Protein Disulfide Isomerase and Host-Pathogen Interaction

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Beatriz S. Stolf

2011-01-01

Full Text Available Reactive oxygen species (ROS production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation and (ii phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

Effect of pharmaceutical potential endocrine disruptor compounds on protein disulfide isomerase reductase activity using di-eosin-oxidized-glutathione.

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Danièle Klett

Full Text Available BACKGROUND: Protein Disulfide Isomerase (PDI in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. METHODOLOGY/PRINCIPAL FINDINGS: Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathione (DiE-GSSG, we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH. Our data show that estrogens (ethynylestradiol and bisphenol-A as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. CONCLUSIONS: The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainly be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

Attachment and entry of Chlamydia have distinct requirements for host protein disulfide isomerase.

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Stephanie Abromaitis

2009-04-01

Full Text Available Chlamydia is an obligate intracellular pathogen that causes a wide range of diseases in humans. Attachment and entry are key processes in infectivity and subsequent pathogenesis of Chlamydia, yet the mechanisms governing these interactions are unknown. It was recently shown that a cell line, CHO6, that is resistant to attachment, and thus infectivity, of multiple Chlamydia species has a defect in protein disulfide isomerase (PDI N-terminal signal sequence processing. Ectopic expression of PDI in CHO6 cells led to restoration of Chlamydia attachment and infectivity; however, the mechanism leading to this recovery was not ascertained. To advance our understanding of the role of PDI in Chlamydia infection, we used RNA interference to establish that cellular PDI is essential for bacterial attachment to cells, making PDI the only host protein identified as necessary for attachment of multiple species of Chlamydia. Genetic complementation and PDI-specific inhibitors were used to determine that cell surface PDI enzymatic activity is required for bacterial entry into cells, but enzymatic function was not required for bacterial attachment. We further determined that it is a PDI-mediated reduction at the cell surface that triggers bacterial uptake. While PDI is necessary for Chlamydia attachment to cells, the bacteria do not appear to utilize plasma membrane-associated PDI as a receptor, suggesting that Chlamydia binds a cell surface protein that requires structural association with PDI. Our findings demonstrate that PDI has two essential and independent roles in the process of chlamydial infectivity: it is structurally required for chlamydial attachment, and the thiol-mediated oxido-reductive function of PDI is necessary for entry.

Protein disulfide isomerase interacts with tau protein and inhibits its fibrillization.

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Li-Rong Xu

Full Text Available BACKGROUND: Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization. METHODOLOGY/PRINCIPAL FINDINGS: As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244-372 monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244-372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244-372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244-372 fibrillization more strongly than full-length human PDI. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.

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Sato, Yoshimi; Kojima, Rieko; Okumura, Masaki; Hagiwara, Masatoshi; Masui, Shoji; Maegawa, Ken-ichi; Saiki, Masatoshi; Horibe, Tomohisa; Suzuki, Mamoru; Inaba, Kenji

2013-01-01

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

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Holst, B; Tachibana, C; Winther, Jakob R.

1997-01-01

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

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Ali Dina

2016-01-01

Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

Synthesis and Experimental Validation of New PDI Inhibitors with Antiproliferative Activity

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Mariateresa Badolato

2017-01-01

Full Text Available Protein disulfide isomerase (PDI is a member of the thioredoxin superfamily of redox enzymes. PDI is a multifunctional protein that catalyzes disulfide bond formation, cleavage, and rearrangement in unfolded or misfolded proteins and functions as a chaperone in the endoplasmic reticulum. Besides acting as a protein folding catalyst, several evidences have suggested that PDI can bind small molecules containing, for example, a phenolic structure, which includes the estrogenic one. Increasing studies indicate that PDI is involved in both physiology and pathophysiology of cells and tissues and is involved in the survival and proliferation of different cancers. Propionic acid carbamoyl methyl amides (PACMAs showed anticancer activity in human ovarian cancer, both in vitro and in vivo, by inhibiting PDI. The inhibition of PDI’s activity may have a therapeutic role, in various diseases, including cancer. In the present study, we designed and synthesized a diversified small library of compounds with the aim of identifying a new class of PDI inhibitors. Most of synthesized compounds showed a good inhibitory potency against PDI and particularly 4-methyl substituted 2,6-di-tert-butylphenol derivatives (8–10 presented an antiproliferative activity in a wide panel of human cancer cell lines, including ovarian ones.

Progranulin, a glycoprotein deficient in frontotemporal dementia, is a novel substrate of several protein disulfide isomerase family proteins.

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Sandra Almeida

Full Text Available The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca(2+-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

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Hongyu Han

Full Text Available Protein disulfide isomerase (PDI and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE according to the expressed sequence tag (EST. The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC. BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells

Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

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Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

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Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias; Malmberg, Karl-Johan; Troye-Blomberg, Marita; Ahlborg, Niklas; Karlsson, Anna; Johansson, Magnus; Lundberg, Mathias

2009-01-01

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.

Protein disulfide isomerases in the endoplasmic reticulum promote anchorage-independent growth of breast cancer cells.

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Wise, Randi; Duhachek-Muggy, Sara; Qi, Yue; Zolkiewski, Michal; Zolkiewska, Anna

2016-06-01

Metastatic breast cancer cells are exposed to stress of detachment from the extracellular matrix (ECM). Cultured breast cancer cells that survive this stress and are capable of anchorage-independent proliferation form mammospheres. The purpose of this study was to explore a link between mammosphere growth, ECM gene expression, and the protein quality control system in the endoplasmic reticulum (ER). We compared the mRNA and protein levels of ER folding factors in SUM159PT and MCF10DCIS.com breast cancer cells grown as mammospheres versus adherent conditions. Publicly available gene expression data for mammospheres formed by primary breast cancer cells and for circulating tumor cells (CTCs) were analyzed to assess the status of ECM/ER folding factor genes in clinically relevant samples. Knock-down of selected protein disulfide isomerase (PDI) family members was performed to examine their roles in SUM159PT mammosphere growth. We found that cells grown as mammospheres had elevated expression of ECM genes and ER folding quality control genes. CTC gene expression data for an index patient indicated that upregulation of ECM and ER folding factor genes occurred at the time of acquired therapy resistance and disease progression. Knock-down of PDI, ERp44, or ERp57, three members of the PDI family with elevated protein levels in mammospheres, in SUM159PT cells partially inhibited the mammosphere growth. Thus, breast cancer cell survival and growth under detachment conditions require enhanced assistance of the ER protein folding machinery. Targeting ER folding factors, in particular members of the PDI family, may improve the therapeutic outcomes in metastatic breast cancer.

Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

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Cody Caba

2018-02-01

Full Text Available Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI, the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin. A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation.

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Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

2018-01-01

Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys 57 and Lys 401 of human PDI in vitro . Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys 57 and Lys 401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys 57 and acLys 401 . The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

Identification of the PDI-family member ERp90 as an interaction partner of ERFAD.

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Jan Riemer

Full Text Available In the endoplasmic reticulum (ER, members of the protein disulfide isomerase (PDI family perform critical functions during protein maturation. Herein, we identify the previously uncharacterized PDI-family member ERp90. In cultured human cells, we find ERp90 to be a soluble ER-luminal glycoprotein that comprises five potential thioredoxin (Trx-like domains. Mature ERp90 contains 10 cysteine residues, of which at least some form intramolecular disulfides. While none of the Trx domains contain a canonical Cys-Xaa-Xaa-Cys active-site motif, other conserved cysteines could endow the protein with redox activity. Importantly, we show that ERp90 co-immunoprecipitates with ERFAD, a flavoprotein involved in ER-associated degradation (ERAD, through what is most likely a direct interaction. We propose that the function of ERp90 is related to substrate recruitment or delivery to the ERAD retrotranslocation machinery by ERFAD.

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

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Ding, Xia; Lv, Zhen-Mei; Zhao, Yang; Min, Hang; Yang, Wei-Jun

2008-01-01

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum.

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Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin

2015-03-01

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment. Copyright © 2014 Elsevier Inc. All rights reserved.

Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

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Minh Tan Nguyen

Full Text Available Human growth hormone (hGH is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, protein disulfide bond isomerase (PDI, and the b'a' domain of PDI (PDIb'a', were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

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Yeon Jeong Kim

Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

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Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

2016-01-01

Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

A novel potential biomarker for metabolic syndrome in Chinese adults: Circulating protein disulfide isomerase family A, member 4.

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Chien, Chu-Yen; Hung, Yi-Jen; Shieh, Yi-Shing; Hsieh, Chang-Hsun; Lu, Chieh-Hua; Lin, Fu-Huang; Su, Sheng-Chiang; Lee, Chien-Hsing

2017-01-01

Protein disulfide isomerase (PDI) family members are specific endoplasmic reticulum proteins that are involved in the pathogenesis of numerous diseases including neurodegenerative diseases, cancer and obesity. However, the metabolic effects of PDIA4 remain unclear in humans. The aims of this study were to investigate the associations of serum PDIA4 with the metabolic syndrome (MetS) and its components in Chinese adults. A total of 669 adults (399 men and 270 women) were recruited. Serum PDIA4 concentrations and biochemical variables were recorded. Insulin sensitivity and β-cell function were examined by homeostasis model assessment. MetS was defined based on the modified National Cholesterol Education Program Adult Treatment Panel III criteria for Asia Pacific. The participants with MetS had significantly higher serum PDIA4 levels than those without MetS (Pmetabolic syndrome were 67 and 72%, respectively, in male patients and 60 and 78%, respectively, in female patients. Finally, the result showed that PDIA4 had a significantly higher area under the curve compared with blood pressure to detect MetS using receiver operating characteristic analysis. Serum PDIA4 concentrations are closely associated to MetS and its components in Chinese adults.

The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

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Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

2014-01-31

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

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Valentina Castillo

Full Text Available ERp57 (also known as grp58 and PDIA3 is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

A disulphide isomerase gene (PDI-V) from Haynaldia villosa contributes to powdery mildew resistance in common wheat.

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Faheem, Muhammad; Li, Yingbo; Arshad, Muhammad; Jiangyue, Cheng; Jia, Zhao; Wang, Zongkuan; Xiao, Jin; Wang, Haiyan; Cao, Aizhong; Xing, Liping; Yu, Feifei; Zhang, Ruiqi; Xie, Qi; Wang, Xiue

2016-04-13

In this study, we report the contribution of a PDI-like gene from wheat wild relative Haynaldia villosa in combating powdery mildew. PDI-V protein contains two conserved thioredoxin (TRX) active domains (a and a') and an inactive domain (b). PDI-V interacted with E3 ligase CMPG1-V protein, which is a positive regulator of powdery mildew response. PDI-V was mono-ubiquitinated by CMPG1-V without degradation being detected. PDI-V was located on H. villosa chromosome 5V and encoded for a protein located in the endoplasmic reticulum. Bgt infection in leaves of H. villosa induced PDI-V expression. Virus induced gene silencing of PDIs in a T. durum-H. villosa amphiploid compromised the resistance. Single cell transient over-expression of PDI-V or a truncated version containing the active TXR domain a decreased the haustorial index in moderately susceptible wheat cultivar Yangmai 158. Stable transgenic lines over-expressing PDI-V in Yangmai 158 displayed improved powdery mildew resistance at both the seedling and adult stages. By contrast over-expression of point-mutated PDI-V(C57A) did not increase the level of resistance in Yangmai 158. The above results indicate a pivotal role of PDI-V in powdery mildew resistance and showed that conserved TRX domain a is critical for its function.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

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Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

2012-10-01

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

Endoplasmic reticulum-dependent redox reactions control endoplasmic reticulum-associated degradation and pathogen entry.

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Walczak, Christopher P; Bernardi, Kaleena M; Tsai, Billy

2012-04-15

Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host-pathogen interactions. Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions.

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

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Lobstein Julie

2012-05-01

Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

The Role of S-Nitrosylation and S-Glutathionylation of Protein Disulphide Isomerase in Protein Misfolding and Neurodegeneration

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M. Halloran

2013-01-01

Full Text Available Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO- containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.

Arabidopsis cotyledon chloroplast biogenesis factor CYO1 uses glutathione as an electron donor and interacts with PSI (A1 and A2) and PSII (CP43 and CP47) subunits.

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Muranaka, Atsuko; Watanabe, Shunsuke; Sakamoto, Atsushi; Shimada, Hiroshi

2012-08-15

CYO1 is required for thylakoid biogenesis in cotyledons of Arabidopsis thaliana. To elucidate the enzymatic characteristics of CYO1, we analyzed the protein disulfide isomerase (PDI) activity of CYO1 using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The reductase activity of CYO1 increased as a function of Di-E-GSSG, with an apparent K(m) of 824nM and K(cat) of 0.53min(-1). PDI catalyzes dithiol/disulfide interchange reactions, and the cysteine residues in PDI proteins are very important. To analyze the significance of the cysteine residues for the PDI activity of CYO1, we estimated the kinetic parameters of point-mutated CYO1 proteins. C117S, C124S, C135S, and C156S had higher values for K(m) than did wild-type CYO1. C158S had a similar K(m) but a higher K(cat), and C138S and C161S had similar K(m) values but lower K(cat) values than did wild-type CYO1. These results suggested that the cysteine residues at positions 138 and 161 were important for PDI activity. Low PDI activity of CYO1 was observed when NADPH or NADH was used as an electron donor. However, PDI activity was observed with CYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for CYO1 in vivo. Based on analysis with the split-ubiquitin system, CYO1 interacted with the A1 and A2 subunits of PSI and the CP43 and CP47 subunits of PSII. Thus, CYO1 may accelerate the folding of cysteine residue--containing PSI and PSII subunits by repeatedly breaking and creating disulfide bonds. Copyright © 2012 Elsevier GmbH. All rights reserved.

Mechanisms of Neuroprotection by Protein Disulphide Isomerase in Amyotrophic Lateral Sclerosis

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Adam K. Walker

2011-01-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating neurodegenerative disease characterised by the progressive loss of motor neurons, leading to paralysis and death within several years of onset. Although protein misfolding is a key feature of ALS, the upstream triggers of disease remain elusive. Recently, endoplasmic reticulum (ER stress was identified as an early and central feature in ALS disease models as well as in human patient tissues, indicating that ER stress could be an important process in disease pathogenesis. One important chaperone induced by ER stress is protein disulphide isomerase (PDI, which is both upregulated and posttranslationally inhibited by S-nitrosylation in ALS. In this paper, we present evidence from studies of genetics, model organisms, and patient tissues which indicate an active role for PDI and ER stress in ALS disease processes.

Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

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Hironori Kurisaki

Full Text Available Although the autoimmune regulator (Aire knockout (KO mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2, which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

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Kurisaki, Hironori; Nagao, Yukihiro; Nagafuchi, Seiho; Mitsuyama, Masao

2013-01-01

Although the autoimmune regulator (Aire) knockout (KO) mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2), which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

Insight into PreImplantation Factor (PIF* mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP through essential RIKP [corrected] binding site.

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Eytan R Barnea

Full Text Available Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control. Murine embryo (d10 lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS. In silico evaluation examined binding of PIF to critical targets, using mutation analysis.PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90, co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a

Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway*

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Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

2013-01-01

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis. PMID:23979138

A Proteomic Screen Identified Stress-Induced Chaperone Proteins as Targets of Akt Phosphorylation in Mesangial Cells

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Barati, Michelle T.; Rane, Madhavi J.; Klein, Jon B.; McLeish, Kenneth R.

2006-01-01

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90α, Hsp90β, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphoryl...

The unfolded protein response and the role of protein disulphide isomerase in neurodegeneration.

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Emma ePerri

2016-01-01

Full Text Available The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR, distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

Urinary Protein Biomarker Analysis

Science.gov (United States)

2017-10-01

silica emitter via a Valco stainless steel union. Four μL of individual peptide fractions (total volume 20 μL) following PRISM were injected for LC...secreted cement gland protein XAG-2 homolog, AGR2 belongs to the protein disulfide 5 isomerase (PDI) family. The strongest AGR2 expression has...µm C18 column (75 µm i.d. × 10 cm), which was connected to a chemically etched 20 µm i.d. fused-silica emitter via a Valco stainless steel union

Research Perspective: Potential Role of Nitazoxanide in Ovarian Cancer Treatment. Old Drug, New Purpose?

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Jessie Ehrisman

2013-09-01

Full Text Available Among gynecological malignancies epithelial ovarian cancer (EOC is the leading cause of death. Despite improvements in conventional chemotherapy combinations, the overall cure rate has remained mostly stable over the years, and only 10%–15% of patients maintain a complete response following first-line therapy. To improve the efficacy of ovarian cancer chemotherapy it is essential to develop drugs with new mechanisms of action. Compared to normal tissues, protein disulfide isomerase (PDI is overexpressed in ovarian tumors. PDI is a cellular enzyme in the lumen of the endoplasmic reticulum (ER of eukaryotes or the periplasmic region of prokaryotes. This protein catalyzes the formation and breakage of disulphide bonds between cysteine residues in proteins, which affects protein folding. Selective inhibition of PDI activity has been exhibited both in vitro and in vivo anticancer activity in human ovarian cancer models. PDI inhibition caused accumulation of unfolded or misfolded proteins, which led to ER stress and the unfolded protein response (UPR, and in turn resulted in cell death. Nitazoxanide [NTZ: 2-acetyloxy-N-(5-nitro-2-thiazolylbenzamide] is a thiazolide antiparasitic agent with excellent activity against a wide variety of protozoa and helminths. In this article, we propose that NTZ, acting as PDI inhibitor, may be a new and potent addition to the chemotherapeutic strategy against ovarian cancer.

Research Perspective: Potential Role of Nitazoxanide in Ovarian Cancer Treatment. Old Drug, New Purpose?

Energy Technology Data Exchange (ETDEWEB)

Di Santo, Nicola, E-mail: nico.disanto@duke.edu; Ehrisman, Jessie [Division of Gynecologic Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

2013-09-10

Among gynecological malignancies epithelial ovarian cancer (EOC) is the leading cause of death. Despite improvements in conventional chemotherapy combinations, the overall cure rate has remained mostly stable over the years, and only 10%–15% of patients maintain a complete response following first-line therapy. To improve the efficacy of ovarian cancer chemotherapy it is essential to develop drugs with new mechanisms of action. Compared to normal tissues, protein disulfide isomerase (PDI) is overexpressed in ovarian tumors. PDI is a cellular enzyme in the lumen of the endoplasmic reticulum (ER) of eukaryotes or the periplasmic region of prokaryotes. This protein catalyzes the formation and breakage of disulphide bonds between cysteine residues in proteins, which affects protein folding. Selective inhibition of PDI activity has been exhibited both in vitro and in vivo anticancer activity in human ovarian cancer models. PDI inhibition caused accumulation of unfolded or misfolded proteins, which led to ER stress and the unfolded protein response (UPR), and in turn resulted in cell death. Nitazoxanide [NTZ: 2-acetyloxy-N-(5-nitro-2-thiazolyl)benzamide] is a thiazolide antiparasitic agent with excellent activity against a wide variety of protozoa and helminths. In this article, we propose that NTZ, acting as PDI inhibitor, may be a new and potent addition to the chemotherapeutic strategy against ovarian cancer.

Spectromicroscopy of self-assembled protein clusters

Energy Technology Data Exchange (ETDEWEB)

Schonschek, O.; Hormes, J.; Herzog, V. [Univ. of Bonn (Germany)

1997-04-01

The aim of this project is to use synchrotron radiation as a tool to study biomedical questions concerned with the thyroid glands. The biological background is outlined in a recent paper. In short, Thyroglobulin (TG), the precursor protein of the hormone thyroxine, forms large (20 - 500 microns in diameter) clusters in the extracellular lumen of thyrocytes. The process of the cluster formation is still not well understood but is thought to be a main storage mechanism of TG and therefore thyroxine inside the thyroid glands. For human thyroids, the interconnections of the proteins inside the clusters are mainly disulfide bondings. Normally, sulfur bridges are catalyzed by an enzyme called Protein Disulfide Bridge Isomerase (PDI). While this enzyme is supposed to be not present in any extracellular space, the cluster formation of TG takes place in the lumen between the thyrocytes. A possible explanation is the autocatalysis of TG.

Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes.

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Pyott, Shawna M; Schwarze, Ulrike; Christiansen, Helena E; Pepin, Melanie G; Leistritz, Dru F; Dineen, Richard; Harris, Catharine; Burton, Barbara K; Angle, Brad; Kim, Katherine; Sussman, Michael D; Weis, Maryann; Eyre, David R; Russell, David W; McCarthy, Kevin J; Steiner, Robert D; Byers, Peter H

2011-04-15

Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis-trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis-trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis-trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.

Chaperonin GroE-facilitated refolding of disulfide-bonded and reduced Taka-amylase A from Aspergillus oryzae.

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Kawata, Y; Hongo, K; Mizobata, T; Nagai, J

1998-12-01

The refolding characteristics of Taka-amylase A (TAA) from Aspergillus oryzae in the presence of the chaperonin GroE were studied in terms of activity and fluorescence. Disulfide-bonded (intact) TAA and non-disulfide-bonded (reduced) TAA were unfolded in guanidine hydrochloride and refolded by dilution into buffer containing GroE. The intermediates of both intact and reduced enzymes were trapped by GroEL in the absence of nucleotide. Upon addition of nucleotides such as ATP, ADP, CTP or UTP, the intermediates were released from GroEL and recovery of activity was detected. In both cases, the refolding yields in the presence of GroEL and ATP were higher than spontaneous recoveries. Fluorescence studies of intrinsic tryptophan and a hydrophobic probe, 8-anilinonaphthalene-1-sulfonate, suggested that the intermediates trapped by GroEL assumed conformations with different hydrophobic properties. The presence of protein disulfide isomerase or reduced and oxidized forms of glutathione in addition to GroE greatly enhanced the refolding reaction of reduced TAA. These findings suggest that GroE has an ability to recognize folding intermediates of TAA protein and facilitate refolding, regardless of the existence or absence of disulfide bonds in the protein.

PDI Derivative through Fine-Tuning Molecular Structure for Fullerene-Free Organic Solar Cells

KAUST Repository

Sun, Hua

2017-08-10

A perylenediimide (PDI)-based small molecular (SM) acceptor with both an extended π-conjugation and a three dimensional structure concurrently is critical for achieving high performance PDI-based fullerene-free organic solar cells (OSCs). In this work, we designed and synthesized a novel PDI-based SM acceptor possessing both characteristics by fusing PDI units with a spiro core of 4,4’-spirobi[cyclopenta[2,1-b;3,4-b’]dithiophene(SCPDT) through the -position of the thiophene rings. An enhanced strong absorption in the range of 350–520 nm and arisen LUMO energy level of FSP was observed, compared with previous reported acceptor SCPDT-PDI4, in which the PDI units and SCPDT are not fused. OSCs based on PTB7-Th donor and FSP acceptor were fabricated and achieved a power conversion efficiency of up to 8.89% with DPE as an additive. Efficient and complementary photo absorption, favorable phase separation and balanced carrier mobilites in the blend film account for the high photovoltaic performance. This study offers an effective strategy to design high performance PDI-based acceptors.

The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

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Lasecka, Lidia; Baron, Michael D

2014-01-01

Nairobi sheep disease virus (NSDV) of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

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Lidia Lasecka

Full Text Available Nairobi sheep disease virus (NSDV of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER, the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI, an oxidoreductase present in the lumen of the endoplasmic reticulum (ER and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

Characterization of wheat endoplasmic reticulum oxidoreductin 1 and its application in Chinese steamed bread.

Science.gov (United States)

Liu, Guang; Wang, JingJing; Hou, Yi; Huang, Yan-Bo; Wang, JiaJia; Li, Cunzhi; Guo, ShiJun; Li, Lin; Hu, Song-Qing

2018-08-01

This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Electron Acceptors Based on α-Substituted Perylene Diimide (PDI) for Organic Solar Cells

Energy Technology Data Exchange (ETDEWEB)

Zhao, Donglin [Department; Wu, Qinghe [Department; Cai, Zhengxu [Department; Zheng, Tianyue [Department; Chen, Wei [Materials; Institute; Lu, Jessica [Department; Yu, Luping [Department

2016-02-02

Perylene diimide (PDI) derivatives functionalized at the ortho-position (αPPID, αPBDT) were synthesized and used as electron acceptors in non-fullerene organic photovoltaic cells. Because of the good planarity and strong π-stacking of ortho-functionalized PDI, the αPPID and αPBDT exhibit a strong tendency to form aggregates, which endow the materials with high electron mobility. The inverted OPVs employing αPDI-based compounds as the acceptors and PBT7-Th as the donor give the highest power conversion efficiency (PCE) values: 4.92% for αPBDT-based devices and 3.61% for αPPID-based devices, which are, respectively, 39% and 4% higher than that of their β-substituted counterparts βPBDT and βPPID. Charge separation studies show more efficient exciton dissociation at interfaces between αPDI-based compounds and PTB7-Th. The results suggest that α-substituted PDI derivatives are more promising electron acceptors for organic photovoltaic (OPV) components than β-isomers.

Ero1-PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

NARCIS (Netherlands)

Benham, A.M.; Lith, M. van; Sitia, R.; Braakman, I.|info:eu-repo/dai/nl/073923737

2013-01-01

The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide

FAD oxidizes the ERO1-PDI electron transfer chain: The role of membrane integrity

International Nuclear Information System (INIS)

Papp, Eszter; Nardai, Gabor; Mandl, Jozsef; Banhegyi, Gabor; Csermely, Peter

2005-01-01

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1 can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding

Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

International Nuclear Information System (INIS)

Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

2010-01-01

Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

Enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

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Kathrin Mutze

2015-08-01

Full Text Available The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI and type II (ATII cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2 and an increase in enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker, exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC, whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

Glucose (xylose) isomerase production from thermotolerant and ...

African Journals Online (AJOL)

Owner

2012-11-13

Nov 13, 2012 ... in the production of the high fructose corn syrup (HFCS) from corn starch. ... Key words: Glucose isomerase, xylose isomerase, enzyme activity, Klebsiella, ... Soil, water, and manure (five samples each) were collected from.

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

Science.gov (United States)

Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

2014-11-01

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

Effects of ubiquilin 1 on the unfolded protein response.

Science.gov (United States)

Lu, Alice; Hiltunen, Mikko; Romano, Donna M; Soininen, Hilkka; Hyman, Bradley T; Bertram, Lars; Tanzi, Rudolph E

2009-05-01

Previous studies have implicated the unfolded protein response (UPR) in the pathogenesis of Alzheimer's disease (AD). We previously reported that DNA variants in the ubiquilin 1 (UBQLN1) gene increase the risk for AD. Since UBQLN1 has been shown to play a role in the UPR, we assessed the effects of overexpression and downregulation of UBQLN1 splice variants during tunicamycin-induced ER stress. In addition to previously described transcript variants, TV1 and TV2, we identified two novel transcript variants of UBQLN1 in brain: TV3 (lacking exons 2-4) and TV4 (lacking exon 4). Overexpression of TV1-3, but not TV4 significantly decreased the mRNA induction of UPR-inducible genes, C/EBP homologous protein (CHOP), BiP/GRP78, and protein disulfide isomerase (PDI) during the UPR. Stable overexpression of TV1-3, but not TV4, also significantly decreased the induction of CHOP protein and increased cell viability during the UPR. In contrast, downregulation of UBQLN1 did not affect CHOP mRNA induction, but instead increased PDI mRNA levels. These findings suggest that overexpression UBQLN1 transcript variants TV1-3, but not TV4, exert a protective effect during the UPR by attenuating CHOP induction and potentially increasing cell viability.

Selenium Utilization Strategy by Microalgae

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Hiroya Araie

2009-11-01

Full Text Available The diversity of selenoproteins raises the question of why so many life forms require selenium. Selenoproteins are found in bacteria, archaea, and many eukaryotes. In photosynthetic microorganisms, the essential requirement for selenium has been reported in 33 species belonging to six phyla, although its biochemical significance is still unclear. According to genome databases, 20 species are defined as selenoprotein-producing organisms, including five photosynthetic organisms. In a marine coccolithophorid, Emiliania huxleyi (Haptophyta, we recently found unique characteristics of selenium utilization and novel selenoproteins using 75Se-tracer experiments. In E. huxleyi, selenite, not selenate, is the main substrate used and its uptake is driven by an ATP-dependent highaffinity, active transport system. Selenite is immediately metabolized to low-molecular mass compounds and partly converted to at least six selenoproteins, named EhSEP1–6. The most (EhSEP2 and second-most abundant selenoproteins (EhSEP1 are disulfide isomerase (PDI homologous protein and thioredoxin reductase (TR 1, respectively. Involvement of selenium in PDI is unique in this organism, while TR1 is also found in other organisms. In this review, we summarize physiological, biochemical, and molecular aspects of selenium utilization by microalgae and discuss their strategy of selenium utilization.

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

Science.gov (United States)

Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

2017-06-15

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Endoplasmic Reticulum Stress and Associated ROS

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Hafiz Maher Ali Zeeshan

2016-03-01

Full Text Available The endoplasmic reticulum (ER is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS. Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI-endoplasmic reticulum oxidoreductin (ERO-1, glutathione (GSH/glutathione disuphide (GSSG, NADPH oxidase 4 (Nox4, NADPH-P450 reductase (NPR, and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases.

Purification and characterization of the d-xylose isomerase gene from Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Ho, N W.Y.; Rosenfeld, S; Stevis, P; Tsao, G T

1983-11-01

A DNA fragment containing both the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene and the D-xylulokinase (ATP: D-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The D-xylose isomerase gene was separated from the D-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The D-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the D-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first D-xylose isomerase gene that has been isolated and extensively purified from any organism. D-Xylose isomerase, the enzyme product of the D-xylose isomerase gene, is responsible for the conversion of D-xylose to D-xylulose, as well as D-glucose to D-fructose. It is widely believed that yeast cannot ferment D-xylose to ethanol primarily because of the lack of D-xylose isomerase in yeast. D-Xylose isomerase (also known as D-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the D-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting D-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology. 14 references.

Insight into interaction mechanism of the inhibitor pDI5W with MDM2 based on molecular dynamics

International Nuclear Information System (INIS)

Chen Jianzhong; Liang Zhiqiang; Wang Wei; Liu Jinqing; Zhang Qinggang; Liu Xiaoyang

2012-01-01

The p53-MDM2 interaction has been an important target of drug design curing cancers. In this work, molecular dynamics (MD) simulation coupled with molecular mechanics/Poisson Boltzmann surface area method (MM-PBSA) was performed to calculate the binding free energy of peptide inhibitor pDI6W to MDM2. The results show that van der Waals energy is the dominant factor of the pDI6W— MDM2 interaction. Cross-correlation matrix calculated suggests that the main motion of the residues in MMDM2 induced by the inhibitor binding is anti-correlation motion. The calculations of residue-residue interactions between pDI6W and MDM2 not only prove that five residues Phe19', Trp22', Trp23', Leu26' and Thr27' from pDI6W can produce strong interaction with MDM2, but also show that CH-π, CH-CH and π-π interactions drive the binding of pDI6W in the hydrophobic cleft of MDM2. This study can provide theoretical helps for anti-cancer drug designs. (authors)

Quantification of thiols and disulfides

DEFF Research Database (Denmark)

Winther, Jakob R.; Thorpe, Colin

2014-01-01

lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions.......Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great...

Additional disulfide bonds in insulin

DEFF Research Database (Denmark)

Vinther, Tine N; Pettersson, Ingrid; Huus, Kasper

2015-01-01

The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin's B......-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the Cβ cut-off distance had...... in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus...

Diphenylphenoxy-Thiophene-PDI Dimers as Acceptors for OPV Applications with Open Circuit Voltage Approaching 1 Volt

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Caterina Stenta

2018-03-01

Full Text Available Two new perylenediimides (PDIs have been developed for use as electron acceptors in solution-processed bulk heterojunction solar cells. The compounds were designed to exhibit maximal solubility in organic solvents, and reduced aggregation in the solid state. In order to achieve this, diphenylphenoxy groups were used to functionalize a monomeric PDI core, and two PDI dimers were bridged with either one or two thiophene units. In photovoltaic devices prepared using PDI dimers and a monomer in conjunction with PTB7, it was found that the formation of crystalline domains in either the acceptor or donor was completely suppressed. Atomic force microscopy, X-ray diffraction, charge carrier mobility measurements and recombination kinetics studies all suggest that the lack of crystallinity in the active layer induces a significant drop in electron mobility. Significant surface recombination losses associated with a lack of segregation in the material were also identified as a significant loss mechanism. Finally, the monomeric PDI was found to have sub-optimum LUMO energy matching the cathode contact, thus limiting charge carrier extraction. Despite these setbacks, all PDIs produced high open circuit voltages, reaching almost 1 V in one particular case.

The NSW radiation therapy PDY educational program. A review of the 2002 and 2003 programs

International Nuclear Information System (INIS)

Beldham-Collins, Rachael

2005-01-01

Each month, nine radiation oncology departments in the public health sector in New South Wales host a one-day educational program for graduate practitioners completing their professional development year (PDY). Various topics are presented which are considered relevant to graduates in the transition from the academic environment to full time work in busy clinical departments. This paper presents the findings of a review of the NSW State Radiation Therapy PDY educational program for 2002 and 2003 instigated by the NSW Chiefs Group. The program was evaluated to determine whether it enhanced the learning experiences of graduate practitioners during the PDY. The results indicate the educational program has been successful in enhancing the skills of the radiation therapy graduate practitioners. This finding suggests the program is having a positive impact on the experience of the graduate practitioners within radiation therapy in NSW. Copyright (2005) Australian Institute of Radiography

Note: Durability analysis of optical fiber hydrogen sensor based on Pd-Y alloy film.

Science.gov (United States)

Huang, Peng-cheng; Chen, You-ping; Zhang, Gang; Song, Han; Liu, Yi

2016-02-01

The Pd-Y alloy sensing film has an excellent property for hydrogen detection, but just for one month, the sensing film's property decreases seriously. To study the failure of the sensing film, the XPS spectra analysis was used to explore the chemical content of the Pd-Y alloy film, and analysis results demonstrate that the yttrium was oxidized. The paper presented that such an oxidized process was the potential reason of the failure of the sensing film. By understanding the reason of the failure of the sensing film better, we could improve the manufacturing process to enhance the property of hydrogen sensor.

Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry

DEFF Research Database (Denmark)

Cramer, Christian N; Haselmann, Kim F; Olsen, Jesper V

2016-01-01

in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide...... link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we...... established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions....

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

Science.gov (United States)

Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

2017-02-01

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

Experimentación del uso didáctico de la pizarra digital interactiva (PDI en el aula: plan formativo y resultados

Directory of Open Access Journals (Sweden)

Maria DOMINGO COSCOLLOLA

2014-03-01

Full Text Available En la investigación que presentamos, Mimio Interactive España, pretendemos ir impulsando la integración de las Tecnologías de la Información y de la Comunicación (TIC, en las aulas, a partir del uso de la pizarra digital interactiva (PDI Mimio con un plan de formación permanente y contextualizado. Este uso de la pdi se complementa con 3 accesorios (una tableta interactiva inalámbrica Wacom, un ratón inalámbrico Gyration y un teclado inalámbrico Gyration. Mimio Interactive España es una investigación colaborativa próxima a los planteamientos de la investigaciónacción donde participan 25 centros de educación primaria y secundaria de España durante un curso académico (13 centros son de primaria y 12 centros son de secundaria. En los seminarios que se realizan en cada uno de estos centros, el profesorado participante recibe progresivamente formación didáctica y tecnológica orientada hacia la experimentación y la reflexión de la práctica docente con el uso de la PDI. Aportamos ideas significativas sobre el plan de formación impartido desde esta investigación. También, aportamos resultados sobre la experimentación del profesorado con la PDI en sus aulas. Concretamente, resultados sobre la intensidad de uso de la PDI, sobre el uso de los accesorios de la PDI, sobre las actividades más habituales que se realizan en el aula, sobre las ventajas y los inconvenientes del uso de la PDI en la práctica educativa y sobre las valoraciones de los aprendizajes del alumnado con el uso de la PDI en el proceso de enseñanza y aprendizaje realizado en el aula. En las conclusiones, se analizan y se valoran los resultados para ir avanzando en la práctica docente del siglo XXI. Un reto importante de hoy para todos.

Amino Acid Patterns around Disulfide Bonds

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Brett Drury

2010-11-01

Full Text Available Disulfide bonds provide an inexhaustible source of information on molecular evolution and biological specificity. In this work, we described the amino acid composition around disulfide bonds in a set of disulfide-rich proteins using appropriate descriptors, based on ANOVA (for all twenty natural amino acids or classes of amino acids clustered according to their chemical similarities and Scheffé (for the disulfide-rich proteins superfamilies statistics. We found that weakly hydrophilic and aromatic amino acids are quite abundant in the regions around disulfide bonds, contrary to aliphatic and hydrophobic amino acids. The density distributions (as a function of the distance to the center of the disulfide bonds for all defined entities presented an overall unimodal behavior: the densities are null at short distances, have maxima at intermediate distances and decrease for long distances. In the end, the amino acid environment around the disulfide bonds was found to be different for different superfamilies, allowing the clustering of proteins in a biologically relevant way, suggesting that this type of chemical information might be used as a tool to assess the relationship between very divergent sets of disulfide-rich proteins.

Marine Stratocumulus Properties from the FPDR - PDI as a Function of Aerosol during ORACLES

Science.gov (United States)

Small Griswold, J. D.; Heikkila, A.

2016-12-01

Aerosol-cloud interactions in the southeastern Atlantic (SEA) region were investigated during year 1 of the ObseRvations of Aerosols above CLouds and their intEractionS (ORACLES) field project in Aug-Sept 2016. This region is of interest due to seasonally persistent marine stratocumulus cloud decks that are an important component of the climate system due to their radiative and hydrologic impacts. The SEA deck is unique due to the interactions between these clouds and transported biomass burning aerosol during the July-October fire season. These biomass burning aerosol play multiple roles in modifying the cloud deck through interactions with radiation as absorbing aerosol and through modifications to cloud microphysical properties as cloud condensation nuclei. This work uses in situcloud data obtained with a Flight Probe Dual Range - Phase Doppler Interferometer (FPDR - PDI), standard aerosol instrumentation on board the NASA P-3, and reanalysis data to investigate Aerosol-Cloud Interactions (ACI). The FPDR - PDI provides unique cloud microphysical observations of individual cloud drop arrivals allowing for the computation of a variety of microphysical cloud properties including individual drop size, cloud drop number concentration, cloud drop size distributions, liquid water content, and cloud thickness. The FPDR - PDI measurement technique also provides droplet spacing and drop velocity information which is used to investigate turbulence and entrainment mixing processes. We use aerosol information such as average background aerosol amount (low, mid, high) and location relative to cloud (above or mixing) to sort FPDR - PDI cloud properties. To control for meteorological co-variances we further sort the data within aerosol categories by lower tropospheric stability, vertical velocity, and surface wind direction. We then determine general marine stratocumulus cloud characteristics under each of the various aerosol categories to investigate ACI in the SEA.

EFP1 is an ER stress-induced glycoprotein which interacts with the pro-apoptotic protein Par-4

Directory of Open Access Journals (Sweden)

Sarah Appel

2009-05-01

Full Text Available Sarah Appel1,2,6, Susanne Vetterkind1,2,6, Ansgar Koplin1,3, Barbara Maertens1,4, Meike Boosen1,5, Ute Preuss11The Institute of Genetics, University of Bonn, Bonn, Germany; 2Department of Health Sciences, Sargent College of Health and Rehabilitation Sciences, Boston University, Boston, MA, USA; 3Center for Molecular Biology Heidelberg (ZMBH, Heidelberg, Germany; 4Institute of Biochemistry II, University of Cologne, Cologne, Germany; 5Institute of Pharmacology and Toxicology, University Hospital of Johann Wolfgang Goethe-University Frankfurt am Main, Frankfurt am Main, Germany; 6These authors contributed equally to this work.Abstract: We have isolated the rat ortholog of EFP1 (EF-hand binding protein 1 as a novel interaction partner of the pro-apoptotic protein Par-4 (prostate apoptosis response-4. Rat EFP1 contains two thioredoxin domains, the COOH-terminal one harboring a CGFC motif, and has a similar protein domain structure as members of the protein disulfide isomerase (PDI family. In REF52.2 and CHO cells, EFP1 colocalized with the endoplasmic reticulum (ER marker PDI. Furthermore, EFP1 possesses catalytic activity as demonstrated by an insulin disulfide reduction assay. Western blot analysis revealed two EFP1 protein bands of approximately 136 and 155 kDa, representing different glycosylation states of the protein. Complex formation between EFP1 and Par-4 was confirmed in vitro and in vivo by co-immunoprecipitation, dot blot overlay and pull-down experiments. In CHO cells, coexpression of EFP1 and Par-4 resulted in enhanced Par-4-mediated apoptosis, which required the catalytic activity of EFP1. Interestingly, EFP1 was specifically upregulated in NIH3T3 cells after induction of ER stress by thapsigargin, tunicamycin, and brefeldin A, but not by agents that induce oxidative stress or ER-independent apoptosis. Furthermore, we could show that the induction of apoptosis by Ca2+ stress-inducing agents was significantly decreased after si

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

International Nuclear Information System (INIS)

Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

2013-01-01

Crystals of E. coli triosephosphate isomerase were obtained as a contaminant and its structure was determined to 1.85 Å resolution. Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure

Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

Science.gov (United States)

Jin, Li-Qun; Xu, Qi; Liu, Zhi-Qiang; Jia, Dong-Xu; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2017-09-01

Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min -1 . The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

Neurological findings in triosephosphate isomerase deficiency

NARCIS (Netherlands)

Poll-The, B. T.; Aicardi, J.; Girot, R.; Rosa, R.

1985-01-01

Two siblings with hemolytic anemia caused by triosephosphate isomerase deficiency developed a progressive neurological syndrome featuring dystonic movements, tremor, pyramidal tract signs, and evidence of spinal motor neuron involvement. Intelligence was unaffected. The findings in these patients

Dye adsorbates BrPDI, BrGly, and BrAsp on anatase TiO2(001) for dye-sensitized solar cell applications

Science.gov (United States)

Çakır, D.; Gülseren, O.; Mete, E.; Ellialtıoǧlu, Ş.

2009-07-01

Using the first-principles plane-wave pseudopotential method within density functional theory, we systematically investigated the interaction of perylenediimide (PDI)-based dye compounds (BrPDI, BrGly, and BrAsp) with both unreconstructed (UR) and reconstructed (RC) anatase TiO2(001) surfaces. All dye molecules form strong chemical bonds with surface in the most favorable adsorption structures. In UR-BrGly, RC-BrGly, and RC-BrAsp cases, we have observed that highest occupied molecular orbital and lowest unoccupied molecular orbital levels of molecules appear within band gap and conduction-band region, respectively. Moreover, we have obtained a gap narrowing upon adsorption of BrPDI on the RC surface. Because of the reduction in effective band gap of surface-dye system and possibly achieving the visible-light activity, these results are valuable for photovoltaic and photocatalytic applications. We have also considered the effects of hydration of surface to the binding of BrPDI. It has been found that the binding energy drops significantly for the completely hydrated surfaces.

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

International Nuclear Information System (INIS)

Premkumar, Lakshmanane; Heras, Begoña; Duprez, Wilko; Walden, Patricia; Halili, Maria; Kurth, Fabian; Fairlie, David P.; Martin, Jennifer L.

2013-01-01

The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

Energy Technology Data Exchange (ETDEWEB)

Premkumar, Lakshmanane, E-mail: p.lakshmanane@imb.uq.edu.au; Heras, Begoña; Duprez, Wilko; Walden, Patricia; Halili, Maria; Kurth, Fabian; Fairlie, David P.; Martin, Jennifer L., E-mail: p.lakshmanane@imb.uq.edu.au [University of Queensland, St Lucia, QLD 4067 (Australia)

2013-10-01

The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited

Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidusd-arabinose isomerase

International Nuclear Information System (INIS)

Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

2008-01-01

Recombinant B. pallidusd-arabinose isomerase was crystallized and diffraction data were collected to 2.3 Å resolution. d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidusd-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-altrose. Recombinant B. pallidusd-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution

Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

DEFF Research Database (Denmark)

Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

2010-01-01

Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...... has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions...

Multiple ways to make disulfides

DEFF Research Database (Denmark)

Bulleid, Neil J; Ellgaard, Lars

2011-01-01

Our concept of how disulfides form in proteins entering the secretory pathway has changed dramatically in recent years. The discovery of endoplasmic reticulum (ER) oxidoreductin 1 (ERO1) was followed by the demonstration that this enzyme couples oxygen reduction to de novo formation of disulfides...

Screening and selection of wild strains for L-arabinose isomerase production

Directory of Open Access Journals (Sweden)

R. M. Manzo

2013-12-01

Full Text Available The majority of L-arabinose isomerases have been isolated by recombinant techniques, but this methodology implies a reduced technological application. For this reason, 29 bacterial strains, some of them previously characterized as L-arabinose isomerase producers, were assayed as L-arabinose fermenting strains by employing conveniently designed culture media with 0.5% (w/v L-arabinose as main carbon source. From all evaluated bacterial strains, Enterococcus faecium DBFIQ ID: E36, Enterococcus faecium DBFIQ ID: ETW4 and Pediococcus acidilactici ATCC ID: 8042 were, in this order, the best L-arabinose fermenting strains. Afterwards, to assay L-arabinose metabolization and L-arabinose isomerase activity, cell-free extract and saline precipitated cell-free extract of the three bacterial cultures were obtained and the production of ketoses was determined by the cysteine carbazole sulfuric acid method. Results showed that the greater the L-arabinose metabolization ability, the higher the enzymatic activity achieved, so Enterococcus faecium DBFIQ ID: E36 was selected to continue with production, purification and characterization studies. This work thus describes a simple microbiological method for the selection of L-arabinose fermenting bacteria for the potential production of the enzyme L-arabinose isomerase.

Widespread Disulfide Bonding in Proteins from Thermophilic Archaea

Directory of Open Access Journals (Sweden)

Julien Jorda

2011-01-01

Full Text Available Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

Widespread disulfide bonding in proteins from thermophilic archaea.

Science.gov (United States)

Jorda, Julien; Yeates, Todd O

2011-01-01

Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

Science.gov (United States)

Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

2018-01-01

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

Autoinducer-2 Quorum Sensing Contributes to Regulation of Microcin PDI in Escherichia coli

Directory of Open Access Journals (Sweden)

Shao-Yeh Lu

2017-12-01

Full Text Available The Escherichia coli quorum sensing (QS signal molecule, autoinducer-2 (AI-2, reaches its maximum concentration during mid-to-late growth phase after which it quickly degrades during stationary phase. This pattern of AI-2 concentration coincides with the up- then down-regulation of a recently described microcin PDI (mccPDI effector protein (McpM. To determine if there is a functional relationship between these systems, a prototypical mccPDI-expressing strain of E. coli 25 was used to generate ΔluxS, ΔlsrACDBFG (Δlsr, and ΔlsrR mutant strains that are deficient in AI-2 production, transportation, and AI-2 transport regulation, respectively. Trans-complementation, RT-qPCR, and western blot assays were used to detect changes of microcin expression and synthesis under co-culture and monoculture conditions. Compared to the wild-type strain, the AI-2-deficient strain (ΔluxS and -uptake negative strain (Δlsr were >1,000-fold less inhibitory to susceptible bacteria (P < 0.05. With in trans complementation of luxS, the AI-2 deficient mutant reduced the susceptible E. coli population by 4-log, which was within 1-log of the wild-type phenotype. RT-qPCR and western blot results for the AI-2 deficient E. coli 25 showed a 5-fold reduction in mcpM transcription with an average 2-h delay in McpM synthesis. Furthermore, overexpression of sRNA micC and micF (both involved in porin protein regulation was correlated with mcpM regulation, consistent with a possible link between QS and mcpM regulation. This is the direct first evidence that microcin regulation can be linked to quorum sensing in a Gram-negative bacterium.

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae.

Science.gov (United States)

Miller, Kristen P; Gowtham, Yogender Kumar; Henson, J Michael; Harcum, Sarah W

2012-01-01

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

21 CFR 862.1570 - Phosphohexose isomerase test system.

Science.gov (United States)

2010-04-01

.... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma. (b...

The secreted l-arabinose isomerase displays anti-hyperglycemic effects in mice

OpenAIRE

Rhimi, Moez; Bermudez-Humaran, Luis G.; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, H?la; Langella, Philippe; Maguin, Emmanuelle

2015-01-01

Background The l-arabinose isomerase is an intracellular enzyme which converts l-arabinose into l-ribulose in living systems and d-galactose into d-tagatose in industrial processes and at industrial scales. d-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The d-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive l-arabinose isomerase should be thermoactive and a...

CuI-Catalyzed: One-Pot Synthesis of Diaryl Disulfides from Aryl Halides and Carbon Disulfide

Directory of Open Access Journals (Sweden)

Mohammad Soleiman-Beigi

2013-01-01

Full Text Available A new application of carbon disulfide in the presence of KF/Al2O3 is reported for the synthesis of organic symmetrical diaryl disulfides. These products were synthesized by one-pot reaction of aryl halides with the in situ generated trithiocarbonate ion in the presence of copper under air atmosphere.

How thioredoxin dissociates its mixed disulfide.

Directory of Open Access Journals (Sweden)

Goedele Roos

2009-08-01

Full Text Available The dissociation mechanism of the thioredoxin (Trx mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC, was used. In this structure, a Cys29(Trx-Cys89(ArsC intermediate disulfide is formed by the nucleophilic attack of Cys29(Trx on the exposed Cys82(ArsC-Cys89(ArsC in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32(Trx in contact with Cys29(Trx. Cys32(Trx is activated for its nucleophilic attack by hydrogen bonds, and Cys32(Trx is found to be more reactive than Cys82(ArsC. Additionally, Cys32(Trx directs its nucleophilic attack on the more susceptible Cys29(Trx and not on Cys89(ArsC. This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.

Purification, crystallization and preliminary crytallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus

NARCIS (Netherlands)

Akerboom, A.P.; Turnbull, A.P.; Hargreaves, D.; Fischer, M.; Geus, de D.; Sedelnikova, S.E.; Berrisford, J.M.; Baker, P.J.; Verhees, C.H.; Oost, van der J.; Rice, D.W.

2003-01-01

The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

International Nuclear Information System (INIS)

Lobley, Carina M. C.; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E.; Nettleship, Joanne E.; Brandao-Neto, Jose; Owens, Raymond J.; O’Toole, Paul W.; Walsh, Martin A.

2012-01-01

The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures. The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography

Radioiodine-labeled disulfide: a novel radiotracer for evaluation of tumor uptake

Energy Technology Data Exchange (ETDEWEB)

Ryu, E. K.; Choi, Y. S.; Byun, S. S.; Baek, J. Y.; Lee, K. H.; Kim, S. E.; Choi, Y.; Kim, B. T. [Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

2002-07-01

Diallyl disulfide found in garlic has been known to inhibit the growth of various cancer cells. In this study, iodine-substituted disulfides were synthesized and their growth inhibitory effects on cancer cells (SUN C5 and MCF-7) were investigated. Dibenzyl disulfide was labeled with {sup 123}I/{sup 125}I for evaluation of tumor uptake. Halogen-substituted disulfides were synthesized using 2,2'-dithiobis(benzothiazole) and one equivalent each of the corresponding thiols. Growth inhibition studies were performed on cancer cells that were grown at 37 .deg. C for 48 hr prior to exposure to the disulfides. Radioiodine-labeled disulfide was prepared by halogen exchange reaction on the 4-bromodibenzyl disulfide in the presence of Na{sup 123}I/{sup 125}I and CuCl at 150 .deg. C for 60 min, followed by HPLC purification. Uptake of the radioactivity to SUN C5 cells was measured as a function of time, and inhibition studies were performed in the presence of either S-methyl methanethiosulfonate (MMTS) or diallyl disulfide. Disulfides were synthesized in the high yields (90%). Tumor growth inhibition studies by the 3 iododisulfides showed the inhibition (>95%) comparable to diallyl disulfide (100%). Cu(I)-assisted radioiodination gave 4-{sup 123}I/{sup 125}I-iododibenzyl disulfide in overall 30-40% radiochemical yield and with high specific activity. Cell uptake studies of the radiolabeled disulfide showed a time-dependent increase of the uptake (4-fold increase from 15 min to 2 hr). Both MMTS, a glutathione depleting agent, and diallyl disulfide reduced the uptake of the radioactivity in a dose-dependent manner. Inhibition studies suggest that uptake of disulfide to the tumor cells could be mediated by thiol-disulfide exchange. This study demonstrates that radioiodine-labeled dibenzyl disulfide may be useful for evaluation of tumor uptake.

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

DEFF Research Database (Denmark)

Spetzler, J C; Westphal, V; Winther, Jakob R.

1998-01-01

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Directory of Open Access Journals (Sweden)

W. Lucca-Junior

2009-02-01

Full Text Available Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18 shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated wild-type human GnRHR (hGnRHR or mutant GnRHR (Cys14Ala and Cys200Ala and pcDNA3.1 without insert (empty vector or ERp18 cDNA (75 ng/well, pre-loaded for 18 h with 1 µCi myo-[2-3H(N]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Qualification according to PDI's techniques UT EPRI methodology Phased Array for the inspection of vessels of PWR reactor with advanced robotic equipment; Cualificacion segun metodologia PDI de EPRI de te cnicas UT Phased Array para la inspeccion de vasijas de reactor PWR con eq uipos roboticos avanzados

Energy Technology Data Exchange (ETDEWEB)

Gadea, J. R.; Gonzalez, P.; Fernandez, F.

2014-07-01

The techniques and procedures qualified in the program EPRI PDI are directly applicable in plants whose reference code is ASME XI - specifically the Appendix VIII-, mainly USA and countries in which it is established American PWR technology. While countries with reactors in operation technology ABB (Sweden) or type VVER (Finland and Eastern countries) requires a qualification of specific technical type ENIQ, PDI qualification is a valuable reference since it allows to deal with such qualifications with guarantees. (Author)

Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

DEFF Research Database (Denmark)

Westphal, V; Darby, N J; Winther, Jakob R.

1999-01-01

to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast......-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast....

Dynamic combinatorial chemistry with diselenides and disulfides in water

DEFF Research Database (Denmark)

Rasmussen, Brian; Sørensen, Anne; Gotfredsen, Henrik

2014-01-01

Diselenide exchange is introduced as a reversible reaction in dynamic combinatorial chemistry in water. At neutral pH, diselenides are found to mix with disulfides and form dynamic combinatorial libraries of diselenides, disulfides, and selenenylsulfides. This journal is......Diselenide exchange is introduced as a reversible reaction in dynamic combinatorial chemistry in water. At neutral pH, diselenides are found to mix with disulfides and form dynamic combinatorial libraries of diselenides, disulfides, and selenenylsulfides. This journal is...

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Directory of Open Access Journals (Sweden)

Hahn-Hägerdal Bärbel

2008-10-01

Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

Science.gov (United States)

Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

2006-07-01

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

Soft Computing Methods for Disulfide Connectivity Prediction.

Science.gov (United States)

Márquez-Chamorro, Alfonso E; Aguilar-Ruiz, Jesús S

2015-01-01

The problem of protein structure prediction (PSP) is one of the main challenges in structural bioinformatics. To tackle this problem, PSP can be divided into several subproblems. One of these subproblems is the prediction of disulfide bonds. The disulfide connectivity prediction problem consists in identifying which nonadjacent cysteines would be cross-linked from all possible candidates. Determining the disulfide bond connectivity between the cysteines of a protein is desirable as a previous step of the 3D PSP, as the protein conformational search space is highly reduced. The most representative soft computing approaches for the disulfide bonds connectivity prediction problem of the last decade are summarized in this paper. Certain aspects, such as the different methodologies based on soft computing approaches (artificial neural network or support vector machine) or features of the algorithms, are used for the classification of these methods.

The synthesis of unsymmetric disulfides for use as radio-protectives

International Nuclear Information System (INIS)

Chang, S.H.H.

1988-01-01

Unsymmetric disulfides with radioprotective potential were synthesized by linking biomolecules, and related substances, to known radio-protective aminothiols via a disulfide bond. The biomolecules used in this research include mercaptoalcohols, mercaptopyridines and mercaptophenothiazines. Unsymmetric disulfides were synthesized by reacting two thiols with diethyl azodicarboxylate sequentially at low temperature. The reactions of thiols with thiosulfinate were studied as an alternative for synthesizing disulfides. A cross-linked polystyrene was thiolated by different reagents. The thiolation of polymers is part of a methodological study using solid phase synthesis to synthesize unsymmetric disulfides

Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity

DEFF Research Database (Denmark)

Nørgaard, P; Winther, Jakob R.

2001-01-01

to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight...... into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild...

75 FR 8080 - Proposed Collection; Comment Request; Investigating the Causes of Post Donation Information (PDI...

Science.gov (United States)

2010-02-23

.... Donors often fail to report a risk that would have resulted in deferral. This deferral risk may be... risk may be at the next donation event, many examples of PDI are not disclosed nor discovered until... into donor understanding of the screening process and their feelings about the process and blood...

A single disulfide bond disruption in the β3 integrin subunit promotes thiol/disulfide exchange, a molecular dynamics study.

Directory of Open Access Journals (Sweden)

Lihie Levin

Full Text Available The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys(567-Cys(581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.

Widespread Disulfide Bonding in Proteins from Thermophilic Archaea

OpenAIRE

Jorda, Julien; Yeates, Todd O.

2011-01-01

Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaea...

Thiol-disulfide exchange in peptides derived from human growth hormone.

Science.gov (United States)

Chandrasekhar, Saradha; Epling, Daniel E; Sophocleous, Andreas M; Topp, Elizabeth M

2014-04-01

Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Kinetic and Thermodynamic Aspects of Cellular Thiol-Disulfide Redox Regulation

DEFF Research Database (Denmark)

Jensen, Kristine Steen; Hansen, Rosa Erritzøe; Winther, Jakob R

2009-01-01

. In the cytosol regulatory disulfide bonds are typically formed in spite of the prevailing reducing conditions and may thereby function as redox switches. Such disulfide bonds are protected from enzymatic reduction by kinetic barriers and are thus allowed to exist long enough to elicit the signal. Factors......Regulation of intracellular thiol-disulfide redox status is an essential part of cellular homeostasis. This involves the regulation of both oxidative and reductive pathways, production of oxidant scavengers and, importantly, the ability of cells to respond to changes in the redox environment...... that affect the rate of thiol-disulfide exchange and stability of disulfide bonds are discussed within the framework of the underlying chemical foundations. This includes the effect of thiol acidity (pKa), the local electrostatic environment, molecular strain and entropy. Even though a thiol-disulfide...

Steric effects in peptide and protein exchange with activated disulfides.

Science.gov (United States)

Kerr, Jason; Schlosser, Jessica L; Griffin, Donald R; Wong, Darice Y; Kasko, Andrea M

2013-08-12

Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (β-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

Science.gov (United States)

Kim, P; Yoon, S H; Roh, H J; Choi, J H

2001-01-01

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

Chemoreactomic analysis of thiamine disulfide, thiamine hydrochloride, and benfotiamine molecules

Directory of Open Access Journals (Sweden)

O. A. Gromova

2017-01-01

Full Text Available Objective: to analyze the interactions that could indicate the potential pharmacological properties of the molecules of thiamin, thiamine disulfide, and others.Material and methods. The investigators simulated the properties of thiamine disulfide (bistiamin versus those of the reference molecules of thiamin hydrochloride and benfotiamine. The study was performed using chemoreactomic simulation that is the newest area in post-genome pharmacology.Results and discussion. Chemoreactomic analysis has shown that thiamine disulfide can inhibit the molecular receptors involved in blood pressure regulation: adrenoceptors, vasopressin receptor, and angiotensin receptor. Thiamine disulfide can inhibit the reuptake of serotonin, increase its levels, inhibit benzodiazepine receptor and dopamine reuptake, and enhance neuronal acetylcholine release to a large extent than benfotiamine. These molecular effects are consistent with the sedative and anticonvulsant action profile of thiamine disulfide. Simulation has indicated that thiamine disulfide has neuroprotective, anti-inflammatory, normolipidemic, and antitumor activities.Conclusion. The simulation results are confirmed by the available clinical and experimental findings and indicate the virtually unstudied molecular mechanisms of action of thiamine disulfide, benfotiamine, and thiamin hydrochloride. 

Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

Science.gov (United States)

Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

2016-07-01

Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

Bacterial L-arabinose isomerases: industrial application for D-tagatose production.

Science.gov (United States)

Boudebbouze, Samira; Maguin, Emmanuelle; Rhimi, Moez

2011-12-01

D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.

Impaired Thiol-Disulfide Balance in Acute Brucellosis.

Science.gov (United States)

Kolgelier, Servet; Ergin, Merve; Demir, Lutfi Saltuk; Inkaya, Ahmet Cagkan; Aktug Demir, Nazlim; Alisik, Murat; Erel, Ozcan

2017-05-24

The objective of this study was to examine a novel profile: thiol-disulfide homeostasis in acute brucellosis. The study included 90 patients with acute brucellosis, and 27 healthy controls. Thiol-disulfide profile tests were analyzed by a recently developed method, and ceruloplasmin levels were determined. Native thiol levels were 256.72 ± 48.20 μmol/L in the acute brucellosis group and 461.13 ± 45.37 μmol/L in the healthy group, and total thiol levels were 298.58 ± 51.78 μmol/L in the acute brucellosis group and 504.83 ± 51.05 μmol/L in the healthy group (p brucellosis than in the healthy controls (p brucellosis. The strong associations between thiol-disulfide parameters and a positive acute-phase reactant reflected the disruption of the balance between the antioxidant and oxidant systems. Since thiol groups act as anti-inflammatory mediators, the alteration in the thiol-disulfide homeostasis may be involved in brucellosis.

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2012-08-01

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase.

Science.gov (United States)

Mishra, Abha; Debnath Das, Meera

2002-01-01

pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60 degrees C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.

Effects of macromolecular crowding on protein conformational changes.

Directory of Open Access Journals (Sweden)

Hao Dong

2010-07-01

Full Text Available Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of "test" proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI, adenylate kinase (AdK, orotidine phosphate decarboxylase (ODCase, Trp repressor (TrpR, hemoglobin, DNA beta-glucosyltransferase, and Ap(4A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 A radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20-44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration. Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues and TrpR (98 residues]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will

On the photostability of the disulfide bond

DEFF Research Database (Denmark)

Stephansen, Anne Boutrup; Larsen, Martin Alex Bjørn; Klein, Liv Bærenholdt

2014-01-01

Photostability is an essential property of molecular building blocks of nature. Disulfides are central in the structure determination of proteins, which is in striking contradiction to the result that the S-S bond is a photochemically labile structural entity that cleaves to form free radicals upon...... on a sub 50 fs timescale without further ado. In a cyclic motif resembling the cysteine-disulfide bond in proteins, light can perturb the S-S bond to generate short-lived diradicaloid species, but the sulfur atoms are conformationally restricted by the ring that prevents the sulfur atoms from flying apart...... the photostability of disulfide-bonds must be ascribed a cyclic structural arrangement....

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

Science.gov (United States)

Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

Energy Technology Data Exchange (ETDEWEB)

Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

2011-12-14

Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

Characteristics of chalcone isomerase promoter in crabapple leaves ...

African Journals Online (AJOL)

Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of plants and chalcone isomerase (CHI) is one of the key enzymes in anthocyanin biosynthetic pathway. What characteristic is CHI promoter known as the regulation sequence of CHI gene, has been rarely investigated. We isolated A ...

Brain MRI findings of carbon disulfide poisoning

International Nuclear Information System (INIS)

Cha, Joo Hee; Kim, Mi Jung; Yim, Sang Hyuk; Kim, Sam Soo; Han, Heon; Kim, Rok Ho

2002-01-01

To evaluate the findings of brain MRI in patients with carbon disulfide poisoning. Ninety-one patients who had suffered carbon disulfide poisoning [male:female=87:4; age, 32-74 (mean 53.3) years] were included in this study. To determine the extent of white matter hyperintensity (Grade 0-V) and lacunar infarction, T2-weighted MR imaging of the brain was performed. T2-weighted images depicted white matter hyperintensity in 70 patients (76.9%) and lacunar infarcts in 27 (29.7%). In these patients, the prevalent findings at T2-weighted MR imaging of the brain were white matter hyperintensity and lacunar infarcts. Disturbance of the cardiovascular system by carbon disulfide might account for these results

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.

Science.gov (United States)

Lakbub, Jude C; Shipman, Joshua T; Desaire, Heather

2018-04-01

Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass

Escherichia coli rpiA gene encoding ribose phosphate isomerase A

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Maigaard, Marianne

1993-01-01

The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque......The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...

Interaction of p53 with prolyl isomerases: Healthy and unhealthy relationships.

Science.gov (United States)

Mantovani, Fiamma; Zannini, Alessandro; Rustighi, Alessandra; Del Sal, Giannino

2015-10-01

The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets. Copyright © 2015 Elsevier B.V. All rights reserved.

JB_054_Supplementary_Tables.docx

Indian Academy of Sciences (India)

LENOVO

TRI 10032, T. aestivum L. var. aestivum, Mexico, Spring, Q4 ... TRI 10311, T. aestivum L. var. aestivum, Japan, Spring, Q2 ..... precursor (sucrose export defective 1), dihydrolipoamide dehydrogenase precursor, disulfide-isomerase precursor.

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

Science.gov (United States)

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

Cloning and characterization of peptidylprolyl isomerase B in the ...

African Journals Online (AJOL)

Peptidylprolyl isomerases (PPIases) play essential roles in protein folding and are implicated in immune response and cell cycle control. Our previous proteomic analysis indicated that Bombyx mori PPIases may be involved in anti- Bombyx mori nucleopolyhedrovirus (BmNPV) response. To help investigate this mechanism, ...

Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

International Nuclear Information System (INIS)

Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

2007-01-01

The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution. Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I2 1 2 1 2 1 , with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å

Measurement of glutathione-protein mixed disulfides

International Nuclear Information System (INIS)

Livesey, J.C.; Reed, D.J.

1984-01-01

The development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols has been undertaken. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein has been found when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in the chromatography system with a retention volume similar to cysteine

Structures and related properties of helical, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

Pagel, Mark D. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

1993-11-01

The three dimensional structure of several peptides were determined by NMR spectroscopy and distance geometry calculations. Each peptide formed a predictable, rigid structure, consisting of an α-helix, a "scaffold" region which packed along one face of the helix, and two disulfide bridges which covalently connect the helix and scaffold regions. The peptide Apa-M5 was designed to constrain the M5 peptide from MLCK in a helical geometry using the apamin disulfide scaffold. This scaffold constrains the N- terminal end of the helix with two disulfide bridges and a reverse turn. Like the M5 peptide, Apa-M5 was found to bind calmodulin in a Ca²⁺-dependent 1:1 stoichiometry. However, the dissociation constant of the (Apa-M5)-calmodulin complex, 107 nM, was 100-fold higher than the dissociation constant of the M5-calmodulin complex. This difference was due to a putative steric overlap between the Apa-M5 scaffold and calmodulin. The peptide Apa-Cro was designed to replace the large structural protein matrix of λ Cro with the apamin disulfide scaffold. However, Apa-Cro did not bind the consensus DNA operator half-site of λ Cro, probably due to a steric overlap between the Apa-Cro disulfide framework and the DNA. The amino acid sequence of the scaffold-disulfide bridge arrangement of the peptide Max was derived from the core sequence of scyllatoxin, which contains an α-helix constrained at the C-terminal end by two disulfide bridges and a two-stranded βsheet scaffold. Max was shown to fold with >84% yield to form a predictable, stable structure that is similar to scyllatoxin. The folding and stability properties of Max make this scaffold and disulfide bridge arrangement an ideal candidate for the development of hybrid sequence peptides. The dynamics of a fraying C-terminal end of the helix of the peptide Apa-AlaN was determined by analysis of ¹⁵N NMR relaxation properties.

Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

OpenAIRE

Maruta, Takanori; Yonemitsu, Miki; Yabuta, Yukinori; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

2008-01-01

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 ...

Thiol/disulfide redox states in signaling and sensing

Science.gov (United States)

Go, Young-Mi; Jones, Dean P.

2015-01-01

Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady-states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling, and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities. PMID:23356510

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide

NARCIS (Netherlands)

Langedijk, J.P.M.; Veelen, van P.A.; Schaaper, W.M.M.; Ru, de A.H.; Meloen, R.H.; Hulst, M.M.

2002-01-01

Erns is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. Erns is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine

Quantifying the global cellular thiol-disulfide status

DEFF Research Database (Denmark)

Hansen, Rosa E; Roth, Doris; Winther, Jakob R

2009-01-01

It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been...... determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data...... cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active...

Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets.

Science.gov (United States)

Indu, S; Kochat, V; Thakurela, S; Ramakrishnan, C; Varadarajan, Raghavan

2011-01-01

Cross-strand disulfides bridge two cysteines in a registered pair of antiparallel β-strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross-strand disulfides. Seventy-six cross-strand disulfides were found of which 75 and 1 occurred at non-hydrogen-bonded (NHB) and hydrogen-bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T(m). All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG⁰ = -3.3 to -6.7 kcal/mol). The data demonstrate that introduction of cross-strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity. © 2010 Wiley-Liss, Inc.

A molybdenum disulfide/carbon nanotube heterogeneous complementary inverter.

Science.gov (United States)

Huang, Jun; Somu, Sivasubramanian; Busnaina, Ahmed

2012-08-24

We report a simple, bottom-up/top-down approach for integrating drastically different nanoscale building blocks to form a heterogeneous complementary inverter circuit based on layered molybdenum disulfide and carbon nanotube (CNT) bundles. The fabricated CNT/MoS(2) inverter is composed of n-type molybdenum disulfide (MOS(2)) and p-type CNT transistors, with a high voltage gain of 1.3. The CNT channels are fabricated using directed assembly while the layered molybdenum disulfide channels are fabricated by mechanical exfoliation. This bottom-up fabrication approach for integrating various nanoscale elements with unique characteristics provides an alternative cost-effective methodology to complementary metal-oxide-semiconductors, laying the foundation for the realization of high performance logic circuits.

Development of a Multimarker Urine Test for Prostate Cancer

Science.gov (United States)

2017-10-01

connected to a chemically etched 20 μm i.d. fused-silica emitter via a Valco stainless steel union. Four μL of individual peptide fractions (total...isomerase family A, member 17 (PD1A17 or member 17); or secreted cement gland protein XAG-2 homolog, AGR2 belongs to the protein disulfide 5 isomerase...silica emitter via a Valco stainless steel union. Four microliters of individual peptide fractions (total volume 20 µL) following PRISM were

Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.

Directory of Open Access Journals (Sweden)

Joel C Watts

2009-10-01

Full Text Available The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP(C interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd and Shadoo (Sprn, two mammalian PrP(C paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP(C and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP(C with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP(Sc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP(C organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.

Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.

Science.gov (United States)

Watts, Joel C; Huo, Hairu; Bai, Yu; Ehsani, Sepehr; Jeon, Amy Hye Won; Won, Amy Hye; Shi, Tujin; Daude, Nathalie; Lau, Agnes; Young, Rebecca; Xu, Lei; Carlson, George A; Williams, David; Westaway, David; Schmitt-Ulms, Gerold

2009-10-01

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C)) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP(C) interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP(C) paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP(C) and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP(C) with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP(Sc). A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP(C) organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.

Endoplasmic reticulum (ER Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

Directory of Open Access Journals (Sweden)

Thomas eSimmen

2014-10-01

Full Text Available Endoplasmic reticulum (ER chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed bulk flow, ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER. However, solid tumors are characterized by the increased production of reactive oxygen species (ROS, combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response (UPR upregulate ER chaperones and oxidoreductases. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the production of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an eat-me signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI, Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies.

Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

Directory of Open Access Journals (Sweden)

Michelle T. Barati

2016-01-01

Full Text Available The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG growth conditions as compared to isoosmotic/normal glucose control (NG⁎ conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1 proteins involved in cell survival/cell signaling and (2 proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB, supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase, suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

Impairment of visual function and retinal ER stress activation in Wfs1-deficient mice.

Directory of Open Access Journals (Sweden)

Delphine Bonnet Wersinger

Full Text Available Wolfram syndrome is an early onset genetic disease (1/180,000 featuring diabetes mellitus and optic neuropathy, associated to mutations in the WFS1 gene. Wfs1-/- mouse model shows pancreatic beta cell atrophy, but its visual performance has not been investigated, prompting us to study its visual function and histopathology of the retina and optic nerve. Electroretinogram and visual evoked potentials (VEPs were performed in Wfs1-/- and Wfs1+/+ mice at 3, 6, 9 and 12 months of age. Fundi were pictured with Micron III apparatus. Retinal ganglion cell (RGC abundance was determined from Brn3a immunolabeling of retinal sections. RGC axonal loss was quantified by electron microscopy in transversal optic nerve sections. Endoplasmic reticulum stress was assessed using immunoglobulin binding protein (BiP, protein disulfide isomerase (PDI and inositol-requiring enzyme 1 alpha (Ire1α markers. Electroretinograms amplitudes were slightly reduced and latencies increased with time in Wfs1-/- mice. Similarly, VEPs showed decreased N+P amplitudes and increased N-wave latency. Analysis of unfolded protein response signaling revealed an activation of endoplasmic reticulum stress in Wfs1-/- mutant mouse retinas. Altogether, progressive VEPs alterations with minimal neuronal cell loss suggest functional alteration of the action potential in the Wfs1-/- optic pathways.

Obtaining mutants of Streptomyces griseoflavus strain 1339, producers of glucose isomerase, following gamma irradiation

International Nuclear Information System (INIS)

Dzhedzheva, G.; Stoeva, N.; Stojchev, M.

1990-01-01

A water suspension of Streptomyces griseoflavus strain 1339 spores of a density of 8.7.10 6 spores/cm 3 is gamma irradiated ( 60 Co, RHM-γ-20, 30.3 Gy/min). The survival of Streptomyces griseoflavus strain 1339 spores was determined depending on radiation doses, exposure times and incubation temperature. Five major morphological types of colonies were isolated, characterized by different levels of glucose isomerase activity. Maximum specific glucose isomerase activity (GIU/g) was attained after the third gamma irradiation step using a dose of 3000 Gy. 2 tabs., 3 figs., 7 refs

UV Photofragmentation Dynamics of Protonated Cystine: Disulfide Bond Rupture.

Science.gov (United States)

Soorkia, Satchin; Dehon, Christophe; Kumar, S Sunil; Pedrazzani, Mélanie; Frantzen, Emilie; Lucas, Bruno; Barat, Michel; Fayeton, Jacqueline A; Jouvet, Christophe

2014-04-03

Disulfide bonds (S-S) play a central role in stabilizing the native structure of proteins against denaturation. Experimentally, identification of these linkages in peptide and protein structure characterization remains challenging. UV photodissociation (UVPD) can be a valuable tool in identifying disulfide linkages. Here, the S-S bond acts as a UV chromophore and absorption of one UV photon corresponds to a σ-σ* transition. We have investigated the photodissociation dynamics of protonated cystine, which is a dimer of two cysteines linked by a disulfide bridge, at 263 nm (4.7 eV) using a multicoincidence technique in which fragments coming from the same fragmentation event are detected. Two types of bond cleavages are observed corresponding to the disulfide (S-S) and adjacent C-S bond ruptures. We show that the S-S cleavage leads to three different fragment ions via three different fragmentation mechanisms. The UVPD results are compared to collision-induced dissociation (CID) and electron-induced dissociation (EID) studies.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

OpenAIRE

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-01-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally...

Modification of molybdenum disulfide in methanol solvent for hydrogen evolution reaction

Science.gov (United States)

Niyitanga, Theophile; Jeong, Hae Kyung

2018-05-01

Molybdenum disulfide is a promising catalyst to replace the expensive platinum as an electrocatalyst but needs to be modified to present excellent electrocatalytic properties. Herein, we successfully modify molybdenum disulfide in methanol solvent for hydrogen evolution reaction by using a simple hydrothermal method. Overpotential reduced to -0.6 V from -1.5 V, and energy band gap decreased from 1.73 eV to 1.58 eV after the modification. The modified molybdenum disulfide also demonstrated lower resistance (42 Ω) at high frequency (1000 kHz) compared with that (240 Ω) of the precursor, showing that conductivity of the modified molybdenum disulfide has improved.

Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

2014-01-01

Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein...... reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" (12C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems....... extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide...

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.

Directory of Open Access Journals (Sweden)

Minh Tan Nguyen

Full Text Available Human vascular endothelial growth factor (VEGF is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF. We created seven N-terminal fusion tag constructs with hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, human protein disulfide isomerase (PDI, and the b'a' domain of PDI (PDIb'a', and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from Bacillus coagulans NL01.

Science.gov (United States)

Zheng, Zhaojuan; Lin, Xi; Jiang, Ting; Ye, Weihua; Ouyang, Jia

2016-08-01

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid. The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7-8 but with a high temperature maximum of 80-85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni(2+) and Co(2+), respectively. Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

Thermodynamic and mechanical effects of disulfide bonds in CXCLl7 chemokine

Science.gov (United States)

Singer, Christopher

Chemokines are a family of signaling proteins mainly responsible for the chemotaxis of leukocytes, where their biological activity is modulated by their oligomerization state. Here, the dynamics and thermodynamic stability are characterized in monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines. The effects of dimerization and disulfide bond formation are investigated using computational methods that include molecular dynamics (MD) simulations and the Distance Constraint Model (DCM). A consistent picture emerges for the effect of dimerization and role of the Cys5-Cys31 and Cys7- Cys47 disulfide bonds. Surprisingly, neither disulfide bond is critical for maintaining structural stability in the monomer or dimer, although the monomer is destabilized more than the dimer upon removal of disulfide bonds. Instead, it is found that disulfide bonds influence the native state dynamics as well as modulates the relative stability between monomer and dimer. The combined analysis elucidates how CXCL7 is mechanically stable as a monomer, and how upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present in each domain, and the homodimer is least stable relative to its two monomers. These results suggest the highly conserved disulfide bonds in chemokines facilitate a structural mechanism for distinguishing functional characteristics between monomer and dimer.

Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

Science.gov (United States)

Medraño-Fernandez, Iria; Fagioli, Claudio; Mezghrani, Alexandre; Otsu, Mieko; Sitia, Roberto

2014-04-01

To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

Protein disulfide bond generation in Escherichia coli DsbB–DsbA

International Nuclear Information System (INIS)

Inaba, Kenji

2008-01-01

The crystal structure of the DsbB–DsbA–ubiquinone ternary complex has revealed a mechanism of protein disulfide bond generation in Escherichia coli. Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB–DsbA–ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA–DsbB oxidative and the DsbC–DsbD reductive pathways has also been proposed

Preventing disulfide bond formation weakens non-covalent forces among lysozyme aggregates.

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Vijay Kumar Ravi

Full Text Available Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ∼50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.

The effect of tensile stress on the conformational free energy landscape of disulfide bonds.

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Padmesh Anjukandi

Full Text Available Disulfide bridges are no longer considered to merely stabilize protein structure, but are increasingly recognized to play a functional role in many regulatory biomolecular processes. Recent studies have uncovered that the redox activity of native disulfides depends on their C-C-S-S dihedrals, χ2 and χ'2. Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force-clamp spectroscopy and computer simulation. The χ2 and χ'2 angles have been found to change from conformations that are open to nucleophilic attack to sterically hindered, so-called closed states upon exerting tensile stress. In view of the growing evidence of the importance of C-C-S-S dihedrals in tuning the reactivity of disulfides, here we present a systematic study of the conformational diversity of disulfides as a function of tensile stress. With the help of force-clamp metadynamics simulations, we show that tensile stress brings about a large stabilization of the closed conformers, thereby giving rise to drastic changes in the conformational free energy landscape of disulfides. Statistical analysis shows that native TDi, DO and interchain Ig protein disulfides prefer open conformations, whereas the intrachain disulfide bridges in Ig proteins favor closed conformations. Correlating mechanical stress with the distance between the two a-carbons of the disulfide moiety reveals that the strain of intrachain Ig protein disulfides corresponds to a mechanical activation of about 100 pN. Such mechanical activation leads to a severalfold increase of the rate of the elementary redox S(N2 reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides.

[Deficiency of triosephosphate isomerase. Apropos of 2 new cases].

Science.gov (United States)

Delso Martínez, M C; Uriel Miñana, P; Pérez Lugmus, G; Giménez Mas, J A; Baldellou Vázquez, A

1983-08-01

Two siblings, born of a no consanguineous couple, a female and a male, affected by a severe and progressive neurological disease and chronic hemolytic anemia are presented. Their clinical, hematological, biochemical and pathological studies are discussed. One of the patients showed a triosephosphate isomerase deficiency and the carrier condition of their parents was tested. Commentaries about physiopathology of this disease are made.

Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

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W Wang

2009-01-01

Full Text Available Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl-phosphine (TCEP in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry

Science.gov (United States)

Switzar, Linda; Nicolardi, Simone; Rutten, Julie W.; Oberstein, Saskia A. J. Lesnik; Aartsma-Rus, Annemieke; van der Burgt, Yuri E. M.

2016-01-01

Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S-S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the "parent" disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.

Large area synthesis, characterization, and anisotropic etching of two dimensional tungsten disulfide films

International Nuclear Information System (INIS)

Mutlu, Zafer; Ozkan, Mihrimah; Ozkan, Cengiz S.

2016-01-01

Emergent properties of tungsten disulfide at the quantum confinement limit hold promise for electronic and optoelectronic applications. Here we report on the large area synthesis of atomically thin tungsten disulfide films with strong photoluminescence properties via sulfurization of the pre-deposited tungsten films. Detailed characterization of the pre-deposited tungsten films and tungsten disulfide films are performed using microscopy and spectroscopy methods. By directly heating tungsten disulfide films in air, we have shown that the films tend to be etched into a series of triangular shaped pits with the same orientations, revealing the anisotropic etching behavior of tungsten disulfide edges. Moreover, the dimensions of the triangular pits increase with the number of layers, suggesting a thickness dependent behavior of etching in tungsten disulfide films. This method offers a promising new avenue for engineering the edge structures of tungsten disulfide films. - Highlights: • Large-scale synthesis of WS_2 films is achieved via sulfurization of W films. • Annealing of W films leads to a substantial improvement in the quality of WS_2 films. • WS_2 films show laser power dependent photoluminescence characteristics. • WS_2 films are etched with well-oriented triangular pits upon annealing in air. • Anisotropic oxidative etching is greatly affected by the thickness of WS_2 films.

Analysis of Disulfide Bond Formation

NARCIS (Netherlands)

Braakman, Ineke; Lamriben, Lydia; van Zadelhoff, Guus; Hebert, Daniel N.

2017-01-01

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive

Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations

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Yusuke Nakatsu

2016-09-01

Full Text Available Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14. Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer’s disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions.

SAXS-WAXS studies of the low-resolution structure in solution of xylose/glucose isomerase from Streptomyces rubiginosus

Science.gov (United States)

Kozak, Maciej; Taube, Michał

2009-10-01

The structure and conformation of molecule of xylose/glucose isomerase from Streptomyces rubiginosus in solution (at pH 6 and 7.6; with and without the substrate) has been studied by small- and wide-angle scattering of synchrotron radiation (SAXS-WAXS). On the basis of the SAXS-WAXS data, the low-resolution structure in solution has been reconstructed using ab inito methods. A comparison of the models of glucose isomerase shows only small differences between the model in solution and the crystal structure.

Electrostatic influence of local cysteine environments on disulfide exchange kinetics.

Science.gov (United States)

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

1981-11-10

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

Intradomain Confinement of Disulfides in the Folding of Two Consecutive Modules of the LDL Receptor.

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Juan Martínez-Oliván

Full Text Available The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial" reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.

Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

DEFF Research Database (Denmark)

Maeda, Kenji; Hägglund, Per; Finnie, Christine

2006-01-01

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure...... of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays...... a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares...

Edge eigen-stress and eigen-displacement of armchair molybdenum disulfide nanoribbons

Energy Technology Data Exchange (ETDEWEB)

Wu, Quan; Li, Xi [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (MOE), University of Science and Technology Beijing, Beijing 100083 (China); Volinsky, Alex A., E-mail: volinsky@usf.edu [Department of Mechanical Engineering, University of South Florida, Tampa, FL 33620 (United States); Su, Yanjing, E-mail: yjsu@ustb.edu.cn [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (MOE), University of Science and Technology Beijing, Beijing 100083 (China)

2017-05-10

Edge effects on mechanical properties of armchair molybdenum disulfide nanoribbons were investigated using first principles calculations. The edge eigen-stress model was applied to explain the relaxation process of forming molybdenum disulfide nanoribbon. Edge effects on surface atoms fluctuation degree were obtained from each fully relaxed nanoribbon with different width. Changes of the relaxed armchair molybdenum disulfide nanoribbons structure can be expressed using hexagonal perimeters pattern. Based on the thickness change, relaxed armchair molybdenum disulfide nanoribbons tensile/compression tests were simulated, providing intrinsic edge elastic parameters, such as eigen-stress, Young's modulus and Poisson's ratio. - Highlights: • Edge effects on mechanical properties of armchair MoS{sub 2} nanoribbons were investigated. • Structure changes of different width armchair MoS{sub 2} nanoribbons were obtained. • Tensile/compressive tests were conducted to determine elastic constants. • Mechanical properties are compared for two and three dimensional conditions.

Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a.

Science.gov (United States)

Yamagami, T; Funatsu, G; Ishiguro, M

2000-06-01

The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.

Disulfide bond within mu-calpain active site inhibits activity and autolysis.

Science.gov (United States)

Lametsch, René; Lonergan, Steven; Huff-Lonergan, Elisabeth

2008-09-01

Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

International Nuclear Information System (INIS)

Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

1980-01-01

N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[ 14 C 2 ]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene

Antiviral propierties of 5,5'-dithiobis-2-nitrobenzoic acid and bacitracin against T-tropic human immunodeficiency virus type 1

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Rodriguez-Padilla Cristina

2011-03-01

Full Text Available Abstract Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB are agents known to inhibit protein disulfide isomerase (PDI, a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI. Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-β-gal (CD4 and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.

Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

Science.gov (United States)

Mast, Jan; Thiel, Heinz-Jürgen; König, Matthias

2014-01-01

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis. PMID:24352462

Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

Science.gov (United States)

Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A

2015-11-15

The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.

Studies on the production of glucose isomerase by Bacillus licheniformis

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Nwokoro Ogbonnaya

2015-09-01

Full Text Available This work reports the effects of some culture conditions on the production of glucose isomerase by Bacillus licheniformis. The bacterium was selected based on the release of 3.62 mg/mL fructose from the fermentation of glucose. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in a medium containing 0.5% xylose and 1% glycerol (specific activity = 6.88 U/mg protein. Media containing only xylose or glucose gave lower enzyme productivies (specific activities= 4.60 and 2.35 U/mg protein respectively. The effects of nitrogen substrates on glucose isomerase production showed that yeast extract supported maximum enzyme activity (specific activity = 5.24 U/mg protein. Lowest enzyme activity was observed with sodium trioxonitrate (specific activity = 2.44 U/mg protein. In general, organic nitrogen substrates supported higher enzyme productivity than inorganic nitrogen substrates. Best enzyme activity was observed in the presence of Mg2+ (specific activity = 6.85 U/mg protein while Hg2+ was inhibitory (specific activity = 1.02 U/mg protein. The optimum pH for best enzyme activity was 6.0 while optimum temperature for enzyme production was 50ºC.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

Science.gov (United States)

Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

2012-12-01

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis

NARCIS (Netherlands)

Kouwen, Thijs R. H. M.; Dubois, Jean-Yves F.; Freudl, Roland; Quax, Wim J.; van Dijl, Jan Maarten

2008-01-01

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of

Identification of the PDI-family member ERp90 as an interaction partner of ERFAD

DEFF Research Database (Denmark)

Riemer, Jan; Hansen, Henning G; Appenzeller-Herzog, C.

2011-01-01

that comprises five potential thioredoxin (Trx)-like domains. Mature ERp90 contains 10 cysteine residues, of which at least some form intramolecular disulfides. While none of the Trx domains contain a canonical Cys-Xaa-Xaa-Cys active-site motif, other conserved cysteines could endow the protein with redox...

Glucose(xylose isomerase production by Streptomyces sp. CH7 grown on agricultural residues

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Kankiya Chanitnun

2012-09-01

Full Text Available Streptomyces sp. CH7 was found to efficiently produce glucose(xylose isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

Inhibition of carbon disulfide on bio-desulfurization in the process of ...

African Journals Online (AJOL)

Biological desulfurization is a novel technology for the removal of hydrogen sulfide from some biogas or sour gas, in which there are always a certain amounts of carbon disulfide together with much hydrogen sulfide. Nowadays, carbon disulfide is found to have negative effect on the biological desulfurization, but seldom ...

Insulin analog with additional disulfide bond has increased stability and preserved activity

DEFF Research Database (Denmark)

Vinther, Tine N.; Norrman, Mathias; Ribel, Ulla

2013-01-01

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide...... bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin...... (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation...

Disulfide Bridges: Bringing Together Frustrated Structure in a Bioactive Peptide.

Science.gov (United States)

Zhang, Yi; Schulten, Klaus; Gruebele, Martin; Bansal, Paramjit S; Wilson, David; Daly, Norelle L

2016-04-26

Disulfide bridges are commonly found covalent bonds that are usually believed to maintain structural stability of proteins. Here, we investigate the influence of disulfide bridges on protein dynamics through molecular dynamics simulations on the cysteine-rich trypsin inhibitor MCoTI-II with three disulfide bridges. Correlation analysis of the reduced cyclic peptide shows that two of the three disulfide distances (Cys(11)-Cys(23) and Cys(17)-Cys(29)) are anticorrelated within ∼1 μs of bridge formation or dissolution: when the peptide is in nativelike structures and one of the distances shortens to allow bond formation, the other tends to lengthen. Simulations over longer timescales, when the denatured state is less structured, do not show the anticorrelation. We propose that the native state contains structural elements that frustrate one another's folding, and that the two bridges are critical for snapping the frustrated native structure into place. In contrast, the Cys(4)-Cys(21) bridge is predicted to form together with either of the other two bridges. Indeed, experimental chromatography and nuclear magnetic resonance data show that an engineered peptide with the Cys(4)-Cys(21) bridge deleted can still fold into its near-native structure even in its noncyclic form, confirming the lesser role of the Cys(4)-Cys(21) bridge. The results highlight the importance of disulfide bridges in a small bioactive peptide to bring together frustrated structure in addition to maintaining protein structural stability. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Chemoreactomic analysis of thiamine disulfide, thiamine hydrochloride, and benfotiamine molecules

OpenAIRE

O. A. Gromova; I. Yu. Torshin; L. V. Stakhovskaya; L. E. Fedotova

2017-01-01

Objective: to analyze the interactions that could indicate the potential pharmacological properties of the molecules of thiamin, thiamine disulfide, and others.Material and methods. The investigators simulated the properties of thiamine disulfide (bistiamin) versus those of the reference molecules of thiamin hydrochloride and benfotiamine. The study was performed using chemoreactomic simulation that is the newest area in post-genome pharmacology.Results and discussion. Chemoreactomic analysis...

Identification of thioredoxin target disulfides in proteins released from barley aleurone layers

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, J.; Yang, Fen

2010-01-01

Thioredoxins are ubiquitous disulfide reductases involved in a wide range of cellular processes including DNA synthesis, oxidative stress response and apoptosis. In cereal seeds thioredoxins are proposed to facilitate the germination process by reducing disulfide bonds in storage proteins and other...

O PDI da UFT: instrumento burocrático ou instrumento de gestão? Uma breve análise

Directory of Open Access Journals (Sweden)

Míriam Lucas da Silva Parente

2015-12-01

Full Text Available Diante do desafio em se adequar às constantes e rápidas transformações do mundo contemporâneo, as Instituições Federais de Ensino Superior (IFES vem desenvolvendo novos processos de gestão frente a situação atual. Neste trabalho, destacou-se o Plano de Desenvolvimento Institucional (PDI como instrumento de planejamento estratégico das IFES, criado pelo Decreto nº 5.773/2006. A pesquisa consistiu em uma análise documental dos PDIs da Universidade Federal do Tocantins (UFT com o objetivo de avaliar se os PDIs da Universidade estão condizentes com as exigências legais. Através da análise, observou-se uma evolução e aperfeiçoamento na elaboração do PDI, do ponto de vista de que este deixa de ser apenas um instrumento burocrático, para se tornar um instrumento de gestão. PALAVRAS-CHAVE: IFES. PDI. Planejamento Estratégico. UFT.   ABSTRACT Faced with the challenge in adjusting to constant and rapid changes of the contemporary world, the Federal Institutions of Higher-Education (IFES has developed new front management processes the current situation. In this work, the highlight was the Institutional Development Plan (PDI as a strategic planning tool of IFES, created by Decree No. 5.773/2006. The research consisted of a desk review of the IDPs from the Federal University of Tocantins, in order to assess whether the IDPs University are consistent with the legal requirements. Through the analysis, there was an evolution and improvement in the preparation of PDI from the point of view that this is no longer just a bureaucratic instrument to become a management tool. KEYWORDS: IFES. PDI. Strategic planning. UFT.     RESUMEN Ante el reto de adaptarse a los cambios constantes y rápidos del mundo contemporáneo, las Instituciones Federales de Educación Superior-IFES ha desarrollado una nueva gestión ante los procesos de la situación actual. En este trabajo, lo más destacado fue el Plan de Desarrollo Institucional (PDI como

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

Science.gov (United States)

Lee, Jung-Kul; Pan, Cheol-Ho

2013-01-01

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

Directory of Open Access Journals (Sweden)

Woo-Suk Jung

Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase.

Science.gov (United States)

Sultana, Ishrat; Mizanur, Rahman Md; Takeshita, Kei; Takada, Goro; Izumori, Ken

2003-01-01

Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.

Characterization of Disulfide-Linked Peptides Using Tandem Mass Spectrometry Coupled with Automated Data Analysis Software

Science.gov (United States)

Liang, Zhidan; McGuinness, Kenneth N.; Crespo, Alejandro; Zhong, Wendy

2018-05-01

Disulfide bond formation is critical for maintaining structure stability and function of many peptides and proteins. Mass spectrometry has become an important tool for the elucidation of molecular connectivity. However, the interpretation of the tandem mass spectral data of disulfide-linked peptides has been a major challenge due to the lack of appropriate tools. Developing proper data analysis software is essential to quickly characterize disulfide-linked peptides. A thorough and in-depth understanding of how disulfide-linked peptides fragment in mass spectrometer is a key in developing software to interpret the tandem mass spectra of these peptides. Two model peptides with inter- and intra-chain disulfide linkages were used to study fragmentation behavior in both collisional-activated dissociation (CAD) and electron-based dissociation (ExD) experiments. Fragments generated from CAD and ExD can be categorized into three major types, which result from different S-S and C-S bond cleavage patterns. DiSulFinder is a computer algorithm that was newly developed based on the fragmentation observed in these peptides. The software is vendor neutral and capable of quickly and accurately identifying a variety of fragments generated from disulfide-linked peptides. DiSulFinder identifies peptide backbone fragments with S-S and C-S bond cleavages and, more importantly, can also identify fragments with the S-S bond still intact to aid disulfide linkage determination. With the assistance of this software, more comprehensive disulfide connectivity characterization can be achieved. [Figure not available: see fulltext.

Characterization of Disulfide-Linked Peptides Using Tandem Mass Spectrometry Coupled with Automated Data Analysis Software.

Science.gov (United States)

Liang, Zhidan; McGuinness, Kenneth N; Crespo, Alejandro; Zhong, Wendy

2018-01-25

Disulfide bond formation is critical for maintaining structure stability and function of many peptides and proteins. Mass spectrometry has become an important tool for the elucidation of molecular connectivity. However, the interpretation of the tandem mass spectral data of disulfide-linked peptides has been a major challenge due to the lack of appropriate tools. Developing proper data analysis software is essential to quickly characterize disulfide-linked peptides. A thorough and in-depth understanding of how disulfide-linked peptides fragment in mass spectrometer is a key in developing software to interpret the tandem mass spectra of these peptides. Two model peptides with inter- and intra-chain disulfide linkages were used to study fragmentation behavior in both collisional-activated dissociation (CAD) and electron-based dissociation (ExD) experiments. Fragments generated from CAD and ExD can be categorized into three major types, which result from different S-S and C-S bond cleavage patterns. DiSulFinder is a computer algorithm that was newly developed based on the fragmentation observed in these peptides. The software is vendor neutral and capable of quickly and accurately identifying a variety of fragments generated from disulfide-linked peptides. DiSulFinder identifies peptide backbone fragments with S-S and C-S bond cleavages and, more importantly, can also identify fragments with the S-S bond still intact to aid disulfide linkage determination. With the assistance of this software, more comprehensive disulfide connectivity characterization can be achieved. Graphical Abstract ᅟ.

The Chemistry of Alk-1-yn-1-yl DisulfidesA Review

DEFF Research Database (Denmark)

Senning, Alexander Erich Eugen

2009-01-01

The preparation and the properties of the elusive alk-1-yn-1-yl disulfides are reviewed, including the most recent quantum chemical findings with regard to their reactivity.......The preparation and the properties of the elusive alk-1-yn-1-yl disulfides are reviewed, including the most recent quantum chemical findings with regard to their reactivity....

Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

Science.gov (United States)

Roh, H J; Kim, P; Park, Y C; Choi, J H

2000-02-01

D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

Complete Mapping of Complex Disulfide Patterns with Closely-Spaced Cysteines by In-Source Reduction and Data-Dependent Mass Spectrometry

DEFF Research Database (Denmark)

Cramer, Christian N; Kelstrup, Christian D; Olsen, Jesper V

2017-01-01

bonds are present in complicated patterns. This includes the presence of disulfide bonds in nested patterns and closely spaced cysteines. Unambiguous mapping of such disulfide bonds typically requires advanced MS approaches. In this study, we exploited in-source reduction (ISR) of disulfide bonds during...... the electrospray ionization process to facilitate disulfide bond assignments. We successfully developed a LC-ISR-MS/MS methodology to use as an online and fully automated partial reduction procedure. Postcolumn partial reduction by ISR provided fast and easy identification of peptides involved in disulfide bonding......Mapping of disulfide bonds is an essential part of protein characterization to ensure correct cysteine pairings. For this, mass spectrometry (MS) is the most widely used technique due to fast and accurate characterization. However, MS-based disulfide mapping is challenged when multiple disulfide...

Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

OpenAIRE

Hahn, F M; Baker, J A; Poulter, C D

1996-01-01

Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic...

Crystal structure of Pyrococcus furiosus phosphoglucose isomerase: Implications for substrate binding and catalysis

NARCIS (Netherlands)

Berrisford, J.M.; Akerboom, A.P.; Turnbull, A.P.; Geus, de D.; Sedelnikova, S.E.; Staton, I.; McLeod, C.W.; Verhees, C.H.; Oost, van der J.; Rice, D.W.; Baker, P.J.

2003-01-01

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between D-fructose 6-phosphate and D-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-Angstrom

Solvent Induced Disulfide Bond Formation in 2,5-dimercapto-1,3,4-thiadiazole

OpenAIRE

Palanisamy Kalimuthu; Palraj Kalimuthu; S. Abraham John

2007-01-01

Disulfide bond formation is the decisive event in the protein folding to determine the conformation and stability of protein. To achieve this disulfide bond formation in vitro, we took 2,5-dimercapto-1,3,4-thiadiazole (DMcT) as a model compound. We found that disulfide bond formation takes place between two sulfhydryl groups of DMcT molecules in methanol. UV-Vis, FT-IR and mass spectroscopic as well as cyclic voltammetry were used to monitor the course of reaction. We proposed a mechanism for...

Selective removal of heavy metal ions by disulfide linked polymer networks

Energy Technology Data Exchange (ETDEWEB)

Ko, Dongah [Department of Environmental Engineering, Technical University of Denmark, Miljøvej 113, 2800 Kgs. Lyngby (Denmark); Lee, Joo Sung [Graduate School of EEWS, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141 (Korea, Republic of); Patel, Hasmukh A. [Department of Chemistry, Northwestern University, Evanston, IL 60208 (United States); Jakobsen, Mogens H. [Department of Micro and Nano technology, Technical University of Denmark, Ørsteds Plads, 345B, 2800 Kgs. Lyngby (Denmark); Hwang, Yuhoon [Department of Environmental Engineering, Seoul National University of Science and Technology, 232 Gongreung-ro, Nowon-gu, Seoul 01811 (Korea, Republic of); Yavuz, Cafer T. [Graduate School of EEWS, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141 (Korea, Republic of); Hansen, Hans Chr. Bruun [Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Andersen, Henrik R., E-mail: henrik@ndersen.net [Department of Environmental Engineering, Technical University of Denmark, Miljøvej 113, 2800 Kgs. Lyngby (Denmark)

2017-06-15

Highlights: • Disulfide/thiol polymer networks are promising as sorbent for heavy metals. • Rapid sorption and high Langmuir affinity constant (a{sub L}) for stormwater treatment. • Selective sorption for copper, cadmium, and zinc in the presence of calcium. • Reusability likely due to structure stability of disulfide linked polymer networks. - Abstract: Heavy metal contaminated surface water is one of the oldest pollution problems, which is critical to ecosystems and human health. We devised disulfide linked polymer networks and employed as a sorbent for removing heavy metal ions from contaminated water. Although the polymer network material has a moderate surface area, it demonstrated cadmium removal efficiency equivalent to highly porous activated carbon while it showed 16 times faster sorption kinetics compared to activated carbon, owing to the high affinity of cadmium towards disulfide and thiol functionality in the polymer network. The metal sorption mechanism on polymer network was studied by sorption kinetics, effect of pH, and metal complexation. We observed that the metal ions–copper, cadmium, and zinc showed high binding affinity in polymer network, even in the presence of competing cations like calcium in water.

Electrochemistry-assisted top-down characterization of disulfide-containing proteins.

Science.gov (United States)

Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D; Chen, Hao

2012-04-17

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

International Nuclear Information System (INIS)

Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

2016-01-01

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

Science.gov (United States)

Jørgensen, F; Hansen, O C; Stougaard, P

2004-06-01

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases

NARCIS (Netherlands)

Zoldak, G.; Aumuller, T.; Lucke, C.; Hritz, J.; Oostenbrink, C.; Fischer, G.; Schmid, F.X.

2009-01-01

To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula

New thiol-responsive mono-cleavable block copolymer micelles labeled with single disulfides.

Science.gov (United States)

Sourkohi, Behnoush Khorsand; Schmidt, Rolf; Oh, Jung Kwon

2011-10-18

Thiol-responsive symmetric triblock copolymers having single disulfide linkages in the middle blocks (called mono-cleavable block copolymers, ss-ABP(2)) were synthesized by atom transfer radical polymerization in the presence of a disulfide-labeled difunctional Br-initiator. These brush-like triblock copolymers consist of a hydrophobic polyacrylate block having pendent oligo(propylene oxide) and a hydrophilic polymethacrylate block having pendent oligo(ethylene oxide). Gel permeation chromatography and (1)H NMR results confirmed the synthesis of well-defined mono-cleavable block copolymers and revealed that polymerizations were well controlled. Because of amphiphilic nature, these copolymers self-assembled to form colloidally stable micelles above critical micellar concentration of 0.032 mg · mL(-1). In response to reductive reactions, disulfides in thiol-responsive micelles were cleaved. Atomic force microscopy and dynamic light scattering analysis suggested that the cleavage of disulfides caused dissociation of micelles to smaller-sized assembled structures in water. Moreover, in a biomedical perspective, the mono-cleavable block copolymer micelles are not cytotoxic and thus biocompatible. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry

DEFF Research Database (Denmark)

Mysling, Simon; Salbo, Rune; Ploug, Michael

2014-01-01

Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically...... of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify...... some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions....

Generation of a Multicomponent Library of Disulfide Donor-Acceptor Architectures Using Dynamic Combinatorial Chemistry.

Science.gov (United States)

Drożdż, Wojciech; Kołodziejski, Michał; Markiewicz, Grzegorz; Jenczak, Anna; Stefankiewicz, Artur R

2015-07-17

We describe here the generation of new donor-acceptor disulfide architectures obtained in aqueous solution at physiological pH. The application of a dynamic combinatorial chemistry approach allowed us to generate a large number of new disulfide macrocyclic architectures together with a new type of [2]catenanes consisting of four distinct components. Up to fifteen types of structurally-distinct dynamic architectures have been generated through one-pot disulfide exchange reactions between four thiol-functionalized aqueous components. The distribution of disulfide products formed was found to be strongly dependent on the structural features of the thiol components employed. This work not only constitutes a success in the synthesis of topologically- and morphologically-complex targets, but it may also open new horizons for the use of this methodology in the construction of molecular machines.

Dissecting the role of disulfide bonds on the amyloid formation of insulin

International Nuclear Information System (INIS)

Li, Yang; Gong, Hao; Sun, Yue; Yan, Juan; Cheng, Biao; Zhang, Xin; Huang, Jing; Yu, Mengying; Guo, Yu; Zheng, Ling; Huang, Kun

2012-01-01

Highlights: ► We dissect how individual disulfide bond affects the amyloidogenicity of insulin. ► A controlled reduction system for insulin is established in this study. ► Disulfide breakage is associated with unfolding and increased amyloidogenicity. ► Breakage of A6-A11 is associated with significantly increased cytotoxicity. ► Analogs without A6-A11 have a higher potency to form high order toxic oligomers. -- Abstract: Disulfide bonds play a critical role in the stability and folding of proteins. Here, we used insulin as a model system, to investigate the role of its individual disulfide bond during the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) was applied to reduce two of the three disulfide bonds in porcine insulin and the reduced disulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modified insulin analogs, INS-2 (lack of A6-A11), INS-3 (lack of A7-B7) and INS-6 (lack of both A6-A11 and A7-B7), were obtained. Far-UV circular dichroism (CD) spectroscopy results indicated that the secondary structure of INS-2 was the closest to insulin under neutral conditions, followed by INS-3 and INS-6, whereas in an acidic solution all analogs were essentially unfolded. To test how these modifications affect the amyloidogenicity of insulin, thioflavin-T (ThT) fluorescence and transmission electronic microscopy (TEM) were performed. Our results showed that all analogs were more prone to aggregation than insulin, with the order of aggregation rates being INS-6 > INS-3 > INS-2. Cross-linking of unmodified proteins (PICUP) assay results showed that analogs without A6-A11 (INS-2 and INS-6) have a higher potential for oligomerization than insulin and INS-3, which is accompanied with a higher cytotoxicity as the hemolytic assays of human erythrocytes suggested. The results indicated that breakage of A7-B7 induced more unfolding of the insulin structure and a higher amyloidogenicity than breakage of A6-A11, but breakage of A6

Crystallization and preliminary X-ray diffraction studies of l-rhamnose isomerase from Pseudomonas stutzeri

International Nuclear Information System (INIS)

Yoshida, Hiromi; Wayoon, Poonperm; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

2006-01-01

Recombinant l-rhamnose isomerase from P. stutzeri has been crystallized. Diffraction data have been collected to 2.0 Å resolution. l-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeril-RhI) catalyzes not only the reversible isomerization of l-rhamnose to l-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeril-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 Å, β = 106.8°. Diffraction data have been collected to 2.0 Å resolution. The molecular weight of the purified P. stutzeril-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI–TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules

Bothrops jararaca venom metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels during envenomation.

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Karine M Yamashita

2014-05-01

Full Text Available BACKGROUND/AIMS: Bleeding tendency, coagulopathy and platelet disorders are recurrent manifestations in snakebites occurring worldwide. We reasoned that by damaging tissues and/or activating cells at the site of the bite and systemically, snake venom toxins might release or decrypt tissue factor (TF, resulting in activation of blood coagulation and aggravation of the bleeding tendency. Thus, we addressed (a whether TF and protein disulfide isomerase (PDI, an oxireductase involved in TF encryption/decryption, were altered in experimental snake envenomation; (b the involvement and significance of snake venom metalloproteinases (SVMP and serine proteinases (SVSP to hemostatic disturbances. METHODS/PRINCIPAL FINDINGS: Crude Bothrops jararaca venom (BjV was preincubated with Na2-EDTA or AEBSF, which are inhibitors of SVMP and SVSP, respectively, and injected subcutaneously or intravenously into rats to analyze the contribution of local lesion to the development of hemostatic disturbances. Samples of blood, lung and skin were collected and analyzed at 3 and 6 h. Platelet counts were markedly diminished in rats, and neither Na2-EDTA nor AEBSF could effectively abrogate this fall. However, Na2-EDTA markedly reduced plasma fibrinogen consumption and hemorrhage at the site of BjV inoculation. Na2-EDTA also abolished the marked elevation in TF levels in plasma at 3 and 6 h, by both administration routes. Moreover, increased TF activity was also noticed in lung and skin tissue samples at 6 h. However, factor VII levels did not decrease over time. PDI expression in skin was normal at 3 h, and downregulated at 6 h in all groups treated with BjV. CONCLUSIONS: SVMP induce coagulopathy, hemorrhage and increased TF levels in plasma, but neither SVMP nor SVSP are directly involved in thrombocytopenia. High levels of TF in plasma and TF decryption occur during snake envenomation, like true disseminated intravascular coagulation syndrome, and might be implicated in

Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

Science.gov (United States)

Landel, C C; Potthoff, S J; Nardulli, A M; Kushner, P J; Greene, G L

1997-01-01

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis

Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli.

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Maria Elena Ortiz-Soto

Full Text Available Sialyltransferases (STs are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs. Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl-α-d-galactopyranoside.

Unraveling the cellular response to oxidative stress in the endoplasmic reticulum

DEFF Research Database (Denmark)

Hansen, Henning Gram

, disulfide bonds are predominantly generated by the two isoforms of the ER oxidoreductin-1 (Ero1) family: Ero1α and Ero1β. Both enzymes oxidize the active-site cysteines in protein disulfide isomerases (PDIs), which in turn introduce disulfide bonds into newly synthesized proteins. Ero1 is re......-oxidized by molecular oxygen and this step generates hydrogen peroxide: a reactive oxygen species. Intramolecular disulfide bonds tightly regulate the oxidase activity of Ero1α. Whereas the regulatory mechanisms that regulate Ero1α activity are well understood, the overall cellular response to oxidative stress....... Interestingly, depletion of GPx8 in cells induced expression of an antioxidant response marker only in the presence of Ero1. These findings imply that GPx8 is an important scavenger of Ero1-generated hydrogen peroxide, and thus provides a critical function in negotiating oxidative stress originating from...

Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Thorsen, Michael; Kielland-Brandt, Morten C

2007-01-01

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-c......Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma...... antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS...

The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

OpenAIRE

Hoskins, J.T.; Zhou, Z.; Harding, P.A.

2008-01-01

A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

Radiation-induced cleavage of disulfide bonds in proteins. Clivage radiolytique des ponts disulfure des proteines

Energy Technology Data Exchange (ETDEWEB)

Favaudon, V; Tourbez, H; Lhoste, J M [Paris-11 Univ., 91 - Orsay (FR); Houee-Levin, C [Paris-5 Univ., 75 (FR)

1991-06-01

The reduction of the disulfide bonds in apo-Riboflavin-Binding Protein (apoRBP) by the CO{sub 2}{sup -}{center dot} radical occurred under {gamma}-ray irradiation as a chain reaction whose efficiency increased upon acidification of the medium. Pulse-radiolysis analysis showed a rapid one-electron oxidation of the disulfide bonds yielding the anionic or protonated form of the disulfide radical. The main decay path of this radical under acidic conditions consisted of the rapid formation of a thiyl radical intermediate in equilibrium with the closed, cyclic form. At pH 8 the disulfide radical anion decayed via intramolecular and/or intermolecular routes including disproportionation, protein-protein crosslinking, non-dismutative recombination processes, and reaction with sulfhydryl groups in pre-reduced systems.

An analytic study of molybdenum disulfide nanofluids using the modern approach of Atangana-Baleanu fractional derivatives

Science.gov (United States)

Ali Abro, Kashif; Hussain, Mukkarum; Mahmood Baig, Mirza

2017-10-01

The significance of the different shapes of molybdenum disulfide nanoparticles contained in ethylene glycol has recently attracted researchers, because of the numerical or experimental analyses on the shapes of molybdenum disulfide and the lack of fractionalized analytic approaches. This work is dedicated to examining the shape impacts of molybdenum disulfide nanofluids in the mixed convection flow with magnetic field and a porous medium. Ethylene glycol is chosen as the base fluid in which molybdenum disulfide nanoparticles are suspended. Non-spherically shaped molybdenum disulfide nanoparticles, namely, platelet, blade, cylinder and brick, are utilized in this analysis. The modeling of the problem is characterized by employing the modern approach of Atangana-Baleanu fractional derivatives and the governing partial differential equations are solved via Laplace transforms with inversion. Solutions are obtained for temperature distribution and velocity field and expressed in terms of compact form of M-function, Mba(T) . In the end, a figures are drawn to compare the different non-spherically shaped molybdenum disulfide nanoparticles. Furthermore, the Atangana-Baleanu fractional derivatives model has been compared with ordinary derivatives models and discussed graphically by setting various rheological parameters.

Synthesis of tetraalkyl thiuram disulfides using different oxidants in recycling solvent mixture

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Milosavljević Milutin M.

2012-01-01

Full Text Available A new optimized laboratory synthesis of tetraalkyl thiuram disulfides, starting from dialkyl amines and carbon disulfide in presence of three oxidants (hydrogen peroxide, potassium peroxodisulfate and sodium hypochlorite and appropriate reaction medium: two mixtures of isopropyl alcohol - water used in two consecutive syntheses, was presented in this work. First synthesis was performed in a recycled azeotropic mixture of isopropyl alcohol - water 87.7% - 12.3%, and second in a filtrate obtained after first synthesis, which was a mixture of isopropyl alcohol - water 70.4% - 29.6%. After the second synthesis and filtration, recycled azeotropic mixture isopropyl alcohol - water 87.7% - 12.3% was regenerated from the filtrate by rectification. Considering this, the technology for beneficial use of recycling isopropyl alcohol - water mixture as reaction medium for tetraalkyl thiuram disulfides synthesis was developed. Such concept contributes to extraordinary economical benefit of implemented optimal laboratory synthesis at semi-industrial level. High yields of tetraalkyl thiuram disulfides syntheses were obtained at both laboratory and semiindustrial level. Structure and purity of synthesized compounds were confirmed by elemental analysis, as well as FTIR, 1H and 13C NMR, and MS spectral data.

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

Science.gov (United States)

2011-01-01

Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we reported the purification and the

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

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Rhimi Moez

2011-11-01

Full Text Available Abstract Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we

Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond.

Science.gov (United States)

Liu, Jinny L; Goldman, Ellen R; Zabetakis, Dan; Walper, Scott A; Turner, Kendrick B; Shriver-Lake, Lisa C; Anderson, George P

2015-10-09

Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. To fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. In this work, we tested the hypothesis that decreasing the isoelectric point of single domain antibody extra disulfide bond mutants whose production fell due to the incorporation of the extra disulfide bond would lead to recovery of the protein yield, while maintaining the favorable melting temperature and affinity. Introduction of negative charges into a disulfide bond mutant of a single domain antibody specific for the L1 antigen of the vaccinia virus led to approximately 3.5-fold increase of protein production to 14 mg/L, while affinity and melting temperature was maintained. In addition, refolding following heat denaturation improved from 15 to 70 %. It also maintained nearly 100 % of its binding function after heating to 85 °C for an hour at 1 mg/mL. Disappointingly, the replacement of neutral or positively charged amino acids with negatively charged ones to lower the isoelectric point of two anti-toxin single domain antibodies stabilized with a second disulfide bond yielded only slight increases in protein production. Nonetheless, for one of these binders the charge change itself stabilized the structure equivalent to disulfide bond addition, thus providing an alternative route to stabilization which is not accompanied by loss in production. The ability to produce high affinity, stable single domain antibodies is critical for their utility. While the addition of a second disulfide bond is a proven method for enhancing stability of single domain antibodies, it frequently comes at the cost of reduced

ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis

Science.gov (United States)

Somlo, Diane R.M.; Kim, Geun Hyang; Prescianotto-Baschong, Cristina; Sun, Shengyi; Beuret, Nicole; Long, Qiaoming; Rutishauser, Jonas

2017-01-01

Peptide hormones are crucial regulators of many aspects of human physiology. Mutations that alter these signaling peptides are associated with physiological imbalances that underlie diseases. However, the conformational maturation of peptide hormone precursors (prohormones) in the ER remains largely unexplored. Here, we report that conformational maturation of proAVP, the precursor for the antidiuretic hormone arginine-vasopressin, within the ER requires the ER-associated degradation (ERAD) activity of the Sel1L-Hrd1 protein complex. Serum hyperosmolality induces expression of both ERAD components and proAVP in AVP-producing neurons. Mice with global or AVP neuron–specific ablation of Se1L-Hrd1 ERAD progressively developed polyuria and polydipsia, characteristics of diabetes insipidus. Mechanistically, we found that ERAD deficiency causes marked ER retention and aggregation of a large proportion of all proAVP protein. Further, we show that proAVP is an endogenous substrate of Sel1L-Hrd1 ERAD. The inability to clear misfolded proAVP with highly reactive cysteine thiols in the absence of Sel1L-Hrd1 ERAD causes proAVP to accumulate and participate in inappropriate intermolecular disulfide–bonded aggregates, promoted by the enzymatic activity of protein disulfide isomerase (PDI). This study highlights a pathway linking ERAD to prohormone conformational maturation in neuroendocrine cells, expanding the role of ERAD in providing a conducive ER environment for nascent proteins to reach proper conformation. PMID:28920920

On the relevance of sophisticated structural annotations for disulfide connectivity pattern prediction.

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Julien Becker

Full Text Available Disulfide bridges strongly constrain the native structure of many proteins and predicting their formation is therefore a key sub-problem of protein structure and function inference. Most recently proposed approaches for this prediction problem adopt the following pipeline: first they enrich the primary sequence with structural annotations, second they apply a binary classifier to each candidate pair of cysteines to predict disulfide bonding probabilities and finally, they use a maximum weight graph matching algorithm to derive the predicted disulfide connectivity pattern of a protein. In this paper, we adopt this three step pipeline and propose an extensive study of the relevance of various structural annotations and feature encodings. In particular, we consider five kinds of structural annotations, among which three are novel in the context of disulfide bridge prediction. So as to be usable by machine learning algorithms, these annotations must be encoded into features. For this purpose, we propose four different feature encodings based on local windows and on different kinds of histograms. The combination of structural annotations with these possible encodings leads to a large number of possible feature functions. In order to identify a minimal subset of relevant feature functions among those, we propose an efficient and interpretable feature function selection scheme, designed so as to avoid any form of overfitting. We apply this scheme on top of three supervised learning algorithms: k-nearest neighbors, support vector machines and extremely randomized trees. Our results indicate that the use of only the PSSM (position-specific scoring matrix together with the CSP (cysteine separation profile are sufficient to construct a high performance disulfide pattern predictor and that extremely randomized trees reach a disulfide pattern prediction accuracy of [Formula: see text] on the benchmark dataset SPX[Formula: see text], which corresponds to

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

Science.gov (United States)

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-01-01

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins

International Nuclear Information System (INIS)

Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune

2012-01-01

NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U- 13 C, 15 N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β- 13 C; α,β- 2 H 2 ] Cys and (2R, 3R)-[β- 13 C; α,β- 2 H 2 ] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ 2 and χ 3 , can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.

Alpha-cyclodextrins reversibly capped with disulfide bonds

Czech Academy of Sciences Publication Activity Database

Kumprecht, Lukáš; Buděšínský, Miloš; Bouř, Petr; Kraus, Tomáš

2010-01-01

Roč. 34, č. 10 (2010), s. 2254-2260 ISSN 1144-0546 R&D Projects: GA AV ČR IAA400550810 Institutional research plan: CEZ:AV0Z40550506 Keywords : cyclodextrins * disulfide bond * dynamic covalent bond Subject RIV: CC - Organic Chemistry Impact factor: 2.631, year: 2010

Photo-reduction on the rupture of disulfide bonds and the related protein assembling

Science.gov (United States)

Wang, Wei

It has been found that many proteins can self-assemble into nanoscale assemblies when they unfold or partially unfold under harsh conditions, such as low pH, high temperature, or the presence of denaturants, and so on. These nanoscale assemblies can have some applications such as the drug-delivery systems (DDSs). Here we report a study that a very physical way, the UV illumination, can be used to facilitate the formation of protein fibrils and nanoparticles under native conditions by breaking disulfide bonds in some disulfide-containing proteins. By controlling the intensity of UV light and the illumination time, we realized the preparation of self-assembly nanoparticles which encapsulate the anticancer drug doxorubicin (DOX) and can be used as the DDS for inhibiting the growth of tumor. The formation of fibrillary assemblies was also observed. The rupture of disulfide bonds through photo-reduction process due to the effect of tryptophan and tyrosine was studied, and the physical mechanism of the assembling of the related disulfide-containing proteins was also discussed. We thank the financial support from NSF of China and the 973 project.

The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

International Nuclear Information System (INIS)

Hoskins, J.T.; Zhou, Z.; Harding, P.A.

2008-01-01

A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser 108/121 , HB-EGF-Cys/Ser 116/132 , and HB-EGF-Cys/Ser 134/143 ) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M r of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF 134/143 proteins competed with 125 I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser 134/143 antagonizes EGFRs

Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

Science.gov (United States)

Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

2016-10-01

To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation.

Science.gov (United States)

Morris, D P; Phatnani, H P; Greenleaf, A L

1999-10-29

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.

Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

Energy Technology Data Exchange (ETDEWEB)

Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

1988-09-26

The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu

DEFF Research Database (Denmark)

Hansen, Rosa E; Østergaard, Henrik; Winther, Jakob R

2005-01-01

K(a) value of Cys149, as well as favorable electrostatic interactions with the negatively charged reagents. The results presented here show that the electrostatic milieu of cysteine thiols in proteins can have substantial effects on the rates of the thiol-disulfide exchange reactions.......The thiol-disulfide exchange reaction plays a central role in the formation of disulfide bonds in newly synthesized proteins and is involved in many aspects of cellular metabolism. Because the thiolate form of the cysteine residue is the key reactive species, its electrostatic milieu is thought...... surface. We have studied properties of vicinal cysteine residues in proteins using a model system based on redox-sensitive yellow fluorescent protein (rxYFP). In this system, the formation of a disulfide bond between two cysteines Cys149 and Cys202 is accompanied by a 2.2-fold decrease in fluorescence...

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

Science.gov (United States)

2014-01-01

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

Biochemical properties of L-arabinose isomerase from Clostridium hylemonae to produce D-tagatose as a functional sweetener.

Science.gov (United States)

Nguyen, Tien-Kieu; Hong, Moon-Gi; Chang, Pahn-Shick; Lee, Byung-Hoo; Yoo, Sang-Ho

2018-01-01

d-Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA, encoding l-arabinose isomerase (l-AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l-AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7-7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-1) of l-AI from C. hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d-tagatose using the C. hylemonae l-arabinose isomerase at 60°C reached approximately 46% from 10 mM of d-galactose after 2 h. From these results, it is suggested that the l-arabinose isomerase from C. hylemonae could be utilized as a potential enzyme for d-tagatose production due to its high conversion yield at an industrially competitive temperature.

Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

Science.gov (United States)

Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

2009-05-01

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

2008-01-01

Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Science.gov (United States)

Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

2015-04-16

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Inhibiting prolyl isomerase activity by hybrid organic-inorganic molecules containing rhodium(II) fragments.

Science.gov (United States)

Coughlin, Jane M; Kundu, Rituparna; Cooper, Julian C; Ball, Zachary T

2014-11-15

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site. Copyright © 2014 Elsevier Ltd. All rights reserved.

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

Directory of Open Access Journals (Sweden)

Hatahet Feras

2010-09-01

Full Text Available Abstract Background The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3 pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Conclusions Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.

Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

Science.gov (United States)

Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

2016-06-01

The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. Copyright © 2016 Elsevier Ltd. All rights reserved.

In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes

International Nuclear Information System (INIS)

Liavonchanka, Alena; Hornung, Ellen; Feussner, Ivo; Rudolph, Markus

2006-01-01

Low iodide concentrations were sufficient to allow SAD and SIRAS phasing of cubic crystals of a novel fatty acid isomerase using Cu Kα radiation. The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy- or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellow-coloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2 1 3, with a unit-cell parameter of 160.4 Å, and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å, β = 115.8°. Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å. Initial phases were obtained by SIRAS from in-house data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M

Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry

OpenAIRE

Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R. Ogorzalek; Julian, Ryan R.; Loo, Joseph A.

2015-01-01

The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in comb...

Photodegradable, Photoadaptable Hydrogels via Radical-Mediated Disulfide Fragmentation Reaction.

Science.gov (United States)

Fairbanks, Benjamin D; Singh, Samir P; Bowman, Christopher N; Anseth, Kristi S

2011-04-26

Various techniques have been adopted to impart a biological responsiveness to synthetic hydrogels for the delivery of therapeutic agents as well as the study and manipulation of biological processes and tissue development. Such techniques and materials include polyelectrolyte gels that swell and deswell with changes in pH, thermosensitive gels that contract at physiological temperatures, and peptide cross-linked hydrogels that degrade upon peptidolysis by cell-secreted enzymes. Herein we report a unique approach to photochemically deform and degrade disulfide cross-linked hydrogels, mitigating the challenges of light attenuation and low quantum yield, permitting the degradation of hydrogels up to 2 mm thick within 120 s at low light intensities (10 mW/cm(2) at 365 nm). Hydrogels were formed by the oxidation of thiol-functionalized 4-armed poly(ethylene glycol) macromolecules. These disulfide cross-linked hydrogels were then swollen in a lithium acylphosphinate photoinitiator solution. Upon exposure to light, photogenerated radicals initiate multiple fragmentation and disulfide exchange reactions, permitting and promoting photodeformation, photowelding, and photodegradation. This novel, but simple, approach to generate photoadaptable hydrogels portends the study of cellular response to mechanically and topographically dynamic substrates as well as novel encapsulations by the welding of solid substrates. The principles and techniques described herein hold implications for more than hydrogel materials but also for photoadaptable polymers more generally.

Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

International Nuclear Information System (INIS)

Nickbarg, E.B.; Knowles, J.R.

1988-01-01

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)- 3 H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

Science.gov (United States)

Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

2017-01-01

Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii.

Science.gov (United States)

Luong, Truc Thanh; Tirgar, Reyhaneh; Reardon-Robinson, Melissa E; Joachimiak, Andrzej; Osipiuk, Jerzy; Ton-That, Hung

2018-05-01

The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbA Cd ). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbA Cm ) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro , we demonstrated that MdbA Cm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbA Cm in the C. diphtheriae Δ mdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbA Cm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in Actinobacteria IMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide

Simultaneous electrochemical determination of L-cysteine and L-cysteine disulfide at carbon ionic liquid electrode.

Science.gov (United States)

Safavi, Afsaneh; Ahmadi, Raheleh; Mahyari, Farzaneh Aghakhani

2014-04-01

A linear sweep voltammetric method is used for direct simultaneous determination of L-cysteine and L-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for L-cysteine (0.62 V) and L-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0-450 and 5.0-700 μM and detection limits were estimated to be 0.298 and 4.258 μM for L-cysteine and L-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of L-cysteine and L-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

Science.gov (United States)

Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

2016-11-25

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

The road to the first, fully active and more stable human insulin variant with an additional disulfide bond

DEFF Research Database (Denmark)

Vinther, Tine N.; Kjeldsen, Thomas B.; Jensen, Knud Jørgen

2015-01-01

Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non-covalent oligomerization. All known insulin...... variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra-chain bond in the A-chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We...... addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four-disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs...

Photoinduced Cross-Linking of Dynamic Poly(disulfide) Films via Thiol Oxidative Coupling.

Science.gov (United States)

Feillée, Noémi; Chemtob, Abraham; Ley, Christian; Croutxé-Barghorn, Céline; Allonas, Xavier; Ponche, Arnaud; Le Nouen, Didier; Majjad, Hicham; Jacomine, Léandro

2016-01-01

Initially developed as an elastomer with an excellent record of barrier and chemical resistance properties, poly(disulfide) has experienced a revival linked to the dynamic nature of the S-S covalent bond. A novel photobase-catalyzed oxidative polymerization of multifunctional thiols to poly(disulfide) network is reported. Based solely on air oxidation, the single-step process is triggered by the photodecarboxylation of a xanthone acetic acid liberating a strong bicyclic guanidine base. Starting with a 1 μm thick film based on trithiol poly(ethylene oxide) oligomer, the UV-mediated oxidation of thiols to disulfides occurs in a matter of minutes both selectively, i.e., without overoxidation, and quantitatively as assessed by a range of spectroscopic techniques. Thiolate formation and film thickness determine the reaction rates and yield. Spatial control of the photopolymerization serves to generate robust micropatterns, while the reductive cleavage of S-S bridges allows the recycling of 40% of the initial thiol groups. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New analogs of the CART peptide with anorexigenic potency: the importance of individual disulfide bridges.

Science.gov (United States)

Blechová, Miroslava; Nagelová, Veronika; Záková, Lenka; Demianová, Zuzana; Zelezná, Blanka; Maletínská, Lenka

2013-01-01

The CART (cocaine- and amphetamine-regulated transcript) peptide is an anorexigenic neuropeptide that acts in the hypothalamus. The receptor and the mechanism of action of this peptide are still unknown. In our previous study, we showed that the CART peptide binds specifically to PC12 rat pheochromocytoma cells in both the native and differentiated into neuronal phenotype. Two biologically active forms, CART(55-102) and CART(61-102), with equal biological activity, contain three disulfide bridges. To clarify the importance of each of these disulfide bridges in maintaining the biological activity of CART(61-102), an Ala scan at particular S-S bridges forming cysteines was performed, and analogs with only one or two disulfide bridges were synthesized. In this study, a stabilized CART(61-102) analog with norleucine instead of methionine at position 67 was also prepared and was found to bind to PC12 cells with an anorexigenic potency similar to that of CART(61-102). The binding study revealed that out of all analogs tested, [Ala(68,86)]CART(61-102), which contains two disulfide bridges (positions 74-94 and 88-101), preserved a high affinity to both native PC12 cells and those that had been differentiated into neurons. In food intake and behavioral tests with mice after intracerebroventricular administration, this analog showed strong and long-lasting anorexigenic potency. Therefore, the disulfide bridge between cysteines 68 and 86 in CART(61-102) can be omitted without a loss of biological activity, but the preservation of two other disulfide bridges and the full-length peptide are essential for biological activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

Science.gov (United States)

Lopez-Zavala, Alonso A.; Carrasco-Miranda, Jesus S.; Ramirez-Aguirre, Claudia D.; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G.; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S.; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

2016-01-01

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation. PMID:27614148

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

Science.gov (United States)

Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

2007-02-02

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

DEFF Research Database (Denmark)

Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

2001-01-01

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom crystal structure of the oxidized protein revealed a disulfide bond- induced distortion of the beta -barrel, as well...... the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway....

Study of the helium cross-section of unsymmetric disulfide self-assembled monolayers on Au(111)

Energy Technology Data Exchange (ETDEWEB)

Albayrak, Erol [Department of Materials and Metallurgical Engineering, Ahi Evran University, Kırşehir 40000 (Turkey); Karabuga, Semistan [Department of Chemistry, Kahramanmaraş Sütçü İmam University, Kahramanmaraş 46030 (Turkey); Bracco, Gianangelo [CNR-IMEM and Department of Physics, University of Genoa, Via Dodecaneso 33, Genoa 16146 (Italy); Danışman, M. Fatih, E-mail: danisman@metu.edu.tr [Department of Chemistry, Middle East Technical University, Ankara 06800 (Turkey)

2016-12-30

Highlights: • Unsymmetrtic disulfide (HDD and HOD) self assembled monolayers were grown on Au(111) by supersonic molecular beam deposition. • Helium scattering cross sections for these two different unsymmetric disulfides were determined. • A common low temperature film phase was observed for the studied disulfides. - Abstract: We have investigated the formation of self-assembled monolayers (SAMs) of 11-hydroxyundecyl decyl disulfide (CH{sub 3}-(CH{sub 2}){sub 9}-S-S-(CH{sub 2}){sub 11}-OH, HDD) and 11-hydroxyundecyl octadecyl disulfide (CH{sub 3}-(CH{sub 2}){sub 17}-S-S-(CH{sub 2}){sub 11}-OH, HOD) produced by supersonic molecular beam deposition (SMBD). The study has been carried out by means of helium diffraction at very low film coverage. In this regime helium single molecule cross sections have been estimated in a temperature range between 100 K and 450 K. The results show a different behavior above 300 K that has been interpreted as the starting of mobility with the formation of two thiolate moieties either linked by a gold adatom or distant enough to prevent cross section overlapping. Finally, helium diffraction patterns measured at 80 K for the SAMs grown at 200 K are discussed and the results support the proposed hypothesis of molecular dissociation based on the cross section data.

Changes in Thiol-Disulfide Homeostasis of the Body to Surgical Trauma in Laparoscopic Cholecystectomy Patients.

Science.gov (United States)

Polat, Murat; Ozcan, Onder; Sahan, Leyla; Üstündag-Budak, Yasemin; Alisik, Murat; Yilmaz, Nigar; Erel, Özcan

2016-12-01

We aimed to investigate the short-term effect of laparoscopic surgery on serum thiol-disulfide homeostasis levels as a marker of oxidant stress of surgical trauma in elective laparoscopic cholecystectomy patients. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfides were determined with a novel automated assay. Total antioxidant capacity (measured as the ferric-reducing ability of plasma) and serum ischemia modified albumin, expressed as absorbance units assayed by the albumin cobalt binding test, were determined. The major findings of the present study were that native thiol (283 ± 45 versus 241 ± 61 μmol/L), total thiol (313 ± 49 versus 263 ± 67 μmol/L), and disulfide (14.9 ± 4.6 versus 11.0 ± 6.1 μmol/L) levels were decreased significantly during operation and although they increased, they did not return to preoperation levels 24 hours after laparoscopic surgery compared to the levels at baseline. Disulfide/native thiol and disulfide/total thiol levels did not change during laparoscopic surgery. The decrease in plasma level of native and total thiol groups suggests impairment of the antioxidant capacity of plasma; however, the delicate balance between the different redox forms of thiols was maintained during surgery.

Effect of gamma irradiation on whole-cell glucose isomerase. Pt.1

International Nuclear Information System (INIS)

Bachman, S.; Gebicka, L.

1984-01-01

Gamma-rays induced inactivation of Actinoplanes missouriensis and Streptomyces olivaceus glucose isomerase has been studied. This enzyme exhibits high resistance against ionizing radiation. The D 37 value was found to be equal to 131 kGy for Actinoplanes missouriensis cells and 88 kGy for Streptomyces olivaceus cells when irradiated in the dry state in the presence of air. Mg 2+ ions do not affect the radiosensitivity of the enzyme in cells, while the addition of Co 2+ ions to the cell suspension increases its stability against ionizing radiation. (orig.) [de

Dissecting molecular interactions involved in recognition of target disulfides by the barley thioredoxin system

DEFF Research Database (Denmark)

Björnberg, Olof; Maeda, Kenji; Svensson, Birte

2012-01-01

Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α-amylase/subtilisin inhibi......Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α...... thioredoxin reductase. HvTrxh2 M88G and M88A adjacent to the invariant cis-proline lost efficiency in both BASI disulfide reduction and recycling by thioredoxin reductase. These effects were further pronounced in M88P lacking a backbone NH group. Remarkably, HvTrxh2 E86R in the same loop displayed overall...... retained catalytic properties, with the exception of a 3-fold increased activity toward BASI. From the 104VGA106 loop, a backbone hydrogen bond donated by A106 appears to be important for target disulfide recognition as A106P lost 90% activity toward BASI but was efficiently recycled by thioredoxin...

Thiol/Disulfide system plays a crucial role in redox protection in the acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum.

Directory of Open Access Journals (Sweden)

Javiera Norambuena

Full Text Available Thiol/disulfide systems are involved in the maintenance of the redox status of proteins and other molecules that contain thiol/disulfide groups. Leptospirillum ferriphilum DSM14647, an acidophilic bacterium that uses Fe(2+ as electron donor, and withstands very high concentrations of iron and other redox active metals, is a good model to study how acidophiles preserve the thiol/disulfide balance. We studied the composition of thiol/disulfide systems and their role in the oxidative stress response in this extremophile bacterium. Bioinformatic analysis using genomic data and enzymatic assays using protein extracts from cells grown under oxidative stress revealed that the major thiol/disulfide system from L. ferriphilum are a cytoplasmic thioredoxin system (composed by thioredoxins Trx and thioredoxin reductase TR, periplasmic thiol oxidation system (DsbA/DsbB and a c-type cytochrome maturation system (DsbD/DsbE. Upon exposure of L. ferriphilum to reactive oxygen species (ROS-generating compounds, transcriptional activation of the genes encoding Trxs and the TR enzyme, which results in an increase of the corresponding activity, was observed. Altogether these data suggest that the thioredoxin-based thiol/disulfide system plays an important role in redox protection of L. ferriphilum favoring the survival of this microorganism under extreme environmental oxidative conditions.

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase.

Science.gov (United States)

Rhimi, Moez; Chouayekh, Hichem; Gouillouard, Isabelle; Maguin, Emmanuelle; Bejar, Samir

2011-02-01

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process. Copyright © 2010 Elsevier Ltd. All rights reserved.

Evaluation of dynamic serum thiol/disulfide homeostasis in locally advanced and metastatic gastric cancer

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Mutlu Hizal

2018-04-01

Full Text Available Background: Gastric cancer is one the most diagnosed cancer and the third leading cause of death from cancer worldwide. As an indicator of antioxidant capacity thiol/disulfide homeostasis regulates detoxification, cell signal mechanisms, apoptosis, transcription and antioxidant defense mechanisms. Disregulation of thiol/disulfide homeostasis identified in other cancer types by recent data. In this study, we aimed to evaluate the thiol/disulfide homeostasis in advanced gastric cancer patients. Methods: The patients who diagnosed with gastric cancer and healthy control subjects were included to study. Serum samples for the thiol-disulphide test were obtained at the time of diagnosis. Thiol-disulphide homeostasis tests were measured by the automated spectrophotometric method. Thiol-disulphide homeostasis was also measured according to clinical and laboratory features. Results: Thirty newly diagnosed advanced gastric adenocarcinoma patients and 28 healthy controls were enrolled in the study. The native thiol (NT and total thiol (TT levels of patients' group were significantly lower compared with controls (p = 0.001 and p < 0.001. In the CEA high (≥5.4 ng/ml group, DS/NT ratio were higher compared with CEA low (<5.4 ng/ml group (p = 0.024. In CA.19-9 high (≥28.3 kU/L group, both DS and DS/NT ratio were significantly higher compared with a CA19-9 low(<28.3 kU/L group (p < 0.05 both. The correlation between CEA and DS levels was also significant (p = 0.02. There was also a positive correlation between CEA levels and DS/NT ratio (p = 0.01. Conclusion: Derangements of thiol/disulfide homeostasis may have a role in gastric cancer pathogenesis and the higher level of oxidative stress may relate to extensive and aggressiveness of the advanced disease. The diagnostic and prognostic values of thiol/disulfide products need to identify with further studies. Keywords: Thiol, Disulfide, Oxidative stress, Gastric cancer, Metastatic

A preliminary X-ray study of sedoheptulose-7-phosphate isomerase from Burkholderia pseudomallei

International Nuclear Information System (INIS)

Kim, Mi-Sun; Shin, Dong Hae

2009-01-01

Sedoheptulose-7-phosphate isomerase (GmhA) from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Sedoheptulose-7-phosphate isomerase (GmhA) converts d-sedoheptulose 7-phosphate to d,d-heptose 7-phosphate. This is the first step in the biosynthesis pathway of NDP-heptose, which is responsible for the pleiotropic phenotype. This biosynthesis pathway is the target of inhibitors to increase the membrane permeability of Gram-negative pathogens or of adjuvants working synergistically with known antibiotics. Burkholderia pseudomallei is the causative agent of melioidosis, a seriously invasive disease in animals and humans in tropical and subtropical areas. GmhA from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 1.9 Å resolution. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 61.3, b = 84.2, c = 142.3 Å. A full structural determination is under way in order to provide insights into the structure–function relationships of this protein

Photo-responsive liquid crystalline epoxy networks with exchangeable disulfide bonds

Energy Technology Data Exchange (ETDEWEB)

Li, Yuzhan [Washington State Univ., Pullman, WA (United States); Zhang, Yuehong [Washington State Univ., Pullman, WA (United States); Rios, Orlando [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Keum, Jong K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Kessler, Michael R. [Washington State Univ., Pullman, WA (United States); North Dakota State Univ., Fargo, ND (United States)

2017-07-27

The increasing demand for intelligent materials has driven the development of polymers with a variety of functionalities. However, combining multiple functionalities within one polymer is still challenging because of the difficulties encountered in coordinating different functional building blocks during fabrication. In this work, we demonstrate the fabrication of a multifunctional liquid crystalline epoxy network (LCEN) using the combination of thermotropic liquid crystals, photo-responsive azobenzene molecules, and exchangeable disulfide bonds. In addition to shape memory behavior enabled by the reversible liquid crystalline phase transition and photo-induced bending behavior resulting from the photo-responsive azobenzene molecules, the introduction of dynamic disulfide bonds into the LCEN resulted in a structurally dynamic network, allowing the reshaping, repairing, and recycling of the material.

Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction

DEFF Research Database (Denmark)

Jensen, Johanne Mørch; Hägglund, Per; Christensen, Hans Erik Mølager

2012-01-01

and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass......Barley limit dextrinase (LD) that catalyses hydrolysis of α-1,6 glucosidic linkages in starch-derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM-protein family) and has four disulfide bridges...... spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function....

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

Science.gov (United States)

Zavodszky, M; Chen, C W; Huang, J K; Zolkiewski, M; Wen, L; Krishnamoorthi, R

2001-01-01

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen

Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

Science.gov (United States)

Inoue, H; Hirobe, M

1987-05-29

The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

The crystal structure of a multifunctional protein: Phosphoglucose isomerase/autocrine motility factor/neuroleukin

OpenAIRE

Sun, Yuh-Ju; Chou, Chia-Cheng; Chen, Wei-Shone; Wu, Rong-Tsun; Meng, Menghsiao; Hsiao, Chwan-Deng

1999-01-01

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-Å resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm tha...

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

DEFF Research Database (Denmark)

Barba Espin, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per

2014-01-01

respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping...... and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat...... shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions...

Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans

International Nuclear Information System (INIS)

Doan, Thi-Ngoc-Thanh; Prabhu, Ponnandy; Kim, Jin-Kwang; Ahn, Yeh-Jin; Natarajan, Sampath; Kang, Lin-Woo; Park, Geon Tae; Lim, Sang-Boem; Lee, Jung-Kul

2010-01-01

l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution. l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å 3 Da −1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method

Electrical Transport Properties of Polycrystalline Monolayer Molybdenum Disulfide

Science.gov (United States)

2014-07-14

Lou, Sina Najmaei, Matin Amani, Matthew L. Chin, Zheng Se. TASK NUMBER Liu Sf. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAMES AND ADDRESSES 8...Transport Properties of Polycrystalline Monolayer Molybdenum Disulfide Sina Najmaei,t.§ Matin Ama ni,M Matthew L. Chin,* Zhe ng liu/ ·"·v: A. Gle n

Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways.

Science.gov (United States)

Nagy, Péter

2013-05-01

Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidation, a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their reduction, isomerisation, and interconversion as well as their production reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biology. Redox homeostasis, a thermodynamic parameter that determines which reactions can occur in cellular compartments, is also balanced by the thiol-disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. This review is focused on the kinetics and mechanisms of thiol-disulfide substitution and redox reactions. It summarizes the challenges and advances that are associated with kinetic investigations in small molecular and enzymatic systems from a rigorous chemical perspective using biological examples. The most important parameters that influence reaction rates are discussed in detail. Kinetic studies of proteins are more challenging than small molecules, and quite often investigators are forced to sacrifice the rigor of the experimental approach to obtain the important kinetic and mechanistic information. However, recent technological advances allow a more comprehensive analysis of enzymatic systems via using the systematic kinetics apparatus that was developed for small molecule reactions, which is expected to provide further insight into the cell's machinery.

Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

International Nuclear Information System (INIS)

Nemoto, Naoto; Tsutsui, Chihiro; Yamaguchi, Junichi; Ueno, Shingo; Machida, Masayuki; Kobayashi, Toshikatsu; Sakai, Takafumi

2012-01-01

Highlights: ► Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. ► Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. ► Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method “cDNA display”. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

Anterior gradient 2 is induced in cutaneous wound and promotes wound healing through its adhesion domain.

Science.gov (United States)

Zhu, Qi; Mangukiya, Hitesh Bhagavanbhai; Mashausi, Dhahiri Saidi; Guo, Hao; Negi, Hema; Merugu, Siva Bharath; Wu, Zhenghua; Li, Dawei

2017-09-01

Anterior gradient 2 (AGR2), a member of protein disulfide isomerase (PDI) family, is both located in cytoplasm and secreted into extracellular matrix. The orthologs of AGR2 have been linked to limb regeneration in newt and wound healing in zebrafish. In mammals, AGR2 influences multiple cell signaling pathways in tumor formation and in normal cell functions related to new tissue formation like angiogenesis. However, the function of AGR2 in mammalian wound healing remains unknown. This study aimed to investigate AGR2 expression and its function during skin wound healing and the possible application of external AGR2 in cutaneous wound to accelerate the healing process. Our results showed that AGR2 expression was induced in the migrating epidermal tongue and hyperplastic epidermis after skin excision. Topical application of recombinant AGR2 significantly accelerated wound-healing process by increasing the migration of keratinocytes (Kera.) and the recruitment of fibroblasts (Fibro.) near the wounded area. External AGR2 also promoted the migration of Kera. and Fibro. in vitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating Fibro., leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of Kera. and Fibro. migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds. © 2017 Federation of European Biochemical Societies.

The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

Directory of Open Access Journals (Sweden)

Ann De Vos

2015-04-01

Full Text Available Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability.

Science.gov (United States)

Peters, Shirley J; Smales, C Mark; Henry, Alistair J; Stephens, Paul E; West, Shauna; Humphreys, David P

2012-07-13

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.

Reduction of the number of defect signals in pressure vessel welds by a phased array ultrasonic test technology qualified beforehand in a blind test according to PDI specifications

International Nuclear Information System (INIS)

Mohr, F.

2007-01-01

In German-language countries, ultrasonic testing of reactor pressure vessel welds in the context of recurrent inspection is based on the KTA rules. This test philosophy is based on the recording of all data of a test section and repeated comparison of these data at regular intervals. Each and every change during operation is displayed. There are many components in which no changes are observed over longer periods of time. Optimisation of the test procedure and test periods requires accurate knowledge of the component condition. This necessitates accurate data of available defects. However, current techniques only provide data for comparative analysis on the basis of reflectivity. Data on the length and depth of a relevant defect can only be obtained by qualified sizing techniques. The PDI programme provides exact rules for qualification of techniques for a given application. Using a PDI qualification with personal blind tests for all data evaluators, one obtains a basis for accurate defect dimensioning and thus for optimisation. In cooperation with KKL, IntelligeNDT AREVA in 2006 successfully underwent the PDI qualification process for phased array testing of longitudinal and circumferential welds in reactor pressure vessels. In addition to this qualification, a comparison was made with the results of the conventionally applied, KTA-oriented test procedure. One of the key elements of qualification is the characterisation of defects, i.e. the distinction between relevant and non-relevant data, which will help to reduce the displayed data. The contribution presents the results and experience of the qualification as well as a comparison of standard testing with a tandem function with the results of phased array testing. (orig.)

Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.

Directory of Open Access Journals (Sweden)

Julie K Klint

Full Text Available Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.

Mechanism of ultraviolet light induced catabolite repression of L-arabinose isomerase

Energy Technology Data Exchange (ETDEWEB)

Bhatnagar, D; Bhattacharya, A K [Banaras Hindu Univ. (India). Inst. of Medical Sciences

1982-12-01

An attempt has been made to find out how U.V. irradiation of E.coli B/r cells causes catabolite repression to inhibit L-arabinose isomerase synthesis. The results presented show that U.V. irradiation leads to a lowering of the cellular cyclic AMP level and of the cyclic AMP binding activity. Unlike catabolite repression by glucose, no small molecular weight compound is involved in U.V. light induced inhibition of the binding activity. It is therefore concluded that the mechanism of catabolite repression induced by U.V. appears to be different from that of the catabolite repression by glucose.

Abiotic synthesis of organic compounds from carbon disulfide under hydrothermal conditions.

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Rushdi, Ahmed I; Simoneit, Bernd R T

2005-12-01

Abiotic formation of organic compounds under hydrothermal conditions is of interest to bio, geo-, and cosmochemists. Oceanic sulfur-rich hydrothermal systems have been proposed as settings for the abiotic synthesis of organic compounds. Carbon disulfide is a common component of magmatic and hot spring gases, and is present in marine and terrestrial hydrothermal systems. Thus, its reactivity should be considered as another carbon source in addition to carbon dioxide in reductive aqueous thermosynthesis. We have examined the formation of organic compounds in aqueous solutions of carbon disulfide and oxalic acid at 175 degrees C for 5 and 72 h. The synthesis products from carbon disulfide in acidic aqueous solutions yielded a series of organic sulfur compounds. The major compounds after 5 h of reaction included dimethyl polysulfides (54.5%), methyl perthioacetate (27.6%), dimethyl trithiocarbonate (6.8%), trithianes (2.7%), hexathiepane (1.4%), trithiolanes (0.8%), and trithiacycloheptanes (0.3%). The main compounds after 72 h of reaction consisted of trithiacycloheptanes (39.4%), pentathiepane (11.6%), tetrathiocyclooctanes (11.5%), trithiolanes (10.6%), tetrathianes (4.4%), trithianes (1.2%), dimethyl trisulfide (1.1%), and numerous minor compounds. It is concluded that the abiotic formation of aliphatic straight-chain and cyclic polysulfides is possible under hydrothermal conditions and warrants further studies.

Kinetic study of the interaction of glutathione with four antitumor disulfides: possible mechanism for cellular glutathione depletion.

Science.gov (United States)

Kirkpatrick, D L

1989-01-01

The reactions between the cellular tripeptide, glutathione (GSH) and four disulfide derivatives of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) (compounds 1-4) were studied kinetically. The decyl and phenyl derivatives of 6-MP and 6-TG were reacted with GSH in phosphate buffer (pH 7.4 or 6.0) at 25.0 degrees C and were monitored spectrophotometrically by observing the release of 6-MP and 6-TG. Second order kinetics were observed, with rate constants of 142, 564, 4174 and 429 M-1 s-1 being measured for compounds 1-4, respectively. When the reactions were carried out in the presence of GSH-S-transferase the rates were enhanced 1.3-5.4 times those observed in the absence of enzyme. Products of the reactions were isolated by chromatography and tentatively identified by TLC or fast atom bombardment mass spectrometry. It was observed that GSH reacted with each disulfide in a 1:1 manner, forming a mixed disulfide between GSH and decanethiol or thiophenol while releasing 6-MP or 6-TG. It was concluded that the reported depletion of GSH from EMT6 cells after exposure to these disulfides could be due to their reaction with GSH, and the formation of the mixed disulfides.

Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail

International Nuclear Information System (INIS)

Dy, Catherine Y.; Buczek, Pawel; Imperial, Julita S.; Bulaj, Grzegorz; Horvath, Martin P.

2006-01-01

Most Kunitz proteins like BPTI and α-dendrotoxin are stabilized by three disulfide bonds. The crystal structure shows how subtle repacking of non-covalent interactions may compensate for disulfide bond loss in a naturally occurring two-disulfide variant, conkunitzin-S1, the first discovered member of a new conotoxin family. Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50–200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine–cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as α-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and α-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 3 10 –β–β–α Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in α-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed the

Selenocysteine in thiol/disulfide-like exchange reactions.

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Hondal, Robert J; Marino, Stefano M; Gladyshev, Vadim N

2013-05-01

Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

N-glycosylation and disulfide bonding affects GPRC6A receptor expression, function, and dimerization

DEFF Research Database (Denmark)

Nørskov-Lauritsen, Lenea; Jørgensen, Stine; Bräuner-Osborne, Hans

2015-01-01

Investigation of post-translational modifications of receptor proteins is important for our understanding of receptor pharmacology and disease physiology. However, our knowledge about post-translational modifications of class C G protein-coupled receptors and how these modifications regulate expr...... covalently linked dimers through cysteine disulfide linkage in the extracellular amino-terminal domain and here we show that GPRC6A indeed is a homodimer and that a disulfide bridge between the C131 residues is formed....

Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans

International Nuclear Information System (INIS)

Wang, Chen; Fan, Xuexin; Cao, Xiaofang; Liu, Xiang; Li, Lanfen; Su, Xiaodong

2012-01-01

Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses. Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni 2+ -chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

Science.gov (United States)

Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

2013-08-01

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

International Nuclear Information System (INIS)

Mori, Tadashi; Hidaka, Masafumi; Lin, Yi-Chin; Yoshizawa, Ibuki; Okabe, Takayoshi; Egashira, Shinichiro; Kojima, Hirotatsu; Nagano, Tetsuo; Koketsu, Mamoru; Takamiya, Mari; Uchida, Takafumi

2011-01-01

Research highlights: → A Pin1 (prolyl isomerase) inhibitor, TME-001, has been discovered by using a new established high-throughput screening method. → The TME-001 showed a cell-active inhibition with lower cytotoxic effect than known Pin1 inhibitors. → Kinetic analyses revealed that the TME-001 is the first compound that exhibits dual inhibition of Pin1 and another type of prolyl isomerase, cyclophilin. → Thus, similarities of structure and reaction mechanism between Pin1 and cyclophilin are proposed. -- Abstract: Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC 50 = 6.1 μM) and cyclophilin, another type of PPIase, (IC 50 = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Science.gov (United States)

Do, Bich Hang; Nguyen, Minh Tan; Song, Jung-A; Park, Sangsu; Yoo, Jiwon; Jang, Jaepyeong; Lee, Sunju; So, Seoungjun; Yoon, Yejin; Kim, Inki; Lee, Kyungjin; Jang, Yeon Jin; Choe, Han

2017-12-28

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli . In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

Redox Regulation in Amyotrophic Lateral Sclerosis

Science.gov (United States)

Parakh, Sonam; Spencer, Damian M.; Halloran, Mark A.; Soo, Kai Y.; Atkin, Julie D.

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from the death of upper and lower motor neurons. Due to a lack of effective treatment, it is imperative to understand the underlying mechanisms and processes involved in disease progression. Regulations in cellular reduction/oxidation (redox) processes are being increasingly implicated in disease. Here we discuss the possible involvement of redox dysregulation in the pathophysiology of ALS, either as a cause of cellular abnormalities or a consequence. We focus on its possible role in oxidative stress, protein misfolding, glutamate excitotoxicity, lipid peroxidation and cholesterol esterification, mitochondrial dysfunction, impaired axonal transport and neurofilament aggregation, autophagic stress, and endoplasmic reticulum (ER) stress. We also speculate that an ER chaperone protein disulphide isomerase (PDI) could play a key role in this dysregulation. PDI is essential for normal protein folding by oxidation and reduction of disulphide bonds, and hence any disruption to this process may have consequences for motor neurons. Addressing the mechanism underlying redox regulation and dysregulation may therefore help to unravel the molecular mechanism involved in ALS. PMID:23533690

Hindered disulfide bonds to regulate release rate of model drug from mesoporous silica.

Science.gov (United States)

Nadrah, Peter; Maver, Uroš; Jemec, Anita; Tišler, Tatjana; Bele, Marjan; Dražić, Goran; Benčina, Mojca; Pintar, Albin; Planinšek, Odon; Gaberšček, Miran

2013-05-01

With the advancement of drug delivery systems based on mesoporous silica nanoparticles (MSNs), a simple and efficient method regulating the drug release kinetics is needed. We developed redox-responsive release systems with three levels of hindrance around the disulfide bond. A model drug (rhodamine B dye) was loaded into MSNs' mesoporous voids. The pore opening was capped with β-cyclodextrin in order to prevent leakage of drug. Indeed, in absence of a reducing agent the systems exhibited little leakage, while the addition of dithiothreitol cleaved the disulfide bonds and enabled the release of cargo. The release rate and the amount of released dye were tuned by the level of hindrance around disulfide bonds, with the increased hindrance causing a decrease in the release rate as well as in the amount of released drug. Thus, we demonstrated the ability of the present mesoporous systems to intrinsically control the release rate and the amount of the released cargo by only minor structural variations. Furthermore, an in vivo experiment on zebrafish confirmed that the present model delivery system is nonteratogenic.

Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways

Science.gov (United States)

2013-01-01

Abstract Significance: Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidation, a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their reduction, isomerisation, and interconversion as well as their production reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biology. Redox homeostasis, a thermodynamic parameter that determines which reactions can occur in cellular compartments, is also balanced by the thiol–disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. Critical Issues: This review is focused on the kinetics and mechanisms of thiol–disulfide substitution and redox reactions. It summarizes the challenges and advances that are associated with kinetic investigations in small molecular and enzymatic systems from a rigorous chemical perspective using biological examples. The most important parameters that influence reaction rates are discussed in detail. Recent Advances and Future Directions: Kinetic studies of proteins are more challenging than small molecules, and quite often investigators are forced to sacrifice the rigor of the experimental approach to obtain the important kinetic and mechanistic information. However, recent technological advances allow a more comprehensive analysis of enzymatic systems via using the systematic kinetics apparatus that was developed for small molecule reactions, which is expected to provide further insight into the cell's machinery. Antioxid. Redox Signal. 18, 1623–1641. PMID:23075118

Crystallographic Studies Evidencing the High Energy Tolerance to Disrupting the Interface Disulfide Bond of Thioredoxin 1 from White Leg Shrimp Litopenaeus vannamei

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Adam A. Campos-Acevedo

2014-12-01

Full Text Available Thioredoxin (Trx is a small 12-kDa redox protein that catalyzes the reduction of disulfide bonds in proteins from different biological systems. A recent study of the crystal structure of white leg shrimp thioredoxin 1 from Litopenaeus vannamei (LvTrx revealed a dimeric form of the protein mediated by a covalent link through a disulfide bond between Cys73 from each monomer. In the present study, X-ray-induced damage in the catalytic and the interface disulfide bond of LvTrx was studied at atomic resolution at different transmission energies of 8% and 27%, 12.8 keV at 100 K in the beamline I-24 at Diamond Light Source. We found that at an absorbed dose of 32 MGy, the X-ray induces the cleavage of the disulfide bond of each catalytic site; however, the interface disulfide bond was cleaved at an X-ray adsorbed dose of 85 MGy; despite being the most solvent-exposed disulfide bond in LvTrx (~50 Å2. This result clearly established that the interface disulfide bond is very stable and, therefore, less susceptible to being reduced by X-rays. In fact, these studies open the possibility of the existence in solution of a dimeric LvTrx.

Crystallographic studies evidencing the high energy tolerance to disrupting the interface disulfide bond of thioredoxin 1 from white leg shrimp Litopenaeus vannamei.

Science.gov (United States)

Campos-Acevedo, Adam A; Rudiño-Piñera, Enrique

2014-12-15

Thioredoxin (Trx) is a small 12-kDa redox protein that catalyzes the reduction of disulfide bonds in proteins from different biological systems. A recent study of the crystal structure of white leg shrimp thioredoxin 1 from Litopenaeus vannamei (LvTrx) revealed a dimeric form of the protein mediated by a covalent link through a disulfide bond between Cys73 from each monomer. In the present study, X-ray-induced damage in the catalytic and the interface disulfide bond of LvTrx was studied at atomic resolution at different transmission energies of 8% and 27%, 12.8 keV at 100 K in the beamline I-24 at Diamond Light Source. We found that at an absorbed dose of 32 MGy, the X-ray induces the cleavage of the disulfide bond of each catalytic site; however, the interface disulfide bond was cleaved at an X-ray adsorbed dose of 85 MGy; despite being the most solvent-exposed disulfide bond in LvTrx (~50 Å2). This result clearly established that the interface disulfide bond is very stable and, therefore, less susceptible to being reduced by X-rays. In fact, these studies open the possibility of the existence in solution of a dimeric LvTrx.

Increased Functional Half-life of Fibroblast Growth Factor-1 by Recovering a Vestigial Disulfide Bond

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Jihun Lee

2010-12-01

Full Text Available The fibroblast growth factor (FGF family of proteins contains an absolutely conserved Cys residue at position 83 that is present as a buried free cysteine. We have previously shown that mutation of the structurally adjacent residue, Ala66, to cysteine results in the formation of a stabilizing disulfide bond in FGF-1. This result suggests that the conserved free cysteine residue at position 83 in the FGF family of proteins represents a vestigial half-cystine. Here, we characterize the functional half-life and mitogenic activity of the oxidized form of the Ala66Cys mutation to identify the effect of the recovered vestigial disulfide bond between Cys83 and Cys66 upon the cellular function of FGF-1. The results show that the mitogenic activity of this mutant is significantly increased and that its functional half-life is greatly extended. These favorable effects are conferred by the formation of a disulfide bond that simultaneously increases thermodynamic stability of the protein and removes a reactive buried thiol at position 83. Recovering this vestigial disulfide by introducing a cysteine at position 66 is a potentially useful protein engineering strategy to improve the functional half-life of other FGF family members.

Dynamic thiol/disulfide homeostasis and effects of smoking on homeostasis parameters in patients with psoriasis.

Science.gov (United States)

Emre, Selma; Demirseren, Duriye Deniz; Alisik, Murat; Aktas, Akin; Neselioglu, Salim; Erel, Ozcan

2017-12-01

Recently, increased reactive oxygen species (ROS), reduced antioxidant capacity, and oxidative stress have been suggested in the pathogenesis of psoriasis. The aim of this study to evaluate the thiol/disulfide homeostasis in patients with psoriasis. Ninety patients with psoriasis who did not receive any systemic treatment in the last six  months were included in the study. Seventy-six age and gender-matched healthy volunteers served as control group. Thiol/disulfide homeostasis was measured in venous blood samples obtained from patient and control groups. Native thiol and total thiol levels were significantly higher in patients than in control group. When thiol/disulfide hemostasis parameters and clinical and demographic characteristics were compared, a negative correlation was detected between native thiol and total thiol with age. The levels of total thiols had also negative correlation with PASI and duration of the disease. When we divided the patients into smokers and non-smokers, native thiol and total thiol levels were significantly higher in smokers than in controls, whereas native thiol and total thiol levels were comparable in non-smoker patients and controls. Thiol/disulfide balance shifted towards thiol in psoriasis patients and this may be responsible for increased keratinocyte proliferation in the pathogenesis of psoriasis.

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

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Alberto Guevara-Flores

Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

Characterization of cyclic peptides containing disulfide bonds

OpenAIRE

Johnson, Mindy; Liu, Mingtao; Struble, Elaine; Hettiarachchi, Kanthi

2015-01-01

Unlike linear peptides, analysis of cyclic peptides containing disulfide bonds is not straightforward and demands indirect methods to achieve a rigorous proof of structure. Three peptides that belong to this category, p-Cl-Phe-DPDPE, DPDPE, and CTOP, were analyzed and the results are presented in this paper. The great potential of two dimensional NMR and ESI tandem mass spectrometry was harnessed during the course of peptide characterizations. A new RP-HPLC method for the analysis of trifluor...

Genome sequence of carboxylesterase, carboxylase and xylose isomerase producing alkaliphilic haloarchaeon Haloterrigena turkmenica WANU15

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Samy Selim

2016-03-01

Full Text Available We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000. Keywords: Soda Lake, Haloterrigena turkmenica, Carboxylesterase, Carboxylase, Xylose isomerase, Whole genome sequencing

Selective removal of heavy metal ions by disulfide linked polymer networks

DEFF Research Database (Denmark)

Ko, Dongah; Sung Lee, Joo; Patel, Hasmukh A.

2017-01-01

Heavy metal contaminated surface water is one of the oldest pollution problems, which is critical to ecosystems and human health. We devised disulfide linked polymer networks and employed as a sorbent for removing heavy metal ions from contaminated water. Although the polymer network material has...... a moderate surface area, it demonstrated cadmium removal efficiency equivalent to highly porous activated carbon while it showed 16 times faster sorption kinetics compared to activated carbon, owing to the high affinity of cadmium towards disulfide and thiol functionality in the polymer network. The metal...... sorption mechanism on polymer network was studied by sorption kinetics, effect of pH, and metal complexation. We observed that the metal ions―copper, cadmium, and zinc showed high binding affinity in polymer network, even in the presence of competing cations like calcium in water....

CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site.

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Helena Kellett-Clarke

Full Text Available CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA, a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the-LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.

Engineering a disulfide bond in the lid hinge region of Rhizopus chinensis lipase: increased thermostability and altered acyl chain length specificity.

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Xiao-Wei Yu

Full Text Available The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for "interfacial activation" is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the "Disulfide by Design" algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t(1/2 value at 60°C and a 7°C increase of T(m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k(cat and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.

Disulfide scrambling in superoxide dismutase 1 reduces its cytotoxic effect in cultured cells and promotes protein aggregation.

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Lina Leinartaitė

Full Text Available Mutations in the gene coding for superoxide dismutase 1 (SOD1 are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS. These mutations are believed to result in a "gain of toxic function", leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn(2+ site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn(2+. This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.

Crystal Structure of Escherichia coli L-Arabinose Isomerase (ECAI), The Putative Target of Biological Tagatose Production

Energy Technology Data Exchange (ETDEWEB)

Manjasetty,B.; Chance, M.

2006-01-01

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 Angstroms resolution. The subunit structure of ECAI is organized into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.

Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

International Nuclear Information System (INIS)

Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

2005-01-01

The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

Science.gov (United States)

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-10-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

Characterization of an L-arabinose isomerase from Bacillus thermoglucosidasius for D-tagatose production.

Science.gov (United States)

Seo, Myung-Ji

2013-01-01

L-Arabinose isomerase from Bacillus thermoglucosidasius KCTC 1828 (BTAI) was expressed in Escherichia coli. The optimal temperature and pH for the activity of the purified BTAI were 40 °C and pH 7.0. The Mn(2+) ion was an activator of BTAI activity. The kinetic parameters of BTAI for D-galactose were a K(m) of 175 mM and a k(cat)/K(m) of 2.8 mM(-1)min(-1). The conversion ratio by BTAI to D-tagatose reached 45.6% at 40 °C.

Role of the Disulfide Bond in Prion Protein Amyloid Formation: A Thermodynamic and Kinetic Analysis.

Science.gov (United States)

Honda, Ryo

2018-02-27

Prion diseases are associated with the structural conversion of prion protein (PrP) to a β-sheet-rich aggregate, PrP Sc . Previous studies have indicated that a reduction of the disulfide bond linking C179 and C214 of PrP yields an amyloidlike β-rich aggregate in vitro. To gain mechanistic insights into the reduction-induced aggregation, here I characterized how disulfide bond reduction modulates the protein folding/misfolding landscape of PrP, by examining 1) the equilibrium stabilities of the native (N) and aggregated states relative to the unfolded (U) state, 2) the transition barrier separating the U and aggregated states, and 3) the final structure of amyloidlike misfolded aggregates. Kinetic and thermodynamic experiments revealed that disulfide bond reduction decreases the equilibrium stabilities of both the N and aggregated states by ∼3 kcal/mol, without changing either the amyloidlike aggregate structure, at least at the secondary structural level, or the transition barrier of aggregation. Therefore, disulfide bond reduction modulates the protein folding/misfolding landscape by entropically stabilizing disordered states, including the U and transition state of aggregation. This also indicates that the equilibrium stability of the N state, but not the transition barrier of aggregation, is the dominant factor determining the reduction-induced aggregation of PrP. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow

DEFF Research Database (Denmark)

Trabjerg, Esben; Jakobsen, Rasmus Uffe; Mysling, Simon

2015-01-01

Analysis of disulfide-bonded proteins by HDX-MS requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench......-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction...... the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and Nerve Growth Factor-β (NGF). Several different parameters (flow rate, applied square wave potential as well as the type of labeling- and quench buffer) were...

Rhodium-Catalyzed Insertion Reaction of PhP Group of Pentaphenylcyclopentaphosphine with Acyclic and Cyclic Disulfides.

Science.gov (United States)

Arisawa, Mieko; Sawahata, Kyosuke; Yamada, Tomoki; Sarkar, Debayan; Yamaguchi, Masahiko

2018-02-16

Organophosphorus compounds with a phosphorus atom attached to a phenyl group and two organothio/organoseleno groups were synthesized using the rhodium-catalyzed insertion reaction of the PhP group of pentaphenylcyclopentaphosphine (PhP) 5 with acyclic disulfides and diselenides. The method was applied to the synthesis of heterocyclic compounds containing the S-P-S group by the reaction of (PhP) 5 and cyclic disulfides such as 1,2-dithietes, 1,2-dithiocane, 1,4,5-dithiopane, and 1,2-dithiolanes.

Polimorfisme Enzim Glucose-6-Phosphate Isomerase pada Tiga Populasi Tuna Sirip Kuning (Thunnus albacares)

OpenAIRE

Permana, Gusti Ngurah; Hutapea, Jhon H.; Moria, Sari Budi; Haryanti, Haryanti

2006-01-01

Samples of yellowfin tuna (Thunnus albacares) were taken from three locations Bali, North Sulawesi and North Maluku. The glucose-6-phosphate isomerase (GPI) was analyzed from liver using allozyme electrophoresis method. Polymorphism of GPI enzyme was observed and four alleles (A, B ,C, D) were found in Bali population, three alleles (A,B,C) were found in North Maluku and North Sulawesi populations. Heterozygosity values, from Bali, North Maluku and North Sulawesi were 0.419; 0.417; 0.143 resp...

Mechanism of thioredoxin-catalyzed disulfide reduction. Activation of the buried thiol and role of the variable active-site residues

NARCIS (Netherlands)

Carvalho, A.P.; Swart, M.; van Stralen, J.N.P.; Fernandes, P.A.; Ramos, M.E.; Bickelhaupt, F.M.

2008-01-01

Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in other proteins. We have theoretically explored this reaction mechanism, both in the gas phase and in water, using density functional theory. The mechanism of disulfide

Regional cerebral blood flow after long-term exposure to carbon disulfide

International Nuclear Information System (INIS)

Aaserud, O.; Russell, D.; Nyberg-Hansen, R.; Joergensen, E.B.; Gjerstad, L.; Rootwelt, K.; Nakstad, P.; Hommeren, O.J.; Tvedt, B.

1992-01-01

Sixteen former rayon viscose workers were investigated four years after the exposure to carbon disulfide was discontinued. Median age was 58 years (range 43-65 years), median exposure time was 17 years (range 10-35 years). Encephalopathy was diagnosed in altogether 14 workers. To further explore pathophysiological mechanisms, cerebrovascular investigations were employed. Doppler ultrasound examination of the precerebral vessels in 15 workers showed a slight stenosis of the left internal carotid artery in one. Regional cerebral blood flow investigation (rCBF) with single photon emission computerized tomography (SPECT) with Xenon-133 gas was performed in 14. There was no significant difference from a control group. Regional side-to-side asymmetries beyond reference limits were demonstrated in eight workers. The abnormalities were modest, but may indicate a tendency toward focal blood flow disturbances in workers with long-term exposure to carbon disulfide. (au)

Rethinking Cysteine Protective Groups: S-Alkylsulfonyl-l-Cysteines for Chemoselective Disulfide Formation.

Science.gov (United States)

Schäfer, Olga; Huesmann, David; Muhl, Christian; Barz, Matthias

2016-12-12

The ability to reversibly cross-link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S-alkylsulfonyl-l-cysteines and N-carboxy anhydrides (NCA) thereof for peptide synthesis. The S-alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well-defined homo- as well as block co-polymers. Yet, thiols react immediately with the S-alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Autonomic healable waterborne organic-inorganic polyurethane hybrids based on aromatic disulfide moieties

Directory of Open Access Journals (Sweden)

R. H. Aguirresarobe

2017-04-01

Full Text Available Aromatic disulfide dynamic structures were incorporated as chain extenders in waterborne organic-inorganic polyurethane hybrids in order to provide autonomic healable characteristics. The synthesis was carried out following the acetone process methodology and the influence of the introduction of the healing agents in the polymer dispersion stability was analyzed. After the crosslinking process at room temperature, organic-inorganic hybrid films, which presented autonomic healing characteristics, were obtained. These features were evaluated by means of stress-strain tests and the films showed repetitive healing abilities. Thus, the optimum healing time at room temperature (25 °C as well as the influence of different parameters in the healing efficiency, such the aromatic disulfide concentration or the physical properties of the polymer matrix were analyzed.

Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase.

Science.gov (United States)

Liu, Dongsheng; Cowburn, David

2016-01-01

The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.

Differential Labeling of Free and Disulfide-Bound Thiol Functions in Proteins

NARCIS (Netherlands)

Seiwert, B.; Hayen, H.; Karst, U.

2008-01-01

A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid

Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

Science.gov (United States)

Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...

Coexpression of β-D-galactosidase and L-arabinose isomerase in the production of D-tagatose: a functional sweetener.

Science.gov (United States)

Zhan, Yijing; Xu, Zheng; Li, Sha; Liu, Xiaoliu; Xu, Lu; Feng, Xiaohai; Xu, Hong

2014-03-19

The functional sweetener, d-tagatose, is commonly transformed from galactose by l-arabinose isomerase. To make use of a much cheaper starting material, lactose, hydrolization, and isomerization are required to take place collaboratively. Therefore, a single-step method involving β-d-galactosidase was explored for d-tagatose production. The two vital genes, β-d-galactosidase gene (lacZ) and l-arabinose isomerase mutant gene (araA') were extracted separately from Escherichia coli strains and incorporated into E. coli simultaneously. This gave us E. coli-ZY, a recombinant producing strain capable of coexpressing the two key enzymes. The resulted cells exhibited maximum d-tagatose producing activity at 34 °C and pH 6.5 and in the presence of borate, 10 mM Fe(2+), and 1 mM Mn(2+). Further monitoring showed that the recombinant cells could hydrolyze more than 95% lactose and convert 43% d-galactose into d-tagatose. This research has verified the feasibility of single-step d-tagatose fermentation, thereby laying down the foundation for industrial usage of lactose.

L-Rhamnose isomerase and its use for biotechnological production of rare sugars.

Science.gov (United States)

Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2016-04-01

L-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between L-rhamnose and L-rhamnulose, plays an important role in microbial L-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of L-rhamnulose and various rare sugars including D-allose, D-gulose, L-lyxose, L-mannose, L-talose, and L-galactose were also reviewed.

Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: possible new targets of pharmacologic agents.

Science.gov (United States)

Nagy, Lajos; Nagata, Miki; Szabo, Sandor

2007-04-14

To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate. Rats were given 1 mL of 75% ethanol, 25% NaCl, 0.6 mol/L HCl, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver. Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCl or NaCl exposure, while oxidized glutathione (GSSG) concentrations increased, except by HCl and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed, NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals. No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations. Our modified methods are now suitable for direct measurements of major protein and non-protein thiols/disulfides in the gastric mucosa or liver. A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: Possible new targets of pharmacologic agents

Institute of Scientific and Technical Information of China (English)

Lajos Nagy; Miki Nagata; Sandor Szabo

2007-01-01

AIM: To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate.METHODS: Rats were given 1 mL of 75% ethanol, 25%NaCl, 0.6 mol/L HCI, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver.RESULTS: Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCI or NaCl exposure,while oxidized glutathione (GSSG) concentrations increased, except by HCI and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed,NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals.No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations.CONCLUSION: Our modified methods are now suitable for direct measurements of major protein and nonprotein thiols/disulfides in the gastric mucosa or liver.A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.

Directory of Open Access Journals (Sweden)

Rugmani Padmanabhan Iyer

Full Text Available SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT and Tris(2-carboxyethylphosphine (TCEP, indicating the possible involvement of disulfide bridge(s. Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

Science.gov (United States)

Inforzato, Antonio; Rivieccio, Vincenzo; Morreale, Antonio P; Bastone, Antonio; Salustri, Antonietta; Scarchilli, Laura; Verdoliva, Antonio; Vincenti, Silvia; Gallo, Grazia; Chiapparino, Caterina; Pacello, Lucrezia; Nucera, Eleonora; Serlupi-Crescenzi, Ottaviano; Day, Anthony J; Bottazzi, Barbara; Mantovani, Alberto; De Santis, Rita; Salvatori, Giovanni

2008-04-11

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

Folding and activity of hybrid sequence, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E. (Univ. of California, Berkeley (USA))

1990-08-01

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a {beta}-turn and an {alpha}-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the {alpha}-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences.

Folding and activity of hybrid sequence, disulfide-stabilized peptides

International Nuclear Information System (INIS)

Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E.

1990-01-01

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a β-turn and an α-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the α-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences

Thermoinactivation Mechanism of Glucose Isomerase

Science.gov (United States)

Lim, Leng Hong; Saville, Bradley A.

In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

Conversion of a disulfide bond into a thioacetal group during echinomycin biosynthesis

Energy Technology Data Exchange (ETDEWEB)

Hotta, Kinya; Keegan, Ronan M.; Ranganathan, Soumya; Fang, Minyi; Bibby, Jaclyn; Winn, Martyn D.; Sato, Michio; Lian, Mingzhu; Watanabe, Kenji; Rigden, Daniel J.; Kim, Chu-Young (Liverpool); (Daresbury); (NU Singapore); (Shizuoka); (RAL)

2013-12-02

Echinomycin is a nonribosomal depsipeptide natural product with a range of interesting bioactivities that make it an important target for drug discovery and development. It contains a thioacetal bridge, a unique chemical motif derived from the disulfide bond of its precursor antibiotic triostin A by the action of an S-adenosyl-L-methionine-dependent methyltransferase, Ecm18. The crystal structure of Ecm18 in complex with its reaction products S-adenosyl-L-homocysteine and echinomycin was determined at 1.50 Å resolution. Phasing was achieved using a new molecular replacement package called AMPLE, which automatically derives search models from structure predictions based on ab initio protein modelling. Structural analysis indicates that a combination of proximity effects, medium effects, and catalysis by strain drives the unique transformation of the disulfide bond into the thioacetal linkage.

Characterization of the triple-component linoleic acid isomerase in Lactobacillus plantarum ZS2058 by genetic manipulation.

Science.gov (United States)

Yang, B; Qi, H; Gu, Z; Zhang, H; Chen, W; Chen, H; Chen, Y Q

2017-11-01

To assess the mechanism for conjugated linoleic acid (CLA) production in Lactobacillus plantarum ZS2058. CLA has attracted great interests for decades due to its health-associated benefits including anticancer, anti-atherogenic, anti-obesity and modulation of the immune system. A number of microbial CLA producers were widely reported including lactic acid bacteria. Lactobacillus plantarum ZS2058, an isolate from Chinese traditional fermented food, could convert LA to CLA with various intermediates. To characterize the genetic determinants for generating CLA, a cre-lox-based system was utilized to delete the genes encoding myosin cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC) in Lact. plantarum ZS2058, respectively. Neither intermediate was detected in the corresponding gene deletion mutant. Meanwhile all those mutants could recover the ability to convert linoleic acid to CLA when the corresponding gene was completed. The results indicated that CLA production was a multiple-step reaction catalysed by triple-component linoleate isomerase system encoded by mcra, dh and dc. Multicomponent linoleic acid isomerase provided important results for illustration unique mechanism for CLA production in Lact. plantarum ZS2058. Lactobacilli with CLA production ability offer novel opportunities for functional food development. © 2017 The Society for Applied Microbiology.

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

Science.gov (United States)

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

Science.gov (United States)

Liu, Shuzhen; Liu, Hua; Johnston, Andrea; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui; Wang, Zhigao

2017-09-05

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry

Science.gov (United States)

Wormwood, Kelly L.; Ngounou Wetie, Armand Gatien; Gomez, Marcus Vinicius; Ju, Yue; Kowalski, Paul; Mihasan, Marius; Darie, Costel C.

2018-05-01

Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. [Figure not available: see fulltext.

Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry

Science.gov (United States)

Wormwood, Kelly L.; Ngounou Wetie, Armand Gatien; Gomez, Marcus Vinicius; Ju, Yue; Kowalski, Paul; Mihasan, Marius; Darie, Costel C.

2018-04-01

Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. [Figure not available: see fulltext.

Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds.

Science.gov (United States)

Rueda, Daniel; Sheen, Patricia; Gilman, Robert H; Bueno, Carlos; Santos, Marco; Pando-Robles, Victoria; Batista, Cesar V; Zimic, Mirko

2014-12-01

Recombinant wild-pyrazinamidase from H37Rv Mycobacterium tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

NARCIS (Netherlands)

Snijder, Joost; Van De Waterbeemd, Michiel; Glover, Matthew S.; Shi, Liuqing; Clemmer, David E.; Heck, Albert J R

2015-01-01

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions.

Disulfide-functional poly(amido amine)s with tunable degradability for gene delivery

NARCIS (Netherlands)

Elzes, M. Rachel; Akeroyd, Niels; Engbersen, Johan F. J.; Paulusse, Jos M. J.

2016-01-01

Controlled degradability in response to the local environment is one of the most effective strategies to achieve spatiotemporal release of genes from a polymeric carrier. Exploiting the differences in reduction potential between the extracellular and intracellular environment, disulfides are

Biosynthesis of collagen in the lung of the mouse after X-irradiation

International Nuclear Information System (INIS)

Walklin, C.M.; Law, M.P.

1986-01-01

Increases in the activities of both prolyl-4-hydroxylase (P-4-Hase) and protein disulphide isomerase (PDI) were observed as early as 1 month after 5-9 Gy. Maximal increases were observed at 6-7 months after 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual but by 1 year after irradiation they had reached levels similar to those after 9 Gy. Collagen types were analysed at 2, 7.5 and 15 months. Results are shown for 7.5 and 15 months after 9 Gy. Although the total collagen content was increased, the ratio of collagen type I to III was normal. (UK)

Helicobacter pylori Peptidyl Prolyl Isomerase Expression Is Associated with the Severity of Gastritis.

Science.gov (United States)

Oghalaie, Akbar; Saberi, Samaneh; Esmaeili, Maryam; Ebrahimzadeh, Fatemeh; Barkhordari, Farzaneh; Ghamarian, Abdolreza; Tashakoripoor, Mohammad; Abdirad, Afshin; Eshagh Hosseini, Mahmoud; Khalaj, Vahid; Mohammadi, Marjan

2016-12-01

Helicobacter pylori secretory peptidyl prolyl isomerase, HP0175, is progressively identified as a pro-inflammatory and pro-carcinogenic protein, which serves to link H. pylori infection to its more severe clinical outcomes. Here, we have analyzed host HP0175-specific antibody responses in relation to the severity of gastritis. The HP0175 gene fragment was PCR-amplified, cloned, expressed and purified by Ni-NTA affinity chromatography. Serum antigen-specific antibody responses of non-ulcer dyspeptic patients (N = 176) against recombinant HP0175 were detected by western blotting. The infection status of these subjects was determined by rapid urease test, culture, histology, and serology. The grade of inflammation and stage of atrophy were scored blindly according to the OLGA staging system. The recombinant HP0175 (rHP0175) was expressed as a ~35 kDa protein and its identity was confirmed by western blotting using anti-6X His tag antibody and pooled H. pylori-positive sera. Serum IgG antibodies against rHP0175 segregated our patients into two similar-sized groups of sero-positives (90/176, 51.1 %) and sero-negatives (86/176, 48.9 %). The former presented with higher grades of gastric inflammation (OR = 4.4, 95 % CI = 1.9-9.9, P = 0.001) and stages of gastric atrophy (OR = 18.3, 95 %CI = 1.4-246.6, P = 0.028). Our findings lend further support to the pro-inflammatory nature of H. pylori peptidyl prolyl isomerase (HP0175) and recommends this antigen as a non-invasive serum biomarker of the severity of H. pylori-associated gastritis.

Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

Energy Technology Data Exchange (ETDEWEB)

P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

2011-12-31

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

Glucose isomerization in simulated moving bed reactor by Glucose isomerase

Directory of Open Access Journals (Sweden)

Eduardo Alberto Borges da Silva

2006-05-01

Full Text Available Studies were carried out on the production of high-fructose syrup by Simulated Moving Bed (SMB technology. A mathematical model and numerical methodology were used to predict the behavior and performance of the simulated moving bed reactors and to verify some important aspects for application of this technology in the isomerization process. The developed algorithm used the strategy that considered equivalences between simulated moving bed reactors and true moving bed reactors. The kinetic parameters of the enzymatic reaction were obtained experimentally using discontinuous reactors by the Lineweaver-Burk technique. Mass transfer effects in the reaction conversion using the immobilized enzyme glucose isomerase were investigated. In the SMB reactive system, the operational variable flow rate of feed stream was evaluated to determine its influence on system performance. Results showed that there were some flow rate values at which greater purities could be obtained.Neste trabalho a tecnologia de Leito Móvel Simulado (LMS reativo é aplicada no processo de isomerização da glicose visando à produção de xarope concentrado de frutose. É apresentada a modelagem matemática e uma metodologia numérica para predizer o comportamento e o desempenho de unidades reativas de leito móvel simulado para verificar alguns aspectos importantes para o emprego desta tecnologia no processo de isomerização. O algoritmo desenvolvido utiliza a abordagem que considera as equivalências entre as unidades reativas de leito móvel simulado e leito móvel verdadeiro. Parâmetros cinéticos da reação enzimática são obtidos experimentalmente usando reatores em batelada pela técnica Lineweaver-Burk. Efeitos da transferência de massa na conversão de reação usando a enzima imobilizada glicose isomerase são verificados. No sistema reativo de LMS, a variável operacional vazão da corrente de alimentação é avaliada para conhecer o efeito de sua influência no

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.

Science.gov (United States)

Zheng, Naiyu; Zeng, Jianing; Manney, Amy; Williams, Lakenya; Aubry, Anne-Françoise; Voronin, Kimberly; Buzescu, Adela; Zhang, Yan J; Allentoff, Alban; Xu, Carrie; Shen, Hongwu; Warner, William; Arnold, Mark E

2016-04-15

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus

DEFF Research Database (Denmark)

Einer-Jensen, Katja; Nielsen, Thomas Krogh; Roepstorff, Peter

1998-01-01

were analyzed by mass spectrometry before and after chemical reduction, and six disulfide bonds were identified: Cys29-Cys339, Cys44-Cys295, Cys90-Cys132, Cys172-Cys177, Cys195-Cys265, and Cys231-CyS236. Mass spectrometric analysis in combination with glycosidases allowed characterization of the glycan...... of the protein, The present study was initiated to identify the disulfide bonds and other structural aspects relevant to vaccine design. The N-terminal amino acid residue was identified as being a pyroglutamic acid, corresponding to Gln21 of the primary transcript, Peptides from endoproteinase-degraded G protein...... cysteine residues are situated at conserved positions, This finding suggests that there might be some common disulfide bonding pattern among the six rhabdoviruses....

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-06-15

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

Identification and characterization of a novel L-arabinose isomerase from Anoxybacillus flavithermus useful in D-tagatose production.

Science.gov (United States)

Li, Yanjun; Zhu, Yueming; Liu, Anjun; Sun, Yuanxia

2011-05-01

D-Tagatose is a highly functional rare ketohexose and many attempts have been made to convert D-galactose into the valuable D-tagatose using L-arabinose isomerase (L-AI). In this study, a thermophilic strain possessing L-AI gene was isolated from hot spring sludge and identified as Anoxybacillus flavithermus based on its physio-biochemical characterization and phylogenetic analysis of its 16s rRNA gene. Furthermore, the gene encoding L-AI from A. flavithermus (AFAI) was cloned and expressed at a high level in E. coli BL21(DE3). L-AI had a molecular weight of 55,876 Da, an optimum pH of 10.5 and temperature of 95°C. The results showed that the conversion equilibrium shifted to more D-tagatose from D-galactose by raising the reaction temperatures and adding borate. A 60% conversion of D-galactose to D-tagatose was observed at an isomerization temperature of 95°C with borate. The catalytic efficiency (k (cat) /K (m)) for D-galactose with borate was 9.47 mM(-1) min(-1), twice as much as that without borate. Our results indicate that AFAI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for D-galactose, suggesting its great potential for producing D-tagatose.

CO2·- radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation

International Nuclear Information System (INIS)

Favaudon, V.; Tourbez, H.; Lhoste, J-M.; Houee-Levin, C.

1990-01-01

Disulfide bond reduction by the CO 2 ·- radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under γ-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pK a around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO 2 ·- proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO 2 ·- uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, appeared to be the major protein radical species formed under acidic conditions. Formation of the disulfide radical cation, phenoxyl radical Tyr-O · disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical

Human cyclophilin B: A second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence

International Nuclear Information System (INIS)

Price, E.R.; Zydowsky, L.D.; Jin, Mingjie; Baker, C.H.; McKeon, F.D.; Walsh, C.T.

1991-01-01

The authors report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, they show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosprorin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression

Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis

DEFF Research Database (Denmark)

Solem, Christian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

2008-01-01

Triosephosphate isomerase (TPI), which catalyses the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), was studied for its control on glycolysis and mixed acid production in L. lactis subspecies lactis IL1403 and L. lactis subspecies cremoris MG1363. Strains...... metabolites glucose-6-phosphate, fructose-1,6-bisphosphate and DHAP in the IL1403 derivatives were essentially unchanged for TPI activities from 26% to 225%. At a TPI activity of 3%, the level of DHAP increased four times. The finding that an increased level of DHAP coincides with an increase in formate...

The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

OpenAIRE

Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

2013-01-01

The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequ...

The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

International Nuclear Information System (INIS)

Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

2009-01-01

Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana.

Science.gov (United States)

Hong, Young-Ho; Lee, Dong-Woo; Lee, Sang-Jae; Choe, Eun-Ah; Kim, Seong-Bo; Lee, Yoon-Hee; Cheigh, Chan-Ick; Pyun, Yu-Ryang

2007-04-01

Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t (1/2) = 43 days at 70 degrees C) in a continuous recycling mode at 70 degrees C produced 49 and 38 g D-tagatose/l from 180 and 90 g D-galactose/l, respectively, within 12 h.

Thiol/disulfide homeostasis in pregnant women with obstructive sleep apnea syndrome.

Science.gov (United States)

Üstündağ, Yasemin; Demirci, Hakan; Balık, Rifat; Erel, Ozcan; Özaydın, Fahri; Kücük, Bilgen; Ertaş, Dilber; Ustunyurt, Emin

2017-11-27

Repetitive episodes of hypoxia and reoxygenation during sleep in patients with obstructive sleep apnea syndrome (OSAS) resemble an ischemia-reperfusion injury. We aimed to test the hypothesis that oxidative stress occurs in pregnant women with OSAS. We also aimed to compare thiol/disulfide homeostasis with ischemia-modified albumin (IMA) and total antioxidant capacity (TAC) as markers of ischemia-reperfusion injury in pregnant women with and without OSAS and healthy control. This study included 29 pregnant women with OSAS, 30 women without OSAS in the third trimester applying for periodic examinations, and 30 healthy women. Serum IMA and TAC (using the ferric reducing power of plasma method) were measured. Serum thiol/disulfide homeostasis was determined by a novel automated method. The mean age of the pregnant women with OSAS was 31.0 ± 4.7 years with a mean gestational age of 36.5 ± 3.0 weeks. The mean age of pregnant women without OSAS was 29.8 ± 4.9 years with a mean gestational age of 36.9 ± 2.7 weeks. The mean age of the nonpregnant control group was 29.7 ± 6.4 years. Both native thiol (291 ± 29 μmol/L versus 314 ± 30 μmol/L; p = .018) and total thiol (325 ± 32 versus 350 ± 32, p = .025) levels were lower in pregnant women with OSAS compared to pregnant women without OSAS, respectively (p total thiol levels were lower in pregnant women with OSAS compared to those without OSAS. However, dynamic thiol/disulfide homeostasis parameters cannot provide valuable information to discriminate OSAS in pregnant women.

Polymeric redox-responsive delivery systems bearing ammonium salts cross-linked via disulfides

Directory of Open Access Journals (Sweden)

Christian Dollendorf

2013-08-01

Full Text Available A redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm and 2-(dimethylaminoethyl methacrylate (DMAEMA. N,N’-bis(4-chlorobutanoylcystamine was used as disulfide-containing cross-linker to form networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction of disulfide to mercaptanes by use of dithiothreitol (DTT, tris(2-carboxyethylphosphine (TCEP or cysteamine, respectively. The soluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, methylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recognized. Physical properties of the cross-linked materials, e.g., glass transition temperature (Tg, swelling behavior and cloud points (Tc were investigated. Redox-responsive behavior was further analyzed by rheological measurements.

Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

Directory of Open Access Journals (Sweden)

Valero Francisco

2007-07-01

Full Text Available Abstract Background The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1, namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL. Results The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected. Conclusion The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of

{sup 13}C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)

Energy Technology Data Exchange (ETDEWEB)

Takeda, Mitsuhiro [Kumamoto University, Department of Structural BioImaging, Faculty of Life Sciences (Japan); Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

2016-09-15

Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14–Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D {sup 13}C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically {sup 13}C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14–Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m{sub c14} and m{sub c38}). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for {sup 13}C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m{sub c38} isomerization, the {sup 1}H-{sup 13}C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.

Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

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Zeynep A Oztug Durer

Full Text Available Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1 are one of the causes of familial amyotrophic lateral sclerosis (FALS. Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1 formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

OpenAIRE

Klose, Jana; Wendt, Norbert; Kubald, Sybille; Krause, Eberhard; Fechner, Klaus; Beyermann, Michael; Bienert, Michael; Rudolph, Rainer; Rothemund, Sven

2004-01-01

The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2–C3 and C4–C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2–C5 a...

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases.

Science.gov (United States)

Legname, Giuseppe; Virgilio, Tommaso; Bistaffa, Edoardo; De Luca, Chiara Maria Giulia; Catania, Marcella; Zago, Paola; Isopi, Elisa; Campagnani, Ilaria; Tagliavini, Fabrizio; Giaccone, Giorgio; Moda, Fabio

2018-04-20

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1 +/+ ), hemizygous (Pin1 +/- ) or knock-out (Pin1 -/- ) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.

UV-Photochemistry of the Disulfide Bond: Evolution of Early Photoproducts from Picosecond X-ray Absorption Spectroscopy at the Sulfur K-Edge.

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Ochmann, Miguel; Hussain, Abid; von Ahnen, Inga; Cordones, Amy A; Hong, Kiryong; Lee, Jae Hyuk; Ma, Rory; Adamczyk, Katrin; Kim, Tae Kyu; Schoenlein, Robert W; Vendrell, Oriol; Huse, Nils

2018-05-30

We have investigated dimethyl disulfide as the basic moiety for understanding the photochemistry of disulfide bonds, which are central to a broad range of biochemical processes. Picosecond time-resolved X-ray absorption spectroscopy at the sulfur K-edge provides unique element-specific insight into the photochemistry of the disulfide bond initiated by 267 nm femtosecond pulses. We observe a broad but distinct transient induced absorption spectrum which recovers on at least two time scales in the nanosecond range. We employed RASSCF electronic structure calculations to simulate the sulfur-1s transitions of multiple possible chemical species, and identified the methylthiyl and methylperthiyl radicals as the primary reaction products. In addition, we identify disulfur and the CH 2 S thione as the secondary reaction products of the perthiyl radical that are most likely to explain the observed spectral and kinetic signatures of our experiment. Our study underscores the importance of elemental specificity and the potential of time-resolved X-ray spectroscopy to identify short-lived reaction products in complex reaction schemes that underlie the rich photochemistry of disulfide systems.

Protein binding of N-2-mercaptoethyl-1,3-diaminopropane via mixed disulfide formation after oral administration of WR 2721

Energy Technology Data Exchange (ETDEWEB)

Tabachnik, N.F.; Blackburn, P.; Peterson, C.M.; Cerami, A.

1982-02-01

Earlier studies have shown that WR 2721 (H2N-(CH2)3-NH(CH2)2SPO3H2) is converted to its free thiol form, N-2-mercaptoethyl-1,3-diaminopropane (MDP), at the acidic pH of the stomach. MDP is a radioprotective compound and a mucolytic agent capable of decreasing sputum viscosity in the lungs of patients with cystic fibrosis. Conversion of WR 2721 and MDP to the corresponding sulfonic acid (MDP-SO3H) permits quantitative determination of these compounds in physiological fluids by use of an automatic amino acid analyzer. After oral administration of WR 2721 to human patients and rabbits it is converted to MDP and the free thiol form of the drug associates with plasma proteins by mixed disulfide linkage. The plasma proteins serve as a depot and reservoir of MDP for potential exchange at the tissues. When incubated with whole sputum or with purified mucin solutions in vitro, MDP decreased the viscosity of these solutions by reduction of the accessible disulfide bonds of the mucin molecule and was subsequently found in mixed disulfide association with the mucin molecule. The association of MDP with proteins via mixed disulfide linkage has important implications for the development of optimal dose regimens for administration of WR 2721 to patients.

Protein binding of N-2-mercaptoethyl-1,3-diaminopropane via mixed disulfide formation after oral administration of WR 2721

International Nuclear Information System (INIS)

Tabachnik, N.F.; Blackburn, P.; Peterson, C.M.; Cerami, A.

1982-01-01

Earlier studies have shown that WR 2721 [H2N-(CH2)3-NH(CH2)2SPO3H2] is converted to its free thiol form, N-2-mercaptoethyl-1,3-diaminopropane (MDP), at the acidic pH of the stomach. MDP is a radioprotective compound and a mucolytic agent capable of decreasing sputum viscosity in the lungs of patients with cystic fibrosis. Conversion of WR 2721 and MDP to the corresponding sulfonic acid (MDP-SO3H) permits quantitative determination of these compounds in physiological fluids by use of an automatic amino acid analyzer. After oral administration of WR 2721 to human patients and rabbits it is converted to MDP and the free thiol form of the drug associates with plasma proteins by mixed disulfide linkage. The plasma proteins serve as a depot and reservoir of MDP for potential exchange at the tissues. When incubated with whole sputum or with purified mucin solutions in vitro, MDP decreased the viscosity of these solutions by reduction of the accessible disulfide bonds of the mucin molecule and was subsequently found in mixed disulfide association with the mucin molecule. The association of MDP with proteins via mixed disulfide linkage has important implications for the development of optimal dose regimens for administration of WR 2721 to patients

Disulfide bonds in folding and transport of the mouse hepatitis virus glycoproteins

NARCIS (Netherlands)

Horzinek, M.C.; Opstelten, D.-J.E.; Groote, P. de; Vennema, H.; Rottier, P.J.M.

1993-01-01

We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

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Wanarska Marta

2012-08-01

Full Text Available Abstract Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c.

Science.gov (United States)

Wanarska, Marta; Kur, Józef

2012-08-23

D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization

Efficacy of HOCl scavenging by sulfur-containing compounds: antioxidant activity of glutathione disulfide?

NARCIS (Netherlands)

den Hartog, G.J.M.; Haenen, G.R.M.M.; Vegt, E.; van der Vijgh, W.J.F.; Bast, A.

2002-01-01

Efficacy of HOCl scavenging by sulfur-containing compounds: antioxidant activity of glutathione disulfide? den Hartog GJ, Haenen GR, Vegt E, van der Vijgh WJ, Bast A. Department of Pharmacology and Toxicology, Maastricht University, The Netherlands. Hypochlorous acid (HOCl) is a bactericidal

Anterior gradient protein 3 is associated with less aggressive tumors and better outcome of breast cancer patients

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Obacz J

2015-06-01

Full Text Available Joanna Obacz,1 Veronika Brychtova,1 Jan Podhorec,1 Pavel Fabian,2 Petr Dobes,1 Borivoj Vojtesek,1 Roman Hrstka1 1Regional Centre for Applied Molecular Oncology (RECAMO, 2Department of Pathology, Masaryk Memorial Cancer Institute, Brno, Czech Republic Abstract: Anterior gradient protein (AGR 3 is a highly related homologue of pro-oncogenic AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumo-rigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly correlated with estrogen receptor, progesterone receptor (both P<0.0001 as well as low histological grade (P=0.003, and inversely correlated with the level of Ki-67 expression (P<0.0001. In the whole cohort, AGR3 expression was associated with longer progression-free survival (PFS, whereas AGR3-positive subgroup of low-histological grade tumors showed both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients. Keywords: AGR3, patient survival, protein disulfide isomerase, ER-positive breast cancer, immuno­histochemistry

Redox Reactivity of Cerium Oxide Nanoparticles Induces the Formation of Disulfide Bridges in Thiol-Containing Biomolecules.

Science.gov (United States)

Rollin-Genetet, Françoise; Seidel, Caroline; Artells, Ester; Auffan, Mélanie; Thiéry, Alain; Vidaud, Claude

2015-12-21

The redox state of disulfide bonds is implicated in many redox control systems, such as the cysteine-cystine couple. Among proteins, ubiquitous cysteine-rich metallothioneins possess thiolate metal binding groups susceptible to metal exchange in detoxification processes. CeO2 NPs are commonly used in various industrial applications due to their redox properties. These redox properties that enable dual oxidation states (Ce(IV)/Ce(III)) to exist at their surface may act as oxidants for biomolecules. The interaction among metallothioneins, cysteine, and CeO2 NPs was investigated through various biophysical approaches to shed light on the potential effects of the Ce(4+)/Ce(3+) redox system on the thiol groups of these biomolecules. The possible reaction mechanisms include the formation of a disulfide bridge/Ce(III) complex resulting from the interaction between Ce(IV) and the thiol groups, leading to metal unloading from the MTs, depending on their metal content and cluster type. The formation of stable Ce(3+) disulfide complexes has been demonstrated via their fluorescence properties. This work provides the first evidence of thiol concentration-dependent catalytic oxidation mechanisms between pristine CeO2 NPs and thiol-containing biomolecules.

An efficient algorithmic approach for mass spectrometry-based disulfide connectivity determination using multi-ion analysis

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Yen Ten-Yang

2011-02-01

Full Text Available Abstract Background Determining the disulfide (S-S bond pattern in a protein is often crucial for understanding its structure and function. In recent research, mass spectrometry (MS based analysis has been applied to this problem following protein digestion under both partial reduction and non-reduction conditions. However, this paradigm still awaits solutions to certain algorithmic problems fundamental amongst which is the efficient matching of an exponentially growing set of putative S-S bonded structural alternatives to the large amounts of experimental spectrometric data. Current methods circumvent this challenge primarily through simplifications, such as by assuming only the occurrence of certain ion-types (b-ions and y-ions that predominate in the more popular dissociation methods, such as collision-induced dissociation (CID. Unfortunately, this can adversely impact the quality of results. Method We present an algorithmic approach to this problem that can, with high computational efficiency, analyze multiple ions types (a, b, bo, b*, c, x, y, yo, y*, and z and deal with complex bonding topologies, such as inter/intra bonding involving more than two peptides. The proposed approach combines an approximation algorithm-based search formulation with data driven parameter estimation. This formulation considers only those regions of the search space where the correct solution resides with a high likelihood. Putative disulfide bonds thus obtained are finally combined in a globally consistent pattern to yield the overall disulfide bonding topology of the molecule. Additionally, each bond is associated with a confidence score, which aids in interpretation and assimilation of the results. Results The method was tested on nine different eukaryotic Glycosyltransferases possessing disulfide bonding topologies of varying complexity. Its performance was found to be characterized by high efficiency (in terms of time and the fraction of search space

Per-2,3-O-alkylated beta-cyclodextrin duplexes connected with disulfide bonds

Czech Academy of Sciences Publication Activity Database

Tatar, Ameneh; Grishina, Anastasia; Buděšínský, Miloš; Kraus, Tomáš

2017-01-01

Roč. 29, č. 1 (2017), s. 40-48 ISSN 1061-0278 R&D Projects: GA MŠk LD12019 Grant - others:COST(XE) CM1005 Institutional support: RVO:61388963 Keywords : cyclodextrins * inclusion complexes * disulfide bonds Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 1.264, year: 2016

Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

Science.gov (United States)

Zichel, R; Mimran, A; Keren, A; Barnea, A; Steinberger-Levy, I; Marcus, D; Turgeman, A; Reuveny, S

2010-05-01

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response

International Nuclear Information System (INIS)

Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B.

2006-01-01

The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and α-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1α and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis

Cigarette smoke induces an unfolded protein response in the human lung: a proteomic approach.

Science.gov (United States)

Kelsen, Steven G; Duan, Xunbao; Ji, Rong; Perez, Oscar; Liu, Chunli; Merali, Salim

2008-05-01

Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the "unfolded protein response" (UPR). The importance of the UPR lies in its ability to alter expression of a variety of genes involved in antioxidant defense, inflammation, energy metabolism, protein synthesis, apoptosis, and cell cycle regulation. The present study used comparative proteomic technology to test the hypothesis that chronic cigarette smoking induces a UPR in the human lung. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Proteomes of lung samples from chronic cigarette smokers demonstrated 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI); and enzymes involved in antioxidant defense. In cultured human airway epithelial cells, GRP78 and the UPR-regulated basic leucine zipper, transcription factors, ATF4 and Nrf2, which enhance expression of important anti-oxidant genes, increased rapidly (< 24 h) with cigarette smoke extract. These data indicate that cigarette smoke induces a UPR response in the human lung that is rapid in onset, concentration dependent, and at least partially reversible with smoking cessation. We speculate that activation of a UPR by cigarette smoke may protect the lung from oxidant injury and the development of COPD.

Species-Specific Thiol-Disulfide Equilibrium Constant: A Tool To Characterize Redox Transitions of Biological Importance.

Science.gov (United States)

Mirzahosseini, Arash; Somlyay, Máté; Noszál, Béla

2015-08-13

Microscopic redox equilibrium constants, a new species-specific type of physicochemical parameters, were introduced and determined to quantify thiol-disulfide equilibria of biological significance. The thiol-disulfide redox equilibria of glutathione with cysteamine, cysteine, and homocysteine were approached from both sides, and the equilibrium mixtures were analyzed by quantitative NMR methods to characterize the highly composite, co-dependent acid-base and redox equilibria. The directly obtained, pH-dependent, conditional constants were then decomposed by a new evaluation method, resulting in pH-independent, microscopic redox equilibrium constants for the first time. The 80 different, microscopic redox equilibrium constant values show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

Synthesis and stable isotope dilution assay of ethanethiol and diethyl disulfide in wine using solid phase microextraction. Effect of aging on their levels in wine.

Science.gov (United States)

Belancic Majcenovic, Andrea; Schneider, Rémi; Lepoutre, Jean-Paul; Lempereur, Valérie; Baumes, Raymond

2002-11-06

Ethanethiol and diethyl disulfide (DEDS) most often occurred at levels above their olfactive threshold in wines with nauseous sulfur-linked smells. As ethanethiol is very oxidizable and chemically reactive, a stable isotopic dilution analysis of both ethanethiol and its disulfide in wines using solid phase microextraction and GC-MS was developed. The latter involved the determination of the proportion of DEDS formed by oxidation of the thiol during the analysis conditions, which was obtained by the use of two differently labeled disulfide standards. An original synthesis of labeled ethanethiol standards in conditions minimizing oxidation was developed, and the corresponding labeled diethyl disulfides were obtained from these thiols. This analytical method was used to follow the levels of these sulfur compounds during aging in a young red wine spiked with ethanethiol and added with enological tannins, with or without oxygen addition. The total levels of these two sulfur compounds were shown to decrease steadily after 60 days of aging, up to 83%. The effect of oxygen sped this decrease, but the effect of enological tannins was very slight. Residual ethanethiol was detected in its disulfide form from approximately 36% in the nonoxygenated wines to 69% in the oxygenated samples.

Increase in D-tagatose production rate by site-directed mutagenesis of L-arabinose isomerase from Geobacillus thermodenitrificans.

Science.gov (United States)

Oh, Hyo-Jung; Kim, Hye-Jung; Oh, Deok-Kun

2006-02-01

Among single-site mutations of L-arabinose isomerase derived from Geobacillus thermodenitrificans, two mutants were produced having the lowest and highest activities of D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at amino acid 164 and the size of amino acid 475 were important for D-tagatose production. Among double-site mutations, one mutant converted D-galactose into D-tagatose with a yield of 58% whereas the wild type gave 46% D-tagatose conversion after 300 min at 65 degrees C.

Metallic and highly conducting two-dimensional atomic arrays of sulfur enabled by molybdenum disulfide nanotemplate

Science.gov (United States)

Zhu, Shuze; Geng, Xiumei; Han, Yang; Benamara, Mourad; Chen, Liao; Li, Jingxiao; Bilgin, Ismail; Zhu, Hongli

2017-10-01

Element sulfur in nature is an insulating solid. While it has been tested that one-dimensional sulfur chain is metallic and conducting, the investigation on two-dimensional sulfur remains elusive. We report that molybdenum disulfide layers are able to serve as the nanotemplate to facilitate the formation of two-dimensional sulfur. Density functional theory calculations suggest that confined in-between layers of molybdenum disulfide, sulfur atoms are able to form two-dimensional triangular arrays that are highly metallic. As a result, these arrays contribute to the high conductivity and metallic phase of the hybrid structures of molybdenum disulfide layers and two-dimensional sulfur arrays. The experimentally measured conductivity of such hybrid structures reaches up to 223 S/m. Multiple experimental results, including X-ray photoelectron spectroscopy (XPS), transition electron microscope (TEM), selected area electron diffraction (SAED), agree with the computational insights. Due to the excellent conductivity, the current density is linearly proportional to the scan rate until 30,000 mV s-1 without the attendance of conductive additives. Using such hybrid structures as electrode, the two-electrode supercapacitor cells yield a power density of 106 Wh kg-1 and energy density 47.5 Wh kg-1 in ionic liquid electrolytes. Our findings offer new insights into using two-dimensional materials and their Van der Waals heterostructures as nanotemplates to pattern foreign atoms for unprecedented material properties.

Interaction between heavy metals and thiol-linked redox reactions in germination.

Science.gov (United States)

Smiri, M; Chaoui, A; Ferjani, E E

2010-09-15

Thioredoxin (TRX) proteins perform important biological functions in cells by changing the redox state of proteins via dithiol disulfide exchange. Several systems are able to control the activity, stability, and correct folding of enzymes through dithiol/disulfide isomerization reactions including the enzyme protein disulfide-isomerase, the glutathione-dependent glutaredoxin system, and the thioredoxin systems. Plants have devised sophisticated mechanisms to cope with biotic and abiotic stresses imposed by their environment. Among these mechanisms, those collectively referred to as redox reactions induced by endogenous systems. This is of agronomical importance since a better knowledge of the involved mechanisms can offer novel means for crop protection. In the plant life cycle, the seed and seedling stages are key developmental stages conditioning the final yield of crops. Both are very sensitive to heavy metal stress. Plant redox reactions are principally studied on adult plant organs and there is only very scarce informations about the onset of redox regulation at the level of seed germination. In the here presented study, we discussed the importance of redox proteins in plant cell metabolism and defence. Special focus is given to TRX, which are involved in detoxification of ROS and also to their targets.

Preparation and Photoluminescence of Tungsten Disulfide Monolayer

Directory of Open Access Journals (Sweden)

Yanfei Lv

2018-05-01

Full Text Available Tungsten disulfide (WS2 monolayer is a direct band gap semiconductor. The growth of WS2 monolayer hinders the progress of its investigation. In this paper, we prepared the WS2 monolayer through chemical vapor transport deposition. This method makes it easier for the growth of WS2 monolayer through the heterogeneous nucleation-and-growth process. The crystal defects introduced by the heterogeneous nucleation could promote the photoluminescence (PL emission. We observed the strong photoluminescence emission in the WS2 monolayer, as well as thermal quenching, and the PL energy redshift as the temperature increases. We attribute the thermal quenching to the energy or charge transfer of the excitons. The redshift is related to the dipole moment of WS2.

Design, Synthesis and Biological Evaluation of Brain-Targeted Thiamine Disulfide Prodrugs of Ampakine Compound LCX001

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Dian Xiao

2016-04-01

Full Text Available Ampakine compounds have been shown to reverse opiate-induced respiratory depression by activation of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA glutamate receptors. However, their pharmacological exploitations are hindered by low blood-brain barrier (BBB permeability and limited brain distribution. Here, we explored whether thiamine disulfide prodrugs with the ability of “lock-in” can be used to solve these problems. A series of thiamine disulfide prodrugs 7a–7f of ampakine compound LCX001 was synthesized and evaluated. The trials in vitro showed that prodrugs 7e, 7d, 7f possessed a certain stability in plasma and quickly decomposed in brain homogenate by the disulfide reductase. In vivo, prodrug 7e decreased the peripheral distribution of LCX001 and significantly increased brain distribution of LCX001 after i.v. administration. This compound showed 2.23- and 3.29-fold greater increases in the AUC0-t and MRT0-t of LCX001 in brain, respectively, than did LCX001 itself. A preliminary pharmacodynamic study indicated that the required molar dose of prodrug 7e was only one eighth that of LCX001 required to achieve the same effect in mice. These findings provide an important reference to evaluate the clinical outlook of ampakine compounds.

Inhibition of rotavirus ECwt infection in ICR suckling mice by N-acetylcysteine, peroxisome proliferator-activated receptor gamma agonists and cyclooxygenase-2 inhibitors

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Carlos Arturo Guerrero

2013-09-01

Full Text Available Live attenuated vaccines have recently been introduced for preventing rotavirus disease in children. However, alternative strategies for prevention and treatment of rotavirus infection are needed mainly in developing countries where low vaccine coverage occurs. In the present work, N-acetylcysteine (NAC, ascorbic acid (AA, some nonsteroidal anti-inflammatory drugs (NSAIDs and peroxisome proliferator-activated receptor gamma (PPARγ agonists were tested for their ability to interfere with rotavirus ECwt infectivity as detected by the percentage of viral antigen-positive cells of small intestinal villi isolated from ECwt-infected ICR mice. Administration of 6 mg NAC/kg every 8 h for three days following the first diarrhoeal episode reduced viral infectivity by about 90%. Administration of AA, ibuprofen, diclofenac, pioglitazone or rosiglitazone decreased viral infectivity by about 55%, 90%, 35%, 32% and 25%, respectively. ECwt infection of mice increased expression of cyclooxygenase-2, ERp57, Hsc70, NF-κB, Hsp70, protein disulphide isomerase (PDI and PPARγ in intestinal villus cells. NAC treatment of ECwt-infected mice reduced Hsc70 and PDI expression to levels similar to those observed in villi from uninfected control mice. The present results suggest that the drugs tested in the present work could be assayed in preventing or treating rotaviral diarrhoea in children and young animals.

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

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Smith, T; Ferreira, L R; Hebert, C; Norris, K; Sauk, J J

1995-08-04

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for

Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

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Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

2015-09-01

Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. © 2015 Authors.

Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

Science.gov (United States)

Doucet, Alain; Williams, Martin; Gagnon, Mylene C; Sasseville, Maxime; Beauregard, Marc

2002-01-02

Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.

Influence of Disulfide Connectivity on Structure and Bioactivity of α-Conotoxin TxIA

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Yong Wu

2014-01-01

Full Text Available Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs. Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs. Here, we used reversed-phase high performance liquid chromatography (RP-HPLC and electrospray ionization-mass spectrometry (ESI-MS to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3β2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3β2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3β2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3β2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges.

Science.gov (United States)

Tan, Hao; Miao, Renyun; Liu, Tianhai; Cao, Xuelian; Wu, Xiang; Xie, Liyuan; Huang, Zhongqian; Peng, Weihong; Gan, Bingcheng

2016-10-28

A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at physiological temperature (37°C) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.

Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli

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Dönhöfer Alexandra

2011-04-01

Full Text Available Abstract Background In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272. Results Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge. Conclusion A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor.

Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

Science.gov (United States)

Ryu, Se-Ah; Kim, Chang Sup; Kim, Hye-Jung; Baek, Dae Heoun; Oh, Deok-Kun

2003-01-01

D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

1.42 A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

International Nuclear Information System (INIS)

Yun Caihong; Tang Yuehua; Feng Youmin; An Xiaomin; Chang Wenrui; Liang Dongcai

2004-01-01

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1

Reduction-Triggered Transformation of Crosslinking Modules of Disulfide-Containing Micelles with Chemically Tunable Rates.

Science.gov (United States)

Deng, Zhengyu; Yuan, Shuai; Xu, Ronald X; Liang, Haojun; Liu, Shiyong

2018-05-16

A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites for designing delivery nanocarriers and in vivo nanoreactors. We herein report disulfide-crosslinked (DCL) micelles exhibiting reduction-triggered switching of crosslinking modules and synchronized hydrophobic-to-hydrophilic transition. Tumor cell-targeted DCL micelles undergo cytoplasmic milieu-triggered disulfide cleavage and cascade self-immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur due to high local concentrations and suppression of apparent amine pKa within hydrophobic cores, leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles with hydrophilic cores inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective.

Science.gov (United States)

Nazari, Mahboobeh; Hosseinkhani, Saman; Hassani, Leila

2013-02-01

Multi-color bioluminescence is developed using the introduction of single/double disulfide bridges in firefly luciferase. The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase sequence and assay conditions. It has been proposed that the stability of a protein may increase through the introduction of a disulfide bridge that decreases the configurational entropy of unfolding. Single and double disulfide bridges are introduced into Photinus pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (C(81)-A(105)C, L(306)C-L(309)C, P(451)C-V(469)C; C(81)-A(105)C/P(451)C-V(469)C, and A(296)C-A(326)C/P(451)C-V(469)C). By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red or kept in different extents. The bioluminescence color shift occurred with displacement of a critical loop in the luciferase structure without any change in green emitter mutants. Thermodynamic analysis revealed that among mutants, L(306)C-L(309)C shows a remarkable stability against urea denaturation and also a considerable increase in kinetic stability and a clear shift in bioluminescence spectra towards red.

Defect-Mediated Lithium Adsorption and Diffusion on Monolayer Molybdenum Disulfide

OpenAIRE

Sun, Xiaoli; Wang, Zhiguo; Fu, Yong Qing

2015-01-01

Monolayer Molybdenum Disulfide (MoS2) is a promising anode material for lithium ion batteries because of its high capacities. In this work, first principle calculations based on spin density functional theory were performed to investigate adsorption and diffusion of lithium on monolayer MoS2 with defects, such as single- and few-atom vacancies, antisite, and grain boundary. The values of adsorption energies on the monolayer MoS2 with the defects were increased compared to those on the pristin...

Mannose Phosphate Isomerase Isoenzymes in Plutella xylostella Support Common Genetic Bases of Resistance to Bacillus thuringiensis Toxins in Lepidopteran Species

OpenAIRE

Herrero, Salvador; Ferré, Juan; Escriche, Baltasar

2001-01-01

A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

Science.gov (United States)

Kumari, Geeta; Serra, Aida; Shin, Joon; Nguyen, Phuong Q T; Sze, Siu Kwan; Yoon, Ho Sup; Tam, James P

2015-11-25

Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

Science.gov (United States)

Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

2018-06-01

 Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

Thermal ripples in model molybdenum disulfide monolayers

Energy Technology Data Exchange (ETDEWEB)

Remsing, Richard C.; Klein, Michael L. [Institute for Computational Molecular Science, Center for the Computational, Design of Functional Layered Materials, and Department of Chemistry, Temple University, 1925 N. 12th St., 19122, Philadelphia, PA (United States); Waghmare, Umesh V. [Theoretical Sciences Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, 560 064, Jakkur, Bangalore (India)

2017-01-15

Molybdenum disulfide (MoS{sub 2}) monolayers have the potential to revolutionize nanotechnology. To reach this potential, it will be necessary to understand the behavior of this two-dimensional (2D) material on large length scales and under thermal conditions. Herein, we use molecular dynamics (MD) simulations to investigate the nature of the rippling induced by thermal fluctuations in monolayers of the 2H and 1T phases of MoS{sub 2}. The 1T phase is found to be more rigid than the 2H phase. Both monolayer phases are predicted to follow long wavelength scaling behavior typical of systems with anharmonic coupling between vibrational modes as predicted by classic theories of membrane-like systems. (copyright 2017 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

11-Hydroxyundecyl octadecyl disulfide self-assembled monolayers on Au(1 1 1)

Energy Technology Data Exchange (ETDEWEB)

Albayrak, Erol [Department of Materials and Metallurgical Engineering, Ahi Evran University, Kırşehir 40000 (Turkey); Karabuga, Semistan [Department of Chemistry, Kahramanmaraş Sütçü İmam University, Kahramanmaraş 46030 (Turkey); Bracco, Gianangelo [CNR-IMEM and Department of Physics, University of Genoa, via Dodecaneso 33, Genoa 16146 (Italy); Danışman, M. Fatih, E-mail: danisman@metu.edu.tr [Department of Chemistry, Middle East Technical University, Ankara 06800 (Turkey)

2014-08-30

Highlights: • 11-Hydroxyundecyl octadecyl disulfide self-assembled monolayers on Au(1 1 1) surface were grown by supersonic molecular beam deposition. • Two different lying down monolayer phases were observed depending on the substrate temperature. • High temperature monolayer phase has a diffraction pattern similar to that of mercaptoundecanol SAMs. • Desorption from several different chemisorbed and physisorbed states were observed. - Abstract: Here, we report a helium atom diffraction study of 11-hydroxyundecyl octadecyl disulfide (CH{sub 3}-(CH{sub 2}){sub 17}-S-S-(CH{sub 2}){sub 11}-OH, HOD) self-assembled monolayers (SAMs) produced by supersonic molecular beam deposition (SMBD). Two different lying down monolayer phases were observed depending on the substrate temperature. At low temperatures a poorly ordered phase was observed, while the diffraction patterns of the film grown at high temperatures were similar to that of mercaptoundecanol (MUD) SAMs reported previously in the literature. The transition from the low temperature phase to the high temperature phase is due to S-S bond cleavage at the surface. Desorption from several different chemisorbed and physisorbed states were observed with energies in the same range as observed for MUD and octadecanelthiol (ODT) SAMs.

Coupling gold nanoparticles to silica nanoparticles through disulfide bonds for glutathione detection

International Nuclear Information System (INIS)

Shi Yupeng; Zhang Heng; Zhang Zhaomin; Yi Changqing; Yue Zhenfeng; Teng, Kar-Seng; Li Meijin; Yang Mengsu

2013-01-01

Advances in the controlled assembly of nanoscale building blocks have resulted in functional devices which can find applications in electronics, biomedical imaging, drug delivery etc. In this study, novel covalent nanohybrid materials based upon [Ru(bpy) 3 ] 2+ -doped silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs), which could be conditioned as OFF–ON probes for glutathione (GSH) detection, were designed and assembled in sequence, with the disulfide bonds as the bridging elements. The structural and optical properties of the nanohybrid architectures were characterized using transmission electron microscopy, UV–vis spectroscopy and fluorescence spectroscopy, respectively. Zeta potential measurements, x-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were employed to monitor the reaction processes of the SiNPs–S–S–COOH and SiNPs–S–S–AuNPs synthesis. It was found that the covalent nanohybrid architectures were fluorescently dark (OFF state), indicating that SiNPs were effectively quenched by AuNPs. The fluorescence of the OFF–ON probe was resumed (ON state) when the bridge of the disulfide bond was cleaved by reducing reagents such as GSH. This work provides a new platform and strategy for GSH detection using covalent nanohybrid materials. (paper)

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

OpenAIRE

Bi, Hongkai; Wang, Haihong; Cronan, John E.

2013-01-01

In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynt...

Research of the technology of obtaining pure and disperse molybdenum disulfide from molybdenum concentrate

International Nuclear Information System (INIS)

Hovsepyan, A.H.; Israyelyan, S.M.

2009-01-01

The technology of obtaining pure and disperse molybdenum disulfide is worked out. The processes of refinement from the flotation reagents and deslimation by means of decantation, refinement of molybdenite concentrate from impurities by selective leaching methods are studied. The optimal regime of technological process is chosen

Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

Science.gov (United States)

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

2016-01-01

The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Ophthalmological and angiographic findings in workers exposed to carbon disulfide (author's transl)

Energy Technology Data Exchange (ETDEWEB)

Savic, S.

1982-01-01

Microaneurysms are important in the diagnosis of vascular changes caused by carbon disulfide. They can be diagnosed by ophtholmoscopy, angiography or angioscopy. In our opinion even a careful ophthalmoscopic investigation is sufficient for diagnosis, so that angiography is not absolutely necessary for any mass survey. The incidence of microaneurysms correlates with the duration (both daily and total) as well as with the intensity of exposure to carbon disulfide. The quantity correlates closely with the intensity of exposure. The incidence of microaneurysms is not correlated to age; however it was found to be highest in 40-50-year-old men working with staple fibers, whereas in the spinning department it occurred in 50-55-old men. Microaneurysms are found equally frequently in active workers and invalids. There was no difference between the two groups with regard to degenerative changes of the macula. However, the changes found in the eyes of men from the staple fiber department were more pronounced than in those from the spinning department.

Structure of α-conotoxin BuIA: influences of disulfide connectivity on structural dynamics

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Craik David J

2007-04-01

Full Text Available Abstract Background α-Conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in α-conotoxins is variable, leading to the classification of sub-families. BuIA is the only α-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. Results In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. Conclusion These findings are opposite to the general trends observed for α-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

Science.gov (United States)

Asgeirsson, Bjarni; Adalbjörnsson, Björn Vidar; Gylfason, Gudjón Andri

2007-06-01

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

Lithium/disulfide battery R and D

Science.gov (United States)

Kaun, T. D.; Deluca, W.; Lee, J.; Redey, L.; Nelson, P. A.

The focus of molten-salt cell R and D in the past year at Argonne National Laboratory has been on developing an understanding of the excellent performance and stability of a lithium/disulfide cell using LiCl-LiBr-KBr electrolyte. For further improvement, we have initiated development of a rod-electrode cell design and design of cells which can tolerate overdischarge and overcharge abuse. Earlier Li/FeS2 cells offered performance quite below expectations and had high capacity decline rates: 0.10 to 0.25 percent per cycle. Approaches for reducing the capacity decline rates of the earlier cells also reduced cell performance. However, our improved Li/FeS2 cell tests indicate good prospects for attaining cell development goals of specific energy of 200 Wh/kg at a 4-h discharge rate, a specific power of 200 W/kg at 80 percent depth of discharge, and a cycle life of 1000 cycles.

Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

Energy Technology Data Exchange (ETDEWEB)

Kovalevsky, Andrey, E-mail: ayk@lanl.gov [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Hanson, B. Leif [University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606 (United States); Mason, Sax A. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Forsyth, V. Trevor [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keele University, Staffordshire (United Kingdom); Fisher, Zoe [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Mustyakimov, Marat [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Blakeley, Matthew P. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keen, David A. [Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX (United Kingdom); Langan, Paul [Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States)

2012-09-01

A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH. d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni{sup 2+} cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg{sup 2+} ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni{sup 2+} ions occupying the catalytic metal site (M2) were found at two locations, while Mg{sup 2+} in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

Science.gov (United States)

Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani

2012-01-01

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

Energy Technology Data Exchange (ETDEWEB)

Gowda, Giri; Sagurthi, Someswar Rao [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

2008-02-01

The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

Investigation of the deposition and thermal behavior of striped phases of unsymmetric disulfide self-assembled monolayers on Au(111): The case of 11-hydroxyundecyl decyl disulfide

Energy Technology Data Exchange (ETDEWEB)

Albayrak, Erol [Department of Materials and Metallurgical Engineering, Ahi Evran University, Kırşehir 40000 (Turkey); Karabuga, Semistan [Department of Chemistry, Kahramanmaraş Sütçü İmam University, Kahramanmaraş 46030 (Turkey); Bracco, Gianangelo [CNR-IMEM and Department of Physics, University of Genoa, via Dodecaneso 33, Genoa 16146 (Italy); Danışman, M. Fatih, E-mail: danisman@metu.edu.tr [Department of Chemistry, Middle East Technical University, Ankara 06800 (Turkey)

2015-01-07

Self-assembled monolayers (SAMs) of unsymmetric disulfides on Au(111) are used to form mixed SAMs that can be utilized in many applications. Here, we have studied 11-hydroxyundecyl decyl disulfide (CH{sub 3}–(CH{sub 2}){sub 9}–S–S–(CH{sub 2}){sub 11}–OH, HDD) SAMs produced by supersonic molecular beam deposition and characterized by He diffraction. The film growth was monitored at different temperatures up to a coverage which corresponds to a full lying down phase and the diffraction analysis shows that below 250 K the phase is different from the phase measured above 300 K. During the annealing of the film, two phase transitions were observed, at 250 K and 350 K. The overall data suggest that the former is related to an irreversible phase separation of HDD above 250 K to decanethiolate (–S–(CH{sub 2}){sub 9}–CH{sub 3}, DTT) and hydroxyundecylthiolate (–S–(CH{sub 2}){sub 11}–OH, MUDT), while the latter to a reversible melting of the film. Above 450 K, the specular intensity shows an increase related to film desorption and different chemisorbed states were observed with energies in the same range as observed for decanethiol (H–S–(CH{sub 2}){sub 9}–CH{sub 3}, DT) and mercaptoundecanol (H–S–(CH{sub 2}){sub 11}–OH, MUD) SAMs.

Dissulfeto de molibdênio, um material multifuncional e surpreendente Molybdenum disulfide, a multifunctional and remarkable material

Directory of Open Access Journals (Sweden)

Fernando Wypych

2002-02-01

Full Text Available The aim of this work is to review the chemical and physical properties of layered molybdenum disulfide. The three polymorphic/polytypic modifications of the compound were found, the polytypes 2H (molybdenite and 3R are semiconductors while the polymorph 1T is an electronic conductor. 2H-MoS2 has several important industrial applications as hydrotreatment catalysts, energy storage devices, solar cells, solid lubricants, among others. When intercalated, the 2H phase changes to a distorted 1T phase, producing unstable intercalation compounds that can be exfoliated in solution, producing single layers and consequently nanocomposites. The direct synthesis of the 1T phase produces stable intercalation compounds. Recently molybdenum disulfide was prepared as nanotubes and fulerene-like structures that bring new insights in the investigation of this important material.

Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.

Directory of Open Access Journals (Sweden)

Nagesh Pulicherla

Full Text Available The capsid proteins of adeno-associated viruses (AAV have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.

Disulfide high mobility group box-1 causes bladder pain through bladder Toll-like receptor 4.

Science.gov (United States)

Ma, Fei; Kouzoukas, Dimitrios E; Meyer-Siegler, Katherine L; Westlund, Karin N; Hunt, David E; Vera, Pedro L

2017-05-25

Bladder pain is a prominent symptom in several urological conditions (e.g. infection, painful bladder syndrome/interstitial cystitis, cancer). Understanding the mechanism of bladder pain is important, particularly when the pain is not accompanied by bladder pathology. Stimulation of protease activated receptor 4 (PAR4) in the urothelium results in bladder pain through release of urothelial high mobility group box-1 (HMGB1). HGMB1 has two functionally active redox states (disulfide and all-thiol) and it is not known which form elicits bladder pain. Therefore, we investigated whether intravesical administration of specific HMGB1 redox forms caused abdominal mechanical hypersensitivity, micturition changes, and bladder inflammation in female C57BL/6 mice 24 hours post-administration. Moreover, we determined which of the specific HMGB1 receptors, Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), mediate HMGB1-induced changes. Disulfide HMGB1 elicited abdominal mechanical hypersensitivity 24 hours after intravesical (5, 10, 20 μg/150 μl) instillation. In contrast, all-thiol HMGB1 did not produce abdominal mechanical hypersensitivity in any of the doses tested (1, 2, 5, 10, 20 μg/150 μl). Both HMGB1 redox forms caused micturition changes only at the highest dose tested (20 μg/150 μl) while eliciting mild bladder edema and reactive changes at all doses. We subsequently tested whether the effects of intravesical disulfide HMGB1 (10 μg/150 μl; a dose that did not produce inflammation) were prevented by systemic (i.p.) or local (intravesical) administration of either a TLR4 antagonist (TAK-242) or a RAGE antagonist (FPS-ZM1). Systemic administration of either TAK-242 (3 mg/kg) or FPS-ZM1 (10 mg/kg) prevented HMGB1 induced abdominal mechanical hypersensitivity while only intravesical TLR4 antagonist pretreatment (1.5 mg/ml; not RAGE) had this effect. The disulfide form of HMGB1 mediates bladder pain directly (not

Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex.

Science.gov (United States)

Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-06-26

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

Biochemical Characterization of the Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B Complex*

Science.gov (United States)

Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-01-01

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969

Subnanometer Resolution and Enhanced Friction Contrast at the Surface of Perylene Diimide PDI8-CN2 Thin Films in Ambient Conditions.

Science.gov (United States)

Buzio, Renato; Gerbi, Andrea; Barra, Mario; Chiarella, Fabio; Gnecco, Enrico; Cassinese, Antonio

2018-03-13

We report high-resolution surface morphology and friction force maps of polycrystalline organic thin films derived by deposition of the n-type perylene diimide semiconductor PDI8-CN 2 . We show that the in-plane molecular arrangement into ordered, cofacial slip-stacked rows results in a largely anisotropic surface structure, with a characteristic sawtooth corrugation of a few Ångstroms wavelength and height. Load-controlled experiments reveal different types of friction contrast between the alternating sloped and stepped regions, with transitions from atomic-scale dissipative stick-slip to smooth sliding with ultralow friction within the surface unit cell. Notably, such a rich phenomenology is captured under ambient conditions. We demonstrate that friction contrast is well reproduced by numerical simulations assuming a reduced corrugation of the tip-molecule potential nearby the step edges. We propose that the side alkyl chains pack into a compact low-surface-energy overlayer, and friction modulation reflects periodic heterogeneity of chains bending properties and subsurface anchoring to the perylene cores.

Reduction of disulfide bonds in peptides and proteins. Reduction des groupes disulfure dans les peptides et proteines

Energy Technology Data Exchange (ETDEWEB)

Conte, D [Institut Curie, 75 - Paris (France); Houee-Levin, C [Paris-5 Univ., 75 (France)

1993-04-01

We have re-examined the mechanism of disulfide bond reduction in oxidized glutathione by C0[sub 2][sup .-] free radicals. The process appears to be a chain reaction whose initial yield depends on pH and on both peptide and formate ion concentrations, but remains independent on the radiation dose rate. Kinetic schemes drawn from studies on dithiothreitol are unable to account for the results obtained with glutathione and proteins, although the disulfide radical anion is the primary intermediate found with all compounds. The rate constant for its formation from C0[sub 2][sup .-] and glutathione is in the same range as those found using proteins, while decay pathways are somewhat different. Hypotheses are proposed to account for these differences. 6 figs., 2 tabs.

The thiol oxidant dipyridyl disulfide can supply the PDI-Ero1p pathway with additional oxidative equivalents

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Winther, Jakob R

2007-01-01

Membrane-permeant oxidants have become a standard tool for studying eukaryotic organisms because they affect the redox state and the redox regulation of different compartments. The ero1-1 mutant is temperature sensitive (37 degrees C) and cannot grow under anaerobic conditions. Low micromolar con...

Functional Poly(ε-caprolactone)s via Copolymerization of ε-Caprolactone and Pyridyl Disulfide-Containing Cyclic Carbonate: Controlled Synthesis and Facile Access to Reduction-Sensitive Biodegradable Graft Copolymer Micelles

NARCIS (Netherlands)

Chen, Wei; Zou, Yan; Jia, Junna; Meng, Fenghua; Cheng, Ru; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan

2013-01-01

Pyridyl disulfide-functionalized cyclic carbonate (PDSC) monomer was obtained in four straightforward steps from 3-methyl-3-oxetanemethanol and exploited for facile preparation of functional poly(ε-caprolactone) (PCL) containing pendant pyridyl disulfide (PDS) groups via ring-opening

Basal-plane thermal conductivity of few-layer molybdenum disulfide

International Nuclear Information System (INIS)

Jo, Insun; Ou, Eric; Shi, Li; Pettes, Michael Thompson; Wu, Wei

2014-01-01

We report the in-plane thermal conductivity of suspended exfoliated few-layer molybdenum disulfide (MoS 2 ) samples that were measured by suspended micro-devices with integrated resistance thermometers. The obtained room-temperature thermal conductivity values are (44–50) and (48–52) W m −1 K −1 for two samples that are 4 and 7 layers thick, respectively. For both samples, the peak thermal conductivity occurs at a temperature close to 120 K, above which the thermal conductivity is dominated by intrinsic phonon-phonon scattering although phonon scattering by surface disorders can still play an important role in these samples especially at low temperatures

Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to D-tagatose using the enzyme.

Science.gov (United States)

Kim, Byoung-Chan; Lee, Yoon-Hee; Lee, Han-Seung; Lee, Dong-Woo; Choe, Eun-Ah; Pyun, Yu-Ryang

2002-06-18

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

Directory of Open Access Journals (Sweden)

Patrick Schaub

Full Text Available CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40 hydrocarbon substrate.

Synthesis of Reusable Silica Nanosphere-Supported Pt(IV Complex for Formation of Disulfide Bonds in Peptides

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Xiaonan Hou

2017-02-01

Full Text Available Some peptide-based drugs, including oxytocin, vasopressin, ziconotide, pramlintide, nesiritide, and octreotide, contain one intramolecular disulfide bond. A novel and reusable monodispersed silica nanosphere-supported Pt(IV complex (SiO2@TPEA@Pt(IV; TPEA: N-[3-(trimethoxysilylpropyl]ethylenediamine was synthesized via a four-step procedure and was used for the formation of intramolecular disulfide bonds in peptides. Transmission electron microscopy (TEM and chemical mapping results for the Pt(II intermediates and for SiO2@TPEA@Pt(IV show that the silica nanospheres possess a monodisperse spherical structure and contain uniformly-distributed Si, O, C, N, Cl, and Pt. The valence state of Pt on the silica nanospheres was characterized by X-ray photoelectron spectroscopy (XPS. The Pt(IV loaded on SiO2@TPEA@Pt(IV was 0.15 mmol/g, as determined by UV-VIS spectrometry. The formation of intramolecular disulfides in six dithiol-containing peptides of variable lengths by the use of SiO2@TPEA@Pt(IV was investigated, and the relative oxidation yields were determined by high-performance liquid chromatography (HPLC. In addition, peptide 1 (Ac-CPFC-NH2 was utilized to study the reusability of SiO2@TPEA@Pt(IV. No significant decrease in the relative oxidation yield was observed after ten reaction cycles. Moreover, the structure of SiO2@TPEA@Pt(IV after being used for ten cycles was determined to be similar to its initial one, demonstrating the cycling stability of the complex.

Decontamination of Oils Contaminated with Polychlorinated Biphenyls and Dibenzyl Disulfide Using Polar Aprotic Solvents

Czech Academy of Sciences Publication Activity Database

Kaštánek, František; Matějková, Martina; Spáčilová, Lucie; Maléterová, Ywetta; Kaštánek, P.; Šolcová, Olga

2015-01-01

Roč. 4, č. 2 (2015), s. 41-48 ISSN 2319-5967 R&D Projects: GA TA ČR(CZ) TA04020151 Institutional support: RVO:67985858 Keywords : corrosive sulfur * dibenzyl disulfide * polar aprotic solvents Subject RIV: CI - Industrial Chemistry, Chemical Engineering http://www.ijesit.com/Volume%204/Issue%202/IJESIT201502_06.pdf

Protein redox regulation in the thylakoid lumen: the importance of disulfide bonds for violaxanthin de-epoxidase.

Science.gov (United States)

Simionato, Diana; Basso, Stefania; Zaffagnini, Mirko; Lana, Tobia; Marzotto, Francesco; Trost, Paolo; Morosinotto, Tomas

2015-04-02

When exposed to saturating light conditions photosynthetic eukaryotes activate the xanthophyll cycle where the carotenoid violaxanthin is converted into zeaxanthin by the enzyme violaxanthin de-epoxidase (VDE). VDE protein sequence includes 13 cysteine residues, 12 of which are strongly conserved in both land plants and algae. Site directed mutagenesis of Arabidopsis thaliana VDE showed that all these 12 conserved cysteines have a major role in protein function and their mutation leads to a strong reduction of activity. VDE is also shown to be active in its completely oxidized form presenting six disulfide bonds. Redox titration showed that VDE activity is sensitive to variation in redox potential, suggesting the possibility that dithiol/disulfide exchange reactions may represent a mechanism for VDE regulation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Multimolecular Salivary Mucin Complex Is Altered in Saliva of Cigarette Smokers: Detection of Disulfide Bridges by Raman Spectroscopy

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Motoe Taniguchi

2013-01-01

Full Text Available Saliva contains mucins, which protect epithelial cells. We showed a smaller amount of salivary mucin, both MG1 and MG2, in the premenopausal female smokers than in their nonsmoking counterparts. Smokers' MG1, which contains almost 2% cysteine/half cystine in its amino acid residues, turned out to be chemically altered in the nonsmoker’s saliva. The smaller acidic glycoprotein bands were detectable only in smoker’s saliva in the range of 20–25 kDa and at 45 kDa, suggesting that degradation, at least in part, caused the reduction of MG1 mucin. This is in agreement with the previous finding that free radicals in cigarette smoke modify mucins in both sugar and protein moieties. Moreover, proteins such as amylase and albumin are bound to other proteins through disulfide bonds and are identifiable only after reduction with DTT. Confocal laser Raman microspectroscopy identified a disulfide stretch band of significantly stronger intensity per protein in the stimulated saliva of smokers alone. We conclude that the saliva of smokers, especially stimulated saliva, contains significantly more oxidized form of proteins with increased disulfide bridges, that reduces protection for oral epithelium. Raman microspectroscopy can be used for an easy detection of the damaged salivary proteins.

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

Science.gov (United States)

Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

2007-12-01

A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

Mapping the ER Interactome: The P Domains of Calnexin and Calreticulin as Plurivalent Adapters for Foldases and Chaperones.

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Kozlov, Guennadi; Muñoz-Escobar, Juliana; Castro, Karla; Gehring, Kalle

2017-09-05

The lectin chaperones calreticulin (CRT) and calnexin (CNX) contribute to the folding of glycoproteins in the ER by recruiting foldases such as the protein disulfide isomerase ERp57 and the peptidyl prolyl cis-trans isomerase CypB. Recently, CRT was shown to interact with the chaperone ERp29. Here, we show that ERp29 directly binds to the P domain of CNX. Crystal structures of the D domain of ERp29 in complex with the P domains from CRT and calmegin, a tissue-specific CNX homolog, reveal a commonality in the mechanism of binding whereby the tip of the P domain functions as a plurivalent adapter to bind a variety of folding factors. We show that mutation of a single residue, D348 in CNX, abrogates binding to ERp29 as well as ERp57 and CypB. The structural diversity of the accessory factors suggests that these chaperones became specialized for glycoprotein folding through convergent evolution of their P-domain binding sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.

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Kuglstatter, A; Stihle, M; Neumann, C; Müller, C; Schaefer, W; Klein, C; Benz, J

2017-09-01

An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Differential regulation of tissue thiol-disulfide redox status in a murine model of peritonitis

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Benton Shana M

2012-10-01

Full Text Available Abstract Background Glutathione (GSH/glutathione disulfide (GSSG and cysteine (Cys/cystine (CySS are major redox pools with important roles in cytoprotection. We determined the impact of septic peritonitis on thiol-disulfide redox status in mice. Methods FVB/N mice (6–12 week old; 8/group underwent laparotomy with cecal ligation and puncture (CLP or laparotomy alone (control. Sections of ileum, colon, lung and liver were obtained and GSH, GSSG, Cys and CySS concentrations determined by HPLC 24 h after laparotomy. Redox potential [Eh in millivolts (mV] of the GSH/GSSG and Cys/CySS pools was calculated using the Nernst equation. Data were analyzed by ANOVA (mean ± SE. Results GSH/GSSG Eh in ileum, colon, and liver was significantly oxidized in septic mice versus control mice (ileum: septic −202±4 versus control −228±2 mV; colon: -195±8 versus −214±1 mV; and liver: -194±3 vs. -210±1 mV, all Ph was unchanged with CLP, while liver and lung Cys/CySS Eh became significantly more reducing (liver: septic = −103±3 versus control −90±2 mV; lung: -101±5 versus −81±1 mV, each P Conclusions Septic peritonitis induced by CLP oxidizes ileal and colonic GSH/GSSG redox but Cys/CySS Eh remains unchanged in these intestinal tissues. In liver, CLP oxidizes the GSH/GSSG redox pool and CyS/CySS Eh becomes more reducing; in lung, CLP does not alter GSH/GSSG Eh, and Cys/CySS Eh is less oxidized. CLP-induced infection/inflammation differentially regulates major thiol-disulfide redox pools in this murine model.

Intracellular drug delivery nanocarriers of glutathione-responsive degradable block copolymers having pendant disulfide linkages.

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Khorsand, Behnoush; Lapointe, Gabriel; Brett, Christopher; Oh, Jung Kwon

2013-06-10

Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications.

Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

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Roopa Thapar

2015-05-01

Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

Photochemical synthesis of ultrafine organosilicon particles from trimethyl(2-propynyloxy)silane and carbon disulfide

Czech Academy of Sciences Publication Activity Database

Morita, H.; Nozawa, R.; Bastl, Zdeněk; Šubrt, Jan; Pola, Josef

2006-01-01

Roč. 179, 1-2 (2006), s. 142-148 ISSN 1010-6030 Grant - others:MEXT(JP) 767/15085203 Institutional research plan: CEZ:AV0Z40400503; CEZ:AV0Z40320502; CEZ:AV0Z40720504 Keywords : ultrafine particles * photo-polymerization * trimethyl(2-propynyloxy)silane * carbon disulfide Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.098, year: 2006

Cloning of araA Gene Encoding L-Arabinose Isomerase from Marine Geobacillus stearothermophilus Isolated from Tanjung Api, Poso, Indonesia

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DEWI FITRIANI

2010-06-01

Full Text Available L-arabinose isomerase is an enzyme converting D-galactose to D-tagatose. D-tagatose is a potential sweetener-sucrose substitute which has low calorie. This research was to clone and sequence araA gene from marine bacterial strain Geobacillus stearothermophilus isolated from Tanjung Api Poso Indonesia. The amplified araA gene consisted of 1494 bp nucleotides encoding 497 amino acids. DNA alignment analysis showed that the gene had high homology with that of G. stearothermophilus T6. The enzyme had optimum activity at high temperature and alkalin condition.

Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation.

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Valle, Aisel; Pérez-Socas, Luis Benito; Canet, Liem; Hervis, Yadira de la Patria; de Armas-Guitart, German; Martins-de-Sa, Diogo; Lima, Jônatas Cunha Barbosa; Souza, Adolfo Carlos Barros; Barbosa, João Alexandre Ribeiro Gonçalves; de Freitas, Sonia Maria; Pazos, Isabel Fabiola

2018-04-26

The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.

Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the high-density lipoprotein receptor SR-BI.

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Yu, Miao; Lau, Thomas Y; Carr, Steven A; Krieger, Monty

2012-12-18

The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories.

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

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Chengtuo Niu

Full Text Available 1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

International Nuclear Information System (INIS)

Distefano, M.D.; Au, K.G.; Walsh, C.T.

1989-01-01

Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys 135 Cys 140 , catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys 135 (Ala 135 Cys 140 ), Cys 14 (Cys 135 Ala 140 ), or both (Ala 135 Ala 140 ). Additionally, they have made double mutants that lack Cys 135 (Ala 135 Cys 139 Cys 140 ) or Cys 140 (Cys 135 Cys 139 Ala 140 ) but introduce a new Cys in place of Gly 139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH 2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala 135 Cys 139 Cys 14 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala 135 Cys 140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys 135 and Cys 140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate

The influence of the Cys46/Cys55 disulfide bond on the redox and spectroscopic properties of human neuroglobin.

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Bellei, Marzia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Borsari, Marco; Lancellotti, Lidia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio

2018-01-01

Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using UV-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the SS bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (ΔH°' rc ) and entropy (ΔS°' rc ), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate. Copyright © 2017 Elsevier Inc. All rights reserved.

The influence of zinc(II) on thioredoxin/glutathione disulfide exchange: QM/MM studies to explore how zinc(II) accelerates exchange in higher dielectric environments.

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Kurian, Roby; Bruce, Mitchell R M; Bruce, Alice E; Amar, François G

2015-08-01

QM/MM studies were performed to explore the energetics of exchange reactions of glutathione disulfide (GSSG) and the active site of thioredoxin [Cys32-Gly33-Pro34-Cys35] with and without zinc(II), in vacuum and solvated models. The activation energy for exchange, in the absence of zinc, is 29.7 kcal mol(-1) for the solvated model. This is 3.3 kcal mol(-1) higher than the activation energy for exchange in the gas phase, due to ground state stabilization of the active site Cys-32 thiolate in a polar environment. In the presence of zinc, the activation energy for exchange is 4.9 kcal mol(-1) lower than in the absence of zinc (solvated models). The decrease in activation energy is attributed to stabilization of the charge-separated transition state, which has a 4-centered, cyclic arrangement of Zn-S-S-S with an estimated dipole moment of 4.2 D. A difference of 4.9 kcal mol(-1) in activation energy would translate to an increase in rate by a factor of about 4000 for zinc-assisted thiol-disulfide exchange. The calculations are consistent with previously reported experimental results, which indicate that metal-thiolate, disulfide exchange rates increase as a function of solvent dielectric. This trend is opposite to that observed for the influence of the dielectric environment on the rate of thiol-disulfide exchange in the absence of metal. The results suggest a dynamic role for zinc in thiol-disulfide exchange reactions, involving accessible cysteine sites on proteins, which may contribute to redox regulation and mechanistic pathways during oxidative stress.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

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Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

2017-09-01

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore.

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Pedro Jacquez

Full Text Available Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig domain of the anthrax toxin receptor 2 (ANTXR2 inhibited the function of the protective antigen (PA pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.

Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH sensitivity.

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Imani, Mehdi; Hosseinkhani, Saman; Ahmadian, Shahin; Nazari, Mahboobeh

2010-08-01

The thermal sensitivity and pH-sensitive spectral properties of firefly luciferase have hampered its application in a variety of fields. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make two separate mutant enzymes with a single bridge. Even though the A103C/S121C mutant showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability, relative pH insensitivity and 7.3-fold increase of specific activity. Moreover, the bioluminescence emission spectrum of A296C/A326C was resistant against higher temperatures (37 degrees C). Far-UV CD analysis showed slight secondary structure changes for both mutants. Thermal denaturation analysis showed that conformational stabilities of A103C/S121C and A296C/A326C are more than native firefly luciferase. It is proposed that since A296 and A326 are situated in the vicinity of the enzyme active site microenvironment in comparison with A103 and S121, the formation of a disulfide bridge in this region has more impact on enzyme kinetic characteristics.

An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin.

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Serebryany, Eugene; Woodard, Jaie C; Adkar, Bharat V; Shabab, Mohammed; King, Jonathan A; Shakhnovich, Eugene I

2016-09-02

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered "amorphous," and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys(32) and Cys(41), was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin*

Science.gov (United States)

Serebryany, Eugene; Woodard, Jaie C.; Adkar, Bharat V.; Shabab, Mohammed; King, Jonathan A.; Shakhnovich, Eugene I.

2016-01-01

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered “amorphous,” and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. PMID:27417136

Raman Signatures of Polytypism in Molybdenum Disulfide.

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Lee, Jae-Ung; Kim, Kangwon; Han, Songhee; Ryu, Gyeong Hee; Lee, Zonghoon; Cheong, Hyeonsik

2016-02-23

Since the stacking order sensitively affects various physical properties of layered materials, accurate determination of the stacking order is important for studying the basic properties of these materials as well as for device applications. Because 2H-molybdenum disulfide (MoS2) is most common in nature, most studies so far have focused on 2H-MoS2. However, we found that the 2H, 3R, and mixed stacking sequences exist in few-layer MoS2 exfoliated from natural molybdenite crystals. The crystal structures are confirmed by HR-TEM measurements. The Raman signatures of different polytypes are investigated by using three different excitation energies that are nonresonant and resonant with A and C excitons, respectively. The low-frequency breathing and shear modes show distinct differences for each polytype, whereas the high-frequency intralayer modes show little difference. For resonant excitations at 1.96 and 2.81 eV, distinct features are observed that enable determination of the stacking order.

Single Layer Molybdenum Disulfide under Direct Out-of-Plane Compression: Low-Stress Band-Gap Engineering

Czech Academy of Sciences Publication Activity Database

Álvarez, M. P.; del Corro, Elena; Morales-García, A.; Kavan, Ladislav; Kalbáč, Martin; Frank, Otakar

2015-01-01

Roč. 15, č. 5 (2015), s. 3139-3146 ISSN 1530-6984 R&D Projects: GA ČR GA14-15357S; GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : Molybdenum disulfide * band gap engineering * out-of-plane compression Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 13.779, year: 2015

Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

Science.gov (United States)

Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

2011-03-01

Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin.

Science.gov (United States)

Sun, Y J; Chou, C C; Chen, W S; Wu, R T; Meng, M; Hsiao, C D

1999-05-11

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

International Nuclear Information System (INIS)

Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

2011-01-01

With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner

L-Arabinose isomerase and its use for biotechnological production of rare sugars.

Science.gov (United States)

Xu, Zheng; Li, Sha; Feng, Xiaohai; Liang, Jinfeng; Xu, Hong

2014-11-01

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.

Raman investigation of molybdenum disulfide with different polytypes

Science.gov (United States)

Lee, Jae-Ung; Kim, Kangwon; Han, Songhee; Ryu, Gyeong Hee; Lee, Zonghoon; Cheong, Hyeonsik

The Raman spectra of molybdenum disulfide (MoS2) with different polytypes are investigated. Although 2H-MoS2 is most common in nature, the 3R phase can exist due to a small difference in the formation energy. However, only a few studies are reported for the 3R phase, and most studies have focused on the 2H phase. We found the 2H, 3R and mixed phases of exfoliated few-layer MoS2 from natural molybdenite crystals. The crystal structures of 2H- and 3R-MoS2 are confirmed by the HR-TEM measurements. By using 3 different excitation energies, we compared the Raman spectra of different polytypes in detail. We show that the Raman spectroscopy can be used to identify not only the number of layers but also the polytypes of MoS2.

Piezoelectricity in two dimensions: Graphene vs. molybdenum disulfide

Science.gov (United States)

Song, Xiaoxue; Hui, Fei; Knobloch, Theresia; Wang, Bingru; Fan, Zhongchao; Grasser, Tibor; Jing, Xu; Shi, Yuanyuan; Lanza, Mario

2017-08-01

The synthesis of piezoelectric two-dimensional (2D) materials is very attractive for implementing advanced energy harvesters and transducers, as these materials provide enormously large areas for the exploitation of the piezoelectric effect. Among all 2D materials, molybdenum disulfide (MoS2) has shown the largest piezoelectric activity. However, all research papers in this field studied just a single material, and this may raise concerns because different setups could provide different values depending on experimental parameters (e.g., probes used and areas analyzed). By using conductive atomic force microscopy, here we in situ demonstrate that the piezoelectric currents generated in MoS2 are gigantic (65 mA/cm2), while the same experiments in graphene just showed noise currents. These results provide the most reliable comparison yet reported on the piezoelectric effect in graphene and MoS2.

Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

Science.gov (United States)

Jia, Dong-Xu; Wang, Teng; Liu, Zi-Jian; Jin, Li-Qun; Li, Jia-Jia; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

2018-04-04

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fast and efficient green synthesis of thiosulfonate S-esters by microwave-supported permanganate oxidation of symmetrical disulfides

DEFF Research Database (Denmark)

Thi, Luu Thi Xuan; Thi Nguyen, Thao-Tran; Le, Thach Ngoc

2015-01-01

Potassium permanganate absorbed on copper(II) sulfate pentahydrate has been found to be an efficient, inexpensive, and green oxidation agent for the synthesis of “symmetrical” thiosulfonate S-esters by oxidation of the corresponding symmetrical disulfides. The oxidation reactions were carried out...

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris*

Science.gov (United States)

Reardon-Robinson, Melissa E.; Osipiuk, Jerzy; Chang, Chungyu; Wu, Chenggang; Jooya, Neda; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

2015-01-01

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria. PMID:26170452

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

Science.gov (United States)

Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Chang, Chungyu; Wu, Chenggang; Jooya, Neda; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

2015-08-28

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds

Czech Academy of Sciences Publication Activity Database

Pompach, Petr; Man, Petr; Kavan, Daniel; Hofbauerová, Kateřina; Kumar, Vinay; Bezouška, Karel; Havlíček, Vladimír; Novák, Petr

2009-01-01

Roč. 44, č. 11 (2009), s. 1571-1578 ISSN 1076-5174 R&D Projects: GA AV ČR KJB400200501; GA AV ČR IAA5020403; GA AV ČR KJB500200612; GA MŠk LC545; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : disulfide bond * cystamine * gel electrophoresis Subject RIV: CE - Biochemistry Impact factor: 3.411, year: 2009

Growth and characterization of tin disulfide (SnS2) thin film deposited by successive ionic layer adsorption and reaction (SILAR) technique

International Nuclear Information System (INIS)

Deshpande, N.G.; Sagade, A.A.; Gudage, Y.G.; Lokhande, C.D.; Sharma, Ramphal

2007-01-01

Thin films of tin disulfide (SnS 2 ) have been deposited by using low cost successive ionic layer adsorption and reaction (SILAR) technique. The deposition parameters such as SILAR cycles (60), immersion time (20 s), rinsing time (10 s) and deposition temperature (27 o C) were optimized to obtain good quality of films. Physical investigations were made to study the structural, optical and electrical properties. X-ray diffraction (XRD) patterns reveal that the deposited SnS 2 thin films have hexagonal crystal structure. Energy dispersive X-ray analysis (EDAX) indicated elemental ratio close to those for tin disulfide (SnS (2.02) ). Uniform deposition of the material over the entire glass substrate was revealed by scanning electron microscopy (SEM). Atomic force microscopy (AFM) showed the film is uniform and the substrate surface is well covered with small spherical grains merged in each other. A direct band gap of 2.22 eV was obtained. Photoluminescence (PL) showed two strong peaks corresponding to green and red emission. Ag/SnS 2 junction showed Schottky diode like I-V characteristics. The barrier height calculated was 0.22 eV. Thermoelectric power (TEP) properties showed that tin disulfide exhibits n-type conductivity

Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis.

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Kajal Gupta

2010-11-01

Full Text Available C-di-GMP, a bacterial second messenger plays a key role in survival and adaptation of bacteria under different environmental conditions. The level of c-di-GMP is regulated by two opposing activities, namely diguanylate cyclase (DGC and phosphodiesterase (PDE-A exhibited by GGDEF and EAL domain, respectively in the same protein. Previously, we reported a bifunctional GGDEF-EAL domain protein, MSDGC-1 from Mycobacterium smegmatis showing both these activities (Kumar and Chatterji, 2008. In this current report, we have identified and characterized the homologous protein from Mycobacterium tuberculosis (Rv 1354c named as MtbDGC. MtbDGC is also a bifunctional protein, which can synthesize and degrade c-di-GMP in vitro. Further we expressed Mtbdgc in M. smegmatis and it was able to complement the MSDGC-1 knock out strain by restoring the long term survival of M. smegmatis. Another protein Rv 1357c, named as MtbPDE, is an EAL domain protein and degrades c-di-GMP to pGpG in vitro. Rv1354c and 1357c have seven cysteine amino acids in their sequence, distributed along the full length of the protein. Disulfide bonds play an important role in stabilizing protein structure and regulating protein function. By proteolytic digestion and mass spectrometric analysis of MtbDGC, connectivity between cysteine pairs Cys94-Cys584, Cys2-Cys479 and Cys429-Cys614 was determined, whereas the third cysteine (Cys406 from N terminal was found to be free in MtbDGC protein, which was further confirmed by alkylation with iodoacetamide labeling. Bioinformatics modeling investigations also supported the pattern of disulfide connectivity obtained by Mass spectrometric analysis. Cys406 was mutated to serine by site directed mutagenesis and the mutant MtbC406S was not found to be active and was not able to synthesize or degrade c-di-GMP. The disulfide connectivity established here would help further in understanding the structure - function relationship in MtbDGC.

Influence of the degree of crosslinking on the depolymerization of disulfide polymer

International Nuclear Information System (INIS)

Rekalicj, J.V.; Radosavljevicj, D.S.; Popovicj, E.M.; Stashicj, L.

1976-01-01

The action of nucleophilic reagents (hydrogen sulfide ion, dithionite ion and hydrazine) on disulfide polymers prepd. from bis-2-chloroethyl formal and 1,2,3-trichloropropane, taken in various mol rations is studied. The depolymerization efficiency is higher with hydrazine and dithionite than with a mixt. of sodium hydrogen sulfide and sodium sulfite. An interpretation of the results is given, attempting to correlate the content of SH-groups in the obtained product with the same quantity in some defined compds. which can be present after the depolymerization

Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

Science.gov (United States)

Bukowska-Faniband, Ewa; Hederstedt, Lars

2017-07-01

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.

Science.gov (United States)

Rathnayaka, Tharangani; Tawa, Minako; Nakamura, Takashi; Sohya, Shihori; Kuwajima, Kunihiro; Yohda, Masafumi; Kuroda, Yutaka

2011-12-01

Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The (1)H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds. Copyright © 2011. Published by Elsevier B.V.

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

Science.gov (United States)

Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

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Anaïs Noblanc

Full Text Available We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4 and the epididymal glutathione peroxidase 5 (GPx5 activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2O(2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.

Science.gov (United States)

Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A; Salic, Adrian

2011-03-07

The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly.

S center dot center dot center dot N chalcogen bonded complexes of carbon disulfide with diazines. Theoretical study

Czech Academy of Sciences Publication Activity Database

Zierkiewicz, W.; Fanfrlík, Jindřich; Michalczyk, M.; Michalska, D.; Hobza, Pavel

2018-01-01

Roč. 500, Jan 26 (2018), s. 37-44 ISSN 0301-0104 Institutional support: RVO:61388963 Keywords : chalcogen bond * carbon disulfide * diazines * DFT Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 1.767, year: 2016

Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing.

Science.gov (United States)

Trexler-Schmidt, Melody; Sargis, Sandy; Chiu, Jason; Sze-Khoo, Stefanie; Mun, Melissa; Kao, Yung-Hsiang; Laird, Michael W

2010-06-15

In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale-up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibody's interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab-scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO(4))), pre- and post-harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO(4), EDTA, L-cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing-scale. The work described here identifies numerous effective reduction prevention measures from lab-scale studies; several of these methods were then successfully translated into manufacturing processes. 2010 Wiley Periodicals, Inc.

Graphite oxide and molybdenum disulfide composite for hydrogen evolution reaction

Science.gov (United States)

Niyitanga, Theophile; Jeong, Hae Kyung

2017-10-01

Graphite oxide and molybdenum disulfide (GO-MoS2) composite is prepared through a wet process by using hydrolysis of ammonium tetrathiomolybdate, and it exhibits excellent catalytic activity of the hydrogen evolution reaction (HER) with a low overpotential of -0.47 V, which is almost two and three times lower than those of precursor MoS2 and GO. The high performance of HER of the composite attributes to the reduced GO supporting MoS2, providing a conducting network for fast electron transport from MoS2 to electrodes. The composite also shows high stability after 500 cycles, demonstrating a synergistic effect of MoS2 and GO for efficient HER.

Sample limited characterization of a novel disulfide-rich venom peptide toxin from terebrid marine snail Terebra variegata.

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Prachi Anand

Full Text Available Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.

Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by ''Escheridia coli'' RNA polymerase

International Nuclear Information System (INIS)

Radlowski, M.; Job, D.

1994-01-01

The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using ''Escherichia coli'' RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes. (author). 15 refs, 1 fig., 1 tab

Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

Science.gov (United States)

Chiarelli, Nicola; Carini, Giulia; Zoppi, Nicoletta; Ritelli, Marco; Colombi, Marina

2018-01-01

Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of

Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

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Nicola Chiarelli

Full Text Available Vascular Ehlers-Danlos syndrome (vEDS is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII, which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER homeostasis, COLLs folding and extracellular matrix (ECM organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition

Missing Links in Antibody Assembly Control

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Tiziana Anelli

2013-01-01

Full Text Available Fidelity of the humoral immune response requires that quiescent B lymphocytes display membrane bound immunoglobulin M (IgM on B lymphocytes surface as part of the B cell receptor, whose function is to recognize an antigen. At the same time B lymphocytes should not secrete IgM until recognition of the antigen has occurred. The heavy chains of the secretory IgM have a C-terminal tail with a cysteine instead of a membrane anchor, which serves to covalently link the IgM subunits by disulfide bonds to form “pentamers” or “hexamers.” By virtue of the same cysteine, unassembled secretory IgM subunits are recognized and retained (via mixed disulfide bonds by members of the protein disulfide isomerase family, in particular ERp44. This so-called “thiol-mediated retention” bars assembly intermediates from prematurely leaving the cell and thereby exerts quality control on the humoral immune response. In this essay we discuss recent findings on how ERp44 governs such assembly control in a pH-dependent manner, shuttling between the cisGolgi and endoplasmic reticulum, and finally on how pERp1/MZB1, possibly as a co-chaperone of GRP94, may help to overrule the thiol-mediated retention in the activated B cell to give way to antibody secretion.

Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.

Science.gov (United States)

Tarasava, Katsiaryna; Chesnov, Serge; Freisinger, Eva

2016-05-01

Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016. © 2016 Wiley Periodicals, Inc.

Molecular Docking Simulation of Neuraminidase Influenza A Subtype H1N1 with Potential Inhibitor of Disulfide Cyclic Peptide (DNY, NNY, LRL)

Science.gov (United States)

Putra, R. P.; Imaniastuti, R.; Nasution, M. A. F.; Kerami, Djati; Tambunan, U. S. F.

2018-04-01

Oseltamivir resistance as an inhibitor of neuraminidase influenza A virus subtype H1N1 has been reported lately. Therefore, to solve this problem, several kinds of research has been conducted to design and discover disulfide cyclic peptide ligands through molecular docking method, to find the potential inhibitors for neuraminidase H1N1 which then can disturb the virus replication. This research was studied and evaluated the interaction of ligands toward enzyme using molecular docking simulation, which was performed on three disulfide cyclic peptide inhibitors (DNY, LRL, and NNT), along with oseltamivir and zanamivir as the standard ligands using MOE 2008.10 software. The docking simulation shows that all disulfide cyclic peptide ligands have lower Gibbs free binding energies (ΔGbinding) than the standard ligands, with DNY ligand has the lowest ΔGbinding at -7.8544 kcal/mol. Furthermore, these ligands were also had better molecular interactions with neuraminidase than the standards, owing by the hydrogen bonds that were formed during the docking simulation. In the end, we concluded that DNY, LRL and NNT ligands have the potential to be developed as the inhibitor of neuraminidase H1N1.

40 CFR 63.500 - Back-end process provisions-carbon disulfide limitations for styrene butadiene rubber by emulsion...

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 9 2010-07-01 2010-07-01 false Back-end process provisions-carbon disulfide limitations for styrene butadiene rubber by emulsion processes. 63.500 Section 63.500 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR...

The effects of morphology re-arrangements on the pseudocapacitive properties of mesoporous molybdenum disulfide (MoS2) nanoflakes

CSIR Research Space (South Africa)

Khawula, TNY

2016-07-01

Full Text Available Mesoporous molybdenum disulfide (MoS(sub2)) with different morphologies have been prepared via hydrothermal method using different solvents, water or water/acetone mixture. The MoS(sub2) obtained with water alone gave a graphene-like nanoflakes (g...

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

DEFF Research Database (Denmark)

Barington, Line; Rummel, Pia C; Lückmann, Michael

2016-01-01

and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action...... in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only...

A mathematical analysis of Prx2-STAT3 disulfide exchange rate constants for a bimolecular reaction mechanism.

Science.gov (United States)

Langford, Troy F; Deen, William M; Sikes, Hadley D

2018-03-22

Appreciation of peroxiredoxins as the major regulators of H 2 O 2 concentrations in human cells has led to a new understanding of redox signaling. In addition to their status as the primary reducers of H 2 O 2 to water, the oxidized peroxiredoxin byproduct of this reaction has recently been shown capable of participation in H 2 O 2 -mediated signaling pathways through disulfide exchange reactions with the transcription factor STAT3. The dynamics of peroxidase-transcription factor disulfide exchange reactions have not yet been considered in detail with respect to how these reactions fit into the larger network of competing reactions in human cells. In this study, we used a kinetic model of oxidation and reduction reactions related to H 2 O 2 metabolism in the cytosol of human cells to study the dynamics of peroxiredoxin-2 mediated oxidation of the redox-regulated transcription factor STAT3. In combination with previously reported experimental data, the model was used to estimate the rate coefficient of a biomolecular reaction between Prx2 and STAT3 for two sets of assumptions that constitute lower and upper bound cases. Using these estimates, we calculated the relative rates of the reaction of oxidized peroxiredoxin-2 and STAT3 and other competing reactions in the cytosol. These calculations revealed that peroxiredoxin-2-mediated oxidation of STAT3 likely occurs at a much slower rate than competing reactions in the cytosol. This analysis suggests the existence of more complex mechanisms, potentially involving currently unknown protein-protein recognition partners, which facilitate disulfide exchange reactions between peroxiredoxin-2 and STAT3. Copyright © 2018 Elsevier Inc. All rights reserved.

A Role of a Newly Identified Isomerase From Yarrowia lipolytica in Erythritol Catabolism

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Aleksandra M. Mirończuk

2018-05-01

Full Text Available Erythritol is a natural sweetener produced by microorganisms as an osmoprotectant. It belongs to the group of polyols and it can be utilized by the oleaginous yeast Yarrowia lipolytica. Despite the recent identification of the transcription factor of erythritol utilization (EUF1, the metabolic pathway of erythritol catabolism remains unknown. In this study we identified a new gene, YALI0F01628g, involved in erythritol assimilation. In silico analysis showed that YALI0F01628g is a putative isomerase and it is localized in the same region as EUF1. qRT-PCR analysis of Y. lipolytica showed a significant increase in YALI0F01628g expression during growth on erythritol and after overexpression of EUF1. Moreover, the deletion strain ΔF01628 showed significantly impaired erythritol assimilation, whereas synthesis of erythritol remained unchanged. The results showed that YALI0F1628g is involved in erythritol assimilation; thus we named the gene EYI1. Moreover, we suggest the metabolic pathway of erythritol assimilation in yeast Y. lipolytica.

Tuning thermal conductivity in molybdenum disulfide by electrochemical intercalation

Science.gov (United States)

Zhu, Gaohua; Liu, Jun; Zheng, Qiye; Zhang, Ruigang; Li, Dongyao; Banerjee, Debasish; Cahill, David G.

2016-01-01

Thermal conductivity of two-dimensional (2D) materials is of interest for energy storage, nanoelectronics and optoelectronics. Here, we report that the thermal conductivity of molybdenum disulfide can be modified by electrochemical intercalation. We observe distinct behaviour for thin films with vertically aligned basal planes and natural bulk crystals with basal planes aligned parallel to the surface. The thermal conductivity is measured as a function of the degree of lithiation, using time-domain thermoreflectance. The change of thermal conductivity correlates with the lithiation-induced structural and compositional disorder. We further show that the ratio of the in-plane to through-plane thermal conductivity of bulk crystal is enhanced by the disorder. These results suggest that stacking disorder and mixture of phases is an effective mechanism to modify the anisotropic thermal conductivity of 2D materials. PMID:27767030

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

Science.gov (United States)

Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

2011-11-01

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

Quantitative analysis of the erythrocyte membrane proteins in polycythemia vera patients treated with hydroxycarbamide

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Darshana Kottahachchi

2015-06-01

Full Text Available More than 90% of polycythemia vera (PV patients have a mutation in the protein JAK2, which is closely associated with the erythrocyte membrane. With the comparison of 1-D gels of erythrocyte membranes obtained from PV patients treated with hydroxycarbamide and those of untreated controls we observed significant differences in the region of 40–55 kDa. On the basis of the LC–MS/MS analysis of this region we report up-regulation of four protein disulfide isomerases, which was subsequently confirmed by targeted mass spectrometric analysis. In further studies it will be prudent to compare this in patients both treated and not treated with hydroxycarbamide.

Symmetric pseudocapacitors based on molybdenum disulfide (MoS2)-modified carbon nanospheres: correlating physicochemistry and synergistic interaction on energy storage

CSIR Research Space (South Africa)

Khawula, TNY

2016-03-01

Full Text Available Molybdenum disulfide-modified carbon nanospheres (MoS(sub2)/CNS) with two different morphologies (spherical and flower-like) have been synthesized using hydrothermal techniques and investigated as symmetric pseudocapacitors in an aqueous electrolyte...