Smith, Ivan K.

    1977-01-01

    Cysteine transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured on liquid B-5 medium was examined. Transport was linear with time or amount of tissue and had a pH optimum of 4.5. Cysteine transport over a wide concentration range was biphasic. The isotherm, for descriptive convenience, was divided into two segments both of which obeyed Michaelis-Menten kinetics. The Km for high affinity transport was in the range 1.7 × 10−5m(±0.17) while the Km for low affinity transport was in the range 3.5 × 10−4m(±0.13). Maximum velocities were 3 to 6 nmoles/g fresh weight/minute and 13 to 16 nmoles/g fresh weight/minute, respectively. Azide and 2,4-dinitrophenol caused more than 90% inhibition of net transport by either system. N,N′-Dicyclohexylcarbodiimide was not inhibitory while the inhibition by carbonylcyanide m-chlorophenylhydrazone was dependent on the cysteine concentration. Only those compounds that were inhibitory to transport caused significant efflux of labeled material from preloaded cells. Tobacco cells that had been preincubated in iodoacetamide or N-ethylmaleimide did not transport cysteine while similar treatments with dithiothreitol were only slightly inhibitory or had no effect on transport. Transport by either system was, to some extent, inhibited by all other tested amino acids and analogs. Alanine, methionine, and S-methyl cysteine were most effective in inhibiting cysteine transport. Both alanine and methionine were competitive inhibitors of cysteine transport by either system with inhibition constants that were similar to the Km for the particular system. PMID:16660190

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

    NASA Technical Reports Server (NTRS)

    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.

  9. Engineering a Chemical Switch into the Light-driven Proton Pump Proteorhodopsin by Cysteine Mutagenesis and Thiol Modification.

    PubMed

    Harder, Daniel; Hirschi, Stephan; Ucurum, Zöhre; Goers, Roland; Meier, Wolfgang; Müller, Daniel J; Fotiadis, Dimitrios

    2016-07-25

    For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system.

  10. Identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library.

    PubMed

    Shah, Falgun; Mukherjee, Prasenjit; Gut, Jiri; Legac, Jennifer; Rosenthal, Philip J; Tekwani, Babu L; Avery, Mitchell A

    2011-04-25

    Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

  11. Addition of HOBt improves the conversion of thioester-Amine chemical ligation.

    PubMed

    Todorovski, Toni; Suñol, David; Riera, Antoni; Macias, Maria J

    2015-11-01

    The syntheses of large peptides and of those containing non-natural amino acids can be facilitated by the application of convergent approaches, dissecting the native sequence into segments connected through a ligation reaction. We describe an improvement of the ligation protocol used to prepare peptides and proteins without cysteine residues at the ligation junction. We have found that the addition of HOBt to the ligation, improves the conversion of the ligation reaction without affecting the epimerization rate or chemoselectivity, and it can be efficiently used with peptides containing phosphorylated amino acids.

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

    SciTech Connect

    Huvent, Isabelle; Kamah, Amina; Cantrelle, François-Xavier; Barois, Nicolas; Slomianny, Christian; Smet-Nocca, Caroline; Landrieu, Isabelle; Lippens, Guy

    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.

  13. 75 FR 31790 - Determination That Cysteine Hydrochloride Injection, USP, 7.25%, Was Not Withdrawn From Sale for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    ..., 1986. Cysteine HCl is indicated for use as an additive to amino acid solutions to meet the nutritional... added to amino acid solutions to provide a more complete profile of amino acids for protein...

  14. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase.

    PubMed

    Carnevale, S; Rodríguez, M I; Guarnera, E A; Carmona, C; Tanos, T; Angel, S O

    2001-01-01

    Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.

  15. Substrate-assisted cysteine deprotonation in the mechanism of dimethylargininase (DDAH) from Pseudomonas aeruginosa.

    PubMed

    Stone, Everett M; Costello, Alison L; Tierney, David L; Fast, Walter

    2006-05-02

    The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N(omega)-methyl-l-arginine and N(omega),N(omega)-dimethyl-l-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK(a) of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK(a) values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k(cat) values indicates a much lower apparent pK(a) value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.

  16. How cysteine reacts with citral: an unexpected reaction of beta,beta-disubstituted acroleins with cysteine leading to hexahydro-1,4-thiazepines.

    PubMed

    Starkenmann, Christian; Brauchli, Robert; Maurer, Bruno

    2005-11-16

    The reaction of beta,beta-disubstituted acroleins [3-methyl-2-butenal (1), 3-methyl-2-hexenal (2), and citral (3)] with cysteine gave 1:2 adducts of a novel structural type, namely hexahydro-1,4-thiazepines. To the best of our knowledge, the spontaneous formation of a seven-membered heterocycle from the addition of cysteine to alpha,beta-unsaturated aldehydes is unprecedented. The adduct 6 obtained from citral, under acidic conditions, reacted further to give the new bicyclic compound 8.

  17. Structure and function in rhodopsin. Cysteines 65 and 316 are in proximity in a rhodopsin mutant as indicated by disulfide formation and interactions between attached spin labels.

    PubMed

    Yang, K; Farrens, D L; Altenbach, C; Farahbakhsh, Z T; Hubbell, W L; Khorana, H G

    1996-11-12

    To probe proximity relationships between different amino acids in the interhelical loops in the cytoplasmic domain of rhodopsin, we are using a general approach in which two cysteine residues are introduced at different locations. Here we report on the characteristics of one such mutant that contains the naturally occurring cysteine 316 near the cytoplasmic end of helix G and a second cysteine at position 65 (H65C), near the cytoplasmic end of helix A. The mutant protein after expression in COS-1 cells and reconstitution with 11-cis-retinal can be bound to anti-rhodopsin antibody 1D4-Sepharose at pH 6 in a form that contains the two cysteines in the free sulfhydryl form. In this form, the mutant protein reacts as expected with N-ethylmaleimide in the dark at room temperature and can be derivatized with nitroxide spin labels. However, under appropriate conditions, the mutant can be isolated with the cysteines in the disulfide form, which has been characterized by analysis of fragments produced on proteolysis with thermolysin. A study of the interactions between nitroxide spin labels attached to the two cysteine residues in the mutant protein indicates that in the dark state they are within about 10 A of each other. On illumination the distance between the spin labels increases. Collectively, the above results show that, upon folding of the mutant opsin in vivo, cysteines 65 and 316, and by inference, helices A and G, are in proximal locations and move further apart upon photoactivation.

  18. Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.

    PubMed

    Ono, Katsuhiko; Jung, Minkyung; Zhang, Tianli; Tsutsuki, Hiroyasu; Sezaki, Hiroshi; Ihara, Hideshi; Wei, Fan-Yan; Tomizawa, Kazuhito; Akaike, Takaaki; Sawa, Tomohiro

    2017-05-01

    Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized (34)S-labeled L-cysteine from O-acetyl-L-serine and (34)S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur ((34)S) and nitrogen ((15)N) atoms was also achieved by performing enzyme reactions with (15)N-labeled L-serine, acetyl-CoA, and (34)S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared (34)S-labeled N-acetyl-L-cysteine (NAC) by incubating (34)S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using (34)S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology.

  19. Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT.

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing.

    PubMed Central

    Musser, J M; Stockbauer, K; Kapur, V; Rudgers, G W

    1996-01-01

    Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolytic activity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis. PMID:8675287

  2. Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing.

    PubMed

    Musser, J M; Stockbauer, K; Kapur, V; Rudgers, G W

    1996-06-01

    Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolytic activity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis.

  3. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

    PubMed Central

    Yang, Jianhong; Wang, Yuxi; Wang, Taijing; Jiang, Jian; Botting, Catherine H.; Liu, Huanting; Chen, Qiang; Yang, Jinliang; Naismith, James H.; Zhu, Xiaofeng; Chen, Lijuan

    2016-01-01

    Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics. PMID:27357539

  4. Lifespan extension and increased resistance to environmental stressors by N-Acetyl-L-Cysteine in Caenorhabditis elegans

    PubMed Central

    Oh, Seung-Il; Park, Jin-Kook; Park, Sang-Kyu

    2015-01-01

    OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditions induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV), was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes, sod-3 and hsp-16.2, increased significantly following N-acetyl-L-cysteine treatment. CONCLUSION: N-acetyl-L-cysteine supplementation confers a longevity phenotype in C. elegans, possibly through increased resistance to environmental stressors. PMID:26039957

  5. On the Importance of Oxidative Folding in the Evolution of Conotoxins: Cysteine Codon Preservation through Gene Duplication and Adaptation

    PubMed Central

    Steiner, Andrew M.; Bulaj, Grzegorz; Puillandre, Nicolas

    2013-01-01

    Conotoxin genes are among the most rapidly evolving genes currently known; however, despite the well-established hypervariability of the intercysteine loops, the cysteines demonstrate significant conservation, with a site-specific codon bias for each cysteine in a family of conotoxins. Herein we present a novel rationale behind the codon-level conservation of the cysteines that comprise the disulfide scaffold. We analyze cysteine codon conservation using an internal reference and phylogenetic tools; our results suggest that the established codon conservation can be explained as the result of selective pressures linked to the production efficiency and folding of conotoxins, driving the conservation of cysteine at the amino-acid level. The preservation of cysteine has resulted in maintenance of the ancestral codon in most of the daughter lineages, despite the hypervariability of adjacent residues. We propose that the selective pressures acting on the venom components of cone snails involve an interplay of biosynthetic efficiency, activity at the target receptor and the importance of that activity to effective prey immobilization. Functional redundancy in the venom can thus serve as a buffer for the energy expenditure of venom production. PMID:24244311

  6. Detection of l-Cysteine in wheat flour by Raman microspectroscopy combined chemometrics of HCA and PCA.

    PubMed

    Cebi, Nur; Dogan, Canan Ekinci; Develioglu, Ayşen; Yayla, Mediha Esra Altuntop; Sagdic, Osman

    2017-08-01

    l-Cysteine is deliberately added to various flour types since l-Cysteine has enabled favorable baking conditions such as low viscosity, increased elasticity and rise during baking. In Turkey, usage of l-Cysteine as a food additive isn't allowed in wheat flour according to the Turkish Food Codex Regulation on food additives. There is an urgent need for effective methods to detect l-Cysteine in wheat flour. In this study, for the first time, a new, rapid, effective, non-destructive and cost-effective method was developed for detection of l-Cysteine in wheat flour using Raman microscopy. Detection of l-Cysteine in wheat flour was accomplished successfully using Raman microscopy combined chemometrics of PCA (Principal Component Analysis) and HCA (Hierarchical Cluster Analysis). In this work, 500-2000cm(-1) spectral range (fingerprint region) was determined to perform PCA and HCA analysis. l-Cysteine and l-Cystine were determined with detection limit of 0.125% (w/w) in different wheat flour samples.

  7. S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II.

    PubMed

    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.

  8. Overview of existing European food consumption databases: critical aspects in relation to their use for the assessment of dietary exposure to additives, flavourings and residues of food contact materials.

    PubMed

    Le Donne, Cinzia; Piccinelli, Raffaela; Sette, Stefania; Leclercq, Catherine

    2011-03-01

    A critical analysis of existing food consumption databases was performed with particular regard for their current and potential use for the assessment of dietary exposure to additives, flavourings and residues of food contact materials. Within the European Food Consumption Validation project (EFCOVAL), a questionnaire on critical aspects of such datasets was developed and administered to researchers responsible for the collection/analysis of national food consumption data in European countries. Information collected was complemented through a review of the literature and of grey publications in order to provide an inventory of the main food consumption surveys performed in Europe from 1994 to 2007, for a total of 23 countries and 37 surveys. It appeared that existing European food consumption surveys have as a main objective the assessment of nutrient intake in the population. On the other hand, most of the databases were shown to be used also for the purpose of dietary exposure assessment.

  9. Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

    SciTech Connect

    Xia, Xue; Kumru, Ozan S.; Blaber, Sachiko I.; Middaugh, C. Russell; Li, Ling; Ornitz, David M.; Sutherland, Mason A.; Tenorio, Connie A.; Blaber, Michael

    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. 2,2′-Dithiobis(5-nitropyridine) (DTNP) as an Effective and Gentle Deprotectant for Common Cysteine Protecting Groups†

    PubMed Central

    Schroll, Alayne L.; Hondal, Robert J.; Flemer, Stevenson

    2011-01-01

    Of all the commercially-available amino acid derivatives for solid phase peptide synthesis, none has a greater abundance of sidechain protection diversity than cysteine. The high reactivity of the cysteine thiol necessitates its attenuation during peptide construction. Moreover, the propensity of cysteine residues within a peptide or protein sequence to form disulfide connectivity allows the opportunity for the peptide chemist to install these disulfides iteratively as a post-synthetic manipulation through the judicious placement of orthogonal pairs of cysteine S-protection within the peptide's architecture. It is important to continuously discover new vectors of deprotection for these different blocking protocol in order to achieve the highest degree of orthogonality between the removal of one species in the presence of another. We report here a complete investigation of the scope and limitations of the deprotective potential of 2,2′-dithiobis(5-nitropyridine) (DTNP) on a selection of commercially-available Cys S-protecting groups. The gentle conditions of DTNP in a TFA solvent system show a remarkable ability to deprotect some cysteine blocking functionality traditionally removable only by more harsh or forcing conditions. Beyond illustrating the deprotective ability of this reagent cocktail within a cysteine-containing peptide sequence, the utility of this method was further demonstrated through iterative disulfide formation in oxytocin and apamin test peptides. It is shown that this methodology has high potential as a stand-alone cysteine deprotection technique or in further manipulation of disulfide architecture within a more complex cysteine-containing peptide template. PMID:22083608

  11. Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

    PubMed

    Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

    2015-09-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.

  12. Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.

    PubMed

    Klüver, Enno; Schulz-Maronde, Sandra; Scheid, Svenja; Meyer, Bernd; Forssmann, Wolf-Georg; Adermann, Knut

    2005-07-19

    Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

  13. Cysteine Prevents Menopausal Syndromes in Ovariectomized Mouse.

    PubMed

    Han, Na-Ra; Kim, Na-Rae; Kim, Hyung-Min; Jeong, Hyun-Ja

    2016-05-01

    Cysteine (Cys) is well known to be involved in oxidation-reduction reactions, serving as a source of sulfides in the body. Amino acids are known to improve menopausal symptoms and significantly reduce morbidity. This study aims to find an unrevealed effect of Cys with estrogenic and osteogenic actions. Ovariectomized (OVX) mice were treated with Cys daily for 8 weeks. Estrogen-related and osteoporosis-related factors were analyzed in the vagina, serum, and tibia. Cys was treated in estrogen receptor (ER)-positive human osteoblast-like MG-63 cells and ER-positive human breast cancer Michigan Cancer Foundation-7 (MCF-7) cells. Cysteine administration ameliorated overweightness of the body and vaginal atrophy in the OVX mice. Cysteine increased the levels of alkaline phosphatase (ALP) and 17β-estradiol in the serum of the OVX mice and improved the bone mineral density in the OVX mice. In MG-63 cells, Cys increased the proliferation, ERβ messenger RNA (mRNA) expression, and estrogen response element (ERE) activity. Cysteine increased the ALP activity and the phosphorylation of extracellular signal-regulated kinase. In MCF-7 cells, Cys also increased the proliferation, ERβ mRNA expression, and ERE activity. Taken together, these results demonstrated that Cys has estrogenic and osteogenic activities in OVX mice, MG-63 cells, and MCF-7 cells. The novel insights gained here strongly imply the potential use of Cys as a new agent for postmenopausal women.

  14. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  15. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  16. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  17. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  18. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  19. Natural cysteine protease inhibitors in protozoa: Fifteen years of the chagasin family.

    PubMed

    Costa, Tatiana F R; Lima, Ana Paula C A

    2016-03-01

    Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archaea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.

  20. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false L-Cysteine. 184.1271 Section 184.1271 Food and....1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3-mercaptopropanoic acid (C3H7O2NS). (b) The.... (c) The ingredient is used to supply up to 0.009 part of total L-cysteine per 100 parts of flour...

  1. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in §...

  2. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2... to 0.009 part of total L-cysteine per 100 parts of flour in dough as a dough strengthener as...

  3. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in §...

  4. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in §...

  5. Cysteine transport through excitatory amino acid transporter 3 (EAAT3).

    PubMed

    Watts, Spencer D; Torres-Salazar, Delany; Divito, Christopher B; Amara, Susan G

    2014-01-01

    Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest

  6. Posttranslational modification of cysteine in redox signaling and oxidative stress: Focus on s-glutathionylation.

    PubMed

    Mieyal, John J; Chock, P Boon

    2012-03-15

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have become recognized as second messengers for initiating and/or regulating vital cellular signaling pathways, and they are known also as deleterious mediators of cellular stress and cell death. ROS and RNS, and their cross products like peroxynitrite, react primarily with cysteine residues whose oxidative modification leads to functional alterations in the proteins. In this Forum, the collection of six review articles presents a perspective on the broad biological impact of cysteine modifications in health and disease from the molecular to the cellular and organismal levels, focusing in particular on reversible protein-S-glutathionylation and its central role in transducing redox signals as well as protecting proteins from irreversible cysteine oxidation. The Forum review articles consider the role of S-glutationylation in regulation of the peroxiredoxin enzymes, the special redox environment of the mitochondria, redox regulation pertinent to the function of the cardiovascular system, mechanisms of redox-activated apoptosis in the pulmonary system, and the role of glutathionylation in the initiation, propagation, and treatment of neurodegenerative diseases. Several common themes emerge from these reviews; notably, the probability of crosstalk between signaling/regulation mechanisms involving protein-S-nitrosylation and protein-S-glutathionylation, and the need for quantitative analysis of the relationship between specific cysteine modifications and corresponding functional changes in various cellular contexts.

  7. Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-12-01

    Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom}; R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

  8. Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

    PubMed

    Bertelsmann, Holger; Kuehbacher, Markus; Weseloh, Gundolf; Kyriakopoulos, Antonios; Behne, Dietrich

    2007-10-01

    The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.

  9. A novel cysteine-sparing NOTCH3 mutation in a Chinese family with CADASIL.

    PubMed

    Ge, Wei; Kuang, Hanzhe; Wei, Bin; Bo, Le; Xu, Zhice; Xu, Xingshun; Geng, Deqin; Sun, Miao

    2014-01-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL.

  10. Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5

    PubMed Central

    Windisch, Dirk; Hoffmann, Silke; Afonin, Sergii; Vollmer, Stefanie; Benamira, Soraya; Langer, Birgid; Bürck, Jochen; Muhle-Goll, Claudia; Ulrich, Anne S.

    2010-01-01

    The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization. PMID:20858420

  11. Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

    PubMed

    Abegg, Daniel; Frei, Reto; Cerato, Luca; Prasad Hari, Durga; Wang, Chao; Waser, Jerome; Adibekian, Alexander

    2015-09-07

    In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

  12. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines.

    PubMed

    Macpherson, Lindsey J; Dubin, Adrienne E; Evans, Michael J; Marr, Felix; Schultz, Peter G; Cravatt, Benjamin F; Patapoutian, Ardem

    2007-02-01

    The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

  13. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl...

  14. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl...

  15. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl...

  16. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl...

  17. A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough.

    PubMed

    Gao, Xin; Zhang, Qisen; Newberry, Marcus P; Chalmers, Ken J; Mather, Diane E

    2013-03-01

    The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue, which may interfere with glutenin polymerisation through cross-linkage within the Bx7.1 subunit, causing this subunit to act as a chain terminator.

  18. S-carboxymethyl-L-cysteine and it (R/S)-S-oxides in beagle dog plasma and hepatic cytosol.

    PubMed

    Panagopoulos, Panayotis; Mitchell, Stephen C; Steventon, Glyn B

    2015-01-01

    1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-L-cysteine, N-acetyl-S-carboxymethyl-L-cysteine, S-methyl-L-cysteine and N-acetyl-S-methyl-L-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (Km, Vmax) were derived indicating S-carboxymethyl-L-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction. 2. Following oral administration of S-carboxymethyl-L-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form. 3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-L-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.

  19. Local flexibility facilitates oxidization of buried methionine residues.

    PubMed

    Xu, Kuiran; Uversky, Vladimir N; Xue, Bin

    2012-06-01

    In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.

  20. Gas-phase interactions of organotin compounds with cysteine.

    PubMed

    Latrous, Latifa; Salpin, Jean-Yves; Haldys, Violette; Léon, Emmanuelle; Correia, Catarina; Lamsabhi, Al Mokhtar

    2016-11-01

    The gas-phase interactions of cysteine with di-organotin and tri-organotin compounds have been studied by mass spectrometry experiments and quantum calculations. Positive-ion electrospray spectra show that the interaction of di- and tri-organotins with cysteine results in the formation of [(R)2 Sn(Cys-H)](+) and [(R)3 Sn(Cys)](+) ions, respectively. MS/MS spectra of [(R)2 Sn(Cys-H)](+) complexes are characterized by numerous fragmentation processes, notably associated with elimination of NH3 and (C,H2 ,O2 ). Several dissociation routes are characteristic of each given organic species. Upon collision, both the [(R)3 Sn(Gly)](+) and [(R)3 Sn(Cys)](+) complexes are associated with elimination of the intact amino acid, leading to the formation of [(R)3 Sn](+) cation. But for the latter complex, two additional fragmentation processes are observed, associated with the elimination of NH3 and C3 H4 O2 S. Calculations indicate that the interaction between organotins and cysteine is predominantly electrostatic but also exhibits a considerable covalent character, which is slightly more pronounced in tri-organotin complexes. A preferred bidentate interaction of the type -η(2) -S-NH2 , with sulfur and the amino group, is observed. As for the [(R)3 Sn(Cys)](+) complexes, their stability is due to the combination of the hydrogen bond taking place between the amino group and the sulfur lone pair and the interaction between the carboxylic oxygen atom and the metal. Copyright © 2016 John Wiley & Sons, Ltd.

  1. How to improve fertility of African soils? Leguminous fallows (Cameroon), addition of farmyard manure and mineral fertilizer (Kenya), organic residues management and introduction of N2 fixing species in forest plantations (Congo).

    NASA Astrophysics Data System (ADS)

    Koutika, Lydie-Stella; Mareschal, Louis; Mouanda, Cadeau; Epron, Daniel

    2014-05-01

    Most of African soils are inherently infertile and poor in nutrients mainly nitrogen and phosphorus. Several practices are used to improve soil fertility, increase productivity and ensure their sustainability. Soil fertility in the leguminous fallows was evaluated through particulate organic matter (POM), the more active part of soil organic matter (SOM) in Cameroon. The combination of mineral and organic (manure) fertilizers increased microbial P biomass allowing the release of P along the plant growing period in the Kenyan soils. Organic residues management and introduction of nitrogen fixing species (Acacia) were used to improve soil fertility and sustain forest productivity on the coastal plains of Congo. SOM fractionation was made under Pueraria, Mucuna fallows and natural regrowth mainly Chromolaena and under 3 forest plantation treatments installed in previous savanna: 1) no input, 2) normal input, and 3) double input of organic residues. Microbial P biomass and sequential P fractionation were evaluated in high and low P fixing soils. N, C, available P and pH were determined on soil sampled in acacia (100A), eucalypt (100E) and mixed-species (50A:50E) stands. N and P were determined in aboveground litters and in leaves, bark and wood of trees. The two leguminous fallows increased N content in POM fractions i.e., N >1% for Pueraria and Mucuna against N<1% for natural regrowth in the 0-0.10m depth, probably through N input from N2 fixation from the atmosphere (Cameroon).The addition of mineral fertilizers and farmyard manure increases P biomass (4.8 after 2 weeks to 15.2 after 16 weeks), and then decreased to 9.7 mg P g-1 soil (week 32). It also changes the P Hedley fractions partition in the high P fixing Kenyan soil (0-0.10m). After two rotations (14 years), SOM mineralization was the highest in the double input of organic residues treatment (low coarse POM 5.6 g kg-1 of soil and high organo-mineral fraction (OMF) 115 g kg-1 of soil). The introduction of A

  2. Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

    PubMed Central

    Girard, Pierre-Marie; Graindorge, Dany; Smirnova, Violetta; Rigolet, Pascal; Francesconi, Stefania; Scanlon, Susan; Sage, Evelyne

    2013-01-01

    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. PMID:24116071

  3. An FITC-BODIPY FRET couple: application to selective, ratiometric detection and bioimaging of cysteine.

    PubMed

    Ma, Dong Hee; Kim, Dokyoung; Akisawa, Takuya; Lee, Kyung-Ha; Kim, Kyong-Tai; Ahn, Kyo Han

    2015-04-01

    A novel FRET couple of fluorescein is disclosed, and it was readily constructed by conjugating an amino-BODIPY dye, a new FRET donor, with fluorescein isocyanate. Its potential was demonstrated by a fluorescence sensing system for cysteine, which was prepared by introducing acryloyl groups to the fluorescein moiety. The FRET probe exhibited promising ratiometric response to cysteine with high selectivity and sensitivity in a buffer solution containing acetonitrile at a physiological pH of 7.4, but showed slow reactivity. This slow response was solved by addition of a surfactant, thus allowing ratiometric imaging and determination of the endogenous level of cysteine in cells in HEPES buffer, by confocal fluorescence microscopy. Imaging experiments toward various cells suggested that such aryl acrylate type probes are vulnerable to the ubiquitous esterase activity. For the selected C6 cell line, in which the esterase activity was minimal, the ratiometric quantification of cysteine level was demonstrated. The FRET probe was also applied to determine the level of cysteine in human blood plasma.

  4. Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification.

    PubMed

    Daffu, Gurdip K; Lopez, Patricia; Katz, Francine; Vinogradov, Michael; Zhan, Chang-Guo; Landry, Donald W; Macdonald, Joanne

    2015-11-01

    The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

  5. Growth of different morphologies (quantum dots to nanorod) of Ag-nanoparticles: role of cysteine concentrations.

    PubMed

    Khan, Zaheer; Talib, Abou

    2010-03-01

    This work describes an easy chemical method for the preparation of orange-red color silver sol by the cysteine reduction of silver (I) in the presence of cetyltrimethylammonium bromide (CTAB). The obtained sol was found to have very small particles in the order of quantum dots for the first time. Transmission electron microscope (TEM) images show that the silver sol consists of aggregated as well as cross-linking arrangement of spherical silver quantum dots (size in the range ca. addition, we observed the changes in the morphologies of the Ag-nanoparticles from quantum dots to nanorod of diameter 60nm and pearl-necklace shaped which occurred due to the cross-linking aggregation of silver quantum dots. For a certain reaction time, i.e., 100min, the absorbance of reaction mixture first increased until it reached a maximum, then decreased with [cysteine]. The rate of Ag-nanoparticles formation decreases with the increase in [cysteine] whereas [CTAB] and [Ag(+)] have no effect on the reaction rate. Interestingly, at higher [cysteine] (>or=20.0x10(-4)mol dm(-3)), white precipitate was formed instead of transparent silver sol. Cysteine acts as a reducing, cross-linking, stabilizing and buffering agent during the growth of different shape and size of silver nanoparticles.

  6. Dual Labeling Biotin Switch Assay to Reduce Bias Derived from Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection

    PubMed Central

    Chung, Heaseung Sophia; Murray, Christopher I.; Venkatraman, Vidya; Crowgey, Erin L.; Rainer, Peter P.; Cole, Robert N.; Bomgarden, Ryan D.; Rogers, John C.; Balkan, Wayne; Hare, Joshua M.; Kass, David A.; Van Eyk, Jennifer E.

    2016-01-01

    Rationale S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent dataset which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. Objective To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. Methods and Results We obtained SNO-modified cysteine datasets for wild-type and S-nitrosoglutathione reductase (GSNOR) knock-out mouse hearts (GSNOR is a negative regulator of GSNO production) and NO-induced human embryonic kidney cell using two labeling reagents; the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, while the remaining SNO sites were only labeled by one reagent. Characterization of the two distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. Based on this observation, we proposed a parallel dual labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO-sites in GSNOR knock-out hearts. Conclusions Using a protocol comprising two tags for dual labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. PMID:26338901

  7. Hieronymain I, a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae).

    PubMed

    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.

  8. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

    PubMed Central

    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.

    2016-01-01

    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. PMID:27782215

  9. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

    PubMed

    Hernández, Hilda M; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.

  10. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

    NASA Astrophysics Data System (ADS)

    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.

  11. L-Cysteine Metabolism and Fermentation in Microorganisms.

    PubMed

    Takagi, Hiroshi; Ohtsu, Iwao

    2016-11-22

    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.

  12. Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion.

    PubMed

    Santos, André L S; d'Avila-Levy, Claudia M; Dias, Felipe A; Ribeiro, Rachel O; Pereira, Fernanda M; Elias, Camila G R; Souto-Padrón, Thaïs; Lopes, Angela H C S; Alviano, Celuta S; Branquinha, Marta H; Soares, Rosangela M A

    2006-01-01

    In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

  13. Proteomic profiling of L-cysteine induced selenite resistance in Enterobacter sp. YSU

    PubMed Central

    Jasenec, Ashley; Barasa, Nathaniel; Kulkarni, Samatha; Shaik, Nabeel; Moparthi, Swarnalatha; Konda, Venkataramana; Caguiat, Jonathan

    2009-01-01

    Background Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway. Results Growth curves using M-9 minimal medium containing 40 mM selenite and 1 mM sulfate showed that Enterobacter sp. YSU grew when L-cysteine was present but died when it was absent. Differential protein expression analysis by two dimensional gel electrophoresis showed that CysK was present in cultures containing selenite and lacking L-cysteine but absent in cultures containing both selenite and L-cysteine. Additional RT-PCR studies demonstrated that transcripts for the sulfate permease genes, cysA, cysT and cysW, were down-regulated in the presence of L-cysteine. Conclusion L-cysteine appeared to confer selenite resistance upon Enterobacter sp. YSU by decreasing the level of selenite transport into the cell through the non-specific pathway. PMID:19715574

  14. Cysteine cathepsin activity regulation by glycosaminoglycans.

    PubMed

    Novinec, Marko; Lenarčič, Brigita; Turk, Boris

    2014-01-01

    Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail.

  15. Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

    PubMed

    Šmid, Ida; Rotter, Ana; Gruden, Kristina; Brzin, Jože; Buh Gašparič, Meti; Kos, Janko; Žel, Jana; Sabotič, Jerica

    2015-07-01

    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

  16. Accelerated on-column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis.

    PubMed

    Cindrić, Mario; Cepo, Tina; Skrlin, Ana; Vuletić, Marko; Bindila, Laura

    2006-01-01

    The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4,5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4,5-dihydro-1H-imidazole. Complete tagging of the peptides by on

  17. Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase.

    PubMed

    Karim, C B; Marquardt, C G; Stamm, J D; Barany, G; Thomas, D D

    2000-09-05

    Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.

  18. A perfluoroaryl-cysteine S(N)Ar chemistry approach to unprotected peptide stapling.

    PubMed

    Spokoyny, Alexander M; Zou, Yekui; Ling, Jingjing J; Yu, Hongtao; Lin, Yu-Shan; Pentelute, Bradley L

    2013-04-24

    We report the discovery of a facile transformation between perfluoroaromatic molecules and a cysteine thiolate, which is arylated at room temperature. This new approach enabled us to selectively modify cysteine residues in unprotected peptides, providing access to variants containing rigid perfluoroaromatic staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 capsid assembly polyprotein (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chemical stability of the formed staples allowed us to use this motif in the native chemical ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor.

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

    PubMed

    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.

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

    PubMed

    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.

  1. Methylation of cysteine in hemoglobin following exposure to methylating agents

    SciTech Connect

    Bailey, E.; Connors, T.A.; Farmer, P.B.; Gorf, S.M.; Rickard, J.

    1981-06-01

    In addition to reacting with biologically important nucleophilic sites in DNA, alkylating agents also interact with amino acids in proteins. Measurements of the extent of formation of these alkyl amino acids may be used as a means of determining exposure to these compounds. The degree of S-methylation of cysteine in hemoglobin was studied following in vivo exposure of rats to methyl methanesulfonate, dimethylnitrosamine, and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. A linear dose-response curve was observed for methyl methanesulfonate over a 100-fold dose range. For dimethylnitrosamine, there was a threshold of doses where no methylation could be detected, and a curved dose-response curve was obtained. At high doses, the degree of methylation of hemoglobin cysteine was 7-fold lower than that with methyl methanesulfonate. In vivo, no alkylation could be observed with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide; however, the existence of naturally occurring S-methylcysteine in the rat hemoglobin may have overshadowed small increases in alkylation arising from exposure to this compound. The natural occurrence of S-methylcysteine was studied in 13 species, and amounts ranging from 5.6 nmol/g globin (hamster) to 481 nmol/g globin (partridge) were observed. The reason for its occurrence is unknown but is under investigation.

  2. Nephrotoxicity of halogenated alkenyl cysteine-S-conjugates.

    PubMed

    Nagelkerke, J F; Boogaard, P J

    1991-01-01

    In 1916 a relationship was postulated between the occurrence of aplastic anaemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene. The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found. Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity. The haloalkenyl cysteine-S-conjugates (Cys-S-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloalkenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity will be outlined.

  3. Nephrotoxicity of halogenated alkenyl cysteine-S-conjugates

    SciTech Connect

    Nagelkerke, J.F.; Boogaard, P.J. )

    1991-01-01

    In 1916 a relationship was postulated between the occurrence of aplastic anemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene. The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found. Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity. The haloalkenyl cysteine-S-conjugates (Cys-D-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloakenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity is outlined.

  4. Photochemical and Nonphotochemical Transformations of Cysteine with Dissolved Organic Matter.

    PubMed

    Chu, Chiheng; Erickson, Paul R; Lundeen, Rachel A; Stamatelatos, Dimitrios; Alaimo, Peter J; Latch, Douglas E; McNeill, Kristopher

    2016-06-21

    Cysteine (Cys) plays numerous key roles in the biogeochemistry of natural waters. Despite its importance, a full assessment of Cys abiotic transformation kinetics, products and pathways under environmental conditions has not been conducted. This study is a mechanistic evaluation of the photochemical and nonphotochemical (dark) transformations of Cys in solutions containing chromophoric dissolved organic matter (CDOM). The results show that Cys underwent abiotic transformations under both dark and irradiated conditions. Under dark conditions, the transformation rates of Cys were moderate and were highly pH- and temperature-dependent. Under UVA or natural sunlight irradiations, Cys transformation rates were enhanced by up to two orders of magnitude compared to rates under dark conditions. Product analysis indicated cystine and cysteine sulfinic acid were the major photooxidation products. In addition, this study provides an assessment of the contributions of singlet oxygen, hydroxyl radical, hydrogen peroxide, and triplet dissolved organic matter to the CDOM-sensitized photochemical oxidation of Cys. The results suggest that another unknown pathway was dominant in the CDOM-sensitized photodegradation of Cys, which will require further study to identify.

  5. Identifying cysteines and histidines in transition-metal-binding sites using support vector machines and neural networks.

    PubMed

    Passerini, Andrea; Punta, Marco; Ceroni, Alessio; Rost, Burkhard; Frasconi, Paolo

    2006-11-01

    Accurate predictions of metal-binding sites in proteins by using sequence as the only source of information can significantly help in the prediction of protein structure and function, genome annotation, and in the experimental determination of protein structure. Here, we introduce a method for identifying histidines and cysteines that participate in binding of several transition metals and iron complexes. The method predicts histidines as being in either of two states (free or metal bound) and cysteines in either of three states (free, metal bound, or in disulfide bridges). The method uses only sequence information by utilizing position-specific evolutionary profiles as well as more global descriptors such as protein length and amino acid composition. Our solution is based on a two-stage machine-learning approach. The first stage consists of a support vector machine trained to locally classify the binding state of single histidines and cysteines. The second stage consists of a bidirectional recurrent neural network trained to refine local predictions by taking into account dependencies among residues within the same protein. A simple finite state automaton is employed as a postprocessing in the second stage in order to enforce an even number of disulfide-bonded cysteines. We predict histidines and cysteines in transition-metal-binding sites at 73% precision and 61% recall. We observe significant differences in performance depending on the ligand (histidine or cysteine) and on the metal bound. We also predict cysteines participating in disulfide bridges at 86% precision and 87% recall. Results are compared to those that would be obtained by using expert information as represented by PROSITE motifs and, for disulfide bonds, to state-of-the-art methods.

  6. A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2*

    PubMed Central

    Garcia-Mata, Carlos; Wang, Jianwen; Gajdanowicz, Pawel; Gonzalez, Wendy; Hills, Adrian; Donald, Naomi; Riedelsberger, Janin; Amtmann, Anna; Dreyer, Ingo; Blatt, Michael R.

    2010-01-01

    Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca2+ and K+ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K+ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H2O2. We show that H2O2 rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H2O2 and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H2O2-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys168 located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H2O2. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys168 as a critical target for H2O2, and implicate ROS-mediated control of the K+ channel in regulating mineral nutrient partitioning within the plant. PMID:20605786

  7. A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

    PubMed

    Shen, Yulong; Tang, Xiao-Feng; Yokoyama, Hideshi; Matsui, Eriko; Matsui, Ikuo

    2004-01-01

    Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.

  8. Affinity of Avr2 for tomato cysteine protease Rcr3 correlates with the Avr2-triggered Cf-2-mediated hypersensitive response.

    PubMed

    Van't Klooster, John W; Van der Kamp, Marc W; Vervoort, Jacques; Beekwilder, Jules; Boeren, Sjef; Joosten, Matthieu H A J; Thomma, Bart P H J; De Wit, Pierre J G M

    2011-01-01

    The Cladosporium fulvum Avr2 effector is a novel type of cysteine protease inhibitor with eight cysteine residues that are all involved in disulphide bonds. We have produced wild-type Avr2 protein in Pichia pastoris and determined its disulphide bond pattern. By site-directed mutagenesis of all eight cysteine residues, we show that three of the four disulphide bonds are required for Avr2 stability. The six C-terminal amino acid residues of Avr2 contain one disulphide bond that is not embedded in its overall structure. Avr2 is not processed by the tomato cysteine protease Rcr3 and is an uncompetitive inhibitor of Rcr3. We also produced mutant Avr2 proteins in which selected amino acid residues were individually replaced by alanine, and, in one mutant, all six C-terminal amino acid residues were deleted. We determined the inhibitory constant (K(i) ) of these mutants for Rcr3 and their ability to trigger a Cf-2-mediated hypersensitive response (HR) in tomato. We found that the two C-terminal cysteine residues and the six amino acid C-terminal tail of Avr2 are required for both Rcr3 inhibitory activity and the ability to trigger a Cf-2-mediated HR. Individual replacement of the lysine-17, lysine-20 or tyrosine-21 residue by alanine did not affect significantly the biological activity of Avr2. Overall, our data suggest that the affinity of the Avr2 mutants for Rcr3 correlates with their ability to trigger a Cf-2-mediated HR.

  9. Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.

    PubMed

    Lee, C Y; Meighen, E A

    1997-04-04

    The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.

  10. [The effects of cysteines on the function of human glutathion S-transferase pi(GSTp) under cell oxidative stress].

    PubMed

    Sima, Jian; He, Lan; Zhu, Jian; Xue, Bin; Tai, Yi Lin; Zhang, Shuang Quan; Yin, Zhi Min

    2004-06-01

    Site-directed mutagenesis was used to generate three cysteine mutants of GSTp, C(47/101), C(14/47/101) and C(14/47/101/169). GSTp, C(47/101), C(14/47/101) and C(14/47/101/169) were transfected into 293 cells separately and GST activity was determined by using CDNB as substrate. Data showed that each cysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant negative effect. The expression vectors of wide type GSTp and its cysteine mutants were co-transfected with c-Jun, NF-kappaB, or p21 luciferase reporting vector, into 293 cells separately, luciferase activity showed that C(14/47/101) and C(14/47/101/169) can dramatically activate c-Jun and p21 transcriptional activity. Each cysteine mutant can increase endogenous p21 level, and also increased mortality rate of 293 cells when exposed to H2O2. These results suggest that cysteine residues of GSTp play an important role in protecting cells against oxitative stress.

  11. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-09

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  12. Enhanced electrogenerated chemiluminescence of tris(2,2'-bipyridyl)ruthenium(II) system by l-cysteine-capped CdTe quantum dots and its application for the determination of nitrofuran antibiotics.

    PubMed

    Taokaenchan, Narin; Tangkuaram, Tanin; Pookmanee, Pusit; Phaisansuthichol, Sirirat; Kuimalee, Surasak; Satienperakul, Sakchai

    2015-04-15

    This paper reports a new approach to enhance the electrogenerated chemiluminescence (ECL) of the tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) system using resonance energy transfer with l-cysteine-capped cadmium telluride quantum dots (CdTe-QDs) in aqueous solution. The oxidative peak signal of Ru(bpy)3(2+) occurred at a voltage of 1.10V when the potential was cycled between 0.4 and 1.6V using cyclic voltammetry with a carbon screen-printed electrode (SPE) in a 0.11M phosphate buffer at pH 7.50. The l-cysteine-capped CdTe-QDs were synthesized and added into the solution of Ru(bpy)3(2+) to magnify the ECL signal. The ECL emission signal was investigated and the extreme enhancement of the ECL intensity was achieved due to the energy transfer by the l-cysteine-capped CdTe-QDs. It was found that the induced ECL from the Ru(bpy)3(2+) CdTe-QDs system was inhibited by the presence of selected nitrofurans. This quenching effect of nitrofuran antibiotics on the anodic ECL of Ru(bpy)3(2+) CdTe-QDs was found to be selective and concentration dependent and was observed to have a linear relationship over the concentration range 10-100×10(-6)M. The detection limits were found to be 0.40, 0.73 and 0.60µM for furaltadone (FTD), furazolidone (FZD) and nitrofurantoin (NFT). In addition, the proposed ECL method was successfully applied to detect the total residuals of selected nitrofuran residues in animal feed samples with satisfactory results.

  13. Metal-catalyzed oxidation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: inactivation and destabilization by oxidation of active-site cysteines.

    PubMed

    Park, O K; Bauerle, R

    1999-03-01

    The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.

  14. Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry.

    PubMed

    Lu, Meiling; Wang, Hailin; Wang, Zhongwen; Li, Xing-Fang; Le, X Chris

    2008-08-01

    Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA (III)), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13alpha and Cys-125beta. Cys-13alpha was bound to DMA (III) in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125beta was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13alpha > Cys-111alpha > Cys-104alpha and Cys-13alpha > Cys-125beta > Cys-93beta. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.

  15. Formation of a Stabilized Cysteine Sulfinic Acid Is Critical for the Mitochondrial Function of the Parkinsonism Protein DJ-1*

    PubMed Central

    Blackinton, Jeff; Lakshminarasimhan, Mahadevan; Thomas, Kelly J.; Ahmad, Rili; Greggio, Elisa; Raza, Ashraf S.; Cookson, Mark R.; Wilson, Mark A.

    2009-01-01

    The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1. PMID:19124468

  16. Cy-preds: An algorithm and a web service for the analysis and prediction of cysteine reactivity.

    PubMed

    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.

  17. Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

    PubMed Central

    Hong, Yeon-Chul; Hwang, Mi-Yul; Yun, Ho-Cheol; Yu, Hak-Sun; Kong, Hyun-Hee; Yong, Tai-Soon

    2002-01-01

    We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoeba healyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitis patient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR using degenerate oligonucleotide primers designed on the basis of conserved amino acids franking the active sites of cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) was composed of 330 amino acids with signal sequence, a proposed pro-domain and a predicted active site made up of the catalytic residues, Cys25, His159, and Asn175. Deduced amino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1 peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1 mRNA expression and virulence of Acanthamoeba, but the gene was expressed at higher level in amoebae isolated from soil than amoeba from clinical samples. These findings raise the possibility that Ahcp1 protein may play a role in protein metabolism and digestion of phagocytosed bacteria or host tissue debris rather than in invasion of amoebae into host tissue. PMID:11949209

  18. Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain.

    PubMed

    McShan, Danielle; Kathman, Stefan; Lowe, Brittiney; Xu, Ziyang; Zhan, Jennifer; Statsyuk, Alexander; Ogungbe, Ifedayo Victor

    2015-10-15

    Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic assays will further expand the use of the covalent tethering method, a simple fragment-based drug discovery technique to discover covalent drug leads.

  19. Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

    PubMed Central

    Lukomski, S; Sreevatsan, S; Amberg, C; Reichardt, W; Woischnik, M; Podbielski, A; Musser, J M

    1997-01-01

    Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions. PMID:9169486

  20. Formation of Hg(II) tetrathiolate complexes with cysteine at neutral pH

    DOE PAGES

    Warner, Thomas; Jalilehvand, Farideh

    2016-01-04

    Mercury(II) ions precipitate from aqueous cysteine (H2Cys) solutions containing H2Cys/Hg(II) mole ratio ≥ 2.0 as Hg(S-HCys)2. In absence of additional cysteine, the precipitate dissolves at pH ~12 with the [Hg(S,N-Cys)2]2- complex dominating. With excess cysteine (H2Cys/Hg(II) mole ratio ≥ 4.0), higher complexes form and the precipitate dissolves at lower pH values. Previously, we found that tetrathiolate [Hg(S-Cys)4]6- complexes form at pH = 11.0; in this work we extend the investigation to pH values of physiological interest. We examined two series of Hg(II)-cysteine solutions in which CHg(II) varied between 8 – 9 mM and 80 – 100 mM, respectively, with H2Cys/Hg(II)more » mole ratios from 4 to ~20. The solutions were prepared in the pH range 7.1 – 8.8, at the pH at which the initial Hg(S-HCys)2 precipitate dissolved. The variations in the Hg(II) speciation were followed by 199Hg NMR, X-ray absorption and Raman spectroscopic techniques. Our results show that in the dilute solutions (CHg(II) = 8 – 9 mM), mixtures of di-, tri- (major) and tetrathiolate complexes exist at moderate cysteine excess (CH2Cys ~ 0.16 M) at pH 7.1. In the more concentrated solutions (CHg(II) = 80 – 100 mM) with high cysteine excess (CH2Cys > 0.9 M), tetrathiolate [Hg(S-cysteinate)4]m-6 (m = 0 – 4) complexes dominate in the pH range 7.3 – 7.8, with lower charge than for the [Hg(S-Cys)4]6- complex due to protonation of some (m) of the amino groups of the coordinated cysteine ligands. In conclusion, the results of this investigation could provide a key to the mechanism of biosorption and accumulation of Hg(II) ions in biological / environmental systems.« less

  1. Formation of Hg(II) tetrathiolate complexes with cysteine at neutral pH

    SciTech Connect

    Warner, Thomas; Jalilehvand, Farideh

    2016-01-04

    Mercury(II) ions precipitate from aqueous cysteine (H2Cys) solutions containing H2Cys/Hg(II) mole ratio ≥ 2.0 as Hg(S-HCys)2. In absence of additional cysteine, the precipitate dissolves at pH ~12 with the [Hg(S,N-Cys)2]2- complex dominating. With excess cysteine (H2Cys/Hg(II) mole ratio ≥ 4.0), higher complexes form and the precipitate dissolves at lower pH values. Previously, we found that tetrathiolate [Hg(S-Cys)4]6- complexes form at pH = 11.0; in this work we extend the investigation to pH values of physiological interest. We examined two series of Hg(II)-cysteine solutions in which CHg(II) varied between 8 – 9 mM and 80 – 100 mM, respectively, with H2Cys/Hg(II) mole ratios from 4 to ~20. The solutions were prepared in the pH range 7.1 – 8.8, at the pH at which the initial Hg(S-HCys)2 precipitate dissolved. The variations in the Hg(II) speciation were followed by 199Hg NMR, X-ray absorption and Raman spectroscopic techniques. Our results show that in the dilute solutions (CHg(II) = 8 – 9 mM), mixtures of di-, tri- (major) and tetrathiolate complexes exist at moderate cysteine excess (CH2Cys ~ 0.16 M) at pH 7.1. In the more concentrated solutions (CHg(II) = 80 – 100 mM) with high cysteine excess (CH2Cys > 0.9 M), tetrathiolate [Hg(S-cysteinate)4]m-6 (m = 0 – 4) complexes dominate in the pH range 7.3 – 7.8, with lower charge than for the [Hg(S-Cys)4]6- complex due to protonation of some (m) of the amino groups of the coordinated cysteine ligands. In conclusion, the results of this investigation could provide a key to the mechanism of biosorption and accumulation of Hg(II) ions in biological / environmental systems.

  2. L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

    PubMed

    Nakatsu, Daiki; Horiuchi, Yuta; Kano, Fumi; Noguchi, Yoshiyuki; Sugawara, Taichi; Takamoto, Iseki; Kubota, Naoto; Kadowaki, Takashi; Murata, Masayuki

    2015-03-10

    Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

  3. Structure of the Autocatalytic Cysteine Protease Domain of Potyvirus Helper-component Proteinase*

    PubMed Central

    Guo, Bihong; Lin, Jinzhong; Ye, Keqiong

    2011-01-01

    The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus. The structure represents a postcleavage state in which the cleaved C terminus remains tightly bound at the active site cleft to prevent trans activity. The structure adopts a compact α/β-fold, which differs from papain-like cysteine proteases and shows weak similarity to nsP2 protease from Venezuelan equine encephalitis alphavirus. Nevertheless, the catalytic cysteine and histidine residues constitute an active site that is highly similar to these in papain-like and nsP2 proteases. HC-Pro recognizes a consensus sequence YXVGG around the cleavage site between the two glycine residues. The structure delineates the sequence specificity at sites P1–P4. Structural modeling and covariation analysis across the Potyviridae family suggest a tryptophan residue accounting for the glycine specificity at site P1′. Moreover, a surface of the protease domain is conserved in potyvirus but not in other genera of the Potyviridae family, likely due to extra functional constrain. The structure provides insight into the catalysis mechanism, cis-acting mode, cleavage site specificity, and other functions of the HC-Pro protease domain. PMID:21543324

  4. Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases.

    PubMed Central

    Lee, K L; Albee, K L; Bernasconi, R J; Edmunds, T

    1997-01-01

    The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain. PMID:9355753

  5. Characterization of Two Cysteine Transfer RNA Genes from Xenopus Laevis

    DTIC Science & Technology

    1984-07-12

    author hereby certifies that the use of any copyrighted material in the dissertation manuscript entitled: "Characterization of two cysteine tRNA genes...Uniformed Services University of the Health Sciences 11 ABSTRACT Title of Thesis: Characterization of Two Cysteine Transfer RNA Genes from Xenopus...method after constructing a set of deletions and reclonlng into the plasmid pUC 8. The DNA fragment is 1737 bp long and contains two cysteine tRNA genes

  6. Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis.

    PubMed

    Wawrzyńska, Anna; Kurzyk, Agata; Mierzwińska, Monika; Płochocka, Danuta; Wieczorek, Grzegorz; Sirko, Agnieszka

    2013-06-01

    Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein-protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation.

  7. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    PubMed

    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.

  8. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    PubMed Central

    Jung, Woongsic; 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. PMID:27097164

  9. DBU-catalyzed transprotection of N-Fmoc-cysteine di- and tripeptides into S-Fm-cysteine di- and tripeptides.

    PubMed

    Katritzky, Alan R; Abo-Dya, Nader E; Abdelmajeid, Abdelmotaal; Tala, Srinivasa R; Amine, M S; El-Feky, Said A

    2011-01-21

    The transprotection of N-Fmoc-cysteine containing di- and tripeptides possessing a free SH group to produce the corresponding S-Fm-cysteine di- and tripeptides bearing a free amino group is accomplished efficiently with DBU in dry THF. The N-Fmoc to S-Fm transformation mechanism is discussed. S-Fm-Cysteine di- and tripeptides readily form amide bonds on coupling with N-(Pg-α-aminoacyl)benzotriazoles and N-(Pg-α-dipeptidoyl)benzotriazoles to give larger peptides.

  10. The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.

    PubMed

    de Groot, Rens; Lane, David A; Crawley, James T B

    2015-03-19

    ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

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

    PubMed Central

    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

  12. Chemical proteomic map of dimethyl fumarate–sensitive cysteines in primary human T cells

    PubMed Central

    Blewett, Megan M.; Xie, Jiji; Zaro, Balyn W.; Backus, Keriann M.; Altman, Amnon; Teijaro, John R.; Cravatt, Benjamin F.

    2016-01-01

    Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear, but may involve the covalent modification of proteins or DMF serving as a pro-drug that is converted to monomethyl fumarate (MMF). Here, we found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF-sensitivity of > 2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase C θ (PKCθ). Furthermore, DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology. PMID:27625306

  13. Transsulfuration is an active pathway for cysteine biosynthesis in Trypanosoma rangeli

    PubMed Central

    2014-01-01

    Background Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress. Methods We cloned and characterised genes coding for a cystathionine β-synthase (CβS) and cysteine synthase (CS), key enzymes of the transsulfuration and assimilatory pathways, respectively, from the hemoflagellate protozoan parasite Trypanosoma rangeli. Results Our results show that T. rangeli CβS (TrCβS), similar to its homologs in T. cruzi, contains the catalytic domain essential for enzymatic activity. Unlike the enzymes in bacteria, plants, and other parasites, T. rangeli CS lacks two of the four lysine residues (Lys26 and Lys184) required for activity. Enzymatic studies using T. rangeli extracts confirmed the absence of CS activity but confirmed the expression of an active CβS. Moreover, CβS biochemical assays revealed that the T. rangeli CβS enzyme also has serine sulfhydrylase activity. Conclusion These findings demonstrate that the RTS pathway is active in T. rangeli, suggesting that this may be the only pathway for cysteine biosynthesis in this parasite. In this sense, the RTS pathway appears to have an important functional role during the insect stage of the life cycle of this protozoan parasite. PMID:24761813

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

    PubMed Central

    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

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

    PubMed

    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. L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

    PubMed Central

    Cooper, Arthur J L; Krasnikov, Boris F; Okuno, Etsuo; Jeitner, Thomas M

    2003-01-01

    Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent

  17. Structure of Leishmania major cysteine synthase

    PubMed Central

    Fyfe, Paul K.; Westrop, Gareth D.; Ramos, Tania; Müller, Sylke; Coombs, Graham H.; Hunter, William N.

    2012-01-01

    Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization. PMID:22750854

  18. Synthesis of ultra-small cysteine-capped gold nanoparticles by pH switching of the Au(I)-cysteine polymer.

    PubMed

    Cappellari, Paula S; Buceta, David; Morales, Gustavo M; Barbero, Cesar A; Sergio Moreno, M; Giovanetti, Lisandro J; Ramallo-López, José Martín; Requejo, Felix G; Craievich, Aldo F; Planes, Gabriel A

    2015-03-01

    We report a synthetic approach for the production of ultra-small (0.6 nm) gold nanoparticles soluble in water with a precise control of the nanoparticle size. Our synthetic approach utilizes a pH-depending Au-cysteine polymer as a quencher for the AuNPs grown. The method extends the synthetic capabilities of nanoparticles with sizes down to 1 nm. In addition to the strict pH control, the existence of free -SH groups present in the mixture of reaction has been observed as a key requirement for the synthesis of small nanoparticles in mild conditions. UV-Vis, SAXS, XANES, EXAFS and HR-TEM, has been used to determinate the particle size, characterization of the gold precursor and gold-cysteine interaction.

  19. Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

    PubMed

    Lorenz, Eric; Schmacht, Maximilian; Senz, Martin

    2016-11-01

    Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29±0.29% for cysteine to 3.65±0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.

  20. Identification of the unpaired cysteine status and complete mapping of the 17 disulfides of recombinant tissue plasminogen activator using LC-MS with electron transfer dissociation/collision induced dissociation.

    PubMed

    Wu, Shiaw-Lin; Jiang, Haitao; Hancock, William S; Karger, Barry L

    2010-06-15

    Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in an approximately 60:40 ratio, respectively. The analysis was conducted using a multifragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID), in combination with a multienzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N- or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID-MS(3) to target partially disulfide-dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress.

  1. Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

    PubMed

    Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

    2016-04-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations

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

    PubMed Central

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

    2017-01-01

    Background: 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. PMID:28230769

  3. A free cysteine prolongs the half-life of a homing peptide and improves its tumor-penetrating activity

    PubMed Central

    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

  4. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks.

    PubMed

    Lavergne, Vincent; Harliwong, Ivon; Jones, Alun; Miller, David; Taft, Ryan J; Alewood, Paul F

    2015-07-21

    Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

  5. Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

    PubMed Central

    Schwartzkopff, Benjamin; Platta, Harald W.; Hasan, Sohel; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-01-01

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p. PMID:26182377

  6. Cystein-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation.

    PubMed

    Schwartzkopff, Benjamin; Platta, Harald W; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-05-14

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide-bonds at two certain lysines and results in proteasomal degradation, or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast to Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast to wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitination enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.

  7. Identification of cysteines involved in the effects of methanethiosulfonate reagents on human equilibrative nucleoside transporter 1.

    PubMed

    Park, Jamie S; Hughes, Scott J; Cunningham, Frances K M; Hammond, James R

    2011-10-01

    Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. Given that selective ENT1 inhibitors, such as nitrobenzylmercaptopurine riboside (NBMPR), bind to the N-terminal half of the ENT1 protein, we hypothesized that one or more of the four cysteine residues in this region were contributing to the effects of the sulfhydryl modifiers. Recombinant human ENT1 (hENT1), and the four cysteine-serine ENT1 mutants, were expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate (MTS) sulfhydryl-modifying reagents. Transporter function was assessed by the binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. The membrane-permeable reagent methyl methanethiosulfonate (MMTS) enhanced [(3)H]NBMPR binding in a pH-dependent manner, but decreased [(3)H]2-chloroadenosine uptake. [2-(Trimethylammonium)ethyl] methane-thiosulfonate (MTSET) (positively charged, membrane-impermeable), but not sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) (negatively charged), inhibited [(3)H]NBMPR binding and enhanced [(3)H]2-chloroadenosine uptake. Mutation of Cys222 in transmembrane (TM) 6 eliminated the effect of MMTS on NBMPR binding. Mutation of Cys193 in TM5 enhanced the ability of MMTS to increase [(3)H]NBMPR binding and attenuated the effects of MMTS and MTSET on [(3)H]2-chloroadenosine uptake. Taken together, these data suggest that Cys222 contributes to the effects of MTS reagents on [(3)H]NBMPR binding, and Cys193 is involved in the effects of these reagents on [(3)H]2-chloroadenosine transport. The results of this study also indicate that the hENT1-C193S mutant may be useful as a MTSET/MTSES-insensitive transporter for future cysteine substitution studies to define the extracellular domains contributing to the binding of substrates and inhibitors to this critical membrane transporter.

  8. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

    PubMed Central

    Lavergne, Vincent; Harliwong, Ivon; Jones, Alun; Miller, David; Taft, Ryan J.; Alewood, Paul F.

    2015-01-01

    Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions. PMID:26150494

  9. Homology modeling, molecular docking and MD simulation studies to investigate role of cysteine protease from Xanthomonas campestris in degradation of Aβ peptide.

    PubMed

    Dhanavade, Maruti J; Jalkute, Chidambar B; Barage, Sagar H; Sonawane, Kailas D

    2013-12-01

    Cysteine protease is known to degrade amyloid beta peptide which is a causative agent of Alzheimer's disease. This cleavage mechanism has not been studied in detail at the atomic level. Hence, a three-dimensional structure of cysteine protease from Xanthomonas campestris was constructed by homology modeling using Geno3D, SWISS-MODEL, and MODELLER 9v7. All the predicted models were analyzed by PROCHECK and PROSA. Three-dimensional model of cysteine protease built by MODELLER 9v7 shows similarity with human cathepsin B crystal structure. This model was then used further for docking and simulation studies. The molecular docking study revealed that Cys17, His87, and Gln88 residues of cysteine protease form an active site pocket similar to human cathepsin B. Then the docked complex was refined by molecular dynamic simulation to confirm its stable behavior over the entire simulation period. The molecular docking and MD simulation studies showed that the sulfhydryl hydrogen atom of Cys17 of cysteine protease interacts with carboxylic oxygen of Lys16 of Aβ peptide indicating the cleavage site. Thus, the cysteine protease model from X. campestris having similarity with human cathepsin B crystal structure may be used as an alternate approach to cleave Aβ peptide a causative agent of Alzheimer's disease.

  10. Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa.

    PubMed

    Cabral, Hamilton; Leopoldino, Andréia M; Tajara, Eloiza H; Greene, Lewis J; Faça, Vitor M; Mateus, Rogério P; Ceron, Carlos R; de Souza Judice, Wagner A; Julianod, Luiz; Bonilla-Rodriguez, Gustavo O

    2006-01-01

    The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).

  11. Unusual hydrogen bonding in L-cysteine hydrogen fluoride.

    PubMed

    Minkov, V S; Ghazaryan, V V; Boldyreva, E V; Petrosyan, A M

    2015-08-01

    L-Cysteine hydrogen fluoride, or bis(L-cysteinium) difluoride-L-cysteine-hydrogen fluoride (1/1/1), 2C3H8NO2S(+)·2F(-)·C3H7NO2S·HF or L-Cys(+)(L-Cys···L-Cys(+))F(-)(F(-)...H-F), provides the first example of a structure with cations of the 'triglycine sulfate' type, i.e. A(+)(A···A(+)) (where A and A(+) are the zwitterionic and cationic states of an amino acid, respectively), without a doubly charged counter-ion. The salt crystallizes in the monoclinic system with the space group P2(1). The dimeric (L-Cys···L-Cys(+)) cation and the dimeric (F(-)···H-F) anion are formed via strong O-H···O or F-H···F hydrogen bonds, respectively, with very short O···O [2.4438 (19) Å] and F···F distances [2.2676 (17) Å]. The F···F distance is significantly shorter than in solid hydrogen fluoride. Additionally, there is another very short hydrogen bond, of O-H···F type, formed by a L-cysteinium cation and a fluoride ion. The corresponding O···F distance of 2.3412 (19) Å seems to be the shortest among O-H···F and F-H···O hydrogen bonds known to date. The single-crystal X-ray diffraction study was complemented by IR spectroscopy. Of special interest was the spectral region of vibrations related to the above-mentioned hydrogen bonds.

  12. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3-mercaptopropanoic acid (C3H7O2NS). (b) The ingredient meets the appropriate part of the specification set forth...

  13. Electrons initiate efficient formation of hydroperoxides from cysteine.

    PubMed

    Gebicki, Janusz M

    2016-09-01

    Amino acid and protein hydroperoxides can constitute a significant hazard if formed in vivo. It has been suggested that cysteine can form hydroperoxides after intramolecular hydrogen transfer to the commonly produced cysteine sulfur-centered radical. The resultant cysteine-derived carbon-centered radicals can react with oxygen at almost diffusion-controlled rate, forming peroxyl radicals which can oxidize other molecules and be reduced to hydroperoxides in the process. No cysteine hydroperoxides have been found so far. In this study, dilute air-saturated cysteine solutions were exposed to radicals generated by ionizing radiation and the hydroperoxides measured by an iodide assay. Of the three primary radicals present, the hydroxyl, hydrogen atoms and hydrated electrons, the first two were ineffective. However, electrons did initiate the generation of hydroperoxides by removing the -SH group and forming cysteine-derived carbon radicals. Under optimal conditions, 100% of the electrons reacting with cysteine produced the hydroperoxides with a 1:1 stoichiometry. Maximum hydroperoxide yields were at pH 5.5, with fairly rapid decline under more acid or alkaline conditions. The hydroperoxides were stable between pH 3 and 7.5, and decomposed in alkaline solutions. The results suggest that formation of cysteine hydroperoxides initiated by electrons is an unlikely event under physiological conditions.

  14. Role of cysteines in mammalian VDAC isoforms' function.

    PubMed

    De Pinto, Vito; Reina, Simona; Gupta, Ankit; Messina, Angela; Mahalakshmi, Radhakrishnan

    2016-08-01

    In this mini-review, we analyze the influence of cysteines in the structure and activity of mitochondrial outer membrane mammalian VDAC isoforms. The three VDAC isoforms show conserved sequences, similar structures and the same gene organization. The meaning of three proteins encoded in different chromosomes must thus be searched for subtle differences at the amino acid level. Among others, cysteine content is noticeable. In humans, VDAC1 has 2, VDAC2 has 9 and VDAC3 has 6 cysteines. Recent works have shown that, at variance from VDAC1, VDAC2 and VDAC3 exhibit cysteines predicted to protrude towards the intermembrane space, making them a preferred target for oxidation by ROS. Mass spectrometry in VDAC3 revealed that a disulfide bridge can be formed and other cysteine oxidations are also detectable. Both VDAC2 and VDAC3 cysteines were mutagenized to highlight their role in vitro and in complementation assays in Δporin1 yeast. Chemico-physical techniques revealed an important function of cysteines in the structural stabilization of the pore. In conclusion, the works available on VDAC cysteines support the notion that the three proteins are paralogs with a similar pore-function and slightly different, but important, ancillary biological functions. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  15. Cysteine sulfoxide derivatives in Petiveria alliacea.

    PubMed

    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.

  16. Cysteine analogues potentiate glucose-induced insulin release in vitro

    SciTech Connect

    Ammon, H.P.; Hehl, K.H.; Enz, G.; Setiadi-Ranti, A.; Verspohl, E.J.

    1986-12-01

    In rat pancreatic islets, cysteine analogues, including glutathione, acetylcysteine, cysteamine, D-penicillamine, L-cysteine ethyl ester, and cysteine-potentiated glucose (11.1 mM) induced insulin secretion in a concentration-dependent manner. Their maximal effects were similar and occurred at approximately 0.05, 0.05, 0.1, 0.5, 1.0, 1.0 mM, respectively. At substimulatory glucose levels (2.8 mM), insulin release was not affected by these compounds. In contrast, thiol compounds, structurally different from cysteine and its analogues, such as mesna, tiopronin, meso-2,3-dimercaptosuccinic acid (DMSA), dimercaprol (BAL), beta-thio-D-glucose, as well as those cysteine analogues that lack a free-thiol group, including L-cystine, cystamine, D-penicillamine disulfide, S-carbocysteine, and S-carbamoyl-L-cysteine, did not enhance insulin release at stimulatory glucose levels (11.1 mM); cystine (5 mM) was inhibitory. These in vitro data indicate that among the thiols tested here, only cysteine and its analogues potentiate glucose-induced insulin secretion, whereas thiols that are structurally not related to cysteine do not. This suggests that a cysteine moiety in the molecule is necessary for the insulinotropic effect. For their synergistic action to glucose, the availability of a sulfhydryl group is also a prerequisite. The maximal synergistic action is similar for all cysteine analogues tested, whereas the potency of action is different, suggesting similarity in the mechanism of action but differences in the affinity to the secretory system.

  17. Cysteine could change the transport mechanism of PVP-coated silver nanoparticles in porous media

    NASA Astrophysics Data System (ADS)

    Yang, X.; Lin, S.; Wiesner, M.

    2012-12-01

    Silver nanoparticles (AgNPs) can hardly be removed by wastewater treatment plant and have big potential to enter groundwater, jeopardizing the water quality & aquatic ecosystem. Most AgNPs have surface coatings such as polyvinylpyrrolidone (PVP) which dominate their transport in porous media. Our previous study shows that PVP may promote the deposition of AgNPs on silica surface by a bridging mechanism. This study further explored how cysteine, a natural organic matter type, may influence the role of the PVP coating on AgNP translocation. Dynamic Light Scattering (DLS) measurement (Figure 1A) shows that the PVP coating rendered the AgNP dispersion high stability during the measuring period (3hrs). Addition of 100 ppm cysteine to the dispersion resulted in a rapid decrease in particle size from 100nm to 52nm within one hour, following which no further decline in particle size occurred. Column experiment results (Figure 1B) show that corresponding to the particle size change was a substantial decrease in particle deposition rates: introduction of 100 ppm cysteine into the particle dispersion resulted in a decrease in AgNP attenuation by the porous medium from 67% to 26%. The decline in particle size suggested that cysteine may have displaced the macromolecular PVP from the particle surface. Desorption of PVP resulted in a weakening or vanish of polymer bridging effect which in turn lowered the deposition rates substantially. This study demonstrated an implication of environmental transformation of coated AgNPs to their mobility in saturated sand aquifers. Acknowledgment Xinyao Yang appreciates the Natural Science Foundation of China (Grant No.:41101475) for covering the registration fee and traveling costs.igure 1 Particle size measurement (A) and breakthrough curves (B) of PVP-coated silver nanoparticle in the absence and presence of cysteine: pH=7.0, ionic strength=1mM, flow rate=1ml/min.

  18. CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

  19. Dealing with the sulfur part of cysteine: four enzymatic steps degrade l-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.

    PubMed

    Höfler, Saskia; Lorenz, Christin; Busch, Tjorven; Brinkkötter, Mascha; Tohge, Takayuki; Fernie, Alisdair R; Braun, Hans-Peter; Hildebrandt, Tatjana M

    2016-07-01

    Amino acid catabolism is essential for adjusting pool sizes of free amino acids and takes part in energy production as well as nutrient remobilization. The carbon skeletons are generally converted to precursors or intermediates of the tricarboxylic acid cycle. In the case of cysteine, the reduced sulfur derived from the thiol group also has to be oxidized in order to prevent accumulation to toxic concentrations. Here we present a mitochondrial sulfur catabolic pathway catalyzing the complete oxidation of l-cysteine to pyruvate and thiosulfate. After transamination to 3-mercaptopyruvate, the sulfhydryl group from l-cysteine is transferred to glutathione by sulfurtransferase 1 and oxidized to sulfite by the sulfur dioxygenase ETHE1. Sulfite is then converted to thiosulfate by addition of a second persulfide group by sulfurtransferase 1. This pathway is most relevant during early embryo development and for vegetative growth under light-limiting conditions. Characterization of a double mutant produced from Arabidopsis thaliana T-DNA insertion lines for ETHE1 and sulfurtransferase 1 revealed that an intermediate of the ETHE1 dependent pathway, most likely a persulfide, interferes with amino acid catabolism and induces early senescence.

  20. Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Cook, Shannon L.; Jackson, Glen P.

    2011-02-01

    The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2-, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile -NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.

  1. S-Sulfhydration: A Cysteine Posttranslational Modification in Plant Systems1

    PubMed Central

    Serna, Antonio

    2015-01-01

    Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems. PMID:25810097

  2. Mutation of cysteine 46 in IKK-beta increases inflammatory responses

    PubMed Central

    Jiang, Zhi Hong; Jiang, Shui Ping; Liu, Yan; Wang, Ting Yu; Yao, Xiao Jun; Su, Xiao Hui; Yan, Feng Gen; Liu, Juan; Leung, Elaine Lai-Han; Yi, Xiao Qin; Wong, Yuen Fan; Zhou, Hua; Liu, Liang

    2015-01-01

    Activation of IκB kinase β (IKK-β) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-β−NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-β. However, mutations in IKK-β have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-β inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-β kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-βC46A transgenic mouse model. We show that a novel IKK-β inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-βC46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-β. PMID:26378659

  3. A Helical Conotoxin from Conus imperialis Has a Novel Cysteine Framework and Defines a New Superfamily*

    PubMed Central

    Ye, Mingyu; Khoo, Keith K.; Xu, Shaoqiong; Zhou, Mi; Boonyalai, Nonlawat; Perugini, Matthew A.; Shao, Xiaoxia; Chi, Chengwu; Galea, Charles A.; Wang, Chunguang; Norton, Raymond S.

    2012-01-01

    Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified. PMID:22399292

  4. Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

    PubMed

    Kumari, Geeta; Serra, Aida; Shin, Joon; Nguyen, Phuong Q T; Sze, Siu Kwan; Yoon, Ho Sup; Tam, James P

    2015-11-25

    Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

  5. Cysteine 893 is a target of regulatory thiol modifications of GluA1 AMPA receptors

    PubMed Central

    von Ossowski, Lotta; Li, Li-Li; Möykkynen, Tommi; Coleman, Sarah K.; Courtney, Michael J.

    2017-01-01

    Recent studies indicate that glutamatergic signaling involves, and is regulated by, thiol modifying and redox-active compounds. In this study, we examined the role of a reactive cysteine residue, Cys-893, in the cytosolic C-terminal tail of GluA1 AMPA receptor as a potential regulatory target. Elimination of the thiol function by substitution of serine for Cys-893 led to increased steady-state expression level and strongly reduced interaction with SAP97, a major cytosolic interaction partner of GluA1 C-terminus. Moreover, we found that of the three cysteine residues in GluA1 C-terminal tail, Cys-893 is the predominant target for S-nitrosylation induced by exogenous nitric oxide donors in cultured cells and lysates. Co-precipitation experiments provided evidence for native association of SAP97 with neuronal nitric oxide synthase (nNOS) and for the potential coupling of Ca2+-permeable GluA1 receptors with nNOS via SAP97. Our results show that Cys-893 can serve as a molecular target for regulatory thiol modifications of GluA1 receptors, including the effects of nitric oxide. PMID:28152104

  6. Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

    PubMed Central

    Gerdol, Marco; Puillandre, Nicolas; Moro, Gianluca De; Guarnaccia, Corrado; Lucafò, Marianna; Benincasa, Monica; Zlatev, Ventislav; Manfrin, Chiara; Torboli, Valentina; Giulianini, Piero Giulio; Sava, Gianni; Venier, Paola; Pallavicini, Alberto

    2015-01-01

    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling. PMID:26201648

  7. Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis.

    PubMed

    Gerdol, Marco; Puillandre, Nicolas; De Moro, Gianluca; Guarnaccia, Corrado; Lucafò, Marianna; Benincasa, Monica; Zlatev, Ventislav; Manfrin, Chiara; Torboli, Valentina; Giulianini, Piero Giulio; Sava, Gianni; Venier, Paola; Pallavicini, Alberto

    2015-07-21

    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

  8. Estimation of whey protein in casein coprecipitate and milk powder by high-performance liquid chromatography quantification of cysteine.

    PubMed

    Ballin, Nicolai Z

    2006-06-14

    An analytical high-performance liquid chromatography (HPLC)-fluorescence method for indirect measuring of whey protein in casein coprecipitate and milk powder was developed. Samples were hydrolyzed with HCl, and cysteyl residues were derivatized with 3,3'-dithiodipropionic acid and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The cysteine content was used to calculate the percentage of whey protein in commercial samples with use of European Union Regulation cysteine reference values in both casein and whey protein. Method validation studies were performed for caseinates and milk powder, and results indicate that the present HPLC approach can be applied as a fast method with a standard deviation of repeatability between 3.3 and 9.5%. Applicability was studied by analysis of 40 commercial caseinate samples, and all complied to European legislation with a content of whey protein not exceeding 5%. Finally, an approach used to estimate the cysteine amount in pure casein by comparison of calculated and experimental values questions the generally accepted cysteine reference value in casein, which is most likely an overestimation.

  9. Effect of cysteine and humic acids on bioavailability of Ag from Ag nanoparticles to a freshwater snail

    USGS Publications Warehouse

    Luoma, Samuel N.; Tasha Stoiber,; Croteau, Marie-Noele; Isabelle Romer,; Ruth Merrifeild,; Lead, Jamie

    2016-01-01

    Metal-based engineered nanoparticles (NPs) will undergo transformations that will affect their bioavailability, toxicity and ecological risk when released to the environment, including interactions with dissolved organic material. The purpose of this paper is to determine how interactions with two different types of organic material affect the bioavailability of silver nanoparticles (AgNPs). Silver uptake rates by the pond snail Lymnaea stagnalis were determined after exposure to 25 nmol l-1 of Ag as PVP AgNPs, PEG AgNPs or AgNO3, in the presence of either Suwannee River humic acid or cysteine, a high-affinity thiol-rich organic ligand. Total uptake rate of Ag from the two NPs was either increased or not strongly affected in the presence of 1 – 10 mg 1-1 humic acid. Humic substances contain relatively few strong ligands for Ag explaining their limited effects on Ag uptake rate. In contrast, Ag uptake rate was substantially reduced by cysteine. Three components of uptake from the AgNPs were quantified in the presence of cysteine using a biodynamic modeling approach: uptake of dissolved Ag released by the AgNPs, uptake of a polymer or large (>3kD) Ag-cysteine complex and uptake of the nanoparticle itself. Addition of 1:1 Ag:cysteine reduced concentrations of dissolved Ag, which contributed to, but did not fully explain the reductions in uptake. A bioavailable Ag-cysteine complex (> 3kD) appeared to be the dominant avenue of uptake from both PVP AgNPs and PEG AgNPs in the presence of cysteine. Quantifying the different avenues of uptake sets the stage for studies to assess toxicity unique to NPs.

  10. IDENTIFICATION OF A MEMBRANE-LOCALIZED CYSTEINE CLUSTER NEAR THE SUBSTRATE BINDING SITES OF THE STREPTOCOCCUS EQUISIMILIS HYALURONAN SYNTHASE

    PubMed Central

    Kumari, Kshama; Weigel, Paul H.

    2005-01-01

    The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wildtype enzyme (Kumari et al., J. Biol. Chem. 277, 13943, 2002). Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but that Cys367 is not. Substrate protection studies of wildtype seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can crosslink vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be crosslinked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367 ) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site. PMID:15616126

  11. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  12. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  13. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  14. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  15. 40 CFR 180.519 - Bromide ion and residual bromine; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bromide ion and residual bromine... Tolerances § 180.519 Bromide ion and residual bromine; tolerances for residues. (a) General. The food additives, bromide ion and residual bromine, may be present in water, potable in accordance with...

  16. L-Cysteine metabolism and its nutritional implications.

    PubMed

    Yin, Jie; Ren, Wenkai; Yang, Guan; Duan, Jielin; Huang, Xingguo; Fang, Rejun; Li, Chongyong; Li, Tiejun; Yin, Yulong; Hou, Yongqing; Kim, Sung Woo; Wu, Guoyao

    2016-01-01

    L-Cysteine is a nutritionally semiessential amino acid and is present mainly in the form of L-cystine in the extracellular space. With the help of a transport system, extracellular L-cystine crosses the plasma membrane and is reduced to L-cysteine within cells by thioredoxin and reduced glutathione (GSH). Intracellular L-cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for production of GSH, hydrogen sulfide (H(2)S), and taurine. L-Cysteine-dependent synthesis of GSH has been investigated in many pathological conditions, while the pathway for L-cysteine metabolism to form H(2)S has received little attention with regard to prevention and treatment of disease in humans. The main objective of this review is to highlight the metabolic pathways of L-cysteine catabolism to GSH, H(2)S, and taurine, with special emphasis on therapeutic and nutritional use of L-cysteine to improve the health and well-being of animals and humans.

  17. Chlorhexidine inhibits the activity of dental cysteine cathepsins.

    PubMed

    Scaffa, P M C; Vidal, C M P; Barros, N; Gesteira, T F; Carmona, A K; Breschi, L; Pashley, D H; Tjäderhane, L; Tersariol, I L S; Nascimento, F D; Carrilho, M R

    2012-04-01

    The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2' of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.

  18. Probes of the Catalytic Site of Cysteine Dioxygenase

    SciTech Connect

    Chai,S.; Bruyere, J.; Maroney, M.

    2006-01-01

    The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the a-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ a-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by {alpha}-ketoglutarate.

  19. Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull.

    PubMed

    Topraggaleh, T R; Shahverdi, A; Rastegarnia, A; Ebrahimi, B; Shafiepour, V; Sharbatoghli, M; Esmaeili, V; Janzamin, E

    2014-09-01

    Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post-thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen-thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen-thawed spermatozoa.

  20. Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis.

    PubMed

    Mitta, G; Hubert, F; Noël, T; Roch, P

    1999-10-01

    We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.

  1. RESIDUAL RISK ASSESSMENTS - RESIDUAL RISK ...

    EPA Pesticide Factsheets

    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.

  2. Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium.

    PubMed Central

    Nakamura, T; Iwahashi, H; Eguchi, Y

    1984-01-01

    S-Sulfocysteine synthase was isolated from Salmonella typhimurium LT-2 to homogeneous form with polyacrylamide gel electrophoresis. The molecular weight of this enzyme was determined to be ca. 55,000. The enzyme consisted of two identically sized subunits, and it contained one pyridoxal phosphate per subunit. The enzyme catalyzed the biosynthesis of cysteine or S-methylcysteine from sulfide or methanethiol and O-acetylserine, respectively, in addition to the formation of S-sulfocysteine from thiosulfate and O-acetylserine. The enzyme is identical to cysteine synthase B. The intracellular level of this enzyme was regulated by lesser extents of the same factors as those effective for cysteine synthase A. Images PMID:6373737

  3. Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle.

    PubMed

    Choi, Ae Ran; Kim, Min-Sik; Kang, Sung Gyun; Lee, Hyun Sook

    2016-01-01

    A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.

  4. Cysteinoyl- and cysteine-containing dipeptidoylbenzotriazoles with free sulfhydryl groups: easy access to N-terminal and internal cysteine peptides.

    PubMed

    Ibrahim, Tarek S; Tala, Srinivasa R; El-Feky, Said A; Abdel-Samii, Zakaria K; Katritzky, Alan R

    2012-08-01

    N-Protected cysteines 4a-c each with a free sulfhydryl group were prepared in 70-75% yields by treatment of L-cysteine with 1-(benzyloxycarbonyl) benzotriazole (Cbz-Bt) 1a, N-(tert-butyloxy-carbonyl)benzotriazole (Boc-Bt) 1b, and 1-(9-fluorenylmethoxy-carbonyl)benzotriazole (Fmoc-Bt) 1c, respectively. N-Protected, free sulfhydryl cysteines 4a-c were then converted into the corresponding N-protected, free sulfhydryl cysteinoylbenzotriazoles 7a-c (70-85%), which on treatment with diverse amino acids and dipeptides afforded the corresponding N-protected, free sulfhydryl N-terminal cysteine dipeptides 8a-e and tripeptides 8f-h in 73-80% yields. N-Protected, free sulfhydryl cysteine-containing dipeptides 9a,b were converted into the corresponding N-protected, free sulfhydryl dipeptidoylbenzotriazoles 10a,b (69-81%), which on treatment with amino acids, dipeptides, and a tripeptide afforded internal cysteine tripeptides 11a-c, tetrapeptides 11d,e and pentapeptide 11f, each containing a N-protected, free sulfhydryl groups in 70-90% yields under mild conditions. Treatment of N-protected, free sulfhydryl cysteinoylbenzotriazole 7a with diamines 12a,b afforded directly the cysteine-containing disulfide-bridged cyclic peptides 14a,b in 50% yields.

  5. A Secreted Protein with Plant-Specific Cysteine-Rich Motif Functions as a Mannose-Binding Lectin That Exhibits Antifungal Activity1[W

    PubMed Central

    Miyakawa, Takuya; Hatano, Ken-ichi; Miyauchi, Yumiko; Suwa, You-ichi; Sawano, Yoriko; Tanokura, Masaru

    2014-01-01

    Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family. PMID:25139159

  6. Oxygen-dependent conjugation of dopa with cysteine catalysed by iron-EDTA complex.

    PubMed

    Ito, S; Fujita, K

    1984-07-15

    Cytotoxicity of catechols has been ascribed to their binding with proteins through sulfhydryl groups. The possibility that iron-protein complexes catalyse this type of covalent binding was studied with a model system. Reaction of dopa and cysteine catalysed by iron-EDTA complexes at physiological pH resulted in the formation of not only cystine but also conjugation products, cysteinyldopas among which 5-S-cysteinyldopa was the major product. The reaction required iron ion, EDTA, and molecular oxygen. Fe3+ and Fe2+ were equally effective, while other transition metal ions examined had no effect on the formation of cysteinyldopas. Catalase, superoxide dismutase, and scavengers of hydroxyl radical inhibited to some extents the formation of 5-S-cysteinyldopa. Addition of both catalase and superoxide dismutase resulted in approximately 60% inhibition. These results indicated that the iron-EDTA-catalysed conjugation of dopa with cysteine was mainly mediated by hydroxyl radical.

  7. The oxidation of cyst(e)ine by mast-cell tumour P815 in culture

    PubMed Central

    Wheldrake, J. F.; Pasternak, C. A.

    1968-01-01

    1. Mast-cell tumour P815 cells oxidize [35S]cyst(e)ine to 35SO42−. 2. Addition of cysteinesulphinate or sulphite decreases the formation of 35SO42−; at the same time [35S]cysteinesulphinate or 35SO32− accumulates. 3. Extracts of P815 cells form sulphite from cysteinesulphinate and 2-oxoglutarate. The Km for cysteinesulphinate is 6·35mm and that for 2-oxoglutarate is 0·165mm. 4. Extracts oxidize sulphite to sulphate. 5. No formation of hydrogen sulphide from cyst(e)ine was detectable. 6. It is concluded that P815 cells oxidize cyst(e)ine to sulphate solely via cysteinesulphinate and sulphite. 7. The concentration of the enzymes catalysing this sequence is unaltered by several variations in the conditions of growth. PMID:4966082

  8. A novel class of cysteine protease inhibitors: solution structure of staphostatin A from Staphylococcus aureus.

    PubMed

    Dubin, Grzegorz; Krajewski, Marcin; Popowicz, Grzegorz; Stec-Niemczyk, Justyna; Bochtler, Matthias; Potempa, Jan; Dubin, Adam; Holak, Tad A

    2003-11-25

    A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of staphostatins, endogenous, highly specific, staphylococcal cysteine protease inhibitors, opened a possibility for structure-based design of low molecular weight analogues. Moreover, the crystal structure of staphostatin B revealed a distinct folding pattern and an unexpected, substrate-like binding mode. The solution structure of staphostatin A reported here confirms that staphostatins constitute a novel, distinct class of cysteine protease inhibitors. In addition, the structure knowledge-based mutagenesis studies shed light on individual structural features of staphostatin A, the inhibition mechanism, and the determinants of distinct specificity of staphostatins toward their target proteases.

  9. Organometallic Palladium Reagents for Cysteine Bioconjugation

    PubMed Central

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

    2015-01-01

    Transition-metal based reactions have found wide use in organic synthesis and are used frequently to functionalize small molecules.1,2 However, there are very few reports of using transition-metal based reactions to modify complex biomolecules3,4, 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 biopolymers. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation reactions. The bioconjugation reaction is rapid and robust under a range of biocompatible 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 new bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as a new set of benchtop reagents for diverse bioconjugation applications. PMID:26511579

  10. Organometallic palladium reagents for cysteine bioconjugation

    NASA Astrophysics Data System (ADS)

    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.

  11. Organometallic palladium reagents for cysteine bioconjugation.

    PubMed

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

    2015-10-29

    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. Developing novel anthelmintics from plant cysteine proteinases

    PubMed Central

    Behnke, Jerzy M; Buttle, David J; Stepek, Gillian; Lowe, Ann; Duce, Ian R

    2008-01-01

    Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock. PMID:18761736

  13. Identification of conformational epitopes and antigen-specific residues at the D/A domains and the extramembrane C-terminal region of E2 glycoprotein of classical swine fever virus.

    PubMed

    Chang, Chia-Yi; Huang, Chin-Cheng; Deng, Ming-Chung; Huang, Yeou-Liang; Lin, Yu-Ju; Liu, Hsin-Meng; Lin, Yeou-Liang; Wang, Fun-In

    2012-09-01

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. Previous studies revealed that both conformation-dependent and linear epitopes are most present within domains B/C/D/A in the N-terminal half of E2. However, studies of antigenicity beyond the B/C domains remain limited. This study revealed that conformational epitopes were present on the D/A domains as well as the proximal C-terminal of E2, since the mutation of cysteine abrogated their bindings to monoclonal antibodies (mAbs). The residue R845 at domain A and E902 at the C-terminal region were critical for specific binding to mAbs, further supporting the presence of antigenic determinants on these regions. Substitutions of cysteines in domains D/A not only abrogated the binding to mAbs directed to D/A, but also affected the binding of the downstream C-terminal region to its specific mAbs, suggesting a close interaction between the two conformational epitopes. Mutations on the five proximal cysteines at positions 869, 877, 893, 896 and 930 in the C-terminal region only affected the binding to its specific mAbs binding sites. In addition, mutation on the three distal C-terminal cysteines at positions 945, 966, and 983 resulted in loss of E2 homodimerization. This study demonstrates new antigenic epitopes on D/A domains and C-terminal of E2 that have not been reported before, and that the nine cysteines in the C-terminal function differently in either maintaining the antigenic structure or in intermolecular dimerization of E2.

  14. Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin.

    PubMed

    Byrne, Dominic P; Wawrzonek, Katarzyna; Jaworska, Anna; Birss, Andrew J; Potempa, Jan; Smalley, John W

    2009-12-14

    The gram-negative oral anaerobe Prevotella intermedia forms an iron(III) protoporphyrin IX pigment from haemoglobin. The bacterium expresses a 90 kDa cysteine protease, InpA (interpain A), a homologue of Streptococcus pyogenes streptopain (SpeB). The role of InpA in haemoglobin breakdown and haem release was investigated. At pH 7.5, InpA mediated oxidation of oxyhaemoglobin to hydroxymethaemoglobin [in which the haem iron is oxidized to the Fe(III) state and which carries OH- as the sixth co-ordinate ligand] by limited proteolysis of globin chains as indicated by SDS/PAGE and MALDI (matrix-assisted laser-desorption ionization)-TOF (time-of-flight) analysis. Prolonged incubation at pH 7.5 did not result in further haemoglobin protein breakdown, but in the formation of a haemoglobin haemichrome (where the haem Fe atom is co-ordinated by another amino acid ligand in addition to the proximal histidine residue) resistant to degradation by InpA. InpA-mediated haem release from hydroxymethaemoglobin-agarose was minimal compared with trypsin at pH 7.5. At pH 6.0, InpA increased oxidation at a rate greater than auto-oxidation, producing aquomethaemoglobin (with water as sixth co-ordinate ligand), and resulted in its complete breakdown and haem loss. Aquomethaemoglobin proteolysis and haem release was prevented by blocking haem dissociation by ligation with azide, whereas InpA proteolysis of haem-free globin was rapid, even at pH 7.5. Both oxidation of oxyhaemoglobin and breakdown of methaemoglobin by InpA were inhibited by the cysteine protease inhibitor E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane]. In summary, we conclude that InpA may play a central role in haem acquisition by mediating oxyhaemoglobin oxidation, and by degrading aquomethaemoglobin in which haem-globin affinity is weakened under acidic conditions.

  15. Pathogenic Cysteine Removal Mutations in FGFR Extracellular Domains Stabilize Receptor Dimers and Perturb the TM Dimer Structure.

    PubMed

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-10-09

    Missense mutations that introduce or remove cysteine residues in receptor tyrosine kinases are believed to cause pathologies by stabilizing the active receptor tyrosine kinase dimers. However, the magnitude of this stabilizing effect has not been measured for full-length receptors. Here, we characterize the dimer stabilities of three full-length fibroblast growth factor receptor (FGFR) mutants harboring pathogenic cysteine substitutions: the C178S FGFR1 mutant, the C342R FGFR2 mutant, and the C228R FGFR3 mutant. We find that the three mutations stabilize the FGFR dimers. We further see that the mutations alter the configuration of the FGFR transmembrane dimers. Thus, both aberrant dimerization and perturbed dimer structure likely contribute to the pathological phenotypes arising due to these mutations.

  16. Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis.

    PubMed

    Charlet, M; Chernysh, S; Philippe, H; Hetru, C; Hoffmann, J A; Bulet, P

    1996-09-06

    We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.

  17. A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic.

    PubMed

    Salazar, Héctor; Llorente, Itzel; Jara-Oseguera, Andrés; García-Villegas, Refugio; Munari, Mika; Gordon, Sharona E; Islas, León D; Rosenbaum, Tamara

    2008-03-01

    Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

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

    PubMed

    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.

  19. Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

    PubMed Central

    Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

    2015-01-01

    How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID

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

    PubMed

    O'Leary, Michael E; Chahine, Mohamed

    2015-01-01

    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 inac