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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. Fluorescent labeling of specific cysteine residues using CyMPL

    PubMed Central

    Puljung, Michael C.

    2012-01-01

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

  3. Role of cysteine residues in pseudouridine synthases of different families.

    PubMed

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  4. Effects of additional cysteine in fish diet on mercury concentration.

    PubMed

    Mok, W J; Hatanaka, Y; Seoka, M; Itoh, T; Tsukamasa, Y; Ando, M

    2014-03-15

    Mercury contamination, especially of seafood, continues to attract public concern. Cysteine, NH2CH(CH2SH)COOH, is a naturally occurring hydrophobic amino acid that contains a thiol group. The purpose of our study was to investigate the use of the additive cysteine in fish diets to reduce mercury concentration in fish, and to observe the effectiveness of dietary cysteine in fish livers. Diets containing 1% and 10% cysteine successfully decreased mercury concentrations in fish compared with the 0% cysteine diet. The liver may have formed excessive lipid droplets or was unable to mobilize lipid stores during exposure to mercury; additional cysteine could help to mobilize excessive lipids in it.

  5. 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 ...

  6. Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry.

    PubMed

    Pilo, Alice L; Zhao, Feifei; McLuckey, Scott A

    2016-09-01

    The thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Additionally, cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric analysis. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidation via selective ion/ion reactions with periodate anions. Multiply protonated peptides containing modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide derivative yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (namely, XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An additional step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically observed upon collision-induced dissociation (CID), they can be used to immediately indicate where in the peptide the PTM was originally located. PMID:27476698

  7. Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry.

    PubMed

    Pilo, Alice L; Zhao, Feifei; McLuckey, Scott A

    2016-09-01

    The thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Additionally, cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric analysis. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidation via selective ion/ion reactions with periodate anions. Multiply protonated peptides containing modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide derivative yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (namely, XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An additional step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically observed upon collision-induced dissociation (CID), they can be used to immediately indicate where in the peptide the PTM was originally located.

  8. 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.

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

    PubMed Central

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

    2013-01-01

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

  10. Evidence for proximal cysteine and lysine residues at or near the active site of arginine kinase of Stichopus japonicus.

    PubMed

    Guo, Qin; Chen, Baoyu; Wang, Xicheng

    2004-12-01

    Inactivation of arginine kinase (AK) of Stichopus japonicus by o-phthalaldehyde (OPTA) was investigated. The modified enzyme showed an absorption peak at 337 nm and a fluorescent emission peak at 410 nm, which are characteristic of an isoindole derivative formed by OPTA binding to a thiol and an amine group in proximity within the enzyme. Loss of enzymatic activity was concomitant with an increase in fluorescence intensity at 410 nm. Stoichiometry studies by Tsou's method showed that among the cysteine residues available for OPTA modification in the enzyme, only one was essential for the enzyme activity. This cysteine residue is located in a highly hydrophobic environment, presumably near ATP and ADP binding region. This conclusion was verified by 5,5 -dithiobis(2-nitrobenzoic acid) modification. In addition, these results were supported by means of electrophoresis and ultraviolet, fluorescence, circular dichroism spectroscopy and fast performance liquid chromatography. Sequence comparison suggested that this essential cysteine residue maybe the conservative Cys274. PMID:15627388

  11. 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

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

    PubMed

    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.

  13. On-line procedures for alkylation of cysteine residues with 3-bromopropylamine prior to protein sequence analysis.

    PubMed

    Jue, R A; Hale, J E

    1994-09-01

    We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.

  14. A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers.

    PubMed

    Bienert, Gerd P; Cavez, Damien; Besserer, Arnaud; Berny, Marie C; Gilis, Dimitri; Rooman, Marianne; Chaumont, François

    2012-07-01

    AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.

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

    PubMed

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

    2012-11-01

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

  16. 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.

  17. 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

  18. 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.

  19. Cysteine 532 and cysteine 545 are the N-ethylmaleimide-reactive residues of the Neurospora plasma membrane H+-ATPase.

    PubMed

    Pardo, J P; Slayman, C W

    1989-06-01

    Previous studies from this laboratory (Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226; Davenport, J. W., and Slayman, C. W. (1988) J. Biol. Chem. 263, 16007-16013) have used the sulfhydryl reagent N-ethylmaleimide (NEM) to define two sites on the Neurospora plasma membrane H+-ATPase: a "fast" site which reacts in several minutes with no loss of enzymatic activity and a "slow" site which reacts in tens of minutes to produce complete inactivation of the enzyme. The slow site is protected when MgATP or MgADP is bound to the catalytic site of the ATPase. The present study demonstrates that the fluorescent reagent 5-[2-iodoacetamido)ethyl)-1-aminonaphthalenesulfonic acid (IAEDANS) can be used to label five of the eight cysteine residues of the Neurospora ATPase (Cys376, Cys409, Cys472, Cys532, Cys545). Tryptic peptides bearing those residues have been purified by high performance liquid chromatography and located within the known primary structure of the ATPase by amino acid analysis and/or sequencing. By pretreating the enzyme with NEM in the presence or absence of MgADP before incubation with IAEDANS, it has been possible to identify the fast NEM site as Cys545 and the slow MgADP-protectable NEM site as Cys532. Both residues lie within the central hydrophilic domain of the protein, close to a highly conserved stretch of amino acids that may be involved in nucleotide binding. However, all five IAEDANS-reactive cysteines can be nearly completely modified by the less bulky sulfhydryl reagent methyl methanethiosulfonate with less than 20% inhibition of enzyme activity; thus, none of the five cysteines can be considered to play a direct role in the reaction cycle of the ATPase.

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

    PubMed

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

    2008-01-01

    Most previously reported mutations in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) result in an odd number of cysteine residues within the epidermal growth factor (EGF)-like repeats in Notch3. We report here R75P mutation in two Japanese CADASIL families not directly involving cysteine residues located within the first EGF-like repeats. Probands in both families had repeated episodes of stroke, depression, dementia as well as T2 high-intensity lesions in the basal ganglia and periventricular white matter, but fewer white matter lesions in the temporal pole on MRI. These families provide new insights into the diagnosis and pathomechanisms of CADASIL. PMID:19043263

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

    PubMed

    Awoonor-Williams, Ernest; Rowley, Christopher N

    2016-09-13

    Methods for the calculation of the pKa ionizable amino acids are valuable tools for understanding pH-dependent properties of proteins. Cysteine is unique among the amino acids because of the chemical reactivity of its thiol group (S-H), which plays an instrumental role in several biochemical and regulatory functions. The acidity of noncatalytic cysteine residues is a factor in their susceptibility to chemical modification. Despite the plethora of existing pKa computing methods, no definitive protocol exists for accurately calculating the pKa's of cysteine residues in proteins. A cysteine pKa test set was developed, which is comprised of 18 cysteine residues in 12 proteins where the pKa's have been determined experimentally and an experimental structure is available. The pKa's of these residues were calculated using three methods that use an implicit solvent model (H++, MCCE, and PROPKA) and an all-atom replica-exchange thermodynamic integration approach with the CHARMM36 and AMBER ff99SB-ILDNP force fields. The models that use implicit solvation methods were generally unreliable in predicting cysteine residue pKa's, with RMSDs between 3.41 and 4.72 pKa units. On average, the explicit solvent methods performed better than the implicit solvent methods. RMSD values of 2.40 and 3.20 were obtained for simulations with the CHARMM36 and AMBER ff99SB-ILDNP force fields, respectively. Further development of these methods is necessary because the performance of the best method is similar to that of the null-model (RMSD = 2.74) and these differences in RMSD are of limited statistical significance given the small size of our test set. PMID:27541839

  2. A Methionine Residue Promotes Hyperoxidation of the Catalytic Cysteine of Mouse Methionine Sulfoxide Reductase A.

    PubMed

    Kim, Geumsoo; Levine, Rodney L

    2016-06-28

    Methionine sulfoxide reductase A (msrA) reduces methionine sulfoxide in proteins back to methionine. Its catalytic cysteine (Cys72-SH) has a low pKa that facilitates oxidation by methionine sulfoxide to cysteine sulfenic acid. If the catalytic cycle proceeds efficiently, the sulfenic acid is reduced back to cysteine at the expense of thioredoxin. However, the sulfenic acid is vulnerable to "irreversible" oxidation to cysteine sulfinic acid that inactivates msrA (hyperoxidation). We observed that human msrA is resistant to hyperoxidation while mouse msrA is readily hyperoxidized by micromolar concentrations of hydrogen peroxide. We investigated the basis of this difference in susceptibility to hyperoxidation and established that it is controlled by the presence or absence of a Met residue in the carboxyl-terminal domain of the enzyme, Met229. This residue is Val in human msrA, and when it was mutated to Met, human msrA became sensitive to hyperoxidation. Conversely, mouse msrA was rendered insensitive to hyperoxidation when Met229 was mutated to Val or one of five other residues. Positioning of the methionine at residue 229 is not critical, as hyperoxidation occurred as long as the methionine was located within the group of 14 carboxyl-terminal residues. The carboxyl domain of msrA is known to be flexible and to have access to the active site, and Met residues are known to form stable, noncovalent bonds with aromatic residues through interaction of the sulfur atom with the aromatic ring. We propose that Met229 forms such a bond with Trp74 at the active site, preventing formation of a protective sulfenylamide with Cys72 sulfenic acid. As a consequence, the sulfenic acid is available for facile, irreversible oxidation to cysteine sulfinic acid. PMID:27259041

  3. Engineering of cysteine residues leads to improved production of a human dipeptidase enzyme in E. coli.

    PubMed

    O'Dwyer, Ronan; Razzaque, Rafia; Hu, Xuejun; Hollingshead, Susan K; Wall, J Gerard

    2009-10-01

    Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human beta-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem beta-lactam antibiotics and can cleave dipeptides containing amino acids in both D: - and L: -configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.

  4. 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...

  5. Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys-34) of BSA. Mapping of the glycation sites of the anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction.

    PubMed

    Demian, Wael L L; Kottari, Naresh; Shiao, Tze Chieh; Randell, Edward; Roy, René; Banoub, Joseph H

    2014-12-01

    We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67,599 and 70,905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier.

  6. Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys-34) of BSA. Mapping of the glycation sites of the anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction.

    PubMed

    Demian, Wael L L; Kottari, Naresh; Shiao, Tze Chieh; Randell, Edward; Roy, René; Banoub, Joseph H

    2014-12-01

    We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67,599 and 70,905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier. PMID:25476939

  7. Redox Sensitivities of Global Cellular Cysteine Residues under Reductive and Oxidative Stress.

    PubMed

    Araki, Kazutaka; Kusano, Hidewo; Sasaki, Naoyuki; Tanaka, Riko; Hatta, Tomohisa; Fukui, Kazuhiko; Natsume, Tohru

    2016-08-01

    The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2), uroporphyrinogen decarboxylase (UROD), and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 μM for 5 min) did not cause global oxidations but instead had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.

  8. Bovine inositol monophosphatase. Modification, identification and mutagenesis of reactive cysteine residues.

    PubMed Central

    Knowles, M R; Gee, N; McAllister, G; Ragan, C I; Greasley, P J; Gore, M G

    1992-01-01

    1. Bovine inositol monophosphatase reacts with thiol reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA). 2. Modification by NEM results in nearly total loss of enzyme activity, whereas modification by IAA causes a slight increase in activity. 3. The loss of activity caused by NEM can be prevented by the inclusion of Ins1P, or better Ins1P and LiCl in the reaction mixture. 4. Two equivalents of p-nitrothiobenzoate (NTB2-) are released from the native enzyme on reaction with DTNB, and six equivalents of NTB2- are released from the SDS-denatured enzyme, suggesting that none of the six cysteine residues per molecule of enzyme is involved in intra- or inter-molecular disulphide bridges. 5. Both NEM and IAA react with two cysteine residues (residues 141 and 184 in the sequence) in a mutually exclusive manner. 6. NEM also reacts stoichiometrically with residue 218. 7. The NEM-induced loss of enzyme activity is accompanied by a 15% decrease in protein fluorescence. 8. A mutant of the enzyme which has an Ala-218 replacement for Cys-218 has full activity and is not sensitive to NEM, showing that the modification of this cysteine by NEM causes inhibition of the native protein by steric effects and that Cys-218 is not essential for activity. PMID:1322134

  9. Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups

    PubMed Central

    Soeda, Yoshiyuki; Yoshikawa, Misato; Almeida, Osborne F. X.; Sumioka, Akio; Maeda, Sumihiro; Osada, Hiroyuki; Kondoh, Yasumitsu; Saito, Akiko; Miyasaka, Tomohiro; Kimura, Tetsuya; Suzuki, Masaaki; Koyama, Hiroko; Yoshiike, Yuji; Sugimoto, Hachiro; Ihara, Yasuo; Takashima, Akihiko

    2015-01-01

    Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies. PMID:26671725

  10. Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase.

    PubMed

    Tinguely, J N; Wermuth, B

    1999-02-01

    Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m). NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione. PMID:10091578

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. Myeloperoxidase Inactivates TIMP-1 by Oxidizing Its N-terminal Cysteine Residue

    PubMed Central

    Wang, Yi; Rosen, Henry; Madtes, David K.; Shao, Baohai; Martin, Thomas R.; Heinecke, Jay W.; Fu, Xiaoyun

    2016-01-01

    An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation. PMID:17726014

  16. Role of cysteine-58 and cysteine-95 residues in the thiol di-sulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti.

    PubMed

    Chauhan, Nikhil; Hoti, S L

    2016-01-01

    Macrophage Migration Inhibitory Factor (MIF) is the first human cytokine reported and was thought to have a central role in the regulation of inflammatory responses. Homologs of this molecule have been reported in bacteria, invertebrates and plants. Apart from cytokine activity, it also has two catalytic activities viz., tautomerase and di-sulfide oxidoreductase, which appear to be involved in immunological functions. The CXXC catalytic site is responsible for di-sulfide oxidoreductase activity of MIF. We have recently reported thiol-disulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti (Wba-MIF-2), although it lacks the CXXC motif. We hypothesized that three conserved cysteine residues might be involved in the formation of di-sulfide oxidoreductase catalytic site. Homology modeling of Wba-MIF-2 showed that among the three cysteine residues, Cys58 and Cys95 residues came in close proximity (3.23Å) in the tertiary structure with pKa value 9, indicating that these residues might play a role in the di-sulfide oxidoreductase catalytic activity. We carried out site directed mutagenesis of these residues (Cys58Ser & Cys95Ser) and expressed mutant proteins in Escherichia coli. The mutant proteins did not show any oxidoreductase activity in the insulin reduction assay, thus indicating that these two cysteine residues are vital for the catalytic activity of Wba-MIF-2. PMID:26432350

  17. 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

  18. 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

  19. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  20. 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

    2015-02-01

    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.

  1. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-02-01

    Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates--both antibody-drug conjugates as well as constructs for in vivo imaging--has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope

  2. Posttranslational heterocyclization of cysteine and serine residues in the antibiotic microcin B17: distributivity and directionality.

    PubMed

    Kelleher, N L; Hendrickson, C L; Walsh, C T

    1999-11-23

    To produce the antibiotic Microcin B17, four Cys and four Ser residues are converted into four thiazoles and four oxazoles by the three subunit Microcin B17 synthetase. High-resolution mass spectrometry (MS) was used to monitor the kinetics of posttranslational heterocyclic ring formation (-20 Da per ring) and demonstrated the accumulation of all intermediates, from one to seven rings, indicating distributive processing. All of the intermediates could be converted by the enzyme to the eight ring product. Enzymatic chemoselectivity (Cys vs Ser cyclization rates) was assessed using iodoacetamido-salicylate to alkylate unreacted cysteines (+193 Da) in the 8 kDa biosynthetic intermediates; three of the first four rings formed were thiazoles, and by the five ring stage, all four of the cysteines had been heterocyclized while three of the original four serines remained uncyclized. Finally, tandem MS using a 9.4 T Fourier transform instrument with electrospray ionization was used to elaborate the major processing pathway: the first two rings formed are at the most amino proximal sites (Cys(41) then Ser(40)) followed by the remaining three cysteines at positions 48, 51, and 55. The cyclization of serines at positions 56, 62, and 65 then follows, with Ser(62) and Ser(65) the last to heterocyclize and the first of these at a slower rate. Thus, despite free dissociation of intermediates after each of seven ring-forming catalytic cycles, there is an overall directionality of ring formation from N-terminal to C-terminal sites. This remarkable regioselectivity is determined more by the substrate than the enzyme, due to a combination of (1) initial high-affinity binding of the posttranslational catalyst to the N-terminal propeptide of substrate 88mer, and (2) a chemoselectivity for thiazole over oxazole formation. This mechanism is consistent with antibiotic biosynthesis in vivo, yielding microcin with six, seven, and eight rings, all with bioactivity.

  3. An additional cysteine in a typical 2-Cys peroxiredoxin of Pseudomonas promotes functional switching between peroxidase and molecular chaperone.

    PubMed

    An, Byung Chull; Lee, Seung Sik; Jung, Hyun Suk; Kim, Jin Young; Lee, Yuno; Lee, Keun Woo; Lee, Sang Yeol; Tripathi, Bhumi Nath; Chung, Byung Yeoup

    2015-09-14

    Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone. PMID:26278368

  4. 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

  5. A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.

    PubMed

    Hansson, Karin; Thämlitz, Ann-Marie; Furie, Bruce; Furie, Barbara C; Stenflo, Johan

    2006-10-24

    Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

  6. 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.

  7. Oxidative post-translational modifications of cysteine residues in plant signal transduction.

    PubMed

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

    2015-05-01

    In plants, fluctuation of the redox balance by altered levels of reactive oxygen species (ROS) can affect many aspects of cellular physiology. ROS homeostasis is governed by a diversified set of antioxidant systems. Perturbation of this homeostasis leads to transient or permanent changes in the redox status and is exploited by plants in different stress signalling mechanisms. Understanding how plants sense ROS and transduce these stimuli into downstream biological responses is still a major challenge. ROS can provoke reversible and irreversible modifications to proteins that act in diverse signalling pathways. These oxidative post-translational modifications (Ox-PTMs) lead to oxidative damage and/or trigger structural alterations in these target proteins. Characterization of the effect of individual Ox-PTMs on individual proteins is the key to a better understanding of how cells interpret the oxidative signals that arise from developmental cues and stress conditions. This review focuses on ROS-mediated Ox-PTMs on cysteine (Cys) residues. The Cys side chain, with its high nucleophilic capacity, appears to be the principle target of ROS. Ox-PTMs on Cys residues participate in various signalling cascades initiated by plant stress hormones. We review the mechanistic aspects and functional consequences of Cys Ox-PTMs on specific target proteins in view of stress signalling events.

  8. 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.

  9. 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. PMID:24625320

  10. Identification of zinc-ligated cysteine residues based on 13Calpha and 13Cbeta chemical shift data.

    PubMed

    Kornhaber, Gregory J; Snyder, David; Moseley, Hunter N B; Montelione, Gaetano T

    2006-04-01

    Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure and/or play critical catalytic roles in the underlying biochemistry. An atom's chemical shift is exquisitely sensitive to its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation. In this study, we demonstrate that overlapped 13Cbeta chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion of the corresponding 13Calpha chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using 287 13Calpha/13Cbeta shift pairs from 79 proteins with known three-dimensional structures, including 86 13Calpha and 13Cbeta shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information, we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines, and Zn-ligated cysteines. Classifying cysteines/cystines with a statistical model incorporating all three phenomena resulted in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron-sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands. We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn

  11. 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.

  12. 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.

  13. Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification.

    PubMed Central

    Kanaya, S; Kimura, S; Katsuda, C; Ikehara, M

    1990-01-01

    The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133. Images Fig. 2. PMID:2171503

  14. Application of iTRAQ Reagents to Relatively Quantify the Reversible Redox State of Cysteine Residues

    PubMed Central

    McDonagh, Brian; Martínez-Acedo, Pablo; Vázquez, Jesús; Padilla, C. Alicia; Sheehan, David; Bárcena, José Antonio

    2012-01-01

    Cysteines are one of the most rarely used amino acids, but when conserved in proteins they often play critical roles in structure, function, or regulation. Reversible cysteine modifications allow for potential redox regulation of proteins. Traditional measurement of the relative absolute quantity of a protein between two samples is not always necessarily proportional to the activity of the protein. We propose application of iTRAQ reagents in combination with a previous thiol selection method to relatively quantify the redox state of cysteines both within and between samples in a single analysis. Our method allows for the identification of the proteins, identification of redox-sensitive cysteines within proteins, and quantification of the redox status of individual cysteine-containing peptides. As a proof of principle, we applied this technique to yeast alcohol dehydrogenase-1 exposed in vitro to H2O2 and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis. PMID:22844595

  15. Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3.

    PubMed

    Chandra, Mintu; Datta, Sunando

    2016-08-01

    The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding. PMID:27485554

  16. 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.

  17. 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. PMID:16497323

  18. Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase.

    PubMed

    Alexeyev, Mikhail F; Winkler, Herbert H

    2002-09-20

    The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches. PMID:12225862

  19. 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

    2015-02-01

    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.

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

    PubMed

    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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:19717178

  2. 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.

  3. Substitution of the cysteine 438 residue in the cytoplasmic tail of the glucagon-like peptide-1 receptor alters signal transduction activity.

    PubMed

    Vázquez, Patricia; Roncero, Isabel; Blázquez, Enrique; Alvarez, Elvira

    2005-04-01

    Several G-protein-coupled receptors contain cysteine residues in the C-terminal tail that may modulate receptor function. In this work we analysed the substitution of Cys438 by alanine in the glucagon-like peptide-1 (GLP-1) receptor (GLPR), which led to a threefold decrease in cAMP production, although endocytosis and cellular redistribution of GLP-1 receptor agonist-induced processes were unaffected. Additionally, cysteine residues in the C-terminal tail of several G-protein-coupled receptors were found to act as substrates for palmitoylation, which might modify the access of protein kinases to this region. His-tagged GLP-1 receptors incorporated 3H-palmitate. Nevertheless, substitution of Cys438 prevented the incorporation of palmitate. Accordingly, we also investigated the effect of substitution of the consensus sequence by protein kinase C (PKC) Ser431/432 in both wild-type and Ala438 GLP-1 receptors. Substitution of Ser431/432 by alanine did not modify the ability of wild-type receptors to stimulate adenylate cyclase or endocytosis and recycling processes. By contrast, the substitution of Ser431/432 by alanine in the receptor containing Ala438 increased the ability to stimulate adenylate cyclase. All types of receptors were mainly internalised through coated pits. Thus, cysteine 438 in the cytoplasmic tail of the GLP-1 receptor would regulate its interaction with G-proteins and the stimulation of adenylyl cyclase. Palmitoylation of this residue might control the access of PKC to Ser431/432.

  4. 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

    2015-11-01

    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.

  5. 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.

  6. Effect of multiple cysteine substitutions on the functionality of human multidrug resistance protein 1 expressed in human embryonic kidney 293 cells: identification of residues essential for function.

    PubMed

    Qin, Lei; Tam, Shui-Pang; Deeley, Roger G

    2012-07-01

    Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17β-estradiol 17-β-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

  7. Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity.

    PubMed

    Wen, L; Miao, Z W; Qing, W D

    1999-08-01

    The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine. PMID:10609644

  8. Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from Entamoeba histolytica Assists in Partial Regaining of Feedback Inhibition by Cysteine

    PubMed Central

    Kumar, Sudhir; Mazumder, Mohit; Dharavath, Sudhaker; Gourinath, S.

    2013-01-01

    The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated in vitro, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by Km, compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3) shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor. PMID:23437075

  9. 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.

  10. Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics.

    PubMed

    Toledo, José Carlos; Audi, Renata; Ogusucu, Renata; Monteiro, Gisele; Netto, Luis Eduardo Soares; Augusto, Ohara

    2011-05-01

    Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M⁻¹ s⁻¹) at pH 7.4 and 25°C.

  11. Ab Initio MD Simulations of the Brønsted Acidity of Glutathione in Aqueous Solutions: Predicting pKa Shifts of the Cysteine Residue.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-12-10

    The tripeptide glutathione (GSH) is one of the most abundant peptides and the major repository for nonprotein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thiol-disulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influence the redox couple, and hence the pKa value of the cysteine residue of GSH is critical to its functioning. Here we report ab initio Car-Parrinello molecular dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. We show that the free-energy landscape for the protonation-deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared with the isolated cysteine amino acid.

  12. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

    PubMed

    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

    2016-10-01

    Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 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 CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 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 CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities. PMID:27571479

  13. 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.

  14. Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson-Calvin cycle enzyme enslaved to its cysteine residues.

    PubMed

    Thieulin-Pardo, Gabriel; Remy, Thérèse; Lignon, Sabrina; Lebrun, Régine; Gontero, Brigitte

    2015-04-01

    Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

  15. Enhancement of ethylenethiourea recoveries in food analyses by addition of cysteine hydrochloride.

    PubMed

    Sack, C A

    1995-01-01

    The effectiveness of cysteine hydrochloride (Cys-HCl) as a preservative of ethylenethiourea (ETU) in product matrixes and during analysis was studied. ETU recoveries were adversely affected by certain product matrixes when fortified directly into the product. Recoveries in 8 selected food items were 0-92% when analyzed 30 min after fortification and 0-51% when analyzed after 24 h. When Cys-HCl was added to product prior to fortification, recoveries increased to 71-95% even after frozen storage for 2-4 weeks. Cys-HCl was added during analysis of 53 untreated items. Recoveries improved an average of 15% with Cys-HCl. Without Cys-HCl, recoveries were erratic (20-98%), but with Cys-HCl, recoveries were 68-113%. Other antioxidants (sodium sulfite, butylated hydroxyanisole, butylated hydroxytoluene, and vitamins A and C) also were evaluated as ETU preservatives. When lettuce was treated first with sodium sulfite and then fortified with ETU, recoveries averaged 86%; without sodium sulfite, they averaged 1%. The other antioxidants were less effective for preserving ETU in lettuce, giving only 8-46% recoveries. The effect of oxidizers (potassium bromate, sodium hypochlorite, and hydrogen peroxide) on ETU recovery was also determined. Recovery of ETU from a baby food product (pears and pineapple) was 82%; with oxidizers, recoveries were 0-8%.

  16. 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.

  17. Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

    SciTech Connect

    Bednar, R.A.; Fried, W.B.; Lock, Y.W.; Pramanik, B. )

    1989-08-25

    Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl{sub 2}. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by ({sup 3}H) N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.

  18. 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

  19. VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

    PubMed

    Reina, Simona; Checchetto, Vanessa; Saletti, Rosaria; Gupta, Ankit; Chaturvedi, Deepti; Guardiani, Carlo; Guarino, Francesca; Scorciapino, Mariano Andrea; Magrì, Andrea; Foti, Salvatore; Ceccarelli, Matteo; Messina, Angela Anna; Mahalakshmi, Radhakrishnan; Szabo, Ildiko; De Pinto, Vito

    2016-01-19

    Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes. PMID:26760765

  20. VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

    PubMed Central

    Saletti, Rosaria; Guardiani, Carlo; Guarino, Francesca; Scorciapino, Mariano Andrea; Magrì, Andrea; Foti, Salvatore; Ceccarelli, Matteo; Messina, Angela Anna; Mahalakshmi, Radhakrishnan; Szabo, Ildiko; De Pinto, Vito

    2016-01-01

    Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes. PMID:26760765

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

    PubMed

    Juvekar, Vinayak; Gong, Young Dae

    2016-02-19

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

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

    PubMed

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

    2015-02-01

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

  3. Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues

    PubMed Central

    2013-01-01

    Background Methanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. The mechanisms by which they sense and respond to oxidizing conditions are unknown. MsvR is a transcription regulatory protein unique to the methanoarchaea. Initially identified and characterized in the methanogen Methanothermobacter thermautotrophicus (Mth), MthMsvR displays differential DNA binding under either oxidizing or reducing conditions. Since MthMsvR regulates a potential oxidative stress operon in M. thermautotrophicus, it was hypothesized that the MsvR family of proteins were redox-sensitive transcription regulators. Results An MsvR homologue from the methanogen Methanosarcina acetivorans, MaMsvR, was overexpressed and purified. The two MsvR proteins bound the same DNA sequence motif found upstream of all known MsvR encoding genes, but unlike MthMsvR, MaMsvR did not bind the promoters of select genes involved in the oxidative stress response. Unlike MthMsvR that bound DNA under both non-reducing and reducing conditions, MaMsvR bound DNA only under reducing conditions. MaMsvR appeared as a dimer in gel filtration chromatography analysis and site-directed mutagenesis suggested that conserved cysteine residues within the V4R domain were involved in conformational rearrangements that impact DNA binding. Conclusions Results presented herein suggest that homodimeric MaMsvR acts as a transcriptional repressor by binding Ma PmsvR under non-reducing conditions. Changing redox conditions promote conformational changes that abrogate binding to Ma PmsvR which likely leads to de-repression. PMID:23865844

  4. 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

  5. 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.

  6. Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue.

    PubMed

    Onishi, H; Maita, T; Miyanishi, T; Watanabe, S; Matsuda, G

    1986-12-01

    A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

  7. Survey of residual solvents in natural food additives by standard addition head-space GC.

    PubMed

    Uematsu, Y; Hirata, K; Suzuki, K; Iida, K; Kamata, K

    2002-04-01

    Residual levels of 12 solvents in 87 natural food additives (66 samples of food colours, 19 samples of natural antioxidants and two natural preservatives) collected between 1997 and 1999 were determined by automated head-space GC using FID, with a porous-polymer (PLOT) column. Calibration curves were prepared by the method of standard addition. Confirmation was by manually injected head-space GC using mass spectrometric detection. 1,2-Dichloroethane was found in turmeric colour (natural food colour) collected in 1997 at the concentrations of 8.6 microg g(-1), but was not found in samples collected in 1998 and 1999. Hexane was found in three samples of dunaliella carotene (11, 72 and 75 microg g(-1)), and in chlorophyll at 93 microg g(-1) (both natural food colours). Acetone was found in turmeric colour, annatto colour, dunaliella carotene, kaoliang colour, cacao colour at a concentration between 8.7 and 42 microg g(-1) (all natural food colours).

  8. Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation.

    PubMed Central

    Jensen, P E; Reid, J D; Hunter, C N

    2000-01-01

    The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg(2+) chelatase, such as the ATPase activity of ChlI, and the formation of ChlI-ChlD-MgATP and ChlH-protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg(2+) chelatase activity, and the formation of the ChlI-ChlD-ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI-ChlD-MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg(2+) ions. The simplest explanation for this is that in an intact Mg(2+) chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI-MgATP association, ATP

  9. Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase.

    PubMed

    Anderson, P M; Korte, J J; Holcomb, T A; Cho, Y G; Son, C M; Sung, Y C

    1994-05-27

    Reaction of the single cysteine residue in each subunit of cyanase with certain SH reagents gives an active decameric derivative that dissociates reversibly to an inactive dimer derivative (Anderson, P. M., Johnson, W. V., Korte, J. J., Xiong, X., Sung, Y.-c., and Fuchs, J. A. (1988) J. Biol. Chem. 263, 5674-5680). Reaction of mixed disulfide dimer derivatives of cyanase with dithiothreitol at 0 degree C results in formation of a disulfide bond between the subunits in the dimer. The disulfide dimer was inactive and did not associate to a decamer; the intersubunit disulfide bond could not be formed when the dimers were associated as a decamer. The two SH groups apparently are in close proximity to each other in the dissociated dimer but not when the dimer is associated to a decamer. Substitution of glycine for the cysteine residue or of tyrosine, asparagine, glycine, valine, or leucine for the single histidine residue in each subunit gave mutant enzymes that were active. However, H113N, H113Y, and C83G were unstable at low temperature and/or ionic strength, dissociating reversibly to an inactive dimer. Efficient reassociation required the presence of bicarbonate or cyanate analog. The results are consistent with a proposed single site per subunit model explaining apparent half-site binding of substrates and the requirement of decameric structure for activity.

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

    PubMed

    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

  11. 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

  12. The dependence of Ag+ block of a potassium channel, murine kir2.1, on a cysteine residue in the selectivity filter.

    PubMed

    Dart, C; Leyland, M L; Barrett-Jolley, R; Shelton, P A; Spencer, P J; Conley, E C; Sutcliffe, M J; Stanfield, P R

    1998-08-15

    Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.

  13. Intra-residue interactions in proteins: interplay between serine or cysteine side chains and backbone conformations, revealed by laser spectroscopy of isolated model peptides.

    PubMed

    Alauddin, Mohammad; Biswal, Himansu S; Gloaguen, Eric; Mons, Michel

    2015-01-21

    Intra-residue interactions play an important role in proteins by influencing local folding of the backbone. Taking advantage of the capability of gas phase experiments to provide relevant information on the intrinsic H-bonding pattern of isolated peptide chains, the intra-residue interactions of serine and cysteine residues, i.e., OH/SH···OC(i) C6 and NH(i···)O/S C5 interactions in Ser/Cys residues, are probed by laser spectroscopy of isolated peptides. The strength of these local side chain-main chain interactions, elegantly documented from their IR spectral features for well-defined conformations of the main chain, demonstrates that a subtle competition exists between the two types of intra-residue bond: the C6 H-bond is the major interaction with Ser, in contrast to Cys where C5 interaction takes over. The restricted number of conformers observed in the gas phase experiment with Ser compared to Cys (where both extended and folded forms are observed) also suggests a significant mediation role of these intra-residue interactions on the competition between the several main chain folding patterns. PMID:25482851

  14. Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state.

    PubMed

    Dotson, P Patrick; Karakashian, Alexander A; Nikolova-Karakashian, Mariana N

    2015-02-01

    Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys(617) to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. PMID:25287744

  15. Cytoplasmic localization and redox cysteine residue of APE1/Ref-1 are associated with its anti-inflammatory activity in cultured endothelial cells.

    PubMed

    Park, Myoung Soo; Kim, Cuk-Seong; Joo, Hee Kyoung; Lee, Yu Ran; Kang, Gun; Kim, Soo Jin; Choi, Sunga; Lee, Sang Do; Park, Jin Bong; Jeon, Byeong Hwa

    2013-11-01

    Apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in base excision DNA repair and transcriptional regulation of gene expression. APE1/Ref-1 is mainly localized in the nucleus, but cytoplasmic localization has also been reported. However, the functional role of cytoplasmic APE1/Ref-1 and its redox cysteine residue are still unknown. We investigated the role of cytoplasmic APE1/Ref-1 on tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) expressions in endothelial cells. Endogenous APE1/Ref-1 was mainly observed in the nucleus, however, cytoplasmic APE1/Ref-1 was increased by TNF-α. Cytoplasmic APE1/Ref-1 expression was not blunted by cycloheximide, a protein synthesis inhibitor, suggesting cytoplasmic translocation of APE1/Ref-1. Transfection of an N-terminus deletion mutant APE1/Ref-1(29-318) inhibited TNF-α-induced VCAM-1 expression, indicating an anti-inflammatory role for APE1/Ref-1 in the cytoplasm. In contrast, redox mutant of APE1/Ref-1 (C65A/C93A) transfection led to increased TNF-α-induced VCAM-1 expression. Our findings suggest cytoplasmic APE1/Ref-1 localization and redox cysteine residues of APE1/Ref-1 are associated with its anti-inflammatory activity in endothelial cells.

  16. Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

    PubMed

    Keskar, S S; Srinivasan, M C; Deshpande, V V

    1989-07-01

    Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.

  17. 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.

  18. 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.

  19. Modification of the cysteine residues in IκBα kinase and NF-κB (p65) by xanthohumol leads to suppression of NF-κB–regulated gene products and potentiation of apoptosis in leukemia cells

    PubMed Central

    Harikumar, Kuzhuvelil B.; Kunnumakkara, Ajaikumar B.; Ahn, Kwang S.; Anand, Preetha; Krishnan, Sunil; Guha, Sushovan

    2009-01-01

    Xanthohumol (XN), a prenylated chalcone isolated from hop plant, exhibits anti-inflammatory, antiproliferative, and antiangiogenic properties through an undefined mechanism. Whether examined by intracellular esterase activity, phosphatidylserine externalization, DNA strand breaks, or caspase activation, we found that XN potentiated tumor necrosis factor–induced apoptosis in leukemia and myeloma cells. This enhancement of apoptosis correlated with down-regulation of nuclear factor-κB (NF-κB) survivin, bcl-xL, XIAP, cIAP1, cIAP2, cylin D1, and c-myc. XN down-regulated both constitutive and inducible NF-κB activation, inhibition of phosphorylation and degradation of IκBα, suppression of p65 nuclear translocation, and NF-κB–dependent reporter gene transcription. XN directly inhibited tumor necrosis factor-induced IκBα kinase (IKK) activation and a reducing agent abolished this inhibition, indicating the role of cysteine residue. XN had no effect on the IKK activity when cysteine residue 179 of IKK was mutated to alanine. XN also directly inhibited binding of p65 to DNA, a reducing agent reversed this effect, and mutation of cysteine residue 38 to serine of p65 abolished this effect. Thus, our results show that modification of cysteine residues of IKK and p65 by XN leads to inhibition of the NF-κB activation pathway, suppression of antiapoptotic gene products, and potentiation of apoptosis in leukemia cells. PMID:18952893

  20. 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.

  1. Comparing the additive composition of smokeless gunpowder and its handgun-fired residues.

    PubMed

    Reardon, M R; MacCrehan, W A; Rowe, W F

    2000-11-01

    Detecting the use of handguns via the determination of the organic additives in smokeless gunpowder residues (OGSR) presents a promising alternative to primer metal residue analysis. Compositional analysis of the gunpowder additives nitroglycerin, diphenylamine, and ethyl centralite provides information that can associate residue samples with unfired gunpowder. We evaluated the composition of seven reloading smokeless gunpowders, both in bulk and as single particles, by ultrasonic solvent extraction/capillary electrophoresis. Handgun-fired residues obtained from three common weapon calibers loaded with the known reloading powders were compared with the unfired powders. In general, the composition of the residues was similar to that found in the unfired powders. For double-base powders, comparing the ratio of the propellant (P) to the total amount of stabilizer (S) for both residue and gunpowder samples proved to be a useful measurement for identification. This P/S ratio demonstrated that the additives in the residues did not greatly change relative to the unfired powder, providing a useful indicator to aid in forensic powder and residue evaluation.

  2. Soil nitrous oxide emissions following crop residue addition: a meta-analysis.

    PubMed

    Chen, Huaihai; Li, Xuechao; Hu, Feng; Shi, Wei

    2013-10-01

    Annual production of crop residues has reached nearly 4 billion metric tons globally. Retention of this large amount of residues on agricultural land can be beneficial to soil C sequestration. Such potential impacts, however, may be offset if residue retention substantially increases soil emissions of N(2)O, a potent greenhouse gas and ozone depletion substance. Residue effects on soil N(2)O emissions have gained considerable attention since early 1990s; yet, it is still a great challenge to predict the magnitude and direction of soil N(2)O emissions following residue amendment. Here, we used a meta-analysis to assess residue impacts on soil N(2)O emissions in relation to soil and residue attributes, i.e., soil pH, soil texture, soil water content, residue C and N input, and residue C : N ratio. Residue effects were negatively associated with C : N ratios, but generally residue amendment could not reduce soil N(2)O emissions, even for C : N ratios well above ca. 30, the threshold for net N immobilization. Residue effects were also comparable to, if not greater than, those of synthetic N fertilizers. In addition, residue effects on soil N(2)O emissions were positively related to the amounts of residue C input as well as residue effects on soil CO(2) respiration. Furthermore, most significant and stimulatory effects occurred at 60-90% soil water-filled pore space and soil pH 7.1-7.8. Stimulatory effects were also present for all soil textures except sand or clay content ≤10%. However, inhibitory effects were found for soils with >90% water-filled pore space. Altogether, our meta-analysis suggests that crop residues played roles beyond N supply for N(2)O production. Perhaps, by stimulating microbial respiration, crop residues enhanced oxygen depletion and therefore promoted anaerobic conditions for denitrification and N(2)O production. Our meta-analysis highlights the necessity to connect the quantity and quality of crop residues with soil properties for predicting

  3. 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.

  4. 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

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

    PubMed

    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.

  6. Intra-membrane Signaling Between the Voltage-Gated Ca2+-Channel and Cysteine Residues of Syntaxin 1A Coordinates Synchronous Release

    PubMed Central

    Bachnoff, Niv; Cohen-Kutner, Moshe; Trus, Michael; Atlas, Daphne

    2013-01-01

    The interaction of syntaxin 1A (Sx1A) with voltage-gated calcium channels (VGCC) is required for depolarization-evoked release. However, it is unclear how the signal is transferred from the channel to the exocytotic machinery and whether assembly of Sx1A and the calcium channel is conformationally linked to triggering synchronous release. Here we demonstrate that depolarization-evoked catecholamine release was decreased in chromaffin cells infected with semliki forest viral vectors encoding Sx1A mutants, Sx1AC271V, or Sx1AC272V, or by direct oxidation of these Sx1A transmembrane (TM) cysteine residues. Mutating or oxidizing these highly conserved Sx1A Cys271 and Cys272 equally disrupted the Sx1A interaction with the channel. The results highlight the functional link between the VGCC and the exocytotic machinery, and attribute the redox sensitivity of the release process to the Sx1A TM C271 and C272. This unique intra-membrane signal-transduction pathway enables fast signaling, and triggers synchronous release by conformational-coupling of the channel with Sx1A. PMID:23567899

  7. Addition of a C-Terminal Cysteine Improves the Anti-Herpes Simplex Virus Activity of a Peptide Containing the Human Immunodeficiency Virus Type 1 TAT Protein Transduction Domain▿

    PubMed Central

    Bultmann, Hermann; Teuton, Jeremy; Brandt, Curtis R.

    2007-01-01

    Previous studies have shown that peptides containing the protein transduction domain (PTD) of the human immunodeficiency virus tat protein (GRKKRRQRRR) were effective inhibitors of herpes simplex virus type 1 (HSV-1) entry (H. Bultmann and C. R. Brandt, J. Biol. Chem. 277:36018-36023, 2002). We now show that the addition of a single cysteine residue to the C terminus of the TAT PTD (TAT-C peptide) improves the antiviral activity against HSV-1 and HSV-2. The principle effect of adding the cysteine was to enable the peptide to inactivate virions and to induce a state of resistance to infection in cells pretreated with peptide. The TAT-C peptide acted extracellularly, immediately blocked entry of adsorbed virus, prevented VP16 translocation to the nucleus, and blocked syncytium formation and cell-cell spread. Thus, TAT-C peptides are fusion inhibitors. The induction of the resistance of cells to infection was rapid, recovered with a half-life of 5 to 6 h, and could be reinduced by peptide treatment. TAT-C bound to heparan sulfate but was a poor competitor for viral attachment. The antiviral activity depended on the net positive charge of the peptide but not on chirality, and a free sulfhydryl group was not essential for antiviral activity because TAT-C dimers were at least as effective as monomers. The unique combination of antiviral activities and low toxicity combine to make TAT-C a strong candidate for further development as a drug to block HSV infection. PMID:17261627

  8. A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification† †Electronic supplementary information (ESI) available: LC-MS, ES-MS, deconvoluted spectra and fluorescence emission spectra for all reactions with proteins described herein. Fluorescence emission spectra of superfolder GFP, all the cysteine mutants and their derivatives are given. See DOI: 10.1039/c3sc51333e Click here for additional data file.

    PubMed Central

    Nathani, Ramiz I.; Moody, Paul; Chudasama, Vijay; Smith, Mark E. B.; Fitzmaurice, Richard J.

    2013-01-01

    Local protein microenvironment is used to control the outcome of reaction between cysteine residues and 2,5-dibromohexanediamide. The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner. Subsequent elaboration with commonly used thiol and alkyne containing reagents affects site-selective protein dual labelling. PMID:24741436

  9. 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.

  10. "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

  11. Additive In Vitro Antiplasmodial Effect of N-Alkyl and N-Benzyl-1,10-Phenanthroline Derivatives and Cysteine Protease Inhibitor E64

    PubMed Central

    Wijayanti, Mahardika Agus; Sholikhah, Eti Nurwening; Hadanu, Ruslin; Jumina, Jumina; Supargiyono, Supargiyono; Mustofa, Mustofa

    2010-01-01

    Potential new targets for antimalarial chemotherapy include parasite proteases, which are required for several cellular functions during the Plasmodium falciparum life cycle. Four new derivatives of N-alkyl and N-benzyl-1,10-phenanthroline have been synthesized. Those are (1)-N-methyl-1,10-phenanthrolinium sulfate, (1)-N-ethyl-1,10-phenanthrolinium sulfate, (1)-N-benzyl-1,10-phenanthrolinium chloride, and (1)-N-benzyl-1,10-phenanthrolinium iodide. Those compounds had potential antiplasmodial activity with IC50 values from 260.42 to 465.38 nM. Cysteine proteinase inhibitor E64 was used to investigate the mechanism of action of N-alkyl and N-benzyl-1,10-phenanthroline derivatives. A modified fixed-ratio isobologram method was used to study the in vitro interactions between the new compounds with either E64 or chloroquine. The interaction between N-alkyl and N-benzyl-1,10-phenanthroline derivatives and E64 was additive as well as their interactions with chloroquine were also additive. Antimalarial mechanism of chloroquine is mainly on the inhibition of hemozoin formation. As the interaction of chloroquine and E64 was additive, the results indicated that these new compounds had a mechanism of action by inhibiting Plasmodium proteases. PMID:22332022

  12. 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.

  13. Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms.

    PubMed Central

    Karlström, A R; Levine, R L

    1991-01-01

    The protease of the human immunodeficiency virus is essential for replication of the virus, and the enzyme is therefore an attractive target for antiviral action. We have found that the viral protease is inhibited by approximately stoichiometric concentrations of copper or mercury ions. Inactivation by Cu2+ was rapid and not reversed by subsequent exposure to EDTA or dithiothreitol. Direct inhibition by Cu2+ required the presence of cysteine residue(s) in the protease. Thus, a synthetic protease lacking cysteine residues was not inhibited by exposure to copper. However, addition of dithiothreitol as an exogenous thiol rendered even the synthetic protease susceptible to inactivation by copper. Oxygen was not required for inactivation of either the wild-type or the synthetic protease. These results provide the basis for the design of novel types of protease inhibitors. PMID:2062837

  14. 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.

  15. 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.

  16. 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. PMID:24971658

  17. 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.

  18. 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.

  19. Cysteine residues in the large extracellular loop (EC2) are essential for the function of the stress-regulated glycoprotein M6a.

    PubMed

    Fuchsova, Beata; Fernández, María E; Alfonso, Julieta; Frasch, Alberto C

    2009-11-13

    Gpm6a was identified as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. Previously we showed that the stress-regulated protein M6a is a key modulator for neurite outgrowth and filopodium/spine formation. In the present work, mutational analysis was used to characterize the action of M6a at the molecular level. We show that four cysteines 162, 174, 192, and 202 within EC2 are functionally crucial sites. The presence of cysteines 162 and 202 is essential for the efficient cell surface expression of the M6a protein. In contrast, cysteines 174 and 192, which form a disulfide bridge as shown by biochemical analysis, are not required for the efficient surface expression of M6a. Their mutation to alanine does not interfere with the localization of M6a to filopodial protrusions in primary hippocampal neurons. The neurons expressing C174A and/or C192A mutants display decreased filopodia number. In non-permeabilized cells, these mutant proteins are not recognized by a function-blocking monoclonal antibody directed to M6a. Moreover, neurons in contact with axons expressing C174A/C192A mutant display significantly lower density of presynaptic clusters over their dendrites. Taken together, this study demonstrates that cysteines in the EC2 domain are critical for the role of M6a in filopodium outgrowth and synaptogenesis. PMID:19737934

  20. Addition of an organic amendment and/or residue mud to bauxite residue sand in order to improve its properties as a growth medium.

    PubMed

    Jones, B E H; Haynes, R J; Phillips, I R

    2012-03-01

    The effects of addition of carbonated residue mud (RMC) or seawater neutralized residue mud (RMS), at two rates, in the presence or absence of added green waste compost, on the chemical, physical and microbial properties of gypsum-treated bauxite residue sand were studied in a laboratory incubation study. The growth of two species commonly used in revegetation of residue sand (Lolium rigidum and Acacia saligna) in the treatments was then studied in a 18-week greenhouse study. Addition of green waste-based compost increased ammonium acetate-extractable (exchangeable) Mg, K and Na. Addition of residue mud at 5 and 10% w/w reduced exchangeable Ca but increased that of Mg and Na (and K for RMS). Concentrations of K, Na, Mg and level of EC in saturation paste extracts were increased by residue mud additions. Concentrations of cations in water extracts were considerably higher than those in saturation paste extracts but trends with treatment were broadly similar. Addition of both compost and residue mud caused a significant decrease in macroporosity with a concomitant increase in mesoporosity and microporosity, available water holding capacity and the quantity of water held at field capacity. Increasing rates of added residue mud reduced the percentage of sample present as discrete sand particles and increased that in aggregated form (particularly in the 1-2 and >10mm diameter ranges). Organic C content, C/N ratio, soluble organic C, microbial biomass C and basal respiration were increased by compost additions. Where compost was added, residue mud additions caused a substantial increase in microbial biomass and basal respiration. L. rigidum grew satisfactorily in all treatments although yields tended to be reduced by additions of mud (especially RMC) particularly in the absence of added compost. Growth of A. saligna was poor in sand alone and mud-amended sand and was greatly promoted by additions of compost. However, in the presence of compost, addition of carbonated

  1. Protein cysteine modifications: (2) reactivity specificity and topics of medicinal chemistry and protein engineering.

    PubMed

    Nagahara, Noriyuki; Matsumura, Tomohiro; Okamoto, Ryo; Kajihara, Yasuhiro

    2009-01-01

    Cysteine (cysteinyl residue) modifications in proteins result in diversity in protein functions. The reaction specificity of a protein with a modified cysteine residue is determined by the overall conditions of the protein, including the spatial position of the cysteine residue, electrostatic interactions between cysteine residue and other charged residues, spatial interactions between the cysteine residue and a chemical compound, electrophilicity of the chemical compound, and the pH of the solution. In cysteine-dependant enzymes, each specific type of cysteine modification characterizes the catalytic mechanism of the enzyme. Recently, the catalytic mechanisms of peroxiredoxins and cysteine proteases, which contain a cysteine residue(s) in their catalytic sites, have been elucidated. In the catalytic process of peroxiredoxins, a sulfenyl intermediate is formed by oxidation of the catalytic cysteine residue. On the other hand, in cysteine proteases, the catalytic cysteine residue reacts with the carboxyl carbon of a peptide substrate to form an intermediate complex via S-alkylation. In this review, we introduce the most current information on the applications of cysteine thiol chemistry for in vitro glycoprotein synthesis. Recently, a glycoprotein (monocyte chemotactic protein-3), containing an intact human complex-type sialyloligosaccharide has been chemically synthesized. The procedure used for this could have applications in the development of new protein-based drugs, including antineoplastic drugs and antibiotics. It can also potentially be applied for improving the half-life and reducing the toxicity of these drugs, and for preventing the development of multidrug resistance.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. [Multi-residue method for determination of veterinary drugs and feed additives in meats by HPLC].

    PubMed

    Chonan, Takao; Fujimoto, Toru; Ueno, Ken-Ichi; Tazawa, Teijiro; Ogawa, Hiroshi

    2007-10-01

    A simple and rapid multi-residue method was developed for the determination of 28 kinds of veterinary drugs and feed additives (drugs) in muscle of cattle, pig and chicken. The drugs were extracted with acetonitrile-water (95:5) in a homogenizer and ultrasonic generator. The extracted solution was poured into an alumina column and the drugs were eluted with acetonitrile-water (90:10). The eluate was washed with n-hexane saturated with acetonitrile and then evaporated. The drugs were separated on a Inertsil ODS-3V column (4.6 mm i.d. x 250 mm) with a gradient system of 0.1% phosphoric acid-acetonitrile as the mobile phase, with monitoring at 280 and 340 nm. The recoveries of the 26 kinds of drugs were over 60% from the meats fortified at 0.1 microg/g, and the quantification limits of most drugs were 0.01 microg/g. This proposed method was found to be effective and suitable for the screening of the above drugs in meats.

  7. Zinc-binding cysteines: diverse functions and structural motifs.

    PubMed

    Pace, Nicholas J; Weerapana, Eranthie

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

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

    PubMed

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

    2014-12-14

    Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2(380) intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of "dangling" water molecules were detected in two different states of the protein using H2 (18)O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm(-1)) and with the other O-H group medium (3440 cm(-1)) to moderately strongly (3300 cm(-1)) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1. PMID:25494778

  10. 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.

  11. Addition of a clay subsoil to a sandy top soil alters CO2 release and the interactions in residue mixtures.

    PubMed

    Shi, Andong; Marschner, Petra

    2013-11-01

    Addition of clay-rich subsoils to sandy top soils is an agricultural management option to increase water and nutrient retention and may also increase organic carbon sequestration by decreasing the decomposition rates. An incubation experiment was carried out in a loamy sand top soil mixed with a clay-rich subsoil (84% clay) at 0, 10 and 30% (w/w) amended with finely ground mature shoot residues of two native perennial grasses and annual barley individually or in 1:1 mixtures of two residues. Extractable C, microbial biomass C, available N and soil pH were analysed at days 0, 3, 14 and 28. Cumulative respiration after 28 days was highest with barley residue and lowest with Wallaby grass at all clay soil addition rates; 30% clay soil addition reduced cumulative respiration, especially with barley alone. In the mixture of native grasses and barley, the measured respiration was lower than expected at a clay soil addition rate of 10%. A synergistic effect (higher than expected cumulative respiration) was only found in mixture of Kangaroo grass and barley at a clay soil addition rate of 30%. Clay soil addition also decreased extractable C, available N and soil pH. The temporal change in microbial biomass C and available N in residue mixtures differed among clay addition rates. In the mixture of Wallaby grass and Kangaroo grass, microbial biomass C (MBC) decreased from day 0 to day 28 at clay soil addition rates of 0 and 10%, whereas at 30% clay MBC increased from day 0 to day 3 and then decreased. Our study shows that addition of a clay-rich subsoil to a loamy sand soil can increase C sequestration by reducing CO2 release and extractable C which are further modulated by the type of residues present individually or as mixtures.

  12. 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.

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

    PubMed

    Mason, Alexander F; Thordarson, Pall

    2016-01-01

    The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers. PMID:27501061

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

    PubMed Central

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

    2015-01-01

    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. PMID:26400108

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

    PubMed Central

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

    1990-01-01

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

  16. Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.

    PubMed Central

    Witte, M M; Dickson, R C

    1988-01-01

    LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought. Images PMID:3146691

  17. One-Step Conjugation Method for Site-Specific Antibody-Drug Conjugates through Reactive Cysteine-Engineered Antibodies.

    PubMed

    Shinmi, Daisuke; Taguchi, Eri; Iwano, Junko; Yamaguchi, Tsuyoshi; Masuda, Kazuhiro; Enokizono, Junichi; Shiraishi, Yasuhisa

    2016-05-18

    Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC. PMID:27074832

  18. 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

    Amara, Amro A; Rehm, Bernd H A

    2003-09-01

    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

  19. 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

    2009-06-01

    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.

  20. 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.

  1. Selenocysteine Positional Variants Reveal Contributions to Copper Binding From Cysteine Residues in Domains 2 And 3 of Human Copper Chaperone for Superoxide Dismutase

    SciTech Connect

    Barry, A.N.; Clark, K.M.; Otoikhian, A.; Donk, W.A.van der; Blackburn, N.J.

    2009-05-11

    The human copper chaperone for superoxide dismutase binds copper both in an Atx1-like MTCQSC motif in domain 1 and via a multinuclear cluster formed by two CXC motifs at the D3 dimer interface. The composition of the Cu(I) cluster has been investigated previously by mutagenesis of the CXC motif, and by construction of a CXU selenocysteine derivative, which has permitted XAS studies at both Cu and Se absorption edges. Here, we report the semisynthesis and spectroscopic characterization of a series of derivatives with the sequences 243-CACA, 243-CAUA, 243-UACA, and 243-UAUA in the D1 double mutant (C22AC25A) background, prepared by expressed protein ligation of Sec-containing tetrapeptides to an hCCS-243 truncation. By varying the position of the Se atom in the CXC motif, we have been able to show that Se is always bridging (2 Se-Cu) rather than terminal (1 Se-Cu). Substitution of both D3 Cys residues by Sec in the UAUA variant does not eliminate the Cu-S contribution, confirming our previous description of the cluster as most likely a Cu{sub 4}S{sub 6} species, and suggesting that D2 Cys residues contribute to the cluster. As predicted by this model, when Cys residues C141, C144, and C227 are mutated to alanine either individually or together as a triple mutant, the cluster nuclearity is dramatically attenuated. These data suggest that Cys residues in D2 of hCCS are involved in the formation, stability, and redox potential of the D3 cluster. The significance of these finding to the SOD1 thiol/disulfide oxidase activity are discussed in terms of a model in which a similar multinuclear cluster may form in the CCS-SOD heterodimer.

  2. Influence of residue and nitrogen fertilizer additions on carbon mineralization in soils with different texture and cropping histories

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 soil sampled from two long-term experiments (clay loam Mollisols in Iowa [IAsoil] and silt loam Ul...

  3. 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

  4. Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay*

    PubMed Central

    Murray, Christopher I.; Uhrigshardt, Helge; O'Meally, Robert N.; Cole, Robert N.; Van Eyk, Jennifer E.

    2012-01-01

    Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT6, is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT6 quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches. PMID:22126794

  5. Effects of Solvent and Residual Water on Enhancing the Reactivity of Six-Membered Silyloxyallyl Cations toward Nucleophilic Addition.

    PubMed

    Malone, Joshua A; Cleveland, Alexander H; Fronczek, Frank R; Kartika, Rendy

    2016-09-01

    A new strategy for the generation of six-membered unsymmetrical silyloxyallyl cations using catalytic mild Brønsted acid is reported. These reactive intermediates were found to readily undergo direct nucleophilic addition with a broad range of nucleophiles to produce various α,α'-disubstituted silyl enol ether structural motifs. The findings also highlight the significance of the solvent effect and residual water in enhancing the reaction rate. PMID:27538538

  6. Structural changes during cysteine desulfurase CsdA and sulfur acceptor CsdE interactions provide insight into the trans-persulfuration.

    PubMed

    Kim, Sunmin; Park, Sangyoun

    2013-09-20

    In Escherichia coli, three cysteine desulfurases (IscS, SufS, and CsdA) initiate the delivery of sulfur for various biological processes such as the biogenesis of Fe-S clusters. The sulfur generated as persulfide on a cysteine residue of cysteine desulfurases is further transferred to Fe-S scaffolds (e.g. IscU) or to intermediate cysteine-containing sulfur acceptors (e.g. TusA, SufE, and CsdE) prior to its utilization. Here, we report the structures of CsdA and the CsdA-CsdE complex, which provide insight into the sulfur transfer mediated by the trans-persulfuration reaction. Analysis of the structures indicates that the conformational flexibility of the active cysteine loop in CsdE is essential for accepting the persulfide from the cysteine of CsdA. Additionally, CsdA and CsdE invoke a different binding mode than those of previously reported cysteine desulfurase (IscS) and sulfur acceptors (TusA and IscU). Moreover, the conservation of interaction-mediating residues between CsdA/SufS and CsdE/SufE further suggests that the SufS-SufE interface likely resembles that of CsdA and CsdE.

  7. Key role of cysteine residues and sulfenic acids in thermal- and H2O2-mediated modification of β-lactoglobulin.

    PubMed

    Krämer, Anna C; Thulstrup, Peter W; Lund, Marianne N; Davies, Michael J

    2016-08-01

    Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of the major whey protein β-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H2O2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H2O2 added before heating depleted thiols, with H2O2 alone, or H2O2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H2O2, and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC-MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H2O2. Studies using circular dichroism and intrinsic fluorescence indicate that H2O2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment.

  8. 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.

  9. Stabilisation/solidification of APC residues from MSW incineration with hydraulic binders and chemical additives.

    PubMed

    Quina, Margarida J; Bordado, João C M; Quinta-Ferreira, Rosa M

    2014-01-15

    This study focuses on the stabilisation/solidification (S/S) treatment of air pollution control (APC) residues from municipal solid waste (MSW) incineration. Six formulations (T1-T6) were tested based on different cements as binders, for the immobilisation of pollutants and to prevent their entering into the environment at unacceptable rates. Soluble phosphates and silicates were considered in some cases to fix heavy metals. The performance of T1-T6 products was measured in terms of initial and final setting times, mechanical strength, total availability and leaching from S/S products. Two monolithic leaching tests were used to estimate emissions of pollutants over 48h and 64 days. The results showed that the setting time was reduced when soluble phosphates were used. Moreover, although all the treatments have met the threshold of 1MPa for unconfined compressive strength, this parameter was significantly reduced due to matrix dissolution during immersion. After three cycles of leaching, the limit of 10% for solubilisation was exceeded for all treatments with the exception of T5 (with phosphates). This study demonstrated that the S/S treatment used at the industrial level can be improved with respect to toxic heavy metals, by using soluble silicates or phosphates, but not regarding soluble salts.

  10. 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.

  11. Cysteines in the Stalk of the Nipah Virus G Glycoprotein Are Located in a Distinct Subdomain Critical for Fusion Activation

    PubMed Central

    Maar, Dianna; Harmon, Brooke; Chu, David; Schulz, Belinda; Aguilar, Hector C.; Lee, Benhur

    2012-01-01

    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. PMID:22496210

  12. [Growth-inhibitory activity of Cladosporium cladosporioides by cysteine].

    PubMed

    Watanabe, Toshihiko; Ueno, Yukihiro; Ogasawara, Ayako; Mikami, Takeshi; Matsumoto, Tatsuji

    2007-07-01

    When Cladosporium cladosporioides was cultured with cysteine, its growth was completely inhibited statically. The growth of C. cladosporioides cultured on potato-dextrose agar plates was also inhibited by the addition of cysteine. The production of ATP in C. cladosporioides was inhibited by cysteine. When a silicone block was incubated with C. cladosporioides, the surface of the block was coated with the biofilm of C. cladosporioides. However, the block containing cysteine was not covered with biofilm. These results indicate that cysteine is useful as a material to prevent the growth of C. cladosporioides.

  13. 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

  14. 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.

  15. Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

    PubMed Central

    Grabarczyk, Daniel B.; Chappell, Paul E.; Eisel, Bianca; Johnson, Steven; Lea, Susan M.; Berks, Ben C.

    2015-01-01

    Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism. PMID:25673696

  16. Mechanism of thiosulfate oxidation in the SoxA family of cysteine-ligated cytochromes.

    PubMed

    Grabarczyk, Daniel B; Chappell, Paul E; Eisel, Bianca; Johnson, Steven; Lea, Susan M; Berks, Ben C

    2015-04-01

    Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.

  17. Assessment of Nano Cellulose from Peach Palm Residue as Potential Food Additive: Part II: Preliminary Studies.

    PubMed

    Andrade, Dayanne Regina Mendes; Mendonça, Márcia Helena; Helm, Cristiane Vieira; Magalhães, Washington L E; de Muniz, Graciela Ines Bonzon; Kestur, Satyanarayana G

    2015-09-01

    High consumption of dietary fibers in the diet is related to the reduction of the risk of non-transmitting of chronic diseases, prevention of the constipation etc. Rich diets in dietary fibers promote beneficial effects for the metabolism. Considering the above and recognizing the multifaceted advantages of nano materials, there have been many attempts in recent times to use the nano materials in the food sector including as food additive. However, whenever new product for human and animal consumption is developed, it has to be tested for their effectiveness regarding improvement in the health of consumers, safety aspects and side effects. However, before it is tried with human beings, normally such materials would be assessed through biological tests on a living organism to understand its effect on health condition of the consumer. Accordingly, based on the authors' finding reported in a previous paper, this paper presents body weight, biochemical (glucose, cholesterol and lipid profile in blood, analysis of feces) and histological tests carried out with biomass based cellulose nano fibrils prepared by the authors for its possible use as food additive. Preliminary results of the study with mice have clearly brought out potential of these fibers for the said purpose. PMID:26344977

  18. Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site.

    PubMed Central

    Takeshita, H; Yasuda, T; Iida, R; Nakajima, T; Mori, S; Mogi, K; Kaneko, Y; Kishi, K

    2001-01-01

    We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40 kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. 'DNase I signature sequences', which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a 'somatomedin B motif' was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family. PMID:11439097

  19. 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.

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

    PubMed

    Hasan, Md Ashraful; Ahn, Won-Gyun; Song, Dong-Keun

    2016-09-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]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 [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]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 [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]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 [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  1. 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

  2. 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.

  3. The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc.

    PubMed

    Gottlieb, Colin D; Zhang, Sheng; Linder, Maurine E

    2015-12-01

    DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.

  4. 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.

  5. 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.

  6. 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. PMID:26791894

  7. 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

  8. 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

  9. 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.

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

    PubMed Central

    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. PMID:26764487

  11. 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.

  12. Cysteine Modification: Probing Channel Structure, Function and Conformational Change.

    PubMed

    Akabas, Myles H

    2015-01-01

    Cysteine substitution has been a powerful tool to investigate the structure and function of proteins. It has been particularly useful for studies of membrane proteins in their native environment, embedded in phospholipid membranes. Among the 20 amino acids, cysteine is uniquely reactive. This reactivity has motivated the synthesis of a wide array of sulfhydryl reactive chemicals. The commercially available array of sulfhydryl reactive reagents has allowed investigators to probe the local steric and electrostatic environment around engineered cysteines and to position fluorescent, paramagnetic and mass probes at specific sites within proteins and for distance measurements between pairs of sites. Probing the reactivity and accessibility of engineered cysteines has been extensively used in Substituted Cysteine Accessibility Method (SCAM) investigations of ion channels, membrane transporters and receptors. These studies have successfully identified the residues lining ion channels, agonist/antagonist and allosteric modulator binding sites, and regions whose conformation changes as proteins transition between different functional states. The thousands of cysteine-substitution mutants reported in the literature demonstrate that, in general, mutation to cysteine is well tolerated. This has allowed systematic studies of residues in transmembrane segments and in other parts of membrane proteins. Finally, by inserting pairs of cysteines and assaying their ability to form disulfide bonds, changes in proximity and mobility relationships between specific positions within a protein can be inferred. Thus, cysteine mutagenesis has provided a wealth of data on the structure of membrane proteins in their functional environment. This data can complement the structural insights obtained from the burgeoning number of crystal structures of detergent solubilized membrane proteins whose functional state is often uncertain. This article will review the use of cysteine mutagenesis to probe

  13. 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.

  14. 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

  15. 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

  16. 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

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

    PubMed

    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.

  18. 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.

  19. 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.

  20. 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. PMID:27521931

  1. Role of residual additives in the cytotoxicity and cytokine release caused by polyvinyl chloride particles in pulmonary cell cultures.

    PubMed

    Xu, Haiyan; Dinsdale, David; Nemery, Benoit; Hoet, Peter H M

    2003-03-01

    Occupational exposure to polyvinyl chloride (PVC) dust has been linked to pulmonary disease. The aim of the present study was to investigate, in vitro, the role of additives in the cytotoxicity and the release of inflammatory mediators caused by PVC particles in different cells. We compared two types of emulsion PVC particles (E3 and E8) with their washed (hence, "additive-free") counterparts (W3 and W8). A positive control (crystalline SiO2, Min-U-Sil) and the pure additives, sodium lauryl sulfate (A3) and sodium alkylbenzenesulfonate (A8), were tested concurrently. Cytotoxicity (MTT assay) was assessed in primary cultures of rat alveolar macrophages, rat type II pneumocytes, and human alveolar macrophages (h-AM), and cultures of the A549 cell line (type II cell-derived) and the differentiated THP-1 cell line (macrophage-like). Hemolytic potential was assessed after a 2-h incubation with human erythrocytes. Cytokine release (IL-8, IL-6, and TNF-alpha) by A549 cells, THP-1 cells, and h-AM, was measured by ELISA after 4, 16, 24 and/or 48 h of exposure. Cytotoxicity and hemolytic activity of the washed particles were abolished or markedly decreased compared with their nonwashed forms. In A549 cells, E3 and E8 (2.5 mg/ml) caused a 3-fold increase in IL-8 release and a more than 10-fold increase in IL-6 release, whereas W3 and W8 did not elicit any significant response at similar concentrations. Compared with Min-U-Sil (0.1, 0.5, and 2.5 mg/ml), the response to E3 and E8 occurred later and was slightly lower (IL-8) or much more pronounced (IL-6). A3 and A8 exhibited similar responses to E3 and E8, at concentrations corresponding to those present in the particles. In conclusion, the in vitro cytotoxicity and inflammatory potential of some PVC particles appear to be mostly due to their residual additives.

  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. 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.

  4. 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. PMID:25591324

  5. Cysteine-Based Redox Switches in Enzymes

    PubMed Central

    Klomsiri, Chananat; Karplus, P. Andrew

    2011-01-01

    Abstract The enzymes involved in metabolism and signaling are regulated by posttranslational modifications that influence their catalytic activity, rates of turnover, and targeting to subcellular locations. Most prominent among these has been phosphorylation/dephosphorylation, but now a distinct class of modification coming to the fore is a set of versatile redox modifications of key cysteine residues. Here we review the chemical, structural, and regulatory aspects of such redox regulation of enzymes and discuss examples of how these regulatory modifications often work in concert with phosphorylation/dephosphorylation events, making redox dependence an integral part of many cell signaling processes. Included are the emerging roles played by peroxiredoxins, a family of cysteine-based peroxidases that now appear to be major players in both antioxidant defense and cell signaling. Antioxid. Redox Signal. 14, 1065–1077. PMID:20799881

  6. N-helix and Cysteines Inter-regulate Human Mitochondrial VDAC-2 Function and Biochemistry*

    PubMed Central

    Maurya, Svetlana Rajkumar; Mahalakshmi, Radhakrishnan

    2015-01-01

    Human voltage-dependent anion channel-2 (hVDAC-2) functions primarily as the crucial anti-apoptotic protein in the outer mitochondrial membrane, and additionally as a gated bidirectional metabolite transporter. The N-terminal helix (NTH), involved in voltage sensing, bears an additional 11-residue extension (NTE) only in hVDAC-2. In this study, we assign a unique role for the NTE as influencing the chaperone-independent refolding kinetics and overall thermodynamic stability of hVDAC-2. Our electrophysiology data shows that the N-helix is crucial for channel activity, whereas NTE sensitizes this isoform to voltage gating. Additionally, hVDAC-2 possesses the highest cysteine content, possibly for regulating reactive oxygen species content. We identify interdependent contributions of the N-helix and cysteines to channel function, and the measured stability in micellar environments with differing physicochemical properties. The evolutionary demand for the NTE in the presence of cysteines clearly emerges from our biochemical and functional studies, providing insight into factors that functionally demarcate hVDAC-2 from the other VDACs. PMID:26487717

  7. 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.

  8. 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. PMID:17373572

  9. 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...

  10. 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. PMID:27509597

  11. 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.

  12. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.

    PubMed

    Stewart-Jones, Guillaume B E; Thomas, Paul V; Chen, Man; Druz, Aliaksandr; Joyce, M Gordon; Kong, Wing-Pui; Sastry, Mallika; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S; McLellan, Jason S; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R; Graham, Barney S; Kwong, Peter D

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. PMID:26098893

  13. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.

    PubMed

    Stewart-Jones, Guillaume B E; Thomas, Paul V; Chen, Man; Druz, Aliaksandr; Joyce, M Gordon; Kong, Wing-Pui; Sastry, Mallika; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S; McLellan, Jason S; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R; Graham, Barney S; Kwong, Peter D

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.

  14. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

    PubMed Central

    Stewart-Jones, Guillaume B. E.; Thomas, Paul V.; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S.; McLellan, Jason S.; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. PMID:26098893

  15. Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology.

    PubMed

    Zhu, Quansheng; Casey, Joseph R

    2007-04-01

    Integral membrane proteins of the plasma membrane span from the inside to the outside of the cell. The primary structural element of integral membrane proteins is their topology: the pattern in which the protein traverses the membrane. A full description of topology, defining which parts of the protein face outside versus inside, goes a long way toward understanding the folding of these proteins. Many approaches have been established to define membrane protein topology. Here, we present the technique of scanning cysteine accessibility mutagenesis (SCAM). This approach uses the unique chemical reactivity of the cysteine sulfhydryl to probe membrane protein structure. Individual cysteine residues are introduced into the target protein by mutagenesis. The ability to chemically react these residues using sulfhydryl-directed reagents (either membrane permeant or impermeant) defines each site as either extracellular or intracellular, thus establishing topology of a location. This analysis performed on many sites in the protein will define the protein's topology. PMID:17367716

  16. 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.

  17. Sequential Copper-Catalyzed Alkyne-Azide Cycloaddition and Thiol-Maleimide Addition for the Synthesis of Photo- and/or Electroactive Fullerodendrimers and Cysteine-Functionalized Fullerene Derivatives.

    PubMed

    Fensterbank, Hélène; Baczko, Krystyna; Constant, Céline; Idttalbe, Najat; Bourdreux, Flavien; Vallée, Anne; Goncalves, Anne-Marie; Méallet-Renault, Rachel; Clavier, Gilles; Wright, Karen; Allard, Emmanuel

    2016-09-16

    In this study, the functionalization of a fullerene building block in a stepwise process by means of the copper-catalyzed alkyne-azide cycloaddition (CuAAC) and thiol-maleimide reactions is reported. Grafting of the fullerene platform with a variety of azido derivatives, including Bodipy, pyrene and ferrocene, was carried out first. These fullerene compounds were then reacted with thiol derivatives to yield sophisticated structures comprising photo- and/or electroactive fullerodendrimers and cysteine-functionalized fullerene assemblies. This strategy, which combines the CuAAC and thiol-maleimide processes, could become more widely adopted in the field of fullerene chemistry. PMID:27518495

  18. Use of generalized additive models and cokriging of spatial residuals to improve land-use regression estimates of nitrogen oxides in Southern California

    PubMed Central

    Li, Lianfa; Wu, Jun; Wilhelm, Michelle; Ritz, Beate

    2012-01-01

    Land-use regression (LUR) models have been developed to estimate spatial distributions of traffic-related pollutants. Several studies have examined spatial autocorrelation among residuals in LUR models, but few utilized spatial residual information in model prediction, or examined the impact of modeling methods, monitoring site selection, or traffic data quality on LUR performance. This study aims to improve spatial models for traffic-related pollutants using generalized additive models (GAM) combined with cokriging of spatial residuals. Specifically, we developed spatial models for nitrogen dioxide (NO2) and nitrogen oxides (NOx) concentrations in Southern California separately for two seasons (summer and winter) based on over 240 sampling locations. Pollutant concentrations were disaggregated into three components: local means, spatial residuals, and normal random residuals. Local means were modeled by GAM. Spatial residuals were cokriged with global residuals at nearby sampling locations that were spatially auto-correlated. We compared this two-stage approach with four commonly-used spatial models: universal kriging, multiple linear LUR and GAM with and without a spatial smoothing term. Leave-one-out cross validation was conducted for model validation and comparison purposes. The results show that our GAM plus cokriging models predicted summer and winter NO2 and NOx concentration surfaces well, with cross validation R2 values ranging from 0.88 to 0.92. While local covariates accounted for partial variance of the measured NO2 and NOx concentrations, spatial autocorrelation accounted for about 20% of the variance. Our spatial GAM model improved R2 considerably compared to the other four approaches. Conclusively, our two-stage model captured summer and winter differences in NO2 and NOx spatial distributions in Southern California well. When sampling location selection cannot be optimized for the intended model and fewer covariates are available as predictors for

  19. 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. PMID:21787927

  20. 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.

  1. Studies on Deprotection of Cysteine and Selenocysteine Side-Chain Protecting Groups†

    PubMed Central

    Harris, Katharine M.; Flemer, Stevenson; Hondal, Robert J.

    2013-01-01

    We present here a simple method for deprotecting p-methoxybenzyl groups and acetamidomethyl groups from the side-chains of cysteine and selenocysteine. This method uses the highly elecrophilic, aromatic disulfides 2,2′-dithiobis(5-nitropyridine) (DTNP) and 2,2′-dithiodipyridine (DTP) dissolved in TFA to effect removal of these heretofore difficult to remove protecting groups. The dissolution of these reagents in TFA in fact serves to “activate” them for the deprotection reaction because protonation of the nitrogen atom of the pyridine ring makes the disulfide bond more electrophilic. Thus these reagents can be added to any standard cleavage cocktail used in peptide synthesis. The p-methoxybenzyl group of selenocysteine is easily removed by DTNP. Only sub-stoichiometric amounts of DTNP are required to cause full removal of the p-methoxybenzyl group, with as little as 0.2 equivalents necessary to effect 70% removal of the protecting group. The ability to remove the p-methoxybenzyl group from cysteine using DTNP required 2 equivalents of DTNP and the addition of thioanisole for complete deprotection. Thioanisole was absolutely required for the reaction in the case of the sulfur-containing amino acids, while it was not required for selenocysteine. The results are consistent with thioanisole acting as a catalyst. The acetamidomethyl group of cysteine can also be removed using DTNP, but required the addition of > 15 equivalents to be effective. DTP was less robust as a deprotection reagent. We also demonstrate that this chemistry can be used in a simultaneous cyclization/deprotection reaction between selenocysteine and cysteine residues protected by p-methoxybenzyl groups to form a selenylsulfide bond, demonstrating future high utility of the deprotection method. PMID:17031870

  2. 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.

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

    PubMed Central

    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. PMID:26642319

  4. Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana.

    PubMed

    Romero, Luis C; Aroca, M Ángeles; Laureano-Marín, Ana M; Moreno, Inmaculada; García, Irene; Gotor, Cecilia

    2014-02-01

    Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor molecule involved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its derivative molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine is synthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed by O-acetylserine(thiol)lyase (OASTL). Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resulting in a complex array of isoforms and subcellular cysteine pools. In recent years, significant progress has been made in Arabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the discovery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCS with S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions. Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signaling molecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essential role in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which is essential for root hair development and plant responses to pathogens.

  5. Complementary roles of specific cysteines in keratin 14 toward the assembly, organization, and dynamics of intermediate filaments in skin keratinocytes.

    PubMed

    Feng, Xia; Coulombe, Pierre A

    2015-09-11

    We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes. In particular, cysteine 367 located in the central α-helical rod domain of keratin 14 is necessary for the formation of a stable perinuclear network of keratin filaments (with type II partner keratin 5) in skin keratinocytes analyzed by static and live cell imaging. Here, we show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, also participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Analysis of K14 variants with single or multiple substitutions of cysteine residues points to a spatial and temporal hierarchy in how Cys-4/Cys-40 and Cys-367 regulate keratin assembly in vitro and filament dynamics in live keratinocytes in culture. Our findings substantiate the importance and complexity of a novel determinant, namely inter-keratin disulfide bonding, for the regulation of several aspects of keratin filaments in surface epithelia.

  6. 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

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

    PubMed

    Dragoni, Ilaria; Guida, Elizabeth; McIntyre, Peter

    2006-12-01

    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.

  8. Orchestrating Redox Signaling Networks Through Regulatory Cysteine Switches

    PubMed Central

    Paulsen, Candice E.; Carroll, Kate S.

    2015-01-01

    Hydrogen peroxide (H2O2) acts as a second messenger that can mediate intracellular signal transduction via chemoselective oxidation of cysteine residues in signaling proteins. This Review presents current mechanistic insights into signal-mediated H2O2 production and highlights recent advances in methods to detect reactive oxygen species (ROS) and cysteine oxidation both in vitro and in cells. Selected examples from the recent literature are used to illustrate the diverse mechanisms by which H2O2 can regulate protein function. The continued development of methods to detect and quantify discrete cysteine oxoforms should further our mechanistic understanding of redox regulation of protein function and may lead to the development of new therapeutic strategies. PMID:19957967

  9. 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.

  10. Orchestrating redox signaling networks through regulatory cysteine switches.

    PubMed

    Paulsen, Candice E; Carroll, Kate S

    2010-01-15

    Hydrogen peroxide (H(2)O(2)) acts as a second messenger that can mediate intracellular signal transduction via chemoselective oxidation of cysteine residues in signaling proteins. This Review presents current mechanistic insights into signal-mediated H(2)O(2) production and highlights recent advances in methods to detect reactive oxygen species (ROS) and cysteine oxidation both in vitro and in cells. Selected examples from the recent literature are used to illustrate the diverse mechanisms by which H(2)O(2) can regulate protein function. The continued development of methods to detect and quantify discrete cysteine oxoforms should further our mechanistic understanding of redox regulation of protein function and may lead to the development of new therapeutic strategies.

  11. 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.

  12. 1-Methylpyridinium-4-(4-phenylmethanethiosulfonate) iodide, MTS-MPP+, a novel scanning cysteine accessibility method (SCAM) reagent for monoamine transporter studies.

    PubMed

    Gallardo-Godoy, Alejandra; Torres-Altoro, Melissa I; White, Kellie J; Barker, Eric L; Nichols, David E

    2007-01-01

    A novel substituted cysteine accessibility method (SCAM) reagent was developed for monoamine uptake transporters. The new reagent, MTS-MPP(+), was a derivative of the neurotoxin and transporter substrate MPP(+). MTS-MPP(+) labeled cysteine residues introduced into the serotonin transporter protein. Although it did not prove to be a substrate, as is MPP(+), it appears to label cysteine residues lining the permeation pore of the transporter more readily than currently available nonspecific SCAM reagents.

  13. 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.

  14. Systematic analysis of the in situ crosstalk of tyrosine modifications reveals no additional natural selection on multiply modified residues

    PubMed Central

    Pan, Zhicheng; Liu, Zexian; Cheng, Han; Wang, Yongbo; Gao, Tianshun; Ullah, Shahid; Ren, Jian; Xue, Yu

    2014-01-01

    Recent studies have indicated that different post-translational modifications (PTMs) synergistically orchestrate specific biological processes by crosstalks. However, the preference of the crosstalk among different PTMs and the evolutionary constraint on the PTM crosstalk need further dissections. In this study, the in situ crosstalk at the same positions among three tyrosine PTMs including sulfation, nitration and phosphorylation were systematically analyzed. The experimentally identified sulfation, nitration and phosphorylation sites were collected and integrated with reliable predictions to perform large-scale analyses of in situ crosstalks. From the results, we observed that the in situ crosstalk between sulfation and nitration is significantly under-represented, whereas both sulfation and nitration prefer to co-occupy with phosphorylation at same tyrosines. Further analyses suggested that sulfation and nitration preferentially co-occur with phosphorylation at specific positions in proteins, and participate in distinct biological processes and functions. More interestingly, the long-term evolutionary analysis indicated that multi-PTM targeting tyrosines didn't show any higher conservation than singly modified ones. Also, the analysis of human genetic variations demonstrated that there is no additional functional constraint on inherited disease, cancer or rare mutations of multiply modified tyrosines. Taken together, our systematic analyses provided a better understanding of the in situ crosstalk among PTMs. PMID:25476580

  15. 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.

  16. 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.

  17. 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. PMID:26967336

  18. Generation and propagation of recombinant mumps viruses exhibiting an additional U residue in the homopolymeric U tract of the F gene-end signal.

    PubMed

    Sauder, Christian J; Ngo, Laurie; Simonyan, Vahan; Cong, Yu; Zhang, Cheryl; Link, Malen; Malik, Tahir; Rubin, Steven A

    2015-08-01

    As a member of the family paramyxoviridae, subfamily paramyxovirinae, the genome of mumps virus (MuV) is postulated to be polyhexameric in length in order to be able to replicate efficiently. While all natural MuV strains sequenced so far obey to this "rule of six," we describe here the isolation of recombinant MuVs that appeared to contain an additional U residue in the homopolymeric tract of the F gene-end signal, resulting in a genome length of 6n + 1. Sequencing of several plaque-purified viruses from these preparations did not reveal the existence of length-correcting mutations, suggesting that they are violators of the rule of six. Employing high-throughput sequencing technology, we provide evidence that the insertion of an additional U residue is mainly the result of the rescue system used that relies on T7 RNA polymerase. Limited in vitro and in vivo testing of the viruses did not reveal any significant impact of the longer genome on virus replication or virulence, suggesting that the rule of six is not a strict requirement for MuV replication.

  19. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  20. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  1. Effects of L-cysteine on Ni-Cu sulfide and marmatite bioleaching by Acidithiobacillus caldus.

    PubMed

    He, Zhiguo; Gao, Fengling; Zhong, Hui; Hu, Yuehua

    2009-02-01

    The effect of L-cysteine in different concentrations on the bioleaching of Ni-Cu sulfide and marmatite were studied with a moderately thermophilic, sulfur-oxidizing bacterium, strain of Acidithiobacillus caldus. X-ray diffraction (XRD) observations showed the change of bioleached solid residues and the effect of L-cysteine on the surface charges of minerals. It was found that adding certain amounts of L-cysteine to the leaching system of Ni-Cu sulfide largely enhanced the leaching rate, while L-cysteine inhibited the bioleaching of marmatite by A. caldus. The mechanism of L-cysteine interaction with mineral surfaces was studied by means of zeta potential determination and IR spectra. PMID:18829304

  2. Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.

    PubMed

    Lee, Chun Pong; Wirtz, Markus; Hell, Rüdiger

    2014-01-01

    Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly supplied [(35)S]cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organello protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ΔpH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.

  3. Cysteine-Free Proteins in the Immunobiology of Arthropod-Borne Diseases

    PubMed Central

    Mejia, J. Santiago; Arthun, Erik N.; Titus, Richard G.

    2010-01-01

    One approach to identify epitopes that could be used in the design of vaccines to control several arthropod-borne diseases simultaneously is to look for common structural features in the secretome of the pathogens that cause them. Using a novel bioinformatics technique, cysteine-abundance and distribution analysis, we found that many different proteins secreted by several arthropod-borne pathogens, including Plasmodium falciparum, Borrelia burgdorferi, and eight species of Proteobacteria, are devoid of cysteine residues. The identification of three cysteine-abundance and distribution patterns in several families of proteins secreted by pathogenic and nonpathogenic Proteobacteria, and not found when the amino acid analyzed was tryptophan, provides evidence of forces restricting the content of cysteine residues in microbial proteins during evolution. We discuss these findings in the context of protein structure and function, antigenicity and immunogenicity, and host-parasite relationships. PMID:20069123

  4. 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.

  5. 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.

  6. Different cysteine proteinases involved in bone resorption and osteoclast formation.

    PubMed

    Brage, M; Abrahamson, M; Lindström, V; Grubb, A; Lerner, U H

    2005-06-01

    Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

  7. 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. PMID:18835094

  8. Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3'-end of an RNA primer.

    PubMed Central

    Zabel, P; Dorssers, L; Wernars, K; Van Kammen, A

    1981-01-01

    An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules. Images PMID:6269049

  9. Amelioration of selenium toxicity by arsenicals and cysteine.

    PubMed

    Lowry, K R; Baker, D H

    1989-04-01

    Young chicks exhibited a 61% reduction in weight gain when a corn-soybean meal diet was supplemented with 15 mg/kg Se provided as Na selenite. The same level of Se provided as selenomethionine depressed weight gain by 32%. Supplementing the high selenite diet with isoarsenous (14 mg/kg As) additions of As2O5, As2O3, phenylarsonic acid, phenylarsine oxide and roxarsone ameliorated the Se-induced growth depression: As2O5 almost totally restored growth rate; As2O3, phenylarsonic acid and phenylarsine oxide gave intermediate responses; and roxarsone gave only a small ameliorative growth response. Arsanilic acid was without effect in stimulating growth rate of selenite-intoxicated chicks. Dietary addition of .4% L-cysteine produced a growth response in selenite intoxicated chicks that was somewhat greater than that obtained with roxarsone; administering both roxarsone and cysteine corrected growth better than either compound given singly. Both roxarsone and As2O5 also effectively ameliorated the Se-toxicity growth depression caused by selenomethionine (15 mg Se/kg) supplementation, but cysteine showed no efficacy against morbidity caused by this form of Se. Liver Se concentration was elevated 10-fold by selenite and 25-fold by selenomethionine supplementation. The arsenic compounds had varying effects on liver Se, whereas cysteine tended to increase Se concentration. These findings suggest that both inorganic and organic arsenicals as well as cysteine ameliorate selenium toxicity by different mechanisms.

  10. A facile and versatile methodology for cysteine specific labeling of proteins with octahedral polypyridyl d6 metal complexes

    PubMed Central

    Dwaraknath, Sudharsan; Tran, Ngoc-Han; Dao, Thanh; Colbert, Alexander; Mullen, Sarah; Nguyen, Angelina; Cortez, Alejandro; Cheruzel, Lionel

    2014-01-01

    We have synthesized and characterized four octahedral polypyridyl d6 metal complexes bearing the 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline ligand (L1) as cysteine specific labeling reagents. The proposed synthetic pathways allow the preparation of the metal complexes containing Re(I), Ru(II), Os(II) and Ir(III) while preserving the epoxide functionality. The complexes were characterized by 1H and 13C NMR, mass spectrometry, UV-visible and luminescence spectroscopies as well as cyclic voltammetry. As proof of concept, a set of non-native single cysteine P450 BM3 heme domain mutants previously developed in our laboratory was used to study the labeling reaction. We demonstrate that the proposed labels can selectively react, often in high yield, with cysteine residues of the protein via the nucleophilic thiol ring opening of the epoxide moiety. In addition, under basic conditions, subsequent loss of a water molecule led to the aromatization of the phenanthroline ring on the protein-bound label compounds, as observed by mass spectrometry and luminescence measurements. PMID:24468675

  11. 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... § 582.5271 Cysteine. (a) Product. Cysteine (L-forms). (b) Conditions of use. This substance is...

  12. 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...

  13. 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

  14. 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.

  15. 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.

  16. Redox regulation of epidermal growth factor receptor signaling through cysteine oxidation.

    PubMed

    Truong, Thu H; Carroll, Kate S

    2012-12-18

    Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a number of human malignancies, making this receptor an important target for the development of anticancer drugs. In addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H(2)O(2) by NADPH-dependent oxidases. In turn, H(2)O(2) functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for the development of new therapeutic strategies for targeting this and other H(2)O(2)-modulated pathways.

  17. Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

    PubMed

    Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

    2003-08-29

    One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

  18. 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.

  19. The antihypertensive effect of cysteine

    PubMed Central

    Vasdev, Sudesh; Singal, Pawan; Gill, Vicki

    2009-01-01

    Hypertension is a leading cause of morbidity and mortality worldwide. Individuals with hypertension are at an increased risk for stroke, heart disease and kidney failure. Essential hypertension results from a combination of genetic and lifestyle factors. One such lifestyle factor is diet, and its role in the control of blood pressure has come under much scrutiny. Just as increased salt and sugar are known to elevate blood pressure, other dietary factors may have antihypertensive effects. Studies including the Optimal Macronutrient Intake to Prevent Heart Disease (OmniHeart) study, Multiple Risk Factor Intervention Trial (MRFIT), International Study of Salt and Blood Pressure (INTERSALT) and Dietary Approaches to Stop Hypertension (DASH) study have demonstrated an inverse relationship between dietary protein and blood pressure. One component of dietary protein that may partially account for its antihypertensive effect is the nonessential amino acid cysteine. Studies in hypertensive humans and animal models of hypertension have shown that N-acetylcysteine, a stable cysteine analogue, lowers blood pressure, which substantiates this idea. Cysteine may exert its antihypertensive effects directly or through its storage form, glutathione, by decreasing oxidative stress, improving insulin resistance and glucose metabolism, lowering advanced glycation end products, and modulating levels of nitric oxide and other vasoactive molecules. Therefore, adopting a balanced diet containing cysteine-rich proteins may be a beneficial lifestyle choice for individuals with hypertension. An example of such a diet is the DASH diet, which is low in salt and saturated fat; includes whole grains, poultry, fish and nuts; and is rich in vegetables, fruits and low-fat dairy products. PMID:22477470

  20. 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.

  1. Discovering mechanisms of signaling-mediated cysteine oxidation.

    PubMed

    Poole, Leslie B; Nelson, Kimberly J

    2008-02-01

    Accumulating evidence reveals hydrogen peroxide as a key player both as a damaging agent and, from emerging evidence over the past decade, as a second messenger in intracellular signaling. This rather mild oxidant acts upon downstream targets within signaling cascades to modulate the activity of a host of enzymes (e.g. phosphatases and kinases) and transcriptional regulators through chemoselective oxidation of cysteine residues. With the recent development of specific detection reagents for hydrogen peroxide and new chemical tools to detect the generation of the initial oxidation product, sulfenic acid, on reactive cysteines within target proteins, the scene is set to gain a better understanding of the mechanisms through which hydrogen peroxide acts as a second messenger in cell signaling.

  2. Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa.

    PubMed Central

    Kekomaki, R; Dawson, B; McFarland, J; Kunicki, T J

    1991-01-01

    The object of this study was to further localize autoantigenic structures on IIb-IIIa and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of IIIa that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to IIIa on intact platelets as well as in an immunoblot assay under nonreduced conditions. Using an immunoblot assay, a carboxy-terminal region of this fragment (33 kD) that contains the cysteine-rich domains of IIIa was found to carry the epitope(s) recognized by the prototype autoantibody RA. The amino-terminal amino acid sequence of the reduced 33-kD fragment, the smallest fragment that retains the RA epitope, is XPSQQDEXSP, and that of the reduced 50-kD fragment is IVQVTFD. This indicates that the 33-kD fragment consists of approximately 175 amino acids beginning at residue 479 and extending at least through residues 636-654, while the 50-kD fragment spans the same region but begins at residue 427. It is apparent that the 33-kD fragment is generated from the 50-kD fragment by additional chymotryptic hydrolysis but remains associated because of the multiple disulfide bonds that are characteristic of this cysteine-rich domain. Sera from 48% of patients with chronic ITP and 2 of 8 patients with acute ITP contain antibodies that bind to the 50-kD fragment in an ELISA. Antibodies of the same specificity are also found in one-third of patients with either secondary immune thrombocytopenia or apparent non-immune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of IIIa is a frequent target of autoantibodies in ITP, but that such antibodies may also be present in cases of thrombocytopenia that cannot be linked to an apparent autoimmune process. Images PMID:1715887

  3. 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).

  4. Crystal structure of the cysteine desulfurase DndA from Streptomyces lividans which is involved in DNA phosphorothioation.

    PubMed

    Chen, Fukun; Zhang, Zhenyi; Lin, Kui; Qian, Tianle; Zhang, Yan; You, Delin; He, Xinyi; Wang, Zhijun; Liang, Jingdan; Deng, Zixin; Wu, Geng

    2012-01-01

    DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA.

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

    PubMed

    1998-01-01

    This report presents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in foods and to recommend maximum levels for such residues in food. The first part of the report considers standards for the performance of studies, residues at the injection site, and several initiatives to promote transparency of the process for setting Maximum Residue Limits (MRLs). A summary follows of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: two anthelminthic agents (moxidectin and tiabendazole), eight antimicrobial agents (ceftiofur, danofloxacin, dihydrostreptomycin, streptomycin, enrofloxacin, flumequine, gentamicin and spiramycin), one glucocorticosteroid (dexamethasone), and two insecticides (cyfluthrin and fluazuron). Annexed to the report are a summary of the Committee's recommendations on these drugs, including Acceptable Daily Intakes and MRL's and further toxicological studies and other information required.

  6. Evaluation of certain veterinary drug residues in food. Sixty-second report of the Joint FAO/WHO Expert Committee on food additives.

    PubMed

    2004-01-01

    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 conclusions on specific toxicological end-points, lipid-soluble residues of veterinary drugs with MRLs in milk, statistical methods for the estimation of MRLs, and the Committee's review and comments on documents provided by Codex Committees. Summaries follow of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: five antibacterial agents (cefuroxime, chloramphenicol, flumequine, lincomycin, pirlimycin), four insecticides (cyhalothrin, cypermethrin and alpha-cypermethrin, doramectin, phoxim), and two production aids (melengestrol acetate, ractopamine). The Committee's comments on chloramphenicol found at low levels in animal products are also summarized. Annexed to the report is a summary of the Committee's recommendations on these drugs, including acceptable daily intakes and proposed maximum residue limits. PMID:15587045

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

    PubMed

    1998-01-01

    This report presents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of residues of certain veterinary drugs in foods and to recommend maximum levels for such residues in food. The first part of the report considers standards for the performance of studies, residues at the injection site, and several initiatives to promote transparency of the process for setting Maximum Residue Limits (MRLs). A summary follows of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: two anthelminthic agents (moxidectin and tiabendazole), eight antimicrobial agents (ceftiofur, danofloxacin, dihydrostreptomycin, streptomycin, enrofloxacin, flumequine, gentamicin and spiramycin), one glucocorticosteroid (dexamethasone), and two insecticides (cyfluthrin and fluazuron). Annexed to the report are a summary of the Committee's recommendations on these drugs, including Acceptable Daily Intakes and MRL's and further toxicological studies and other information required. PMID:9727328

  8. 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.

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

    PubMed

    Tarasava, Katsiaryna; Freisinger, Eva

    2014-12-01

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

  10. 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

  11. 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.

  12. Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria.

    PubMed

    Kundu, S; Sundd, M; Jagannadham, M V

    2000-02-01

    Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases. PMID:10691612

  13. 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. PMID:21195770

  14. Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase].

    PubMed

    Saito, K; Kurosawa, M; Tatsuguchi, K; Takagi, Y; Murakoshi, I

    1994-11-01

    Cysteine synthase [O-acetyl-L-serine(thiol)-lyase, EC 4.2.99.8] (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of L-cysteine from O-acetyl-L-serine (OAS) and hydrogen sulfide. Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K. Saito, N. Miura, M. Yamazaki, H. Horano, I. Murakoshi [1992] Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35S RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 355 promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-biphosphate carboxylase small subunit driven by the CaMV 35S promoter with a sense orientation. These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained. CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2- to 3-fold higher than those of control and pCSK3R plants. CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants. Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant. In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts. The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs. In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine. These results suggested that overaccumulated

  15. Amelioration of bauxite residue sand by intermittent additions of nitrogen fertiliser and leaching fractions: The effect on growth of kikuyu grass and fate of applied nutrients.

    PubMed

    Kaur, Navjot; Phillips, Ian; Fey, Martin V

    2016-04-15

    Bauxite residue, a waste product of aluminium processing operations is characterised by high pH, salinity and exchangeable sodium which hinders sustainable plant growth. The aim of this study was to investigate the uptake form, optimum application rate and timing of nitrogen fertiliser to improve bauxite residue characteristics for plant growth. Kikuyu grass was grown in plastic columns filled with residue sand/carbonated residue mud mixture (20:1) previously amended with gypsum, phosphoric acid and basal nutrients. The experiment was set up as a 4×4 factorial design comprising four levels of applied nitrogen (N) fertiliser (0, 3, 6 and 12mgNkg(-1) residue) and four frequencies of leaching (16, 8 and 4day intervals). We hypothesised that the use of ammonium sulfate fertiliser would increase retention of N within the rhizosphere thereby encouraging more efficient fertiliser use. We found that N uptake by kikuyu grass was enhanced due to leaching of excess salts and alkalinity from the residue profile. It was also concluded that biomass production and associated N uptake by kikuyu grass grown in residue is dependent on the type of fertiliser used.

  16. Amelioration of bauxite residue sand by intermittent additions of nitrogen fertiliser and leaching fractions: The effect on growth of kikuyu grass and fate of applied nutrients.

    PubMed

    Kaur, Navjot; Phillips, Ian; Fey, Martin V

    2016-04-15

    Bauxite residue, a waste product of aluminium processing operations is characterised by high pH, salinity and exchangeable sodium which hinders sustainable plant growth. The aim of this study was to investigate the uptake form, optimum application rate and timing of nitrogen fertiliser to improve bauxite residue characteristics for plant growth. Kikuyu grass was grown in plastic columns filled with residue sand/carbonated residue mud mixture (20:1) previously amended with gypsum, phosphoric acid and basal nutrients. The experiment was set up as a 4×4 factorial design comprising four levels of applied nitrogen (N) fertiliser (0, 3, 6 and 12mgNkg(-1) residue) and four frequencies of leaching (16, 8 and 4day intervals). We hypothesised that the use of ammonium sulfate fertiliser would increase retention of N within the rhizosphere thereby encouraging more efficient fertiliser use. We found that N uptake by kikuyu grass was enhanced due to leaching of excess salts and alkalinity from the residue profile. It was also concluded that biomass production and associated N uptake by kikuyu grass grown in residue is dependent on the type of fertiliser used. PMID:26824271

  17. 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.

  18. Evaluation of certain veterinary drug residues in food. Fiftieth report of the joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    1999-01-01

    This report presents 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. The first part of the report considers the neurotoxicity of anthelminthics belonging to the avermectin and milbemycin classes of compounds and the evaluation policy of the Committee in establishing Maximum Residue Levels (MRLs) for veterinary drugs in food. A summary follows of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: five anthelminthic agents (eprinomectin, febantel, fenbendazole, oxfendazole and moxidectin), seven antimicrobial agents (gentamicin, procaine benzylpenicillin, sarafloxacin, spectinomycin, chlortetracycline, oxytetracycline and tetracycline), three antiprotozoal agents (diclazuril, imidocarb and nicarbazin), one glucocorticosteroid (dexamethasone), one production aid (recombinant bovine somatotropins) and one tranquilizing agent (azaperone). Annexed to the report are a summary of the Committee's recommendations on these drugs, including Acceptable Daily Intakes and MRLs, and further toxicological studies and other information required. PMID:10416362

  19. Evaluation of certain veterinary drug residues in food. Forty-third report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    1995-01-01

    This report presents 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 foods. The first part of the report considers the evaluation of veterinary drugs with a long history of use and the implications of veterinary drug use in aquaculture, and discusses the need for adequate data. A summary follows of the Committee's evaluations of toxicological and residue data on a variety of veterinary drugs: a beta-adrenoceptor-blocking agent (carazolol), seven antimicrobial agents (dihydrostreptomycin and streptomycin, enrofloxacin, gentamicin, neomycin, oxolinic acid and spiramycin) a glucocorticosteroid (dexamethasone), and a tranquilizing agent (azaperone). Annexed to the report are a summary of the Committee's recommendations on these drugs, including Acceptable Daily intakes and Maximum Residue Limits, and a list of further toxicological studies and other information required or desired. PMID:8585227

  20. The Basics of Thiols and Cysteines in Redox Biology and Chemistry

    PubMed Central

    Poole, Leslie B.

    2014-01-01

    Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts functional sites with their specialized properties (e.g., nucleophilicity, high affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low molecular weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes including signal transduction. PMID:25433365

  1. Aspartic acid-promoted highly selective and sensitive colorimetric sensing of cysteine in rat brain.

    PubMed

    Qian, Qin; Deng, Jingjing; Wang, Dalei; Yang, Lifen; Yu, Ping; Mao, Lanqun

    2012-11-01

    Direct selective determination of cysteine in the cerebral system is of great importance because of the crucial roles of cysteine in physiological and pathological processes. In this study, we report a sensitive and selective colorimetric assay for cysteine in the rat brain with gold nanoparticles (Au-NPs) as the signal readout. Initially, Au-NPs synthesized with citrate as the stabilizer are red in color and exhibit absorption at 520 nm. The addition of an aqueous solution (20 μL) of cysteine or aspartic acid alone to a 200 μL Au-NP dispersion causes no aggregation, while the addition of an aqueous solution of cysteine into a Au-NP dispersion containing aspartic acid (1.8 mM) causes the aggregation of Au-NPs and thus results in the color change of the colloid from wine red to blue. These changes are ascribed to the ion pair interaction between aspartic acid and cysteine on the interface between Au-NPs and solution. The concentration of cysteine can be visualized with the naked eye and determined by UV-vis spectroscopy. The signal output shows a linear relationship for cysteine within the concentration range from 0.166 to 1.67 μM with a detection limit of 100 nM. The assay demonstrated here is highly selective and is free from the interference of other natural amino acids and other thiol-containing species as well as the species commonly existing in the brain such as lactate, ascorbic acid, and glucose. The basal dialysate level of cysteine in the microdialysate from the striatum of adult male Sprague-Dawley rats is determined to be around 9.6 ± 2.1 μM. The method demonstrated here is facile but reliable and durable and is envisaged to be applicable to understanding the chemical essence involved in physiological and pathological events associated with cysteine. PMID:23025476

  2. Cysteine metabolism and metal toxicity.

    PubMed

    Quig, D

    1998-08-01

    Chronic, low level exposure to toxic metals is an increasing global problem. The symptoms associated with the slow accumulation of toxic metals are multiple and rather nondescript, and overt expression of toxic effects may not appear until later in life. The sulfhydryl-reactive metals (mercury, cadmium, lead, arsenic) are particularly insidious and can affect a vast array of biochemical and nutritional processes. The primary mechanisms by which the sulfhydryl-reactive metals elicit their toxic effects are summarized. The pro-oxidative effects of the metals are compounded by the fact that the metals also inhibit antioxidative enzymes and deplete intracellular glutathione. The metals also have the potential to disrupt the metabolism and biological activities of many proteins due to their high affinity for free sulfhydryl groups. Cysteine has a pivotal role in inducible, endogenous detoxication mechanisms in the body, and metal exposure taxes cysteine status. The protective effects of glutathione and the metallothioneins are discussed in detail. Basic research pertaining to the transport of toxic metals into the brain is summarized, and a case is made for the use of hydrolyzed whey protein to support metal detoxification and neurological function. Metal exposure also affects essential element status, which can further decrease antioxidation and detoxification processes. Early detection and treatment of metal burden is important for successful detoxification, and optimization of nutritional status is paramount to the prevention and treatment of metal toxicity.

  3. Aminothienopyridazines and Methylene Blue Affect Tau Fibrillization via Cysteine Oxidation*

    PubMed Central

    Crowe, Alex; James, Michael J.; Lee, Virginia M.-Y.; Smith, Amos B.; Trojanowski, John Q.; Ballatore, Carlo; Brunden, Kurt R.

    2013-01-01

    Alzheimer disease and several other neurodegenerative disorders are characterized by the accumulation of intraneuronal fibrils comprised of the protein Tau. Tau is normally a soluble protein that stabilizes microtubules, with splice isoforms that contain either three (3-R) or four (4-R) microtubule binding repeats. The formation of Tau fibrils is thought to result in neuronal damage, and inhibitors of Tau fibrillization may hold promise as therapeutic agents. The process of Tau fibrillization can be replicated in vitro, and a number of small molecules have been identified that inhibit Tau fibril formation. However, little is known about how these molecules affect Tau fibrillization. Here, we examined the mechanism by which the previously described aminothieno pyridazine (ATPZ) series of compounds inhibit Tau fibrillization. Active ATPZs were found to promote the oxidation of the two cysteine residues within 4-R Tau by a redox cycling mechanism, resulting in the formation of a disulfide-containing compact monomer that was refractory to fibrillization. Moreover, the ATPZs facilitated intermolecular disulfide formation between 3-R Tau monomers, leading to dimers that were capable of fibrillization. The ATPZs also caused cysteine oxidation in molecules unrelated to Tau. Interestingly, methylene blue, an inhibitor of Tau fibrillization under evaluation in Alzheimer disease clinical trials, caused a similar oxidation of cysteines in Tau and other molecules. These findings reveal that the ATPZs and methylene blue act by a mechanism that may affect their viability as potential therapeutic agents. PMID:23443659

  4. Characterization of cysteine string protein in rat parotid acinar cells.

    PubMed

    Shimomura, Hiromi; Imai, Akane; Nashida, Tomoko

    2013-10-01

    Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11-112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11-82, containing the J-domain only, nor GST-CSP183-112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.

  5. 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.

  6. Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

    PubMed

    Yuan, Ye; Knaggs, Michael H; Poole, Leslie B; Fetrow, Jacquelyn S; Salsbury, Freddie R

    2010-08-01

    Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (C(p)) supported by a lowered pK(a) in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that C(p) (C52) pK(a) values are highly affected by oligomeric state; an unshifted C(p) pK(a) (approximately 8.3, comparable to the pK(a) of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the C(p)s evolve dynamically asymmetric pK(a)s during the simulations; one subunit's C(p) pK(a) is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same C(p), showing no preference for a lowered pK(a) in either subunit. Two conserved pathways that communicate the asymmetric pK(a)s between C(p)s of different subunits can be identified. In the decamer, all the C(p) pK(a)s are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent C(p) pK(a) shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the C(p) pK(a). In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the C(p) in dimers relative to the monomers. These side chain and backbone interactions

  7. Conformational and oligomeric effects on the cysteine pKa of typaredoxin peroxidase

    PubMed Central

    Yuan, Ye; Knaggs, Michael H.; Poole, Leslie B.; Fetrow, Jacquelyn S.; Salsbury, Freddie R.

    2010-01-01

    Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (Cp) supported by a lowered pKa in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that Cp (C52) pKa values are highly affected by oligomeric state; an unshifted Cp pKa (~ 8.3, comparable to the pKa of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the Cps evolve dynamically asymmetric pKas during the simulations; one subunit’s Cp pKa is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same Cp, showing no preference for a lowered pKa in either subunit. Two conserved pathways that communicate the asymmetric pKas between Cps of different subunits can be identified. In the decamer, all the Cp pKas are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent Cp pKa shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the Cp pKa. In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the Cp in dimers relative to the monomers. These side chain and backbone interactions which contribute to pKa modulation indicate the importance

  8. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development. PMID:22718265

  9. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

  10. 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.

  11. 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

  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. 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).

  15. 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.

  16. 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.

  17. 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

  18. 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. PMID:25810097

  19. Critical aspartic acid residues in pseudouridine synthases.

    PubMed

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  20. Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition.

    PubMed Central

    Wilbourn, B; Nesbeth, D N; Wainwright, L J; Field, M C

    1998-01-01

    Improperly processed secretory proteins are degraded by a hydrolytic system that is associated with the endoplasmic reticulum (ER) and appears to involve re-export of lumenal proteins into the cytoplasm for ultimate degradation by the proteasome. The chimaeric protein hGHDAF28, which contains a crippled glycosylphosphatidylinositol (GPI) C-terminal signal peptide, is degraded by a pathway highly similar to that for other ER-retained proteins and is characterized by formation of disulphide-linked aggregates, failure to reach the Golgi complex and intracellular degradation with a half life of approximately 2 h. Here we show that N-acetyl-leucinal-leucinal-norleucinal, MG-132 and lactacystin, all inhibitors of the proteasome, protect hGHDAF28; hGHDAF28 is still proteolytically cleaved in the presence of lactacystin or MG-132, by the removal of approximately 2 kDa, but the truncated fragment is not processed further. We demonstrate that the ubiquitination system accelerates ER-degradation of hGHDAF28, but is not essential to the process. Overall, these findings indicate that GPI quality control is mediated by the cytoplasmic proteasome. We also show that the presence of a cysteine residue in the GPI signal of hGHDAF28 is required for retention and degradation, as mutation of this residue to serine results in secretion of the fusion protein, implicating thiol-mediated retention as a mechanism for quality control of some GPI signals. Removal of the cysteine also prevents inclusion of hGHDAF28 in disulphide-linked aggregates, indicating that aggregate formation is an additional retention mechanism for this class of protein. Therefore our data suggest that an unpaired terminal cysteine is the retention motif of the hGHDAF28 GPI-processing signal and that additional information may be required for efficient engagement of ER quality control systems by the majority of GPI signals which lack cysteine residues. PMID:9576858

  1. 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

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

    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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Chemical Biology Approaches to Study Protein Cysteine Sulfenylation1

    PubMed Central

    Pan, Jia; Carroll, Kate S.

    2014-01-01

    The oxidation of cysteine thiol side chains by hydrogen peroxide to afford protein sulfenyl modifications is an important mechanism in signal transduction. In addition, aberrant protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments with molecular specificity. In this review, we trace the history of chemical and biological methods that have been developed to detect protein sulfenylation and illustrate how a recent cell-permeable chemical reporter, DYn-2, has been used to detect identify intracellular targets of endogenous H2O2 during growth factor signaling, including the EGF receptor. The array of new tools and methods discussed herein enables the discovery of new biological roles for cysteine sulfenylation in human health and disease. PMID:23576224

  11. 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

    2014-11-01

    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.

  12. 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.

  13. Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from bufo melanostictus.

    PubMed

    Liu, Wa; Ji, Senlin; Zhang, A-Mei; Han, Qinqin; Feng, Yue; Song, Yuzhu

    2013-01-01

    Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

  14. Activity concentrations of 224Ra, 226Ra, 228Ra and 40K radionuclides in refinery products and the additional radiation dose originated from oil residues in Turkey.

    PubMed

    Parmaksiz, A; Agus, Y; Bulgurlu, F; Bulur, E; Yildiz, Ç; Öncü, T

    2013-10-01

    A total of 56 crude oil, refinery product, waste water, sludge and scale samples collected from three refineries were measured by gamma-ray spectrometry. Except for nine samples, all refinery product samples were found to have activity concentrations below the minimum detectable activity (MDA) values. The maximum (224)Ra, (226)Ra, (228)Ra and (40)K activity concentrations in crude oil and refinery product samples were measured as 11.7 ± 4.5, 14.9 ± 3.5, 11.6 ± 4.5, 248.5 ± 18.5 Bq kg(-1), respectively. The maximum (224)Ra, (226)Ra, (228)Ra and (40)K activity concentrations in scale, sludge and water samples were measured as 343.7 ± 11.8, 809.2 ± 29.0, 302.5 ± 21.6, 623.0 ± 80.9 Bq kg(-1), respectively. Radium equivalent activities of the residue samples were calculated up to 1241.8 ± 42.4 Bq kg(-1). The maximum activity concentration index and the alpha index were found to be 4.2 and 4.0, respectively. The annual effective doses of residue samples were calculated below the permitted dose rate for the public, i.e. 1 mSv y(-1).

  15. 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.

  16. Evidence for a role of CETP in HDL remodeling and cholesterol efflux: role of cysteine 13 of CETP.

    PubMed

    Maugeais, Cyrille; Perez, Anne; von der Mark, Elisabeth; Magg, Christine; Pflieger, Philippe; Niesor, Eric J

    2013-11-01

    Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-β-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-β-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.

  17. 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

  18. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  19. Cysteine pK(a) values for the bacterial peroxiredoxin AhpC.

    PubMed

    Nelson, Kimberly J; Parsonage, Derek; Hall, Andrea; Karplus, P Andrew; Poole, Leslie B

    2008-12-01

    Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C(P) (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C(P) within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles. PMID:18986167

  20. Elevation of cysteine consumption in tamoxifen-resistant MCF-7 cells.

    PubMed

    Ryu, Chang Seon; Kwak, Hui Chan; Lee, Ji-Yoon; Oh, Soo Jin; Phuong, Nguyen Thi Thuy; Kang, Keon Wook; Kim, Sang Kyum

    2013-01-15

    Tamoxifen (TAM) resistance is a main cause of therapeutic failure in breast cancers. Although methionine dependency is a phenotypic characteristic of tumor cells, the role of sulfur amino acid metabolism in chemotherapy resistance remains to be elucidated. This study compared metabolite profiles of sulfur amino acid metabolism from methionine to taurine or glutathione (GSH) between normal MCF-7 and TAM-resistant MCF-7 (TAMR-MCF-7) cells. TAMR-MCF-7 cells showed elevated levels and activities of enzymes involved in both transsulfuration from methionine to cysteine and metabolism of cysteine to GSH and taurine. Cysteine concentrations in TAMR-MCF-7 cells and medium conditioned by cell culture for 42h were markedly decreased, while GSH, hypotaurine, and taurine concentrations in the medium were increased. These results show that TAMR-MCF-7 cells display enhanced cysteine utilization. The addition of propargylglycine, a specific cystathionine γ-lyase inhibitor, and buthionine sulfoximine, a specific γ-glutamylcysteine ligase inhibitor, to TAMR-MCF-7 cells, but not to MCF-7 cells, resulted in cytotoxicity after sulfur amino acid deprivation. These results suggest that cell viability of TAMR-MCF-7 cells is affected by inhibition of sulfur amino acid metabolism, particularly cysteine synthesis from homocysteine and GSH synthesis from cysteine. Additionally, the S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in TAMR-MCF-7 cells increased to ~3.6-fold relative to that in MCF-7 cells, a finding that may result from upregulation of methionine adenosyltransferase IIa and S-adenosylhomocysteine hydrolase. In conclusion, this study suggests that TAMR-MCF-7 cells display enhanced cysteine utilization for synthesis of GSH and taurine, and are sensitive to inhibition of cysteine metabolism.

  1. 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

  2. New cysteine-S-conjugate precursors of volatile sulfur compounds in bell peppers (Capsicum annuum L. cultivar).

    PubMed

    Starkenmann, Christian; Niclass, Yvan

    2011-04-13

    The objective of this study was to verify whether the volatile organic sulfur compounds recently discovered in bell pepper (Capsicum annuum, L. cultivars), such as the mercapto-ketones: 4-sulfanyl-2-heptanone and 2-sulfanyl-4-heptanone, the mercapto-alcohols: 4-sulfanyl-2-heptanol and 2-sulfanyl-4-heptanol, and heptane-2,4-dithiol, originate from their corresponding cysteine-S-conjugates. Analysis of aqueous extracts of red and green bell pepper by ultraperformance liquid chromatography-mass spectrometry with electrospray ionization in the positive mode (UPLC-MS ESI(+)) displayed masses corresponding to the expected cysteine-S-conjugates. To confirm this observation, four cysteine-S-conjugates were prepared as authentic samples: S-(3-hydroxy-1-methylhexyl)-L-cysteine, S-(3-hydroxy-1-propylbutyl)-L-cysteine, S-(3-oxo-1-propylbutyl)-L-cysteine, and (2R,2'R)-3,3'-(4-hydroxyheptane-2,6-diyl)bis(sulfanediyl) bis(2-aminopropanoic acid). By comparison with the fragmentation patterns and retention times of synthetic mixtures of cysteine-S-conjugate diastereoisomers, the natural occurrence of cysteine conjugates was confirmed in bell peppers. In addition, the cysteine-S-conjugates from red and green bell pepper extracts were concentrated by ion exchange chromatography and the fractions incubated with a β-lyase (apotryptophanase). The liberated thiols were concentrated by affinity chromatography, and their occurrence, detected by gas chromatography-mass spectrometry, confirmed our predictions. Moreover, 3-sulfanyl-1-hexanol was also detected and the occurrence of S-(1(2-hydroxyethyl)butyl)-L-cysteine confirmed. A quantitative estimation based on external calibration curves, established by UPLC-MS ESI(+) in selected reaction monitoring mode, showed that cysteine-S-conjugates were present at concentrations in the range of 1 to 100 μg/kg (±20%).

  3. Site-directed mutagenesis of HgcA and HgcB reveals amino acid residues important for mercury methylation.

    PubMed

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

    2015-05-01

    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.

  4. 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

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

    DOE PAGES

    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 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

  6. 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.

  7. 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. PMID:27145786

  8. 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.

  9. Application of SCAM (substituted cysteine accessibility method) to gap junction intercellular channels.

    PubMed

    Skerrett, M; Kasperek, E; Cao, F L; Shin, J H; Aronowitz, J; Ahmed, S; Nicholson, B J

    2001-01-01

    The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.

  10. ROSICS: CHEMISTRY AND PROTEOMICS OF CYSTEINE MODIFICATIONS IN REDOX BIOLOGY

    PubMed Central

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

    2015-01-01

    Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1–2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, “ROSics,” for the science which describes the principles of mode of action of ROS at molecular levels. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:184–208, 2015. PMID:24916017

  11. 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.

  12. 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.

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

    SciTech Connect

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

    2006-01-01

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

  14. Cysteine string protein (CSP) and its role in preventing neurodegeneration.

    PubMed

    Burgoyne, Robert D; Morgan, Alan

    2015-04-01

    Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of co-chaperones that localises to neuronal synaptic vesicles. Its name derives from the possession of a string of 12-15 cysteine residues, palmitoylation of which is required for targeting to post-Golgi membranes. The DnaJ domain of CSP enables it to bind client proteins and recruit Hsc70 chaperones, thereby contributing to the maintenance of protein folding in the presynaptic compartment. Mutation of CSP in flies, worms and mice reduces lifespan and causes synaptic dysfunction and neurodegeneration. Furthermore, recent studies have revealed that the neurodegenerative disease, adult onset neuronal ceroid lipofuscinosis, is caused by mutations in the human CSPα-encoding DNAJC5 gene. Accumulating evidence suggests that the major mechanism by which CSP prevents neurodegeneration is by maintaining the conformation of SNAP-25, thereby facilitating its entry into the membrane-fusing SNARE complex. In this review, we focus on the role of CSP in preventing neurodegeneration and discuss how recent studies of this universal neuroprotective chaperone are being translated into potential novel therapeutics for neurodegenerative diseases.

  15. 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

  16. A novel sulfate-reducing bacteria detection method based on inhibition of cysteine protease activity.

    PubMed

    Qi, Peng; Zhang, Dun; Wan, Yi

    2014-11-01

    Sulfate-reducing bacteria (SRB) have been extensively studied in corrosion and environmental science. However, fast enumeration of SRB population is still a difficult task. This work presents a novel specific SRB detection method based on inhibition of cysteine protease activity. The hydrolytic activity of cysteine protease was inhibited by taking advantage of sulfide, the characteristic metabolic product of SRB, to attack active cysteine thiol group in cysteine protease catalytic sites. The active thiol S-sulfhydration process could be used for SRB detection, since the amount of sulfide accumulated in culture medium was highly related with initial bacterial concentration. The working conditions of cysteine protease have been optimized to obtain better detection capability, and the SRB detection performances have been evaluated in this work. The proposed SRB detection method based on inhibition of cysteine protease activity avoided the use of biological recognition elements. In addition, compared with the widely used most probable number (MPN) method which would take up to at least 15days to accomplish whole detection process, the method based on inhibition of papain activity could detect SRB in 2 days, with a detection limit of 5.21×10(2) cfu mL(-1). The detection time for SRB population quantitative analysis was greatly shortened.

  17. Residues in a jellyfish shaker-like channel involved in modulation by external potassium.

    PubMed

    Grigoriev, N G; Spafford, J D; Spencer, A N

    1999-10-01

    The jellyfish gene, jShak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K(+)](out) was increased. The relative modulatory effectiveness of K(+), Rb(+), Cs(+), and Na(+) indicated that a weak-field-strength site is present. Cysteine substituted mutants (L369C and F370C) of an N-terminal truncated construct, (jShak2Delta2-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In comparison with jShak2Delta2-38 and F370C, L369C showed a greater relative increase in peak current when [K(+)](out) was increased from 1 to 100 mM because the affinity of this site was reduced at low [K(+)](out). Increasing [K(+)](out) had little effect on the rate of inactivation of L369C; however, the appearance of a second, hyperbolic component to the inactivation curve for F370C indicated that this mutation had increased the affinity of the low-affinity site by bringing the backbone oxygens closer together. Methanethiosulphonate reagents were used to form positively (MTSET), negatively (MTSES), and neutrally (MTSM) charged side groups on the cysteine-substituted residues at the purported K(+) binding site(s) in the channel mouth and conductance and inactivation kinetic measurements made. The reduced affinity of the site produced by the mutation L369C was probably due to the increased hydrophobicity of cysteine, which changed the relative positions of carbonyl oxygens since MTSES modification did not form a high-field-strength site as might be expected if the cysteine residues project into the pore. Addition of the side chain -CH(2)-S-S-CH(3), which is similar to the side chain of methionine, a conserved residue in many potassium channels, resulted in an increased peak current and reduced inactivation rate, hence a higher affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state confirming that side chains probably rotate

  18. 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.

  19. 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. PMID:27294681

  20. 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.

  1. Chemical Synthesis of Proteins with Non-Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization.

    PubMed

    Reddy, Post Sai; Dery, Shahar; Metanis, Norman

    2016-01-18

    Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site. PMID:26636774

  2. Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill).

    PubMed

    Kimura, M; Ikeda, T; Fukumoto, D; Yamasaki, N; Yonekura, M

    1995-12-01

    The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.

  3. Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine.

    PubMed

    Improta-Brears, T; Ghosh, S; Bell, R M

    1999-08-01

    Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.

  4. Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

    PubMed

    Bade, Veronika N; Nickels, Jochen; Keusekotten, Kirstin; Praefcke, Gerrit J K

    2012-01-01

    The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent

  5. 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.

  6. 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.

  7. 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.

  8. Application of dispersive solid-phase extraction and ultra-fast liquid chromatography-tandem quadrupole mass spectrometry in food additive residue analysis of red wine.

    PubMed

    Chen, Xiao-Hong; Zhao, Yong-Gang; Shen, Hao-Yu; Jin, Mi-Cong

    2012-11-01

    A novel and effective dispersive solid-phase extraction (dSPE) procedure with rapid magnetic separation using ethylenediamine-functionalized magnetic polymer as an adsorbent was developed. The new procedure had excellent clean-up ability for the selective removal of the matrix in red wine. An accurate, simple, and rapid analytical method using ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC-MS/MS) for the simultaneous determination of nine food additives (i.e., acesulfame, saccharin, sodium cyclamate, aspartame, benzoic acid, sorbic acid, stevioside, dehydroacetic acid, and neotame) in red wine was also used and validated. Recoveries ranging from 78.5% to 99.2% with relative standard deviations ranging from 0.46% to 6.3% were obtained using the new method. All target compounds showed good linearities in the tested range with correlation coefficients (r) higher than 0.9993. The limits of quantification for the nine food additives were between 0.10 μg/L and 50.0 μg/L. The proposed dSPE-UFLC-MS/MS method was successfully applied in the food-safety risk monitoring of real red wine in Zhejiang Province, China.

  9. 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.

  10. 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

  11. Phenothiazine-based CaaX competitive inhibitors of human farnesyltransferase bearing a cysteine, methionine, serine or valine moiety as a new family of antitumoral compounds.

    PubMed

    Dumitriu, Gina-Mirabela; Bîcu, Elena; Belei, Dalila; Rigo, Benoît; Dubois, Joëlle; Farce, Amaury; Ghinet, Alina

    2015-10-15

    A new family of CaaX competitive inhibitors of human farnesyltransferase based on phenothiazine and carbazole skeleton bearing a l-cysteine, l-methionine, l-serine or l-valine moiety was designed, synthesized and biologically evaluated. Phenothiazine derivatives proved to be more active than carbazole-based compounds. Phenothiazine 1b with cysteine residue was the most promising inhibitor of human farnesyltransferase in the current study.

  12. 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

  13. 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.

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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. PMID:26494699

  19. Structure and mechanism of mouse cysteine dioxygenase

    PubMed Central

    McCoy, Jason G.; Bailey, Lucas J.; Bitto, Eduard; Bingman, Craig A.; Aceti, David J.; Fox, Brian G.; Phillips, George N.

    2006-01-01

    Cysteine dioxygenase (CDO) catalyzes the oxidation of l-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical β-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme. PMID:16492780

  20. Synthesis of magnetic molecularly imprinted polymers by reversible addition fragmentation chain transfer strategy and its application in the Sudan dyes residue analysis.

    PubMed

    Xie, Xiaoyu; Chen, Liang; Pan, Xiaoyan; Wang, Sicen

    2015-07-31

    Magnetic molecularly imprinted polymers (MMIPs) have become a hotspot owing to the dual functions of target recognition and magnetic separation. In this study, the MMIPs were obtained by the surface-initiated reversible addition fragmentation chain transfer (RAFT) polymerization using Sudan I as the template. The resultant MMIPs were characterized by transmission electron microscope, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, vibrating sample magnetometer, and X-ray diffraction. Benefiting from the controlled/living property of the RAFT strategy, the uniform MIP layer was successfully grafted on the surface of RAFT agent-modified Fe3O4@SiO2 nanoparticles, favoring the fast mass transfer and rapid binding kinetics. The developed MMIPs were used as the solid-phase extraction sorbents to selectively extract four Sudan dyes (Sudan I, II, III, and IV) from chili powder samples. The recoveries of the spiked samples in chili powder samples ranged from 74.1 to 93.3% with RSD lower than 6.4% and the relative standard uncertainty lower than 0.029. This work provided a good platform for the extraction and removal of Sudan dyes in complicated matrixes and demonstrated a bright future for the application of the well-constructed MMIPs in the field of solid-phase extraction. PMID:26077971

  1. 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. PMID:19660785

  2. Synthesis of magnetic molecularly imprinted polymers by reversible addition fragmentation chain transfer strategy and its application in the Sudan dyes residue analysis.

    PubMed

    Xie, Xiaoyu; Chen, Liang; Pan, Xiaoyan; Wang, Sicen

    2015-07-31

    Magnetic molecularly imprinted polymers (MMIPs) have become a hotspot owing to the dual functions of target recognition and magnetic separation. In this study, the MMIPs were obtained by the surface-initiated reversible addition fragmentation chain transfer (RAFT) polymerization using Sudan I as the template. The resultant MMIPs were characterized by transmission electron microscope, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, vibrating sample magnetometer, and X-ray diffraction. Benefiting from the controlled/living property of the RAFT strategy, the uniform MIP layer was successfully grafted on the surface of RAFT agent-modified Fe3O4@SiO2 nanoparticles, favoring the fast mass transfer and rapid binding kinetics. The developed MMIPs were used as the solid-phase extraction sorbents to selectively extract four Sudan dyes (Sudan I, II, III, and IV) from chili powder samples. The recoveries of the spiked samples in chili powder samples ranged from 74.1 to 93.3% with RSD lower than 6.4% and the relative standard uncertainty lower than 0.029. This work provided a good platform for the extraction and removal of Sudan dyes in complicated matrixes and demonstrated a bright future for the application of the well-constructed MMIPs in the field of solid-phase extraction.

  3. Cysteine Substitution of Transmembrane Domain Amino Acids Alters the Ethanol Inhibition of GluN1/GluN2A N-Methyl-d-Aspartate Receptors

    PubMed Central

    Xu, Minfu; Smothers, C. Thetford

    2015-01-01

    N-Methyl-d-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors. PMID:25635140

  4. 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... ingredient is used to supply up to 0.009 part of total L-cysteine per 100 parts of flour in dough as a...

  5. 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 §...

  6. Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues.

    PubMed

    Osz, Katalin; Várnagy, Katalin; Süli-Vargha, Helga; Csámpay, Antal; Sanna, Daniele; Micera, Giovanni; Sóvágó, Imre

    2004-01-01

    Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10. PMID:14659629

  7. 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.

  8. 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...

  9. 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...

  10. 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...

  11. 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.

  12. Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.

    PubMed

    Windisch, Dirk; Hoffmann, Silke; Afonin, Sergii; Vollmer, Stefanie; Benamira, Soraya; Langer, Birgid; Bürck, Jochen; Muhle-Goll, Claudia; Ulrich, Anne S

    2010-09-22

    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.

  13. 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.

  14. Posttranslational Modification of Cysteine in Redox Signaling and Oxidative Stress: Focus on S-Glutathionylation

    PubMed Central

    Chock, P. Boon

    2012-01-01

    Abstract 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. Antioxid. Redox Signal. 16, 471–475. PMID:22136616

  15. N-Acetyl cysteine restores viability and function of rat odontoblast-like cells impaired by polymethylmethacrylate dental resin extract.

    PubMed

    Yamada, Masahiro; Kojima, Norinaga; Att, Wael; Hori, Norio; Suzuki, Takeo; Ogawa, Takahiro

    2009-01-01

    There is concern that dental-resin materials directly loaded on a prepared tooth adversely affect dental pulp tissue by releasing the resin chemicals through dentinal tubes. This study determined whether self-curing polymethyl methacrylate (PMMA)-based dental resin extract adversely affected the viability and function of odontoblast-like cells and whether the cytotoxicity of this resin, if any, could be eliminated by N-acetyl cysteine, an antioxidant amino acid derivative. Odontoblast-like cells isolated from rat maxillary incisor dental pulp tissue were exposed to a PMMA resin extract with or without N-acetyl cysteine for 1 h and then cultured in osteoblastic media. The percentage of viable cells 24 h after seeding was 20% in cells exposed to the resin extract without N-acetyl cysteine, whereas 45% of cells were viable after exposure to the N-acetyl cysteine-supplemented extract. The cells that had been exposed to the extract showed a strong tendency for apoptosis associated with the increased reactive oxygen species production and decreased intracellular glutathione level, which was improved by the addition of N-acetyl cysteine. N-Acetyl cysteine supplementation almost completely restored the significantly reduced alkaline phosphatase activity and matrix mineralization by the resin extract. These results conclusively demonstrated that exposure of odontoblast-like cells to the resin extract impaired the cell viability and function and, more intriguingly, N-acetyl cysteine supplementation to the extract significantly prevented these toxic effects.

  16. Activation Mechanism of the Bacteroides fragilis Cysteine Peptidase, Fragipain.

    PubMed

    Herrou, Julien; Choi, Vivian M; Bubeck Wardenburg, Juliane; Crosson, Sean

    2016-07-26

    Enterotoxigenic Bacteroides fragilis produces a secreted metalloprotease known as B. fragilis toxin (BFT), which contributes to anaerobic sepsis, colitis, and colonic malignancy in mouse models of disease. A C11 family cysteine protease, fragipain (Fpn), directly activates BFT in the B. fragilis cell by removing the BFT prodomain. Fpn is itself a proenzyme and is autoactivated upon cleavage at an arginine residue in its activation loop. We have defined the proteolytic active site of Fpn, demonstrated that Fpn autoactivation can occur by an in trans loop cleavage mechanism, and characterized structural features of the Fpn activation loop that control peptidase activity against several substrates, including BFT. An arginine residue at the autocleavage site determines the fast activation kinetics of Fpn relative to the homologous C11 protease, PmC11, which is cleaved at lysine. Arginine to alanine substitution at the cleavage site ablated peptidase activity, as did partial truncation of the Fpn activation loop. However, complete truncation of the activation loop yielded an uncleaved, pro form of Fpn that was active as a peptidase against both Fpn and BFT substrates. Thus, Fpn can be transformed into an active peptidase in the absence of activation loop cleavage. This study provides insight into the mechanism of fragipain activation and, more generally, defines the role of the C11 activation loop in the control of peptidase activity and substrate specificity.

  17. 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.

  18. 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.

  19. Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

    PubMed Central

    Ernst, Dustin C.; Lambrecht, Jennifer A.; Schomer, Rebecca A.

    2014-01-01

    RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica. The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes. PMID:25002544

  20. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

    PubMed

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir

    2015-02-01

    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions. PMID:25462979

  1. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

    PubMed

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir

    2015-02-01

    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

  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. 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.

  4. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis

    PubMed Central

    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. PMID:25348828

  5. 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. PMID:25348828

  6. 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.

  7. Efficient Palladium-Assisted One-Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis.

    PubMed

    Maity, Suman Kumar; Jbara, Muhammad; Laps, Shay; Brik, Ashraf

    2016-07-01

    The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd(II) complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one-pot fashion by native chemical ligation where the Acm moiety was used to protect the N-terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin-like protein UBL-5, which contains two native Cys residues, was accomplished through the one-pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.

  8. Copper(I) stabilization by cysteine/tryptophan motif in the extracellular domain of Ctr4.

    PubMed

    Okada, Mariko; Miura, Takashi

    2016-06-01

    Copper transporter Ctr4 of fission yeast has a quasi-palindromic sequence rich in cysteine and aromatic amino acid residues, CX4YWNWYX4C (where X represents any amino acid), in the N-terminal extracellular domain. A 24-mer peptide comprising this sequence is bound to Cu(I) through the cysteine thiolate coordination. Luminescence, UV absorption and resonance Raman spectra of the Cu(I)-peptide complex show that at least one of the two tryptophan side chains is located in close proximity to the thiolate-Cu(I) center and interacts with the Cu(I) ion via π-electrons of the indole ring. Although the thiolates and Cu(I) are oxidized to disulfide and Cu(II), respectively, only very slowly in air-saturated solutions, replacements of the tryptophan residues to phenylalanine significantly accelerate the oxidation reactions. The results obtained indicate that the interaction between Cu(I) and tryptophan via π-electrons plays a significant role in protecting the thiolate-Cu(I) center against the oxidation. The cysteine- and tryptophan-rich quasi-palindromic sequence may be a metal binding motif that stabilizes Cu(I) in the oxidizing extracellular environment. PMID:26908286

  9. Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate.

    PubMed

    Jurkowska, Halina; Roman, Heather B; Hirschberger, Lawrence L; Sasakura, Kiyoshi; Nagano, Tetsuo; Hanaoka, Kenjiro; Krijt, Jakub; Stipanuk, Martha H

    2014-05-01

    The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.

  10. 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.

  11. Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha

    SciTech Connect

    Whiteheart, S.W.; Shenbagamurthi, P.; Chen, L.; Cotter, R.J.; Hart, G.W. )

    1989-08-25

    Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with ({sup 3}H) ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major ({sup 3}H)ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release ({sup 3}H)ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues.

  12. 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

  13. The Cysteine-Rich Interdomain Region from the Highly Variable Plasmodium falciparum Erythrocyte Membrane Protein-1 Exhibits a Conserved Structure

    PubMed Central

    Su, Hua-Poo; Makobongo, Morris O.; Moore, Jaime M.; Singh, Sanjay; Miller, Louis H.; Garboczi, David N.

    2008-01-01

    Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1α domains bind CD36. Here we describe the CD36-binding portion of a CIDR1α domain, MC179, as a bundle of three α-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1α three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1α and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1α domains are approximately 100 residues larger and that CIDR1α domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the

  14. 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. PMID:26071808

  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. Synthesis and characterization of a calix[4]arene amphiphilie bearing cysteine and uniform Au nanoparticle formation templated by its four cysteine moieties.

    PubMed

    Fujii, Shota; Sakurai, Kazuo; Okobira, Tadashi; Ohta, Noboru; Takahara, Atsushi

    2013-11-12

    A novel calix[4]arene amphiphilic molecule, denoted by CCaL3, was synthesized and found to form a spherical micelle consisting of 12 molecules at low pH in aqueous solution. Furthermore, uniform Au nanoparticles with 2.0 nm in diameter were synthesized in aqueous solution on the template consisting of the four cysteines of the upper rim of CCaL3. Asymmetric field flow fractionation coupled with light scattering showed that there was no dispersity in the CCaL3 micellar aggregation number. When AuCl4(-) ions were added into the CCaL3 micelle solution, induced circular dichroism (ICD) appeared, indicating appearance of the structural chirality of the CCaL3/AuCl4(-) complex. A combination of electron microscopy and small-angle X-ray scattering showed that helically coiled bilayer sheets were formed upon addition of AuCl4(-). Subsequent reduction with the amine of cysteine moieties led to uniform Au nanoparticles formation with 2.0 nm in diameter on the micellar plate surface. The nanoparticle size was almost equal to the size of cavity constructed by the four cysteines on the calix[4]arene upper rim, indicating that the growth of Au nanoparticles was spatially controlled by the host-guest interaction between the cysteines and Au.

  17. Synthesis and characterization of a calix[4]arene amphiphilie bearing cysteine and uniform Au nanoparticle formation templated by its four cysteine moieties.

    PubMed

    Fujii, Shota; Sakurai, Kazuo; Okobira, Tadashi; Ohta, Noboru; Takahara, Atsushi

    2013-11-12

    A novel calix[4]arene amphiphilic molecule, denoted by CCaL3, was synthesized and found to form a spherical micelle consisting of 12 molecules at low pH in aqueous solution. Furthermore, uniform Au nanoparticles with 2.0 nm in diameter were synthesized in aqueous solution on the template consisting of the four cysteines of the upper rim of CCaL3. Asymmetric field flow fractionation coupled with light scattering showed that there was no dispersity in the CCaL3 micellar aggregation number. When AuCl4(-) ions were added into the CCaL3 micelle solution, induced circular dichroism (ICD) appeared, indicating appearance of the structural chirality of the CCaL3/AuCl4(-) complex. A combination of electron microscopy and small-angle X-ray scattering showed that helically coiled bilayer sheets were formed upon addition of AuCl4(-). Subsequent reduction with the amine of cysteine moieties led to uniform Au nanoparticles formation with 2.0 nm in diameter on the micellar plate surface. The nanoparticle size was almost equal to the size of cavity constructed by the four cysteines on the calix[4]arene upper rim, indicating that the growth of Au nanoparticles was spatially controlled by the host-guest interaction between the cysteines and Au. PMID:24111537

  18. 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. PMID:27172378

  19. 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.

  20. Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.

    PubMed

    Cerny, Christoph; Guntz-Dubini, Renée

    2013-11-15

    Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by β-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by β -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively.

  1. Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.

    PubMed

    Cerny, Christoph; Guntz-Dubini, Renée

    2013-11-15

    Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by β-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by β -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively. PMID:23790889

  2. 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.

  3. CPDadh: A new peptidase family homologous to the cysteine protease domain in bacterial MARTX toxins

    PubMed Central

    Pei, Jimin; Lupardus, Patrick J; Garcia, K Christopher; Grishin, Nick V

    2009-01-01

    A cysteine protease domain (CPD) has been recently discovered in a group of multifunctional, autoprocessing RTX toxins (MARTX) and Clostridium difficile toxins A and B. These CPDs (referred to as CPDmartx) autocleave the toxins to release domains with toxic effects inside host cells. We report identification and computational analysis of CPDadh, a new cysteine peptidase family homologous to CPDmartx. CPDadh and CPDmartx share a Rossmann-like structural core and conserved catalytic residues. In bacteria, domains of the CPDadh family are present at the N-termini of a diverse group of putative cell-cell interaction proteins and at the C-termini of some RHS (recombination hot spot) proteins. In eukaryotes, catalytically inactive members of the CPDadh family are found in cell surface protein NELF (nasal embryonic LHRH factor) and some putative signaling proteins. PMID:19309740

  4. 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.

  5. Determining OMP topology by computation, surface plasmon resonance and cysteine labelling: The test case of OMPG

    SciTech Connect

    Visudtiphole, Virak; Chalton, David A.; Hong Qi; Lakey, Jeremy H. . E-mail: j.h.lakey@ncl.ac.uk

    2006-12-08

    Bacterial outer-membrane proteins (OMP) are important in pathogenicity and the recently solved structure of OmpG provides an excellent test case for topological predictions since it is monomeric. Here we compare the results of applying several computerised structure prediction algorithms to the sequence of OmpG. Furthermore, we probe the OmpG topology by both an established chemical labelling approach and a new method which combines epitope insertion and surface plasmon resonance. The computational approaches are broadly accurate but the exact choice of the number of {beta} strands remains difficult. The algorithms also tend to predict the entire {beta} strand rather than just the transmembrane region. Epitope insertion clearly pinpoints exposed loops but its utility in defining buried or periplasmic sites is less clear cut. Cysteine-mutant labelling is largely confined to exposed residues but one periplasmic cysteine may be labelled by reagents entering via the OmpG pore.

  6. 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

  7. Significance of redox-active cysteines in human FAD synthase isoform 2.

    PubMed

    Miccolis, Angelica; Galluccio, Michele; Nitride, Chiara; Giancaspero, Teresa Anna; Ferranti, Pasquale; Iametti, Stefania; Indiveri, Cesare; Bonomi, Francesco; Barile, Maria

    2014-12-01

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain. PMID:25135855

  8. Molecular cloning and characterization of Vigna mungo processing enzyme 1 (VmPE-1), an asparaginyl endopeptidase possibly involved in post-translational processing of a vacuolar cysteine endopeptidase (SH-EP).

    PubMed

    Okamoto, T; Minamikawa, T

    1999-01-01

    Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxy side of asparagine residues. Vigna mungo processing enzyme 1, termed VmPE-1, occurs in the cotyledons of germinated seeds of V. mungo, and is possibly involved in the post-translational processing of a vacuolar cysteine endopeptidase, designated SH-EP, which degrades seed storage protein. VmPE-1 also showed a substrate specificity to asparagine residues, and its enzymatic activity was inhibited by NEM but not E-64. In addition, purified VmPE-1 had a potential to process the recombinant SH-EP precursor to its intermediate in vitro. cDNA clones for VmPE-1 and its homologue, named VmPE-1A, were identified and sequenced, and their expressions in the cotyledons of V. mungo seedlings and other organs were investigated. VmPE-1 mRNA and SH-EP mRNA were expressed in germinated seeds at the same stage of germination although the enzymatic activity of VmPE-1 rose prior to that of SH-EP. The level of VmPE-1A mRNA continued increasing as germination proceeded. In roots, stems and leaves of fully grown plants, and in hypocotyls, VmPE-1 and VmPE-1A were little expressed. We discuss possible functions of VmPE-1 and VmPE-1A in the cotyledons of germinated seeds.

  9. 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.

  10. Pectin-cysteine conjugate: synthesis and in-vitro evaluation of its potential for drug delivery.

    PubMed

    Majzoob, Sayeh; Atyabi, Fatemeh; Dorkoosh, Farid; Kafedjiiski, Krum; Loretz, Brigitta; Bernkop-Schnürch, Andreas

    2006-12-01

    This study was aimed at improving certain properties of pectin by introduction of thiol moieties on the polymer. Thiolated pectin was synthesized by covalent attachment of cysteine. Pectin-cysteine conjugate was evaluated for its ability to be degraded by pectinolytic enzyme. The toxicity profile of the thiolated polymer in Caco-2-cells, its permeation enhancing effect and its mucoadhesive and swelling properties were studied. Moreover insulin-loaded hydrogel beads of the new polymer were examined for their stability in simulated gastrointestinal conditions and their drug release profile. The new polymer displayed 892.27 +/- 68.68 micromol thiol groups immobilized per g polymer, and proved to have retained its biodegradability, upon addition of Pectinex Ultra SPL in-vitro, determined by viscosity measurements and titration method. Pectin-cysteine showed no severe toxicity in Caco-2 cells, as tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Moreover, the synthesized polymer exhibited a relative permeation enhancement ratio of 1.61 for sodium fluorescein, compared to unmodified pectin. Pectin-cysteine conjugate exhibited approximately 5-fold increased in in-vitro adhesion duration and significantly improved cohesive properties. Zinc pectin-cysteine beads showed improved stability in simulated gastrointestinal media; however, insulin release from these beads followed the same profile as unmodified zinc pectinate beads. Due to favourable safety and biodegradability profile, and improved cohesive and permeation-enhancing properties, pectin-cysteine might be a promising excipient in various transmucosal drug delivery systems.

  11. 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

  12. Formation of a Stabilized Cysteine Sulfinic Acid Is Critical for the Mitochondrial Function of the Parkinsonism Protein DJ-1

    SciTech Connect

    Blackinton, Jeff; Lakshminarasimhan, Mahadevan; Thomas, Kelly J.; Ahmad, Rili; Greggio, Elisa; Raza, Ashraf S.; Cookson, Mark R.; Wilson, Mark A.

    2009-03-02

    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 (Cys{sup 106} in human DJ-1). Mutation of Cys{sup 106} 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 Cys{sup 106} 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 (Glu{sup 18}) in human DJ-1 that alter the oxidative propensity of Cys{sup 106} through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys{sup 106} to be oxidized to Cys{sup 106}-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 Cys{sup 106}-sulfinic acid is a key modification that regulates the protective function of DJ-1.

  13. 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

  14. 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.

  15. A study of low-energy ion induced radiolysis of thiol-containing amino acid cysteine in the solid and aqueous solution states

    NASA Astrophysics Data System (ADS)

    Ke, Zhigang; Huang, Qing; Dang, Bingrong; Lu, Yilin; Yuan, Hang; Zhang, Shuqing; Yu, Zengliang

    2010-09-01

    The radiolysis of cysteine under plasma discharge and irradiation of low-energy ion beam was investigated. The damage of cysteine in aqueous solution under discharge was assessed via the acid ninhydrin reagent and the yield of cystine produced from the reaction was analyzed by FTIR. In addition, the generation of hydrogen sulfide was also identified. The destruction of solid cysteine under low-energy ion beam irradiation was estimated via monitoring IR bands of different functional groups (-SH, -NH 3, -COO -) of cysteine, and the production of cystine from ion-irradiated solid cysteine after dissolution in water was also verified. These results may help us to understand the inactivation of sulphydryl enzymes under direct and indirect interaction with the low-energy ion irradiation.

  16. Differential expression of cysteine desulfurases in soybean

    PubMed Central

    2011-01-01

    Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression. PMID:22099069

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

    PubMed

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

    2013-04-01

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

  18. Causes and Consequences of Cysteine S-Glutathionylation*

    PubMed Central

    Grek, Christina L.; Zhang, Jie; Manevich, Yefim; Townsend, Danyelle M.; Tew, Kenneth D.

    2013-01-01

    Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress. PMID:23861399

  19. Functional cardiovascular action of L-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat.

    PubMed

    Takemoto, Yumi

    2014-04-01

    The endogenous sulfur-containing amino acid L-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to L-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to L-glutamate (10 mM, 34 nl), microinjections of L-cysteine increased ABP and HR dose dependently (3-100 mM, 34 nl). The cardiovascular responses to L-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to L-cysteine. The results indicate that L-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to L-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of L-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.

  20. 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.

  1. Chemical modification of an alpha 3-fucosyltransferase; definition of amino acid residues essential for enzyme activity.

    PubMed

    Britten, C J; Bird, M I

    1997-02-11

    The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta (1-4)GlcNAc-R alpha (1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 microM. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta (1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta (1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to, the substrate-binding site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.

  2. 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. PMID:23602110

  3. Cysteine-reactive covalent capture tags for enrichment of cysteine-containing peptides.

    PubMed

    Giron, Priscille; Dayon, Loïc; Mihala, Nikolett; Sanchez, Jean-Charles; Rose, Keith

    2009-11-01

    Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so-called cysteine-reactive covalent capture tags (C3T), for the isolation of Cys-containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine-containing peptides. The combination of C3T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. PMID:19813279

  4. Phosphorylation of Cysteine String Protein Triggers a Major Conformational Switch.

    PubMed

    Patel, Pryank; Prescott, Gerald R; Burgoyne, Robert D; Lian, Lu-Yun; Morgan, Alan

    2016-08-01

    Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins.

  5. Phosphorylation of Cysteine String Protein Triggers a Major Conformational Switch.

    PubMed

    Patel, Pryank; Prescott, Gerald R; Burgoyne, Robert D; Lian, Lu-Yun; Morgan, Alan

    2016-08-01

    Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins. PMID:27452402

  6. Protein-based biomemory device consisting of the cysteine-modified azurin

    NASA Astrophysics Data System (ADS)

    Choi, Jeong-Woo; Oh, Byung-Keun; Kim, Young Jun; Min, Junhong

    2007-12-01

    We demonstrated a protein-based memory device using recombinant Pseudomonas aeruginosa azurin (azurin), a metalloprotein with a redox property. Azurin was recombined with a cysteine residue to enhance the stability of the self-assembled protein on the gold surface. The memory device characteristics, including the "read," "write," and "erase" functions of the self-assembled azurin layer, were well demonstrated with three distinct electrical states of azurin layers by cyclic voltammetry. The robustness of the protein-based biomemory device was validated by the repeated electrochemical performance of 500000cycles.

  7. The substituted-cysteine accessibility method (SCAM) to elucidate membrane protein structure.

    PubMed

    Liapakis, G; Simpson, M M; Javitch, J A

    2001-05-01

    The substituted-cysteine accessibility method (SCAM) provides an approach to identifying the residues in the membrane-spanning segments that line a channel, transporter, or binding-site crevice. SCAM can also be used to determine differences in the structures of the membrane-spanning segments in different functional states of the proteins, to map electrostatic potential in the membrane-spanning domains, and to size a channel or binding-site crevice. The protocol in this unit describes the use of SCAM to map the binding-site crevice of a G-protein coupled receptor (GPCR) which binds ligand within the transmembrane portion of the receptor.

  8. 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. PMID:27097164

  9. 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.

  10. Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

    PubMed Central

    Chella Krishnan, Karthickeyan; Mukundan, Santhosh; Landero Figueroa, Julio A.; Caruso, Joseph A.

    2014-01-01

    Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues 47Cys and 195His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections. PMID:24799625

  11. 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

  12. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine.

    PubMed

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W

    1994-05-10

    Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Metal binding ability of cysteine-rich peptide domain of ZIP13 Zn2+ ions transporter.

    PubMed

    Potocki, Slawomir; Rowinska-Zyrek, Magdalena; Valensin, Daniela; Krzywoszynska, Karolina; Witkowska, Danuta; Luczkowski, Marek; Kozlowski, Henryk

    2011-07-01

    The coordination modes and thermodynamic stabilities of the complexes of the cysteine-rich N-terminal domain fragment of the ZIP13 zinc transporter (MPGCPCPGCG-NH(2)) with Zn(2+), Cd(2+), Bi(3+), and Ni(2+) have been studied by potentiometric, mass spectrometric, NMR, CD, and UV-vis spectroscopic methods. All of the studied metals had similar binding modes, with the three thiol sulfurs of cysteine residues involved in metal ion coordination. The stability of the complexes formed in solution changes in the series Bi(3+) ≫ Cd(2+) > Zn(2+) > Ni(2+), the strongest being for bismuth and the weakest for nickel. The N-terminal fragment of the human metalothionein-3 (MDPETCPCP-NH(2)) and unique histidine- and cysteine-rich domain of the C-terminus of Helicobacter pyroli HspA protein (Ac-ACCHDHKKH-NH(2)) have been chosen for the comparison studies. It confirmed indirectly which groups were the anchoring ones of ZIP13 domain. Experimental data from all of the used techniques and comparisons allowed us to propose possible coordination modes for all of the studied ZIP13 complexes.

  14. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

    PubMed

    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 prodrug that is converted to monomethyl fumarate (MMF). 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 Cθ (PKCθ). 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

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

    PubMed

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

    2016-08-01

    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

  16. Metal binding ability of cysteine-rich peptide domain of ZIP13 Zn2+ ions transporter.

    PubMed

    Potocki, Slawomir; Rowinska-Zyrek, Magdalena; Valensin, Daniela; Krzywoszynska, Karolina; Witkowska, Danuta; Luczkowski, Marek; Kozlowski, Henryk

    2011-07-01

    The coordination modes and thermodynamic stabilities of the complexes of the cysteine-rich N-terminal domain fragment of the ZIP13 zinc transporter (MPGCPCPGCG-NH(2)) with Zn(2+), Cd(2+), Bi(3+), and Ni(2+) have been studied by potentiometric, mass spectrometric, NMR, CD, and UV-vis spectroscopic methods. All of the studied metals had similar binding modes, with the three thiol sulfurs of cysteine residues involved in metal ion coordination. The stability of the complexes formed in solution changes in the series Bi(3+) ≫ Cd(2+) > Zn(2+) > Ni(2+), the strongest being for bismuth and the weakest for nickel. The N-terminal fragment of the human metalothionein-3 (MDPETCPCP-NH(2)) and unique histidine- and cysteine-rich domain of the C-terminus of Helicobacter pyroli HspA protein (Ac-ACCHDHKKH-NH(2)) have been chosen for the comparison studies. It confirmed indirectly which groups were the anchoring ones of ZIP13 domain. Experimental data from all of the used techniques and comparisons allowed us to propose possible coordination modes for all of the studied ZIP13 complexes. PMID:21630642

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

    PubMed Central

    Hempe, J M; Cousins, R J

    1991-01-01

    The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. We have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPLC and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein [Birkenmeier, E. H. & Gordon, J. I. (1986) Proc. Natl. Acad. Sci. USA 83, 2516-2520]. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient. Images PMID:1946385

  18. 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

  19. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

    PubMed

    Blewett, Megan M; Xie, Jiji; Zaro, Balyn W; Backus, Keriann M; Altman, Amnon; Teijaro, John R; Cravatt, Benjamin F

    2016-09-13

    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 prodrug that is converted to monomethyl fumarate (MMF). 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 Cθ (PKCθ). 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.

  20. 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

  1. 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.

  2. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

    PubMed

    Oancea, E; Teruel, M N; Quest, A F; Meyer, T

    1998-02-01

    Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

  3. To assess whether indoor residual spraying can provide additional protection against clinical malaria over current best practice of long-lasting insecticidal mosquito nets in The Gambia: study protocol for a two-armed cluster-randomised trial

    PubMed Central

    2011-01-01

    Background Recently, there has been mounting interest in scaling-up vector control against malaria in Africa. It needs to be determined if indoor residual spraying (IRS with DDT) will provide significant marginal protection against malaria over current best practice of long-lasting insecticidal nets (LLINs) and prompt treatment in a controlled trial, given that DDT is currently the most persistent insecticide for IRS. Methods A 2 armed cluster-randomised controlled trial will be conducted to assess whether DDT IRS and LLINs combined provide better protection against clinical malaria in children than LLINs alone in rural Gambia. Each cluster will be a village, or a group of small adjacent villages; all clusters will receive LLINs and half will receive IRS in addition. Study children, aged 6 months to 13 years, will be enrolled from all clusters and followed for clinical malaria using passive case detection to estimate malaria incidence for 2 malaria transmission seasons in 2010 and 2011. This will be the primary endpoint. Exposure to malaria parasites will be assessed using light and exit traps followed by detection of Anopheles gambiae species and sporozoite infection. Study children will be surveyed at the end of each transmission season to estimate the prevalence of Plasmodium falciparum infection and the prevalence of anaemia. Discussion Practical issues concerning intervention implementation, as well as the potential benefits and risks of the study, are discussed. Trial Registration ISRCTN01738840 - Spraying And Nets Towards malaria Elimination (SANTE) PMID:21663656

  4. The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis

    PubMed Central

    Lane, David A.; Crawley, James T. B.

    2015-01-01

    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. PMID:25564400

  5. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    PubMed Central

    Bazan, J F; Fletterick, R J

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases. Images PMID:3186696

  6. 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

    2011-11-01

    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.

  7. 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

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

    PubMed

    Liu, Xuehong; Dawson, David C

    2011-11-29

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

  9. 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. PMID:26947058

  10. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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...

  11. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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...

  12. 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.

  13. 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...

  14. 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.

  15. 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

  16. 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

  17. Enhanced biocatalytic production of L-cysteine by Pseudomonas sp. B-3 with in situ product removal using ion-exchange resin.

    PubMed

    Wang, Pu; He, Jun-Yao; Yin, Jiang-Feng

    2015-03-01

    Bioconversion of DL-2-amino-Δ(2)-thiazoline-4-carboxylic acid (DL-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of L-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated L-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve L-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of DL-ATC for the in situ removal of L-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for L-cysteine and low adsorption for DL-ATC, which is a favorable option. With in situ addition of 60 g L(-1) resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L(-1) of DL-ATC was converted to L-cysteine with 90.4 % of yield and 28.6 mmol L(-1 )h(-1) of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of L-cysteine was improved by 2.27-fold using ISPR method.

  18. Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S-Se Bond Cleavage with 266 nm Ultraviolet Photodissociation.

    PubMed

    Parker, W Ryan; Holden, Dustin D; Cotham, Victoria C; Xu, Hua; Brodbelt, Jennifer S

    2016-07-19

    The tremendous number of peptides identified in current bottom-up mass spectrometric workflows, although impressive for high-throughput proteomics, results in little selectivity for more targeted applications. We describe a strategy for cysteine-selective proteomics based on a tagging method that installs a S-Se bond in peptides that is cleavable upon 266 nm ultraviolet photodissociation (UVPD). The alkylating reagent, N-(phenylseleno)phthalimide (NPSP), reacts with free thiols in cysteine residues and attaches a chromogenic benzeneselenol (SePh) group. Upon irradiation of tagged peptides with 266 nm photons, the S-Se bond is selectively cleaved, releasing a benzeneselenol moiety corresponding to a neutral loss of 156 Da per cysteine. Herein we demonstrate a new MS/MS scan mode, UVPDnLossCID, which facilitates selective screening of cysteine-containing peptides. A "prescreening" event occurs by activation of the top N peptide ions by 266 nm UVPD. Peptides exhibiting a neutral loss corresponding to one or more SePh groups are reactivated and sequenced by CID. Because of the low frequency of cysteine in the proteome, unique cysteine-containing peptides may serve as surrogates for entire proteins. UVPDnLossCID does not generate as many peptide spectrum matches (PSMs) as conventional bottom-up methods; however, UVPDnLossCID provides far greater selectivity. PMID:27320857

  19. A free cysteine prolongs the half-life of a homing peptide and improves its tumor-penetrating activity.

    PubMed

    Pang, Hong-Bo; Braun, Gary B; She, Zhi-Gang; Kotamraju, Venkata R; Sugahara, Kazuki N; Teesalu, Tambet; Ruoslahti, Erkki

    2014-02-10

    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.

  20. Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

    PubMed

    Reymond, I; Sergeant, A; Tappaz, M

    1996-06-01

    The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported. PMID:8679699

  1. 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%).

  2. 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.

  3. Vanadium inhibition of serine and cysteine proteases.

    PubMed

    Guerrieri, N; Cerletti, P; De Vincentiis, M; Salvati, A; Scippa, S

    1999-03-01

    A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.

  4. 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.

  5. Quantitation of multiple pathways for the metabolism of nephrotoxic cysteine conjugates using selective inhibitors of L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and cysteine conjugate beta-lyase

    SciTech Connect

    Stevens, J.L.; Hatzinger, P.B.; Hayden, P.J. )

    1989-05-01

    In this study, we have established the selectivity of inhibitors for rat kidney cysteine conjugate beta-lyase and L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and have used these inhibitors to explore the relative roles of these two enzymes in the metabolism of nephrotoxic cysteine conjugates by rat kidney homogenate. In addition, we have investigated the relationship between structure and the metabolism of toxic cysteine conjugates by purified rat kidney L-alpha-hydroxy acid oxidase. With purified enzyme, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBDC) was about four times more active than S(1,2-dichlorovinyl)-L-cysteine (DCVC). Three alkyl conjugates were less active than DCVC. Purified L-alpha-hydroxy acid oxidase was not inhibited by the beta-lyase inhibitor aminooxyacetic acid but was inactivated by 2-hydroxy-3-butynoate. PCBDC metabolism in rat kidney homogenate was inhibited 74% by aminooxyacetic acid and 42% by 2-hydroxy-3-butynoate, whereas DCVC metabolism was inhibited 77% by aminooxyacetic acid and 28% by 2-hydroxy-3-butynoate. However, only aminooxyacetic acid inhibited the binding of {sup 35}S label from ({sup 35}S)DCVC. Based on these results we have reached three conclusions. First, L-alpha-hydroxy acid oxidase plays a significant role in the metabolism of some cysteine conjugates. Second, metabolism of DCVC by L-alpha-hydroxy acid oxidase does not contribute directly to covalent binding. Third, as much as 65% of DCVC may be metabolized to its corresponding alpha-keto acid. The results are discussed with regard to the nephrotoxicity of cysteine conjugates.

  6. 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.

  7. 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...

  8. 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. PMID:27105581

  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,; Jamie Lead,

    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. Crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate.

    PubMed

    Blaesse, M; Kupke, T; Huber, R; Steinbacher, S

    2000-12-01

    Epidermin from Staphylococcus epidermidis Tü3298 is an antimicrobial peptide of the lantibiotic family that contains, amongst other unusual amino acids, S:-[(Z:)- 2-aminovinyl]-D-cysteine. This residue is introduced by post-translational modification of the ribosomally synthesized precursor EpiA. Modification starts with the oxidative decarboxylation of its C-terminal cysteine by the flavoprotein EpiD generating a reactive (Z:)-enethiol intermediate. We have determined the crystal structures of EpiD and EpiD H67N in complex with the substrate pentapeptide DSYTC at 2.5 A resolution. Rossmann-type monomers build up a dodecamer of 23 point symmetry with trimers disposed at the vertices of a tetrahedron. Oligomer formation is essential for binding of flavin mononucleotide and substrate, which is buried by an otherwise disordered substrate recognition clamp. A pocket for the tyrosine residue of the substrate peptide is formed by an induced fit mechanism. The substrate contacts flavin mononucleotide only via Cys-Sgamma, suggesting its oxidation as the initial step. A thioaldehyde intermediate could undergo spontaneous decarboxylation. The unusual substrate recognition mode and the type of chemical reaction performed provide insight into a novel family of flavoproteins. PMID:11101502

  11. 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

  12. 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

  13. 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

    2015-08-01

    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.

  14. Inhibiting the color formation by gradient temperature-elevating Maillard reaction of soybean peptide-xylose system based on interaction of l-cysteine and Amadori compounds.

    PubMed

    Huang, M G; Zhang, X M; Eric, K; Abbas, S; Hayat, K; Liu, P; Xia, S Q; Jia, C S

    2012-05-01

    Light color and savory flavor enhancer are attractive for consumers and food producers. The effect of addition time of l-cysteine on inhibiting color formation was investigated in soybean peptide-xylose system, and the possible pathway was explored. Once dicarbonyl compounds were formed during the Maillard reaction, the addition of l-cysteine had no color-inhibiting effect; if l-cysteine was added immediately after the Amadori compound was formed, the extraordinary color-inhibiting effect was observed. Therefore, an improved way to inhibit color formation was proposed on the basis of the interaction of l-cysteine and Amadori compounds by controlling the addition time of l-cysteine through gradient temperature-elevating Maillard reaction. The system was heated at 80 °C for 60 min to form Amadori compounds, followed by the addition of L-cysteine, and the temperature was raised to 120 °C and held for 110 min. Compared with traditional products, the lightest color product was found desirable by GC/MS analysis and sensory evaluation. The novel method proposed can be a guide for the industrial preparation of light-colored products.

  15. Assembly of the K40 Antigen in Escherichia coli: Identification of a Novel Enzyme Responsible for Addition of l-Serine Residues to the Glycan Backbone and Its Requirement for K40 Polymerization

    PubMed Central

    Amor, Paul A.; Yethon, Jeremy A.; Monteiro, Mario A.; Whitfield, Chris

    1999-01-01

    Escherichia coli O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40LPS backbone structure is composed of a trisaccharide repeating unit of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, an l-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145–161, 1997). Here, we demonstrate that insertional inactivation of orf1 results in the accumulation of a semirough (SR)-K40LPS form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40LPS reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates that orf1 encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of orf3 also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the l-serine substitution of GlcA. These results show that (i) orf3 encodes the enzyme responsible for the addition of the l-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats with l-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy. PMID:9922239

  16. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    PubMed

    Winter, Gal; Cordente, Antonio G; Curtin, Chris

    2014-01-01

    Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes. PMID:25517415

  17. Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

    PubMed Central

    Winter, Gal; Cordente, Antonio G.; Curtin, Chris

    2014-01-01

    Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes. PMID:25517415

  18. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    PubMed

    Winter, Gal; Cordente, Antonio G; Curtin, Chris

    2014-01-01

    Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes.

  19. 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.

  20. 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.

  1. 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. PMID:25929483

  2. Residue chemistry guidelines.

    PubMed

    Olinger, C L; Schmitt, R D; Zager, E

    1993-01-01

    Residue chemistry guidelines are designed to determine what the potential residues in food are and how much may be present as a result of pesticide application, so that a tolerance level may be established. Some requirements are established to assist in the enforcement of tolerances by the USDA, FDA, and the states. I realize I have given you a quick overview of the residue chemistry requirements. There are many documents which are available if you should require more information, such as the Subdivision O Residue Chemistry Guidelines, Standard Evaluation Procedures (which are used by reviewers when evaluating the studies), the Data Reporting Guidelines (which provide guidance on preparing final reports), and the Technical Guidance from Phase III of Reregistration. We have also released various papers on studies when additional guidance is required. Most of these documents are available from NTIS. I hope you will consider this information when auditing residue chemistry studies. As I see the efforts that you, the QA professionals, have made to educate yourselves on residue chemistry studies through programs such as this meeting, I have a little more confidence in answering the question "Do you trust them?" with a "Yes." Thank you.

  3. 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.

  4. NMR-Based Mapping of Disulfide Bridges in Cysteine-Rich Peptides: Application to the μ-Conotoxin SxIIIA*

    PubMed Central

    Walewska, Aleksandra; Skalicky, Jack J.; Davis, Darrell R.; Zhang, Min-Min; Lopez-Vera, Estuardo; Watkins, Maren; Han, Tiffany S.; Yoshikami, Doju; Olivera, Baldomero M.; Bulaj, Grzegorz

    2009-01-01

    Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide bridged conotoxin, μ-SxIIIA (from the venom of Conus striolatus) whose discovery is also reported here for the first time. μ-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with 15N/13C, while the remaining three cysteine residues were incorporated using a mixture of 70%:30% unlabeled:labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple resonance (1H,13C,15N) NMR experiments and 2D [13C,1H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of μ-conotoxins. μ-SxIIIA was found to be a potent blocker of the sodium channel subtype NaV1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides. PMID:18831583

  5. 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.

  6. 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

  7. Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis).

    PubMed

    Malo, C; Gil, L; Gonzalez, N; Martínez, F; Cano, R; de Blas, I; Espinosa, E

    2010-08-01

    Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.

  8. Modeling the Active Sites in Metalloenzymes 5. The Heterolytic Bond Cleavage of H2 in the [NiFe] Hydrogenase of DesulfoWibrio gigas by a Nucleophilic Addition Mechanism

    SciTech Connect

    Niu, Shuqiang; Hall, Michael B.

    2001-11-19

    The H2 activation catalyzed by an Fe(II)-Ni(III) model of the [NiFe] hydrogenase of DesulfoVibrio gigas has been investigated by density functional theory (DFT/B3LYP) calculations on the neutral and anionic active site complexes, [(CO)(CN)2Fe(Mu-SH)2Ni(SH)(SH2)]0 and [(CO)(CN)2Fe(Mu-SH)2Ni(SH)2]-. The results suggest that the reaction proceeds by a nucleophilic addition mechanism that cleaves the H-H bond heterolytically. The terminal cysteine residue Cys530 in the [NiFe] hydrogenase active site of the D. gigas enzyme plays a crucial role in the catalytic process by accepting the proton. The active site is constructed to provide access by this cysteine residue, and this role explains the change in activity observed when this cysteine is replaced by a selenocysteine. Furthermore, the optimized geometry of the transition state in the model bears a striking resemblance to the geometry of the active site as determined by X-ray crystallography.

  9. The maize cystatin CC9 interacts with apoplastic cysteine proteases.

    PubMed

    van der Linde, Karina; Mueller, André N; Hemetsberger, Christoph; Kashani, Farnusch; van der Hoorn, Renier A L; Doehlemann, Gunther

    2012-11-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signaling including PR-gene expression. Therefore the inhibition of apoplastic papain-like cysteine proteases by CC9 is essential to suppress host immunity during U. maydis infection. Here were present new experimental data on the cysteine protease-cystatin interaction and provide an in silco analysis of plant cystatins and the identified apoplastic cysteine proteases.

  10. The metabolism of S-methyl-l-cysteine

    PubMed Central

    Sklan, Naomi M.; Barnsley, E. A.

    1968-01-01

    1. Methylsulphinylacetic acid, 2-hydroxy-3-methylsulphinylpropionic acid and methylmercapturic acid sulphoxide (N-acetyl-S-methyl-l-cysteine S-oxide) were isolated as their dicyclohexylammonium salts from the urine of rats after they had been dosed with S-methyl-l-cysteine. 2. A fourth sulphoxide was isolated but not identified. 3. The excretion of sulphate in the urine of rats dosed with S-methyl-l-cysteine was measured. 4. The metabolism of S-methyl-l-cysteine by the hamster and guinea pig was examined chromatographically. 5. The preparation of the following compounds is reported: (−)-dicyclohexylammonium methyl-mercapturate sulphoxide; the dicyclohexylammonium salts of the optically inactive forms of 2-hydroxy-3-methylthiopropionic acid, 2-hydroxy-3-methyl-sulphinylpropionic acid and methylsulphinylacetic acid. PMID:5641877

  11. 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

  12. Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA.

    PubMed Central

    Diamond, G; Zasloff, M; Eck, H; Brasseur, M; Maloy, W L; Bevins, C L

    1991-01-01

    Extracts of the bovine tracheal mucosa have an abundant peptide with potent antimicrobial activity. The 38-amino acid peptide, which we have named tracheal antimicrobial peptide (TAP), was isolated by a sequential use of size-exclusion, ion-exchange, and reverse-phase chromatographic fractionations using antimicrobial activity as a functional assay. The yield was appr