Sample records for gaf domain cysteine

  1. Interaction of GlnK with the GAF domain of Herbaspirillum seropedicae NifA mediates NH₄⁺-regulation.

    PubMed

    Oliveira, Marco A S; Aquino, Bruno; Bonatto, Ana Claudia; Huergo, Luciano F; Chubatsu, Leda S; Pedrosa, Fábio O; Souza, Emanuel M; Dixon, Ray; Monteiro, Rose A

    2012-04-01

    Nitrogen fixation in Herbaspirillum seropedicae is transcriptionally regulated by NifA, a σ(54) transcriptional activator with three structural domains: an N-terminal GAF domain, a catalytic AAA+ domain and a C-terminal DNA-binding domain. NifA is only active in H. seropedicae when cultures are grown in the absence of fixed nitrogen and at low oxygen tensions. There is evidence that the inactivation of NifA in response to fixed nitrogen is mediated by the regulatory GAF domain. However, the mechanism of NifA repression by the GAF domain, as well as the transduction of nitrogen status to NifA, is not understood. In order to study the regulation of NifA activity by fixed nitrogen independently of oxygen regulation, we constructed a chimeric protein containing the GAF domain of H. seropedicae NifA fused to the AAA+ and C-terminal domains of Azotobacter vinelandii NifA. This chimeric protein (NifAQ1) lacks the cysteine motif found in oxygen sensitive NifA proteins and is not oxygen responsive in vivo. Our results demonstrate that NifAQ1 responds to fixed nitrogen and requires GlnK protein for activity, a behavior similar to H. seropedicae NifA. In addition, protein footprinting analysis indicates that this response probably involves a protein-protein contact between the GAF domain and the GlnK protein. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  2. The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii.

    PubMed

    Yoshimitsu, Kyohei; Takatani, Nobuyuki; Miura, Yukio; Watanabe, Yoshihito; Nakajima, Hiroshi

    2011-09-01

    VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase-2 in Azotobacter vinelandii. VnfA consists of three domains: an N-terminal regulatory domain termed GAF, including a Cys-rich motif; a central domain from the AAA+ family; and a C-terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe-S cluster (presumably of the 3Fe-4S type) as a prosthetic group and the Cys-rich motif were possibly associated with coordination of the Fe-S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: ΔN15(VnfA) and ΔGAF(VnfA) that lack the N-terminal 15 residues and whole GAF domain, respectively, and GAF(VnfA) consisting of only the GAF domain. ΔN15(VnfA) and ΔGAF(VnfA) lost the ability to bind the Fe-S cluster, whereas GAF(VnfA) was still able to bind to the cluster, consistent with the hypothesis that the Cys-rich motif is essential for Fe-S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe-S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain. © 2011 The Authors Journal compilation © 2011 FEBS.

  3. Conformation changes, N-terminal involvement and cGMP signal relay in phosphodiesterase-5 GAF domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, H.; Robinson, H.; Ke, H.

    2010-12-03

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

  4. Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain*

    PubMed Central

    Wang, Huanchen; Robinson, Howard; Ke, Hengming

    2010-01-01

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes. PMID:20861010

  5. Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    H Wang; H Robinson; H Ke

    2011-12-31

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

  6. The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

    PubMed

    Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

    2018-04-09

    The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  7. Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5: Insights into Nucleotide Specificity, Dimerization, and cGMP-Dependent Conformational Change

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Heikaus, Clemens C.; Stout, Joseph R.; Sekharan, Monica R.

    2008-08-15

    Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular NOEs.

  8. Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.

    PubMed

    Hontani, Yusaku; Shcherbakova, Daria M; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav V; Kennis, John T M

    2016-11-18

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

  9. Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

    PubMed Central

    Sotomaior, P.; Araújo, L.M.; Nishikawa, C.Y.; Huergo, L.F.; Monteiro, R.A.; Pedrosa, F.O.; Chubatsu, L.S.; Souza, E.M.

    2012-01-01

    Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate. PMID:22983183

  10. Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense.

    PubMed

    Sotomaior, P; Araújo, L M; Nishikawa, C Y; Huergo, L F; Monteiro, R A; Pedrosa, F O; Chubatsu, L S; Souza, E M

    2012-12-01

    Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

  11. 2.3 Å X-ray Structure of the Heme-Bound GAF Domain of Sensory Histidine Kinase DosT of Mycobacterium tuberculosis†

    PubMed Central

    Podust, Larissa M.; Ioanoviciu, Alexandra; Ortiz de Montellano, Paul R.

    2009-01-01

    Mycobacterium tuberculosis responds to the changes in environmental conditions through a two-component signaling system that detects reduced O2 tension and NO and CO exposures via the heme-binding GAF domains of two sensory histidine kinases, DosT and DevS, and the transcriptional regulator DosR. We report the first x-ray structure of the DosT heme-bound GAF domain (GAFDosT) in both oxy and deoxy forms determined to a resolution of 2.3 Å. In GAFDosT, heme binds in an orientation orthogonal to that in the PAS domains via a highly conserved motif including invariant H147 as a proximal heme axial ligand. On the distal side, invariant Y169 is in stacking interactions with the heme with its long axis parallel and the plane of the ring orthogonal to the heme plane. In one of the two protein monomers in an asymmetric unit, O2 binds as a second axial ligand to the heme iron, and is stabilized via an H-bond to the OH-group of Y169. The structure reveals two small tunnel-connected cavities and a pore on the protein surface that suggest a potential route for O2 access to the sensing pocket. The limited conformational differences observed between differently heme iron-ligated GAFDosT monomers in the asymmetric unit may result from crystal lattice limitations since atmospheric oxygen binding likely occurs in the crystal as a result of x-ray induced Fe3+ photoreduction during diffraction data collection. Determination of the GAFDosT structure sets up a framework in which to address ligand-recognition, discrimination, and signal propagation schemes in the heme-based GAF domains of biological sensors. PMID:18980385

  12. The cysteine-rich domain regulates ADAM protease function in vivo.

    PubMed

    Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

    2002-12-09

    ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

  13. Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

    PubMed

    Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

    2016-12-01

    Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be

  14. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

    PubMed Central

    Lidell, Martin E.; Moncada, Darcy M.; Chadee, Kris; Hansson, Gunnar C.

    2006-01-01

    In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel. PMID:16754877

  15. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel.

    PubMed

    Lidell, Martin E; Moncada, Darcy M; Chadee, Kris; Hansson, Gunnar C

    2006-06-13

    In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.

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

    PubMed Central

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

    2015-01-01

    DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins. PMID:26487721

  17. Laser cooling of BH and GaF: insights from an ab initio study.

    PubMed

    Gao, Yu-feng; Gao, Tao

    2015-04-28

    The feasibility of laser cooling BH and GaF is investigated using ab initio quantum chemistry. The ground state X (1)Σ(+) and first two excited states (3)Π and (1)Π of BH and GaF are calculated using the multireference configuration interaction (MRCI) level of theory. For GaF, the spin-orbit coupling effect is also taken into account in the electronic structure calculations at the MRCI level. Calculated spectroscopic constants for BH and GaF show good agreement with available theoretical and experimental results. The highly diagonal Franck-Condon factors (BH: f00 = 0.9992, f11 = 0.9908, f22 = 0.9235; GaF: f00 = 0.997, f11 = 0.989, f22 = 0.958) for the (1)Π (v' = 0-2) → X (1)Σ(+) (v = 0-2) transitions in BH and GaF are determined, which are found to be in good agreement with the theoretical and experimental data. Radiative lifetime calculations of the (1)Π (v' = 0-2) state (BH: 131, 151, and 187 ns; GaF: 2.26, 2.36, and 2.48 ns) are found to be short enough for rapid laser cooling. The proposed laser cooling schemes that drive the (1)Π (v' = 0) → X (1)Σ(+) (v = 0) transition use just one laser wavelength λ00 (BH: 436 nm, GaF: 209 nm). Though the cooling wavelength of GaF is deep in the UVC, a frequency quadrupled Ti:sapphire laser (189-235 nm) could be capable of generating useful quantities of light at this wavelength. The present results indicate that BH and GaF are two good choices of molecules for laser cooling.

  18. The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

    PubMed

    Wirtz, M; Berkowitz, O; Droux, M; Hell, R

    2001-02-01

    Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

  19. Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins

    PubMed Central

    Pei, Jimin; Grishin, Nick V

    2012-01-01

    Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. PMID:22693159

  20. Photoacoustic and fluorescent imaging GAF2 photoswitchable chromoproteins (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chee, Ryan K.; Li, Yan; Paproski, Robert J.; Campbell, Robert E.; Zemp, Roger J.

    2017-03-01

    Molecular photoacoustic imaging is hindered by hemoglobin background signal. Photoswitchable chromoproteins can be used to obtain images with significantly reduced background signal. Molecular imaging of multiple biological processes via multiple chromoprotiens is difficult due to overlapping imaging spectra. Using a new rate-of-change imaging methodology, we can obtain molecular images with multiple chromoprotiens with overlapping imaging spectra. We also present a new photoswitchable chromoprotein, GAF2, which is significantly smaller than the BphP1 which has shown promise for photoswitchable photoacoustic imaging [Yao et al., Nat. Meth. 13, 67-73 (2016)]. We use BphP1 and GAF2 with photoacoustic (Vevo LAZR, Fujifilm Visualsonics Inc) and fluorescence (In vivo Xtreme, Bruker) imaging systems to show background-free multiplexed images. We image before, after, and during photoconversion to obtain background-free rate-of-change images and compare our results to difference imaging and spectral demixing. After phantom imaging, we inject mice with different chromoprotein-expressing E. coli bacteria to show multiplexed images of bacterial infections. We show distinguishable differences in the rate-of-change between GAF2 and BphP1. We obtain rate-of-change feasibility images and in vivo images in mice showing the ability to differentiate between GAF2 and BphP1 even though they are spectrally similar. We photoconvert both GAF2 and BphP1 using 550nm and 735nm light. Phantom studies suggest a 10-20dB improvement in the rate-of-change and difference images in comparison to images with background. Multiplexed background-free molecular imaging using chromoproteins could prove to be a promising new imaging methodology especially when combined with spectral demixing.

  1. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lupardus, P.J.; Shen, A.; Bogyo, M.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  2. Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica.

    PubMed

    Cancela, Martín; Corvo, Ileana; DA Silva, Edileuza; Teichmann, Aline; Roche, Leda; Díaz, Alvaro; Tort, José Fransisco; Ferreira, Henrique B; Zaha, Arnaldo

    2017-11-01

    Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

  3. Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.

    PubMed Central

    Harrington, Melissa A; Kopito, Ron R

    2002-01-01

    Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance. PMID:11867445

  4. Collecting Information for Rating Global Assessment of Functioning (GAF): Sources of Information and Methods for Information Collection.

    PubMed

    I H, Monrad Aas

    2014-11-01

    Global Assessment of Functioning (GAF) is an assessment instrument that is known worldwide. It is widely used for rating the severity of illness. Results from evaluations in psychiatry should characterize the patients. Rating of GAF is based on collected information. The aim of the study is to identify the factors involved in collecting information that is relevant for rating GAF, and gaps in knowledge where it is likely that further development would play a role for improved scoring. A literature search was conducted with a combination of thorough hand search and search in the bibliographic databases PubMed, PsycINFO, Google Scholar, and Campbell Collaboration Library of Systematic Reviews. Collection of information for rating GAF depends on two fundamental factors: the sources of information and the methods for information collection. Sources of information are patients, informants, health personnel, medical records, letters of referral and police records about violence and substance abuse. Methods for information collection include the many different types of interview - unstructured, semi-structured, structured, interviews for Axis I and II disorders, semistructured interviews for rating GAF, and interviews of informants - as well as instruments for rating symptoms and functioning, and observation. The different sources of information, and methods for collection, frequently result in inconsistencies in the information collected. The variation in collected information, and lack of a generally accepted algorithm for combining collected information, is likely to be important for rated GAF values, but there is a fundamental lack of knowledge about the degree of importance. Research to improve GAF has not reached a high level. Rated GAF values are likely to be influenced by both the sources of information used and the methods employed for information collection, but the lack of research-based information about these influences is fundamental. Further development of

  5. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bradshaw, William J.; Public Health England, Porton Down, Salisbury SP4 0JG; Kirby, Jonathan M.

    2014-07-01

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA)more » into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.« less

  6. Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence

    NASA Astrophysics Data System (ADS)

    Rumyantsev, Konstantin A.; Shcherbakova, Daria M.; Zakharova, Natalia I.; Emelyanov, Alexander V.; Turoverov, Konstantin K.; Verkhusha, Vladislav V.

    2015-12-01

    Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

  7. 42 CFR 414.26 - Determining the GAF.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) Geographic indices. CMS uses the following indices to establish the GAF: (1) An index that reflects one... fee schedule areas as determined under § 414.22(a) and the national average of that work effort. (2... § 414.22(b) compared to the national average of those costs. (3) An index that reflects the relative...

  8. 42 CFR 414.26 - Determining the GAF.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...) Geographic indices. CMS uses the following indices to establish the GAF: (1) An index that reflects one... fee schedule areas as determined under § 414.22(a) and the national average of that work effort. (2... § 414.22(b) compared to the national average of those costs. (3) An index that reflects the relative...

  9. 42 CFR 414.26 - Determining the GAF.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...) Geographic indices. CMS uses the following indices to establish the GAF: (1) An index that reflects one... fee schedule areas as determined under § 414.22(a) and the national average of that work effort. (2... § 414.22(b) compared to the national average of those costs. (3) An index that reflects the relative...

  10. 42 CFR 414.26 - Determining the GAF.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) Geographic indices. CMS uses the following indices to establish the GAF: (1) An index that reflects one... fee schedule areas as determined under § 414.22(a) and the national average of that work effort. (2... § 414.22(b) compared to the national average of those costs. (3) An index that reflects the relative...

  11. 42 CFR 414.26 - Determining the GAF.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...) Geographic indices. CMS uses the following indices to establish the GAF: (1) An index that reflects one... fee schedule areas as determined under § 414.22(a) and the national average of that work effort. (2... § 414.22(b) compared to the national average of those costs. (3) An index that reflects the relative...

  12. Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation.

    PubMed

    Chen, P; Melchior, C; Brons, N H; Schlegel, N; Caen, J; Kieffer, N

    2001-10-19

    We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin

  13. Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

    PubMed Central

    Bischak, Connor G.; Longhi, Sonia; Snead, David M.; Costanzo, Stéphanie; Terrer, Elodie; Londergan, Casey H.

    2010-01-01

    Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) were cyanylated at cysteine and their infrared spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiological partners of NTAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within NTAIL, the C≡N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level. PMID:20816082

  14. Selective inhibition of Erwinia amylovora by the herbicidally-active Germination-Arrest Factor (GAF) produced by Pseudomonas bacteria

    USDA-ARS?s Scientific Manuscript database

    Aims: The Germination-Arrest Factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4-formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. The present study was undertaken to determine if GAF has antimicrobial activity in addition to it...

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

    PubMed

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

    2015-12-30

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

  16. Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

    PubMed Central

    Hansen, M S; Barklis, E

    1995-01-01

    We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

  17. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stiegler, A.; Burden, S; Hubbard, S

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteinsmore » but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.« less

  18. Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

    DOE PAGES

    Travers, Timothy; Lopez Bautista, Cesar Augusto; Van, Que; ...

    2018-05-31

    Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here in this paper, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which ismore » consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.« less

  19. Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Travers, Timothy; Lopez Bautista, Cesar Augusto; Van, Que

    Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here in this paper, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which ismore » consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.« less

  20. Genome-wide search and comparative genomic analysis of the trypsin inhibitor-like cysteine-rich domain-containing peptides.

    PubMed

    Zeng, Xian-Chun; Liu, Yichen; Shi, Wanxia; Zhang, Lei; Luo, Xuesong; Nie, Yao; Yang, Ye

    2014-03-01

    It was shown that peptides containing trypsin inhibitor-like cysteine-rich (TIL) domain are able to inhibit proteinase activities, and thus play important roles in various biological processes, such as immune response and anticoagulation. However, only a limited number of the TIL peptides have been identified and characterized so far; and little has been known about the evolutionary relationships of the genes encoding the TIL peptides. BmKAPi is a TIL domain-containing peptide that was identified from Mesobuthus martensii Karsch. Here, we conducted genome-wide searches for new peptides that are homologous to BmKAPi or possess a cysteine pattern similar to that of BmKAPi. As a result, we identified a total of 80 different TIL peptides from 34 species of arthropods. We found that these peptides can be classified into seven evolutionarily distinct groups. Furthermore, we cloned the genomic sequence of BmKAPi; the genomic sequences of the majority of other TIL peptides were also identified from the GenBank database using bioinformatical approaches. Through phylogenetic and comparative genomic analysis, we found 26 cases of intron gain events occurred in the genes of the TIL peptides; however, no instances of intron loss were observed. Moreover, we found that alternative splicing contributes to the diversification of the TIL peptides. It is interesting to see that four genes of the TIL domain-containing peptides overlap in a DNA region located on the chromosome LG B15 of Bombus terretris. These data suggest that the evolution of the TIL peptide genes are dynamic, which was dominated by intron gain. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Bacterial Cysteine-Inducible Cysteine Resistance Systems

    PubMed Central

    Takumi, Kazuhiro

    2016-01-01

    ABSTRACT Cysteine donates sulfur to macromolecules and occurs naturally in many proteins. Because low concentrations of cysteine are cytotoxic, its intracellular concentration is stringently controlled. In bacteria, cysteine biosynthesis is regulated by feedback inhibition of the activities of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) and is also regulated at the transcriptional level by inducing the cysteine regulon using the master regulator CysB. Here, we describe two novel cysteine-inducible systems that regulate the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for biotechnological, medical, and industrial purposes. One locus, designated ccdA (formerly PAJ_0331), encodes a novel cysteine-inducible cysteine desulfhydrase (CD) that degrades cysteine, and its expression is controlled by the transcriptional regulator encoded by ccdR (formerly PAJ_0332 or ybaO), located just upstream of ccdA. The other locus, designated cefA (formerly PAJ_3026), encodes a novel cysteine-inducible cysteine efflux pump that is controlled by the transcriptional regulator cefR (formerly PAJ_3027), located just upstream of cefA. To our knowledge, this is the first example where the expression of CD and an efflux pump is regulated in response to cysteine and is directly involved in imparting resistance to excess levels of cysteine. We propose that ccdA and cefA function as safety valves that maintain homeostasis when the intra- or extracellular cysteine concentration fluctuates. Our findings contribute important insights into optimizing the production of cysteine and related biomaterials by P. ananatis. IMPORTANCE Because of its toxicity, the bacterial intracellular cysteine level is stringently regulated at biosynthesis. This work describes the identification and characterization of two novel cysteine-inducible systems that regulate, through degradation and

  2. Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk ▿

    PubMed Central

    He, Shihan; Ni, Shuang; Hegde, Shailaja; Wang, Xin; Sharda, Daniel R.; August, Avery; Paulson, Robert F.; Hankey, Pamela A.

    2010-01-01

    Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth. PMID:20016000

  3. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Suzuki, Nobuhiro; Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602; Yamazaki, Yasuo

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinitiesmore » on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.« less

  4. Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

    PubMed

    Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

    2009-01-01

    Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

  5. Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance

    PubMed Central

    Mulinta, Ras; Yao, Sylvia Y. M.; Ng, Amy M. L.; Cass, Carol E.; Young, James D.

    2017-01-01

    The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function. PMID:28385889

  6. Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

    PubMed Central

    Smulski, Cristian R.; Beyrath, Julien; Decossas, Marion; Chekkat, Neila; Wolff, Philippe; Estieu-Gionnet, Karine; Guichard, Gilles; Speiser, Daniel; Schneider, Pascal; Fournel, Sylvie

    2013-01-01

    The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state. PMID:23463508

  7. Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding.

    PubMed

    Visperas, Patrick R; Winger, Jonathan A; Horton, Timothy M; Shah, Neel H; Aum, Diane J; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G; Arkin, Michelle R; Weiss, Arthur; Kuriyan, John

    2015-01-01

    Zeta-chain associated protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling respectively. They are recruited, via their tandem-SH2 (Src-homology domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.

  8. Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

    PubMed

    Boulangé, A; Serveau, C; Brillard, M; Minet, C; Gauthier, F; Diallo, A; Lalmanach, G; Authié, E

    2001-11-01

    The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

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

    PubMed Central

    Xu, Minfu; Smothers, C. Thetford

    2015-01-01

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

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

    PubMed

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

    2013-04-15

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

  11. Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

    PubMed Central

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

    2013-01-01

    IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor. PMID:23282150

  12. Does a web-based feedback training program result in improved reliability in clinicians' ratings of the Global Assessment of Functioning (GAF) Scale?

    PubMed

    Støre-Valen, Jakob; Ryum, Truls; Pedersen, Geir A F; Pripp, Are H; Jose, Paul E; Karterud, Sigmund

    2015-09-01

    The Global Assessment of Functioning (GAF) Scale is used in routine clinical practice and research to estimate symptom and functional severity and longitudinal change. Concerns about poor interrater reliability have been raised, and the present study evaluated the effect of a Web-based GAF training program designed to improve interrater reliability in routine clinical practice. Clinicians rated up to 20 vignettes online, and received deviation scores as immediate feedback (i.e., own scores compared with expert raters) after each rating. Growth curves of absolute SD scores across the vignettes were modeled. A linear mixed effects model, using the clinician's deviation scores from expert raters as the dependent variable, indicated an improvement in reliability during training. Moderation by content of scale (symptoms; functioning), scale range (average; extreme), previous experience with GAF rating, profession, and postgraduate training were assessed. Training reduced deviation scores for inexperienced GAF raters, for individuals in clinical professions other than nursing and medicine, and for individuals with no postgraduate specialization. In addition, training was most beneficial for cases with average severity of symptoms compared with cases with extreme severity. The results support the use of Web-based training with feedback routines as a means to improve the reliability of GAF ratings performed by clinicians in mental health practice. These results especially pertain to clinicians in mental health practice who do not have a masters or doctoral degree. (c) 2015 APA, all rights reserved.

  13. Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

    PubMed

    Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G

    2016-12-09

    A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5  M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

  14. Modification by covalent reaction or oxidation of cysteine residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding

    PubMed Central

    Visperas, Patrick R.; Winger, Jonathan A.; Horton, Timothy M.; Shah, Neel H.; Aum, Diane J.; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G.; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, John

    2015-01-01

    Zeta-chain Associated Protein of 70kDa (ZAP-70) and Spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signaling, respectively. They are recruited, via their tandem-SH2 domains, to doubly-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signaling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys 39 in ZAP-70, Cys 206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of hydrogen peroxide, and these two cysteine residues are also necessary for inhibition by hydrogen peroxide. Our findings suggest a mechanism by which the generation of reactive oxygen species generated during responses to antigen could attenuate signaling through these kinases, and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains. PMID:25287889

  15. Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230*

    PubMed Central

    MacDonald, Nicholas J.; Nguyen, Vu; Shimp, Richard; Reiter, Karine; Herrera, Raul; Burkhardt, Martin; Muratova, Olga; Kumar, Krishan; Aebig, Joan; Rausch, Kelly; Lambert, Lynn; Dawson, Nikiah; Sattabongkot, Jetsumon; Ambroggio, Xavier; Duffy, Patrick E.; Wu, Yimin; Narum, David L.

    2016-01-01

    Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria. PMID:27432885

  16. ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove.

    PubMed

    Antoniou, Antony N; Santos, Susana G; Campbell, Elaine C; Lynch, Sarah; Arosa, Fernando A; Powis, Simon J

    2007-05-15

    The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.

  17. Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli.

    PubMed

    Nonaka, Gen; Takumi, Kazuhiro

    2017-12-01

    Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.

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

    PubMed Central

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

    2011-01-01

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

  19. A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced From Studies With Subunit B of Heterodisulfide Reductase From Methanothermobacter Marburgensis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hamann, N.; Mander, G.J.; Shokes, J.E.

    Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]{sup 3+} cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX{sub 31-39}CCX{sub 35-36}CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusualmore » [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g{sub zyx} = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. {sup 57}Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with {sup 57}Fe hyperfine couplings very similar to that of CoM-HDR. CoM-{sup 33}SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S{sub 3}(O/N){sub 1} geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to

  20. The pepper cysteine/histidine-rich DC1 domain protein CaDC1 binds both RNA and DNA and is required for plant cell death and defense response.

    PubMed

    Hwang, In Sun; Choi, Du Seok; Kim, Nak Hyun; Kim, Dae Sung; Hwang, Byung Kook

    2014-01-01

    Plant defense against microbial pathogens is coordinated by a complex regulatory network. Cysteine/histidine-rich DC1 domain proteins mediate a variety of cellular processes involved in plant growth, development and stress responses. We identified a pepper (Capsicum annuum) cysteine/histidine-rich DC1 domain protein gene, CaDC1, which positively regulates plant defense during microbial infection, based on gene silencing and transient expression in pepper, as well as ectopic expression in Arabidopsis. Induction of CaDC1 by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection was pronounced at both transcriptional and translational levels in pepper leaves. Purified CaDC1 protein bound to both DNA and RNA in vitro, especially in the presence of Zn(2+). CaDC1 was localized to both the nucleus and the cytoplasm, which was required for plant cell death signaling. The nuclear localization of CaDC1 was dependent on the divergent C1 (DC1) domain. CaDC1 silencing in pepper conferred increased susceptibility to Xcv infection, which was accompanied by reduced salicylic acid accumulation and defense-related gene expression. Ectopic expression of CaDC1 in Arabidopsis enhanced resistance to Hyaloperonospora arabidopsidis. CaDC1 binds both RNA and DNA and functions as a positive regulator of plant cell death and SA-dependent defense responses. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  1. Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra.

    PubMed

    Engel, Ulrike; Ozbek, Suat; Streitwolf-Engel, Ruth; Petri, Barbara; Lottspeich, Friedrich; Holstein, Thomas W; Oezbek, Suat; Engel, Ruth

    2002-10-15

    The novel protein Nowa was identified in nematocysts, explosive organelles of Hydra, jellyfish, corals and other CNIDARIA: Biogenesis of these organelles is complex and involves assembly of proteins inside a post-Golgi vesicle to form a double-layered capsule with a long tubule. Nowa is the major component of the outer wall, which is formed very early in morphogenesis. The high molecular weight glycoprotein has a modular structure with an N-terminal sperm coating glycoprotein domain, a central C-type lectin-like domain, and an eightfold repeated cysteine-rich domain at the C-terminus. Interestingly, the cysteine-rich domains are homologous to the cysteine-rich domains of minicollagens. We have previously shown that the cysteines of these minicollagen cysteine-rich domains undergo an isomerization process from intra- to intermolecular disulfide bonds, which mediates the crosslinking of minicollagens to networks in the inner wall of the capsule. The minicollagen cysteine-rich domains present in both proteins provide a potential link between Nowa in the outer wall and minicollagens in the inner wall. We propose a model for nematocyst formation that integrates cytoskeleton rearrangements around the post-Golgi vesicle and protein assembly inside the vesicle to generate a complex structure that is stabilized by intermolecular disulfide bonds.

  2. Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

    PubMed

    Fujishima, Kosuke; Wang, Kendrick M; Palmer, Jesse A; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J

    2018-01-29

    Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

  3. Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

    PubMed Central

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

    2013-01-01

    Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. PMID:23362256

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

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

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

  5. Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO

    PubMed Central

    Sun, Junhui; Xin, Chunlin; Eu, Jerry P.; Stamler, Jonathan S.; Meissner, Gerhard

    2001-01-01

    We have shown previously that at physiologically relevant oxygen tension (pO2 ≈ 10 mmHg), NO S-nitrosylates 1 of ≈50 free cysteines per ryanodine receptor 1 (RyR1) subunit and transduces a calcium-sensitizing effect on the channel by means of calmodulin (CaM). It has been suggested that cysteine-3635 is part of a CaM-binding domain, and its reactivity is attenuated by CaM [Porter Moore, C., Zhang, J. Z., Hamilton, S. L. (1999) J. Biol. Chem. 274, 36831–36834]. Therefore, we tested the hypothesis that the effect of NO was mediated by C3635. The full-length RyR1 single-site C3635A mutant was generated and expressed in HEK293 cells. The mutation resulted in the loss of CaM-dependent NO modulation of channel activity and reduced S-nitrosylation by NO to background levels but did not affect NO-independent channel modulation by CaM or the redox sensitivity of the channel to O2 and glutathione. Our results reveal that different cysteines within the channel have been adapted to serve in nitrosative and oxidative responses, and that S-nitrosylation of the cysteine-containing CaM-binding domain underlies the mechanism of CaM-dependent regulation of RyR1 by NO. PMID:11562475

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

    PubMed Central

    Loo, Shining; Kam, Antony; Xiao, Tianshu; Tam, James P.

    2017-01-01

    Cysteine-rich peptides (CRPs) play important host-defense roles in plants. However, information concerning CRPs in the Cactaceae (cactus) family is limited, with only a single cactus-derived CRP described to date. Here, we report the identification of 15 novel CRPs with three different precursor architectures, bleogens pB1-15 from Pereskia bleo of the Cactaceae family. By combining proteomic and transcriptomic methods, we showed that the prototype, bleogen pB1, contained 36 amino acid residues, a six-cysteine motif typical of the six-cysteine-hevein-like peptide (6C-HLP) family, and a type I two-domain precursor consisting of an endoplasmic reticulum (ER) and a mature domain. In contrast, the precursors of the other 14 bleogens contained a type II three-domain architecture with a propeptide domain inserted between the ER and the mature bleogen domain. Four of these 14 bleogens display a third type of architecture with a tandemly repeating bleogen domain. A search of the Onekp database revealed that <1% plant species possess three different precursor architectures for the biosynthesis of 6C-HLPs, including Lophophora williamsii, Pereskia aculeate, Portulaca cryptopetala, Portulaca oleracea, Portulaca suffruticosa, and Talinum sp. NMR analysis confirmed that bleogen pB1 has cystine-knot disulfide connectivity as well as a two-beta-sheet and a four-loop structural fold that is similar to other 6C-HLPs. Sequence analysis, structural studies, and in silico modeling revealed that bleogen pB1 has a cation-polar-cation motif, a signature heparin-binding motif that was confirmed by heparin affinity chromatography. Cell-based assays showed that bleogen pB1 is non-toxic to mammalian cells but functions as an anti-Candida peptide. Taken together, our findings provide insight into the occurrence, functions and precursor architectures of CRPs in the cactus family. PMID:29312404

  7. Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

    PubMed Central

    Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

    2015-01-01

    How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID

  8. Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

    PubMed

    Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

    2016-04-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations

  9. Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

    PubMed

    Kumari, Geeta; Serra, Aida; Shin, Joon; Nguyen, Phuong Q T; Sze, Siu Kwan; Yoon, Ho Sup; Tam, James P

    2015-11-25

    Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

  10. Expression, purification and characterisation of two variant cysteine peptidases from Trypanosoma congolense with active site substitutions.

    PubMed

    Pillay, Davita; Boulangé, Alain F; Coetzer, Theresa H T

    2010-12-01

    Congopain, the major cysteine peptidase of Trypanosoma congolense is an attractive candidate for an anti-disease vaccine and target for the design of specific inhibitors. A complicating factor for the inclusion of congopain in a vaccine is that multiple variants of congopain are present in the genome of the parasite. In order to determine whether the variant congopain-like genes code for peptidases with enzymatic activities different to those of congopain, two variants were cloned and expressed. Two truncated catalytic domain variants were recombinantly expressed in Pichia pastoris. The two expressed catalytic domain variants differed slightly from one another in substrate preferences and also from that of C2 (the recombinant truncated form of congopain). Surprisingly, a variant with the catalytic triad Ser(25), His(159) and Asn(175) was shown to be active against classical cysteine peptidase substrates and inhibited by E-64, a class-specific cysteine protease inhibitor. Both catalytic domain clones and C2 had pH optima of either 6.0 or 6.5 implying that these congopain-like proteases are likely to be expressed and active in the bloodstream of the host animal. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Oguri, Tamiko; Schneider, Barbara

    2012-01-01

    Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages. PMID:22685283

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

    PubMed Central

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

    2016-01-01

    The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice. PMID:24609271

  13. The CW domain, a structural module shared amongst vertebrates, vertebrate-infecting parasites and higher plants.

    PubMed

    Perry, Jason; Zhao, Yunde

    2003-11-01

    A previously undetected domain, named CW for its conserved cysteine and tryptophan residues, appears to be a four-cysteine zinc-finger motif found exclusively in vertebrates, vertebrate-infecting parasites and higher plants. Of the twelve distinct nuclear protein families that comprise the CW domain-containing superfamily, only the microrchida (MORC) family has begun to be characterized. However, several families contain other domains suggesting a relationship between the CW domain and either chromatin methylation status or early embryonic development.

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

    PubMed

    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.

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

    PubMed Central

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

    2017-01-01

    Phytoaccumulation is a technique to extract metals from soil utilising ability of plants. Cesium is a valuable metal while radioactive isotopes of cesium can be hazardous. In order to establish a more efficient phytoaccumulation system, small molecules which promote plants to accumulate cesium were investigated. Through chemical library screening, 14 chemicals were isolated as ‘cesium accumulators’ in Arabidopsis thaliana. Of those, methyl cysteinate, a derivative of cysteine, was found to function within the plant to accumulate externally supplemented cesium. Moreover, metabolite profiling demonstrated that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation. PMID:28230101

  16. Emergence of CD134 cysteine-rich domain 2 (CRD2)-independent strains of feline immunodeficiency virus (FIV) is associated with disease progression in naturally infected cats.

    PubMed

    Bęczkowski, Paweł M; Techakriengkrai, Navapon; Logan, Nicola; McMonagle, Elizabeth; Litster, Annette; Willett, Brian J; Hosie, Margaret J

    2014-11-28

    Feline immunodeficiency virus (FIV) infection is mediated by sequential interactions with CD134 and CXCR4. Field strains of virus vary in their dependence on cysteine-rich domain 2 (CRD2) of CD134 for infection. Here, we analyse the receptor usage of viral variants in the blood of 39 naturally infected cats, revealing that CRD2-dependent viral variants dominate in early infection, evolving towards CRD2-independence with disease progression. These findings are consistent with a shift in CRD2 of CD134 usage with disease progression.

  17. Quantification of the fluorine containing drug 5-fluorouracil in cancer cells by GaF molecular absorption via high-resolution continuum source molecular absorption spectrometry

    NASA Astrophysics Data System (ADS)

    Krüger, Magnus; Huang, Mao-Dong; Becker-Roß, Helmut; Florek, Stefan; Ott, Ingo; Gust, Ronald

    The development of high-resolution continuum source molecular absorption spectrometry made the quantification of fluorine feasible by measuring the molecular absorption as gallium monofluoride (GaF). Using this new technique, we developed on the example of 5-fluorouracil (5-FU) a graphite furnace method to quantify fluorine in organic molecules. The effect of 5-FU on the generation of the diatomic GaF molecule was investigated. The experimental conditions such as gallium nitrate amount, temperature program, interfering anions (represented as corresponding acids) and calibration for the determination of 5-FU in standard solution and in cellular matrix samples were investigated and optimized. The sample matrix showed no effect on the sensitivity of GaF molecular absorption. A simple calibration curve using an inorganic sodium fluoride solution can conveniently be used for the calibration. The described method is sensitive and the achievable limit of detection is 0.23 ng of 5-FU. In order to establish the concept of "fluorine as a probe in medicinal chemistry" an exemplary application was selected, in which the developed method was successfully demonstrated by performing cellular uptake studies of the 5-FU in human colon carcinoma cells.

  18. Importance of Conserved Cysteine Residues in the Coronavirus Envelope Protein▿

    PubMed Central

    Lopez, Lisa A.; Riffle, Ambere J.; Pike, Steven L.; Gardner, Douglas; Hogue, Brenda G.

    2008-01-01

    Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double- and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triple-mutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production. PMID:18184703

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

    PubMed

    Wible, Ryan S; Sutter, Thomas R

    2017-03-20

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

  20. Light-regulated synthesis of cyclic-di-GMP by a bidomain construct of the cyanobacteriochrome Tlr0924 (SesA) without stable dimerization

    DOE PAGES

    Blain-Hartung, Matthew D.; Rockwell, Nathan Clarke; Lagarias, J. Clark

    2017-10-26

    Here, phytochromes and cyanobacteriochromes (CBCRs) use double bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/Adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit much more diverse photocycles than phytochromes, and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multi-domain extension at its N-terminus. To probemore » the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, CD spectroscopy and size exclusion chromatography data do not support formation of stable dimers in the either the blue-absorbing 15ZP b dark state or the green-absorbing 15EP g photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. Based on these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker region to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytic competent dimers.« less

  1. Light-regulated synthesis of cyclic-di-GMP by a bidomain construct of the cyanobacteriochrome Tlr0924 (SesA) without stable dimerization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blain-Hartung, Matthew D.; Rockwell, Nathan Clarke; Lagarias, J. Clark

    Here, phytochromes and cyanobacteriochromes (CBCRs) use double bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/Adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit much more diverse photocycles than phytochromes, and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multi-domain extension at its N-terminus. To probemore » the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, CD spectroscopy and size exclusion chromatography data do not support formation of stable dimers in the either the blue-absorbing 15ZP b dark state or the green-absorbing 15EP g photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. Based on these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker region to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytic competent dimers.« less

  2. The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

    PubMed Central

    Elvers, Karen T.; Geoghegan, Ivey; Shoemark, Debbie K.; Lampasona, Vito; Bingley, Polly J.; Williams, Alistair J.K.

    2013-01-01

    Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions. PMID:22966073

  3. Fermentative Production of Cysteine by Pantoea ananatis

    PubMed Central

    Takumi, Kazuhiro; Ziyatdinov, Mikhail Kharisovich; Samsonov, Viktor

    2016-01-01

    ABSTRACT Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe. IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to

  4. Fermentative Production of Cysteine by Pantoea ananatis.

    PubMed

    Takumi, Kazuhiro; Ziyatdinov, Mikhail Kharisovich; Samsonov, Viktor; Nonaka, Gen

    2017-03-01

    Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis , an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis , which has fundamental implications for establishing overproduction in this microbe. IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis , an emerging host for the fermentative production of bio-based materials, to

  5. Effect of point mutations on Herbaspirillum seropedicae NifA activity.

    PubMed

    Aquino, B; Stefanello, A A; Oliveira, M A S; Pedrosa, F O; Souza, E M; Monteiro, R A; Chubatsu, L S

    2015-08-01

    NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

  6. Effect of point mutations on Herbaspirillum seropedicae NifA activity

    PubMed Central

    Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S.

    2015-01-01

    NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain. PMID:26176311

  7. Cysteine-Zn2+ complexes: unique molecular switches for inducible nitric oxide synthase-derived NO.

    PubMed

    Kröncke, K D

    2001-11-01

    Nitric oxide (NO) in the low nanomolar range acts as a transcellular messenger molecule to initiate regulatory and physiological responses in nearby target cells via binding to the soluble guanylate cyclase heme moiety. Higher NO concentrations, as synthesized by the inducible NO synthase (iNOS) during inflammatory processes, show additional effects: NO may react with O2, yielding nitrogen oxides like N2O3 that are able to nitrosate thiols. A variety of proteins involved in very different functions of the cell contain cysteine-Zn2+ complexes. Effects of NO on different proteins containing cysteine-Zn2+ domains and playing essential roles during transcription, protein folding, and proteolysis are discussed. It is suggested that iNOS-derived NO acts as a signal molecule targeting cysteine-Zn2+ linkages, thus enabling cells to react toward nitrosative stress.

  8. Probing N-methyl-D-aspartate receptor desensitization with the substituted-cysteine accessibility method.

    PubMed

    Thomas, Christopher G; Krupp, Johannes J; Bagley, Elena E; Bauzon, Reginald; Heinemann, Stephen F; Vissel, Bryce; Westbrook, Gary L

    2006-04-01

    Several forms of macroscopic N-methyl-D-aspartate (NMDA) receptor desensitization affect the amplitude and duration of postsynaptic responses. In addition to its functional significance, desensitization provides one means to examine the conformational coupling of ligand binding to channel gating. Segments flanking the ligand binding domain in the extracellular N terminus of the NMDA receptor NR2 subunit influence the glycine-independent form of desensitization. The NR2A pre-M1 region, the linker between the glutamate binding domain and the channel pore, plays a critical role in desensitization. Thus, we used the substituted-cysteine accessibility method to scan the accessibility of residues in the pre-M1 region and the first transmembrane domain (M1) of NR2A. Cysteine mutants were expressed with NR1 in human embryonic kidney 293 cells and were assayed by whole-cell recording. With activation of the receptor by glutamate and glycine, only a single mutant, V557C, which is located at the beginning of M1, led to irreversible inhibition by the methanethiosulfonate derivative methanethiosulfonate ethyltrimethylammonium (MTSET). The NR2 ligand glutamate was insufficient on its own to induce modification of V557C by MTSET, suggesting that the change in accessibility required channel gating. The rate of MTSET modification of the homologous residue on NR1 (NR1-1a(L562C)/NR2A) was much slower than V557C. We also substituted cysteine in the V557 site of mutant subunits that exhibit either enhanced or reduced desensitization. Modification by MTSET correlated with the degree of desensitization for these subunits, suggesting that V557C is a sensitive detector of desensitization gating.

  9. The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression.

    PubMed

    Kinoshita, Rie; Watanabe, Masami; Huang, Peng; Li, Shun-Ai; Sakaguchi, Masakiyo; Kumon, Hiromi; Futami, Junichiro

    2015-06-01

    Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3β (GSK-3β) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

  10. Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

    PubMed Central

    2012-01-01

    Background Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. Results Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. Conclusions In this work, we showed that Grx1 and

  11. Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

    PubMed

    Thompson, Joanne; Fernandez-Reyes, Delmiro; Sharling, Lisa; Moore, Sally G; Eling, Wijnand M; Kyes, Sue A; Newbold, Christopher I; Kafatos, Fotis C; Janse, Chris J; Waters, Andrew P

    2007-06-01

    The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

  12. CHPA, a Cysteine- and Histidine-Rich-Domain-Containing Protein, Contributes to Maintenance of the Diploid State in Aspergillus nidulans

    PubMed Central

    Sadanandom, Ari; Findlay, Kim; Doonan, John H.; Schulze-Lefert, Paul; Shirasu, Ken

    2004-01-01

    The alternation of eukaryotic life cycles between haploid and diploid phases is crucial for maintaining genetic diversity. In some organisms, the growth and development of haploid and diploid phases are nearly identical, and one might suppose that all genes required for one phase are likely to be critical for the other phase. Here, we show that targeted disruption of the chpA (cysteine- and histidine-rich-domain- [CHORD]-containing protein A) gene in haploid Aspergillus nidulans strains gives rise to chpA knockout haploids and heterozygous diploids but no chpA knockout diploids. A. nidulans chpA heterozygous diploids showed impaired conidiophore development and reduced conidiation. Deletion of chpA from diploid A. nidulans resulted in genome instability and reversion to a haploid state. Thus, our data suggest a vital role for chpA in maintenance of the diploid phase in A. nidulans. Furthermore, the human chpA homolog, Chp-1, was able to complement haploinsufficiency in A. nidulans chpA heterozygotes, suggesting that the function of CHORD-containing proteins is highly conserved in eukaryotes. PMID:15302831

  13. Targeting cysteine-mediated dimerization of the MUC1-C oncoprotein in human cancer cells

    PubMed Central

    RAINA, DEEPAK; AHMAD, REHAN; RAJABI, HASAN; PANCHAMOORTHY, GOVIND; KHARBANDA, SURENDER; KUFE, DONALD

    2012-01-01

    The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas and contributes to the malignant phenotype. The MUC1-C transmembrane subunit contains a CQC motif in the cytoplasmic domain that has been implicated in the formation of dimers and in its oncogenic function. The present study demonstrates that MUC1-C forms dimers in human breast and lung cancer cells. MUC1-C dimerization was detectable in the cytoplasm and was independent of MUC1-N, the N-terminal mucin subunit that extends outside the cell. We show that the MUC1-C cytoplasmic domain forms dimers in vitro that are disrupted by reducing agents. Moreover, dimerization of the MUC1-C subunit in cancer cells was blocked by reducing agents and increased by oxidative stress, supporting involvement of the CQC motif in forming disulfide bonds. In support of these observations, mutation of the MUC1-C CQC motif to AQA completely blocked MUC1-C dimerization. Importantly, this study was performed with MUC1-C devoid of fluorescent proteins, such as GFP, CFP and YFP. In this regard, we show that GFP, CFP and YFP themselves form dimers that are readily detectable with cross-linking agents. The present results further demonstrate that a cell-penetrating peptide that targets the MUC1-C CQC cysteines blocks MUC1-C dimerization in cancer cells. These findings provide definitive evidence that: i) the MUC1-C cytoplasmic domain cysteines are necessary and sufficient for MUC1-C dimerization, and ii) these CQC motif cysteines represent an Achilles’ heel for targeting MUC1-C function. PMID:22200620

  14. Phyletic Distribution and Lineage-Specific Domain Architectures of Archaeal Two-Component Signal Transduction Systems

    PubMed Central

    Makarova, Kira S.; Wolf, Yuri I.

    2017-01-01

    ABSTRACT The two-component signal transduction (TCS) machinery is a key mechanism of sensing environmental changes in the prokaryotic world. TCS systems have been characterized thoroughly in bacteria but to a much lesser extent in archaea. Here, we provide an updated census of more than 2,000 histidine kinases and response regulators encoded in 218 complete archaeal genomes, as well as unfinished genomes available from metagenomic data. We describe the domain architectures of the archaeal TCS components, including several novel output domains, and discuss the evolution of the archaeal TCS machinery. The distribution of TCS systems in archaea is strongly biased, with high levels of abundance in haloarchaea and thaumarchaea but none detected in the sequenced genomes from the phyla Crenarchaeota, Nanoarchaeota, and Korarchaeota. The archaeal sensor histidine kinases are generally similar to their well-studied bacterial counterparts but are often located in the cytoplasm and carry multiple PAS and/or GAF domains. In contrast, archaeal response regulators differ dramatically from the bacterial ones. Most archaeal genomes do not encode any of the major classes of bacterial response regulators, such as the DNA-binding transcriptional regulators of the OmpR/PhoB, NarL/FixJ, NtrC, AgrA/LytR, and ActR/PrrA families and the response regulators with GGDEF and/or EAL output domains. Instead, archaea encode multiple copies of response regulators containing either the stand-alone receiver (REC) domain or combinations of REC with PAS and/or GAF domains. Therefore, the prevailing mechanism of archaeal TCS signaling appears to be via a variety of protein-protein interactions, rather than direct transcriptional regulation. IMPORTANCE Although the Archaea represent a separate domain of life, their signaling systems have been assumed to be closely similar to the bacterial ones. A study of the domain architectures of the archaeal two-component signal transduction (TCS) machinery

  15. L-Cysteine Metabolism and Fermentation in Microorganisms.

    PubMed

    Takagi, Hiroshi; Ohtsu, Iwao

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

  16. SU-E-T-96: Energy Dependence of the New GafChromic- EBT3 Film's Dose Response-Curve.

    PubMed

    Chiu-Tsao, S; Massillon-Jl, G; Domingo-Muñoz, I; Chan, M

    2012-06-01

    To study and compare the dose response curves of the new GafChromic EBT3 film for megavoltage and kilovoltage x-ray beams, with different spatial resolution. Two sets of EBT3 films (lot#A101711-02) were exposed to each x-ray beam (6MV, 15MV and 50kV) at 8 dose values (50-3200cGy). The megavoltage beams were calibrated per AAPM TG-51 protocol while the kilovoltage beam was calibrated following the TG-61 using an ionization chamber calibrated at NIST. Each film piece was scanned three consecutive times in the center of Epson 10000XL flatbed scanner in transmission mode, landscape orientation, 48-bit color at two separate spatial resolutions of 75 and 300 dpi. The data were analyzed using ImageJ and, for each scanned image, a region of interest (ROI) of 2×2cm 2 at the field center was selected to obtain the mean pixel value with its standard deviation in the ROI. For each energy, dose value and spatial resolution, the average netOD and its associated uncertainty were determined. The Student's t-test was performed to evaluate the statistical differences between the netOD/dose values of the three energy modalities, with different color channels and spatial resolutions. The dose response curves for the three energy modalities were compared in three color channels with 75 and 300dpi. Weak energy dependence was found. For doses above 100cGy, no statistical differences were observed between 6 and 15MV beams, regardless of spatial resolution. However, statistical differences were observed between 50kV and the megavoltage beams. The degree of energy dependence (from MV to 50kV) was found to be function of color channel, dose level and spatial resolution. The dose response curves for GafChromic EBT3 films were found to be weakly dependent on the energy of the photon beams from 6MV to 50kV. The degree of energy dependence varies with color channel, dose and spatial resolution. GafChromic EBT3 films were supplied by Ashland Corp. This work was partially supported by DGAPA

  17. Cysteine Cathepsins in Human Carious Dentin

    PubMed Central

    Nascimento, F.D.; Minciotti, C.L.; Geraldeli, S.; Carrilho, M.R.; Pashley, D.H.; Tay, F.R.; Nader, H.B.; Salo, T.; Tjäderhane, L.; Tersariol, I.L.S.

    2011-01-01

    Matrix metalloproteinases (MMPs) are important in dentinal caries, and analysis of recent data demonstrates the presence of other collagen-degrading enzymes, cysteine cathepsins, in human dentin. This study aimed to examine the presence, source, and activity of cysteine cathepsins in human caries. Cathepsin B was detected with immunostaining. Saliva and dentin cysteine cathepsin and MMP activities on caries lesions were analyzed spectrofluorometrically. Immunostaining demonstrated stronger cathepsins B in carious than in healthy dentin. In carious dentin, cysteine cathepsin activity increased with increasing depth and age in chronic lesions, but decreased with age in active lesions. MMP activity decreased with age in both active and chronic lesions. Salivary MMP activities were higher in patients with active than chronic lesions and with increasing lesion depth, while cysteine cathepsin activities showed no differences. The results indicate that, along with MMPs, cysteine cathepsins are important, especially in active and deep caries. PMID:21248362

  18. Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

    PubMed

    Resnick, D; Chatterton, J E; Schwartz, K; Slayter, H; Krieger, M

    1996-10-25

    Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

  19. Cysteine sensing by plasmons of silver nanocubes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Elfassy, Eitan, E-mail: eitan.elfassi@gmail.com; Mastai, Yitzhak, E-mail: Yitzhak.Mastai@biu.ac.il; Salomon, Adi, E-mail: adi.salomon@biu.ac.il

    2016-09-15

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

  20. Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.

    PubMed

    Fernandez, Francisco J; de Vries, Dominique; Peña-Soler, Esther; Coll, Miquel; Christen, Philipp; Gehring, Heinz; Vega, M Cristina

    2012-02-01

    The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. On the Dynamical Behavior of the Cysteine Dioxygenase-l-Cysteine Complex in the Presence of Free Dioxygen and l-Cysteine.

    PubMed

    Pietra, Francesco

    2017-11-01

    In this work, viable models of cysteine dioxygenase (CDO) and its complex with l-cysteine dianion were built for the first time, under strict adherence to the crystal structure from X-ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O 2 ) and l-cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O 2 ), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l-cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

  2. π-Clamp-mediated cysteine conjugation

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  3. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

    USDA-ARS?s Scientific Manuscript database

    LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

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

    PubMed

    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.

  5. L-Cysteine metabolism and its nutritional implications.

    PubMed

    Yin, Jie; Ren, Wenkai; Yang, Guan; Duan, Jielin; Huang, Xingguo; Fang, Rejun; Li, Chongyong; Li, Tiejun; Yin, Yulong; Hou, Yongqing; Kim, Sung Woo; Wu, Guoyao

    2016-01-01

    L-Cysteine is a nutritionally semiessential amino acid and is present mainly in the form of L-cystine in the extracellular space. With the help of a transport system, extracellular L-cystine crosses the plasma membrane and is reduced to L-cysteine within cells by thioredoxin and reduced glutathione (GSH). Intracellular L-cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for production of GSH, hydrogen sulfide (H(2)S), and taurine. L-Cysteine-dependent synthesis of GSH has been investigated in many pathological conditions, while the pathway for L-cysteine metabolism to form H(2)S has received little attention with regard to prevention and treatment of disease in humans. The main objective of this review is to highlight the metabolic pathways of L-cysteine catabolism to GSH, H(2)S, and taurine, with special emphasis on therapeutic and nutritional use of L-cysteine to improve the health and well-being of animals and humans. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A spectroscopic study of the chromatic properties of GafChromicEBT3 films.

    PubMed

    Callens, M; Crijns, W; Simons, V; De Wolf, I; Depuydt, T; Maes, F; Haustermans, K; D'hooge, J; D'Agostino, E; Wevers, M; Pfeiffer, H; Van Den Abeele, K

    2016-03-01

    This work provides an interpretation of the chromatic properties of GafChromicEBT3 films based on the chemical nature of the polydiacetylene (PDA) molecules formed upon interaction with ionizing radiation. The EBT3 films become optically less transparent with increasing radiation dose as a result of the radiation-induced polymerization of diacetylene monomers. In contrast to empirical quantification of the chromatic properties, less attention has been given to the underlying molecular mechanism that induces the strong decrease in transparency. Unlaminated GafChromicEBT3 films were irradiated with a 6 MV photon beam to dose levels up to 20 Gy. The optical absorption properties of the films were investigated using visible (vis) spectroscopy. The presence of PDA molecules in the active layer of the EBT3 films was investigated using Raman spectroscopy, which probes the vibrational modes of the molecules in the layer. The vibrational modes assigned to PDA's were used in a theoretical vis-absorption model to fit our experimental vis-absorption spectra. From the fit parameters, one can assess the relative contribution of different PDA conformations and the length distribution of PDA's in the film. Vis-spectroscopy shows that the optical density increases with dose in the full region of the visible spectrum. The Raman spectrum is dominated by two vibrational modes, most notably by the ν(C≡C) and the ν(C=C) stretching modes of the PDA backbone. By fitting the vis-absorption model to experimental spectra, it is found that the active layer contains two distinct PDA conformations with different absorption properties and reaction kinetics. Furthermore, the mean PDA conjugation length is found to be 2-3 orders of magnitude smaller than the crystals PDA's are embedded in. Vis- and Raman spectroscopy provided more insight into the molecular nature of the radiochromic properties of EBT3 films through the identification of the excited states of PDA and the presence of two PDA

  7. Differential Roles of Cysteine Residues in Cellular Trafficking, Dimerization, and Function of the HDL Receptor, SR-BI *

    PubMed Central

    Hu, Jie; Zhang, Zhonghua; Shen, Wen-Jun; Nomoto, Ann; Azhar, Salman

    2011-01-01

    The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking and SR-BI dimerization. Pretreatment of SR-BI overexpressing COS-7 cells with disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM) modestly, but significantly impaired the SR-BI mediated selective HDL-CE uptake. Treatment of SR-BI overexpressing COS-7 cells with the optimum doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS that specifically react with the free sulfhydryl group of cysteine reduced the SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine and the mutant SR-BIs expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S or C334S of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective-CE uptake, and trafficking to cell surface. Surprisingly, we found that

  8. Blends of cysteine-containing proteins

    NASA Astrophysics Data System (ADS)

    Barone, Justin

    2005-03-01

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

  9. Phyletic Distribution and Lineage-Specific Domain Architectures of Archaeal Two-Component Signal Transduction Systems.

    PubMed

    Galperin, Michael Y; Makarova, Kira S; Wolf, Yuri I; Koonin, Eugene V

    2018-04-01

    The two-component signal transduction (TCS) machinery is a key mechanism of sensing environmental changes in the prokaryotic world. TCS systems have been characterized thoroughly in bacteria but to a much lesser extent in archaea. Here, we provide an updated census of more than 2,000 histidine kinases and response regulators encoded in 218 complete archaeal genomes, as well as unfinished genomes available from metagenomic data. We describe the domain architectures of the archaeal TCS components, including several novel output domains, and discuss the evolution of the archaeal TCS machinery. The distribution of TCS systems in archaea is strongly biased, with high levels of abundance in haloarchaea and thaumarchaea but none detected in the sequenced genomes from the phyla Crenarchaeota , Nanoarchaeota , and Korarchaeota The archaeal sensor histidine kinases are generally similar to their well-studied bacterial counterparts but are often located in the cytoplasm and carry multiple PAS and/or GAF domains. In contrast, archaeal response regulators differ dramatically from the bacterial ones. Most archaeal genomes do not encode any of the major classes of bacterial response regulators, such as the DNA-binding transcriptional regulators of the OmpR/PhoB, NarL/FixJ, NtrC, AgrA/LytR, and ActR/PrrA families and the response regulators with GGDEF and/or EAL output domains. Instead, archaea encode multiple copies of response regulators containing either the stand-alone receiver (REC) domain or combinations of REC with PAS and/or GAF domains. Therefore, the prevailing mechanism of archaeal TCS signaling appears to be via a variety of protein-protein interactions, rather than direct transcriptional regulation. IMPORTANCE Although the Archaea represent a separate domain of life, their signaling systems have been assumed to be closely similar to the bacterial ones. A study of the domain architectures of the archaeal two-component signal transduction (TCS) machinery revealed an

  10. Evaluation of cysteine proteases of Plasmodium vivax as antimalarial drug targets: sequence analysis and sensitivity to cysteine protease inhibitors.

    PubMed

    Na, Byoung-Kuk; Kim, Tong-Soo; Rosenthal, Philip J; Lee, Jong-Koo; Kong, Yoon

    2004-10-01

    Cysteine proteases perform critical roles in the life cycles of malaria parasites. In Plasmodium falciparum, treatment of cysteine protease inhibitors inhibits hemoglobin hydrolysis and blocks the parasite development in vitro and in vivo, suggesting that plasmodial cysteine proteases may be interesting targets for new chemotherapeutics. To determine whether sequence diversity may limit chemotherapy against Plasmodium vivax, we analyzed sequence variations in the genes encoding three cysteine proteases, vivapain-1, -2 and -3, in 22 wild isolates of P. vivax. The sequences were highly conserved among wild isolates. A small number of substitutions leading to amino acid changes were found, while they did not modify essential residues for the function or structure of the enzymes. The substrate specificities and sensitivities to synthetic cysteine protease inhibitors of vivapain-2 and -3 from wild isolates were also very similar. These results support the suggestion that cysteine proteases of P. vivax are promising antimalarial chemotherapeutic targets.

  11. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    PubMed

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

    2011-06-01

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

  13. Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo

    2014-01-01

    ABSTRACT l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled l-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of l-cysteine in E. histolytica. [U-13C3, 15N]l-cysteine was rapidly metabolized into three unknown metabolites, besides l-cystine and l-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of l-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of l-cysteine. Liberation of l-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these l-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress. PMID:25370494

  14. Electrons initiate efficient formation of hydroperoxides from cysteine.

    PubMed

    Gebicki, Janusz M

    2016-09-01

    Amino acid and protein hydroperoxides can constitute a significant hazard if formed in vivo. It has been suggested that cysteine can form hydroperoxides after intramolecular hydrogen transfer to the commonly produced cysteine sulfur-centered radical. The resultant cysteine-derived carbon-centered radicals can react with oxygen at almost diffusion-controlled rate, forming peroxyl radicals which can oxidize other molecules and be reduced to hydroperoxides in the process. No cysteine hydroperoxides have been found so far. In this study, dilute air-saturated cysteine solutions were exposed to radicals generated by ionizing radiation and the hydroperoxides measured by an iodide assay. Of the three primary radicals present, the hydroxyl, hydrogen atoms and hydrated electrons, the first two were ineffective. However, electrons did initiate the generation of hydroperoxides by removing the -SH group and forming cysteine-derived carbon radicals. Under optimal conditions, 100% of the electrons reacting with cysteine produced the hydroperoxides with a 1:1 stoichiometry. Maximum hydroperoxide yields were at pH 5.5, with fairly rapid decline under more acid or alkaline conditions. The hydroperoxides were stable between pH 3 and 7.5, and decomposed in alkaline solutions. The results suggest that formation of cysteine hydroperoxides initiated by electrons is an unlikely event under physiological conditions.

  15. Murein Lytic Enzyme TgaA of Bifidobacterium bifidum MIMBb75 Modulates Dendritic Cell Maturation through Its Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase (CHAP) Amidase Domain

    PubMed Central

    Zanoni, Ivan; Balzaretti, Silvia; Miriani, Matteo; Taverniti, Valentina; De Noni, Ivano; Presti, Ilaria; Stuknyte, Milda; Scarafoni, Alessio; Arioli, Stefania; Iametti, Stefania; Bonomi, Francesco; Mora, Diego; Karp, Matti; Granucci, Francesca

    2014-01-01

    Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system. PMID:24814791

  16. The global cysteine peptidase landscape in parasites

    PubMed Central

    Atkinson, Holly J; Babbitt, Patricia C; Sajid, Mohammed

    2013-01-01

    The accumulation of sequenced genomes has expanded the already sizeable population of cysteine peptidases from parasites. Characterization of a few of these enzymes has ascribed key roles to peptidases in parasite life cycles and also shed light on mechanisms of pathogenesis. Here, we discuss recent observations on the physiological activities of cysteine peptidases of parasitic organisms, paired with a global view of all cysteine peptidases from the MEROPS database grouped by similarity. This snapshot of the landscape of parasite cysteine peptidases is complex and highly populated, which suggests that expansion of research beyond the few ‘model’ parasite peptidases is now timely. PMID:19854678

  17. Neutron response of GafChromic® EBT2 film

    NASA Astrophysics Data System (ADS)

    Hsiao, Ming-Chen; Liu, Yuan-Hao; Chen, Wei-Lin; Jiang, Shiang-Huei

    2013-03-01

    Neutron and gamma-ray mixed field dosimetry remains one of the most challenging topics in radiation dosimetry studies. However, the requirement for accurate mixed field dosimetry is increasing because of the considerable interest in high-energy radiotherapy machines, medical ion beams and BNCT epithermal neutron beams. Therefore, this study investigated the GafChromic® EBT2 film. The linearity, reproducibility, energy dependence and homogeneity of the film were tested in a 60Co medical beam, 6-MV LINAC and 10-MV LINAC. The linearity and self-developing effect of the film irradiated in an epithermal neutron beam were also examined. These basic detector characteristics showed that EBT2 film can be effectively applied in mixed field dosimetry. A general detector response model was developed to determine the neutron relative effectiveness (RE) values. The RE value of fast neutrons varies with neutron spectra. By contrast, the RE value of thermal neutrons was determined as a constant; it is only 32.5% in relation to gamma rays. No synergy effect was observed in this study. The lithium-6 capture reaction dominates the neutron response in the thermal neutron energy range, and the recoil hydrogen dose becomes the dominant component in the fast neutron energy region. Based on this study, the application of the EBT2 film in the neutron and gamma-ray mixed field is feasible.

  18. Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity

    PubMed Central

    Anderson, Mark T.; Mitchell, Lindsay A.

    2017-01-01

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

  19. Mass spectrometric analysis of L-cysteine metabolism: physiological role and fate of L-cysteine in the enteric protozoan parasite Entamoeba histolytica.

    PubMed

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo; Nozaki, Tomoyoshi

    2014-11-04

    L-cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, L-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, L-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled L-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of L-cysteine in E. histolytica. [U-(13)C3, (15)N]L-cysteine was rapidly metabolized into three unknown metabolites, besides L-cystine and L-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of L-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of L-cysteine. Liberation of L-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these L-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress. Amebiasis is a human parasitic disease caused by the protozoan parasite Entamoeba histolytica. In this parasite, L-cysteine is the principal low-molecular-weight thiol and is assumed to play a significant role in supplying the amino acid during trophozoite invasion, particularly when the parasites move from the anaerobic intestinal lumen to highly

  20. The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source.

    PubMed

    Santiago, Margarita; Gardner, Richard C

    2015-07-01

    Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full-length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l-cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l-cystine and some other cysteine conjugates, but not l-cystathionine or l-methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l-cysteine as a nitrogen source, and that overexpression of the gene results in increased H2 S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S-ethyl-l-cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway

    PubMed Central

    Yamamoto, Katsuyoshi

    2015-01-01

    To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor. PMID:26598606

  2. Probes of the catalytic site of cysteine dioxygenase.

    PubMed

    Chai, Sergio C; Bruyere, John R; Maroney, Michael J

    2006-06-09

    The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.

  3. Comparisons of l-cysteine and d-cysteine toxicity in 4-week repeated-dose toxicity studies of rats receiving daily oral administration.

    PubMed

    Shibui, Yusuke; Sakai, Ryosei; Manabe, Yasuhiro; Masuyama, Takeshi

    2017-07-01

    Two 4-week repeated-dose toxicity studies were conducted to evaluate the potential toxicity of l-cysteine and d-cysteine. In one study, three groups of 6 male rats were each administered l-cysteine once daily by gavage at doses of 500, 1,000, or 2,000 mg/kg/day for 28 consecutive days. The control group was administered a 0.5% methylcellulose vehicle solution. The other study followed a similar protocol except that the experimental groups received d-cysteine. Toxicological observations showed that the l-cysteine-treated groups exhibited renal injuries such as basophilic tubules with eosinophilic material in the lumen, and there were increased numbers of basophilic tubules in all treated groups. In 1,000 or 2,000 mg/kg/day-treated groups, salivation and necropsy findings indicative of focal erosion in the stomach mucosa were found. Increases in reticulocyte counts were observed in the 2,000 mg/kg/day-treated group. Toxicological findings obtained for the d-cysteine-treated groups included anemia and renal injuries such as basophilic tubules with eosinophilic material in the lumen, increased numbers of basophilic tubules, and crystal deposition in the medulla in the 2,000 mg/kg/day-treated group. Additional findings included sperm granuloma in the epididymis, necropsy findings suggestive of focal erosion in the stomach mucosa, and salivation in the 1,000 or 2,000 mg/kg/day-treated groups. One rat in the 2,000 mg/kg/day-treated group died due to renal failure. In conclusion, the no-observed-adverse-effect levels (NOAELs) were estimated to be less than 500 mg/kg/day for l-cysteine and 500 mg/kg/day for d-cysteine under our study conditions. The toxicological profiles were similar for l-cysteine and d-cysteine; however, there were slight differences in the dose responses. The mechanisms underlying these differences remain to be determined.

  4. Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

    PubMed Central

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

    2014-01-01

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

  5. Cysteine analogues potentiate glucose-induced insulin release in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ammon, H.P.; Hehl, K.H.; Enz, G.

    1986-12-01

    In rat pancreatic islets, cysteine analogues, including glutathione, acetylcysteine, cysteamine, D-penicillamine, L-cysteine ethyl ester, and cysteine-potentiated glucose (11.1 mM) induced insulin secretion in a concentration-dependent manner. Their maximal effects were similar and occurred at approximately 0.05, 0.05, 0.1, 0.5, 1.0, 1.0 mM, respectively. At substimulatory glucose levels (2.8 mM), insulin release was not affected by these compounds. In contrast, thiol compounds, structurally different from cysteine and its analogues, such as mesna, tiopronin, meso-2,3-dimercaptosuccinic acid (DMSA), dimercaprol (BAL), beta-thio-D-glucose, as well as those cysteine analogues that lack a free-thiol group, including L-cystine, cystamine, D-penicillamine disulfide, S-carbocysteine, and S-carbamoyl-L-cysteine, did not enhancemore » insulin release at stimulatory glucose levels (11.1 mM); cystine (5 mM) was inhibitory. These in vitro data indicate that among the thiols tested here, only cysteine and its analogues potentiate glucose-induced insulin secretion, whereas thiols that are structurally not related to cysteine do not. This suggests that a cysteine moiety in the molecule is necessary for the insulinotropic effect. For their synergistic action to glucose, the availability of a sulfhydryl group is also a prerequisite. The maximal synergistic action is similar for all cysteine analogues tested, whereas the potency of action is different, suggesting similarity in the mechanism of action but differences in the affinity to the secretory system.« less

  6. Intermediate closed state for glycine receptor function revealed by cysteine cross-linking.

    PubMed

    Prevost, Marie S; Moraga-Cid, Gustavo; Van Renterghem, Catherine; Edelstein, Stuart J; Changeux, Jean-Pierre; Corringer, Pierre-Jean

    2013-10-15

    Pentameric ligand-gated ion channels (pLGICs) mediate signal transmission by coupling the binding of extracellular ligands to the opening of their ion channel. Agonist binding elicits activation and desensitization of pLGICs, through several conformational states, that are, thus far, incompletely characterized at the structural level. We previously reported for GLIC, a prokaryotic pLGIC, that cross-linking of a pair of cysteines at both sides of the extracellular and transmembrane domain interface stabilizes a locally closed (LC) X-ray structure. Here, we introduced the homologous pair of cysteines on the human α1 glycine receptor. We show by electrophysiology that cysteine cross-linking produces a gain-of-function phenotype characterized by concomitant constitutive openings, increased agonist potency, and equalization of efficacies of full and partial agonists. However, it also produces a reduction of maximal currents at saturating agonist concentrations without change of the unitary channel conductance, an effect reversed by the positive allosteric modulator propofol. The cross-linking thus favors a unique closed state distinct from the resting and longest-lived desensitized states. Fitting the data according to a three-state allosteric model suggests that it could correspond to a LC conformation. Its plausible assignment to a gating intermediate or a fast-desensitized state is discussed. Overall, our data show that relative movement of two loops at the extracellular-transmembrane interface accompanies orthosteric agonist-mediated gating.

  7. Browning inhibition mechanisms by cysteine, ascorbic acid and citric acid, and identifying PPO-catechol-cysteine reaction products.

    PubMed

    Ali, Hussein M; El-Gizawy, Ahmed M; El-Bassiouny, Rawia E I; Saleh, Mahmoud A

    2015-06-01

    The titled compounds were examined as PPO inhibitors and antibrowning agents; their various mechanisms were investigated and discussed. All compounds reduced significantly both the browning process and PPO activity. Browning index gave strong correlation with PPO activity (r(2) = 0.96, n = 19) indicating that the browning process is mainly enzymatic. Ascorbic acid could reduce the formed quinone instantly to the original substrate (catechol) at high concentration (>1.5 %) while at lower concentrations acted as competitive inhibitor (KI = 0.256 ± 0.067 mM). Cysteine, at higher concentrations (≥1.0 %), reacted with the resulted quinone to give a colorless products while at the low concentrations, cysteine worked as competitive inhibitor (KI = 1.113 ± 0.176 mM). Citric acid acted only as PPO non-competitive inhibitor with KI = 2.074 ± 0.363 mM. The products of PPO-catechole-cysteine reaction could be separation and identification by LC-ESI-MS. Results indicated that the product of the enzymatic oxidation of catechol, quinone, undergoes two successive nucleophilic attacks by cysteine thiol group. Cysteine was condensed with the resulted mono and dithiocatechols to form peptide side chains.

  8. Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

    PubMed

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

    2017-08-15

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

  9. Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.

    PubMed

    Mehrotra, Sonali; B Ningappa, Mylarappa; Raman, Jayalakshmi; Anand, Ranjith P; Balaram, Hemalatha

    2012-04-01

    Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Effect of (L)-cysteine on acetaldehyde self-administration.

    PubMed

    Peana, Alessandra T; Muggironi, Giulia; Fois, Giulia R; Zinellu, Manuel; Sirca, Donatella; Diana, Marco

    2012-08-01

    Acetaldehyde (ACD), the first metabolite of ethanol, has been implicated in several behavioural actions of alcohol, including its reinforcing effects. Recently, we reported that l-cysteine, a sequestrating agent of ACD, reduced oral ethanol self-administration and that ACD was orally self-administered. This study examined the effects of l-cysteine pre-treatment during the acquisition and maintenance phases of ACD (0.2%) self-administration as well as on the deprivation effect after ACD extinction and on a progressive ratio (PR) schedule of reinforcement. In a separate PR schedule of reinforcement, the effect of l-cysteine was assessed on the break-point produced by ethanol (10%). Furthermore, we tested the effect of l-cysteine on saccharin (0.2%) reinforcement. Wistar rats were trained to self-administer ACD by nose poking on a fixed ratio (FR1) schedule in 30-min daily sessions. Responses on an active nose-poke caused delivery of ACD solution, whereas responses on an inactive nose-poke had no consequences. l-cysteine reduced the acquisition (40 mg/kg), the maintenance and the deprivation effect (100 mg/kg) of ACD self-administration. Furthermore, at the same dose, l-cysteine (120 mg/kg) decreased both ACD and ethanol break point. In addition, l-cysteine was unable to suppress the different responses for saccharin, suggesting that its effect did not relate to an unspecific decrease in a general motivational state. Compared to saline, l-cysteine did not modify responses on inactive nose-pokes, suggesting an absence of a non-specific behavioural activation. Taken together, these results could support the hypotheses that ACD possesses reinforcing properties and l-cysteine reduces motivation to self-administer ACD. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2013-01-01

    The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity. PMID:23417561

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

    PubMed Central

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

    2016-01-01

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

  13. Systematic Review of Cysteine-Sparing NOTCH3 Missense Mutations in Patients with Clinical Suspicion of CADASIL.

    PubMed

    Muiño, Elena; Gallego-Fabrega, Cristina; Cullell, Natalia; Carrera, Caty; Torres, Nuria; Krupinski, Jurek; Roquer, Jaume; Montaner, Joan; Fernández-Cadenas, Israel

    2017-09-13

    CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by mutations in the NOTCH3 gene, affecting the number of cysteines in the extracellular domain of the receptor, causing protein misfolding and receptor aggregation. The pathogenic role of cysteine-sparing NOTCH3 missense mutations in patients with typical clinical CADASIL syndrome is unknown. The aim of this article is to describe these mutations to clarify if any could be potentially pathogenic. Articles on cysteine-sparing NOTCH3 missense mutations in patients with clinical suspicion of CADASIL were reviewed. Mutations were considered potentially pathogenic if patients had: (a) typical clinical CADASIL syndrome; (b) diffuse white matter hyperintensities; (c) the 33 NOTCH3 exons analyzed; (d) mutations that were not polymorphisms; and (e) Granular osmiophilic material (GOM) deposits in the skin biopsy. Twenty-five different mutations were listed. Four fulfill the above criteria: p.R61W; p.R75P; p.D80G; and p.R213K. Patients carrying these mutations had typical clinical CADASIL syndrome and diffuse white matter hyperintensities, mostly without anterior temporal pole involvement. Cysteine-sparing NOTCH3 missense mutations are associated with typical clinical CADASIL syndrome and typical magnetic resonance imaging (MRI) findings, although with less involvement of the anterior temporal lobe. Hence, these mutations should be further studied to confirm their pathological role in CADASIL.

  14. Src binds cortactin through an SH2 domain cystine-mediated linkage.

    PubMed

    Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

    2012-12-15

    Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

  15. Src binds cortactin through an SH2 domain cystine-mediated linkage

    PubMed Central

    Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

    2012-01-01

    Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

  16. CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

  17. Modification of Keap1 Cysteine Residues by Sulforaphane

    PubMed Central

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

    2011-01-01

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

  18. Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment.

    PubMed

    Elias, Camila G R; Pereira, Fernanda M; Dias, Felipe A; Silva, Thiago L A; Lopes, Angela H C S; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

    2008-12-01

    We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.

  19. Cysteine-Accelerated Methanogenic Propionate Degradation in Paddy Soil Enrichment.

    PubMed

    Zhuang, Li; Ma, Jinlian; Tang, Jia; Tang, Ziyang; Zhou, Shungui

    2017-05-01

    Propionate degradation is a critical step during the conversion of complex organic matter under methanogenic conditions, and it requires a syntrophic cooperation between propionate-oxidizing bacteria and methanogenic archaea. Increasing evidences suggest that interspecies electron transfer for syntrophic metabolism is not limited to the reducing equivalents of hydrogen and formate. This study tested the ability of an electron shuttle to mediate interspecies electron transfer in syntrophic methanogenesis. We found that cysteine supplementation (100, 400, and 800 μM) accelerated CH 4 production from propionate in paddy soil enrichments. Of the concentrations tested, 100 μM cysteine was the most effective at enhancing propionate degradation to CH 4 , and the rates of CH 4 production and propionate degradation were increased by 109 and 79%, respectively, compared with the cysteine-free control incubations. We eliminated the possibility that the stimulatory effect of cysteine on methanogenesis was attributable to the function of cysteine as a methanogenic substrate in the presence of propionate. The potential catalytic effect involved cysteine serving as an electron carrier to mediate interspecies electron transfer in syntrophic propionate oxidization. The redox potential of cystine/cysteine, which is dependent on the concentration, might be more suitable to facilitate interspecies electron transfer between syntrophic partners at a concentration of 100 μM. Pelotomaculum, obligately syntrophic, propionate-oxidizing bacteria, and hydrogenotrophic methanogens of the family Methanobacteriaceae are predominant in cysteine-mediated methanogenic propionate degradation. The stimulatory effect of cysteine on syntrophic methanogenesis offers remarkable potential for improving the performance of anaerobic digestion and conceptually broaden strategies for interspecies electron transfer in syntrophic metabolism.

  20. A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines.

    PubMed

    Seal, R P; Leighton, B H; Amara, S G

    2000-03-01

    Excitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.

  1. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3-mercaptopropanoic acid (C3H7O2NS). (b) The ingredient meets the appropriate part of the specification set forth in...

  2. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3-mercaptopropanoic acid (C3H7O2NS). (b) The ingredient meets the appropriate part of the specification set forth in...

  3. Beyond the global assessment of functioning: learning from Virginia Apgar.

    PubMed

    Dimsdale, Joel E; Jeste, Dilip V; Patterson, Thomas L

    2010-01-01

    The Global Assessment of Functioning (GAF) scale is widely used in psychiatry, yet it has certain drawbacks. The authors seek to generate further discussion and research around developing an improved successor to the GAF. The authors used the Apgar scale as a template for constructing a possible successor to the GAF. Consulting with 16 colleagues, they selected 5 domains that were felt to be central to functioning in psychiatric patients. Psychiatrists in diverse clinical settings then completed both a GAF and a Psychiatric Apgar scale on 40 patients. The two scales were found to agree significantly. Use of the Psychiatric Apgar, however, provides clearer guidance about assessing functioning. The GAF was a brilliant addition to psychiatric practice. As we develop the next Diagnostic and Statistical Manual, it is pertinent to ask whether the GAF approach could be optimized even further by applying the lessons of Virginia Apgar.

  4. Differential Evolution and Neofunctionalization of Snake Venom Metalloprotease Domains*

    PubMed Central

    Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A.B.; Vetter, Irina; Yang, Daryl C.; Casewell, Nicholas R.; Jackson, Timothy N. W.; Koludarov, Ivan; Alewood, Paul F.; Hodgson, Wayne C.; Lewis, Richard J.; King, Glenn F.; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G.

    2013-01-01

    Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands. PMID:23242553

  5. Differential evolution and neofunctionalization of snake venom metalloprotease domains.

    PubMed

    Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A B; Vetter, Irina; Yang, Daryl C; Yang, Dary C; Casewell, Nicholas R; Jackson, Timothy N W; Koludarov, Ivan; Alewood, Paul F; Hodgson, Wayne C; Lewis, Richard J; King, Glenn F; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G

    2013-03-01

    Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.

  6. Cysteine Scanning Mutagenesis of Transmembrane Domain 10 in Organic Anion Transporting Polypeptide 1B1

    PubMed Central

    2015-01-01

    Organic anion transporting polypeptide (OATP) 1B1 is an important drug transporter expressed in human hepatocytes. Previous studies have indicated that transmembrane (TM) domain 2, 6, 8, 9, and in particular 10 might be part of the substrate binding site/translocation pathway. To explore which amino acids in TM10 are important for substrate transport, we mutated 34 amino acids individually to cysteines, expressed them in HEK293 cells, and determined their surface expression. Transport activity of the two model substrates estrone-3-sulfate and estradiol-17β-glucuronide as well as of the drug substrate valsartan for selected mutants was measured. Except for F534C and F537C, all mutants were expressed at the plasma membrane of HEK293 cells. Mutants Q541C and A549C did not transport estradiol-17β-glucuronide and showed negligible estrone-3-sulfate transport. However, A549C showed normal valsartan transport. Pretreatment with the anionic and cell impermeable sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) affected the transport of each substrate differently. Pretreatment of L545C abolished estrone-3-sulfate uptake almost completely, while it stimulated estradiol-17β-glucuronide uptake. Further analyses revealed that mutant L545C in the absence of MTSES showed biphasic kinetics for estrone-3-sulfate that was converted to monophasic kinetics with a decreased apparent affinity, explaining the previously seen inhibition. In contrast, the apparent affinity for estradiol-17β-glucuronide was not changed by MTSES treatment, but the Vmax value was increased about 4-fold, explaining the previously seen stimulation. Maleimide labeling of L545C was affected by preincubation with estrone-3-sulfate but not with estradiol-17β-glucuronide. These results strongly suggest that L545C is part of the estrone-3-sulfate binding site/translocation pathway but is not directly involved in binding/translocation of estradiol-17β-glucuronide. PMID:24673529

  7. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur

    PubMed Central

    Ueki, Iori

    2010-01-01

    Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine β-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when methionine availability is high. Cysteine is catabolized by several desulfuration reactions that release sulfur in a reduced oxidation state, generating sulfane sulfur or hydrogen sulfide (H2S), which can be further oxidized to sulfate. Cysteine desulfuration is accomplished by alternate reactions catalyzed by cystathionine β-synthase and cystathionine γ-lyase. Cysteine is also catabolized by pathways that require the initial oxidation of the cysteine thiol by cysteine dioxygenase to form cysteinesulfinate. The oxidative pathway leads to production of taurine and sulfate in a ratio of approximately 2:1. Relative metabolism of cysteine by desulfuration versus oxidative pathways is influenced by cysteine dioxygenase activity, which is low in animals fed low-protein diets and high in animals fed excess sulfur amino acids. Thus, desulfuration reactions dominate when cysteine is deficient, whereas oxidative catabolism dominates when cysteine is in excess. In rats consuming a diet with an adequate level of sulfur amino acids, about two thirds of cysteine catabolism occurs by oxidative pathways and one third by desulfuration pathways. Cysteine dioxygenase is robustly regulated in response to cysteine availability and may function to provide a pathway to siphon cysteine to less toxic metabolites than those produced by cysteine desulfuration reactions. PMID:20162368

  8. From the limits of the classical model of sensitometric curves to a realistic model based on the percolation theory for GafChromic EBT films.

    PubMed

    del Moral, F; Vázquez, J A; Ferrero, J J; Willisch, P; Ramírez, R D; Teijeiro, A; López Medina, A; Andrade, B; Vázquez, J; Salvador, F; Medal, D; Salgado, M; Muñoz, V

    2009-09-01

    Modern radiotherapy uses complex treatments that necessitate more complex quality assurance procedures. As a continuous medium, GafChromic EBT films offer suitable features for such verification. However, its sensitometric curve is not fully understood in terms of classical theoretical models. In fact, measured optical densities and those predicted by the classical models differ significantly. This difference increases systematically with wider dose ranges. Thus, achieving the accuracy required for intensity-modulated radiotherapy (IMRT) by classical methods is not possible, plecluding their use. As a result, experimental parametrizations, such as polynomial fits, are replacing phenomenological expressions in modern investigations. This article focuses on identifying new theoretical ways to describe sensitometric curves and on evaluating the quality of fit for experimental data based on four proposed models. A whole mathematical formalism starting with a geometrical version of the classical theory is used to develop new expressions for the sensitometric curves. General results from the percolation theory are also used. A flat-bed-scanner-based method was chosen for the film analysis. Different tests were performed, such as consistency of the numeric results for the proposed model and double examination using data from independent researchers. Results show that the percolation-theory-based model provides the best theoretical explanation for the sensitometric behavior of GafChromic films. The different sizes of active centers or monomer crystals of the film are the basis of this model, allowing acquisition of information about the internal structure of the films. Values for the mean size of the active centers were obtained in accordance with technical specifications. In this model, the dynamics of the interaction between the active centers of GafChromic film and radiation is also characterized by means of its interaction cross-section value. The percolation model

  9. Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

    PubMed

    Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

    2017-07-21

    E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Beyond the Global Assessment of Functioning: Learning From Virginia Apgar

    PubMed Central

    Dimsdale, Joel E.; Jeste, Dilip V.; Patterson, Thomas L.

    2011-01-01

    Background The Global Assessment of Functioning (GAF) scale is widely used in psychiatry, yet it has certain drawbacks. Objective The authors seek to generate further discussion and research around developing an improved successor to the GAF. Method The authors used the Apgar scale as a template for constructing a possible successor to the GAF. Consulting with 16 colleagues, they selected 5 domains that were felt to be central to functioning in psychiatric patients. Psychiatrists in diverse clinical settings then completed both a GAF and a Psychiatric Apgar scale on 40 patients. Results The two scales were found to agree significantly. Use of the Psychiatric Apgar, however, provides clearer guidance about assessing functioning. Conclusion The GAF was a brilliant addition to psychiatric practice. As we develop the next Diagnostic and Statistical Manual, it is pertinent to ask whether the GAF approach could be optimized even further by applying the lessons of Virginia Apgar. PMID:21051684

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-06-09

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

  13. Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

    PubMed Central

    Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

    2014-01-01

    Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

  14. Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

    PubMed

    Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

    2014-03-28

    Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

  15. DevS, a heme-containing two-component oxygen sensor of Mycobacterium tuberculosis.

    PubMed

    Ioanoviciu, Alexandra; Yukl, Erik T; Moënne-Loccoz, Pierre; de Montellano, Paul R Ortiz

    2007-04-10

    Mycobacterium tuberculosis can exist in the actively growing state of the overt disease or in a latent quiescent state that can be induced, among other things, by anaerobiosis. Eradication of the latent state is particularly difficult with the available drugs and requires prolonged treatment. DevS is a member of the DevS-DevR two-component regulatory system that is thought to mediate the cellular response to anaerobiosis. Here we report the cloning, expression, and initial characterization of a truncated version of DevS (DevS642) containing only the N-terminal GAF sensor domain (GAF-A) and of the full-length protein DevS. The DevS truncated construct quantitatively binds heme in a 1:1 stoichiometry, and the complex of the protein with ferrous heme reversibly binds O2, NO, and CO. UV-vis and resonance Raman spectroscopy of the wild-type protein and the H149A mutant confirm that His149 is the proximal ligand to the heme iron atom. While the heme-CO complex is present as two conformers in the GAF-A domain, a single set of [Fe-C-O] vibrations is observed with the full-length protein, suggesting that interactions between domains within DevS influence the distal pocket environment of the heme in the GAF-A domain.

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

    PubMed

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

    2009-12-01

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

  17. Antibacterial Effect of Cysteine-Nitrosothiol and Possible Percursors Thereof

    PubMed Central

    Incze, K.; Farkas, J.; Mihályi, Vilma; Zukál, E.

    1974-01-01

    The postulated intermediate of nitrite-myoglobin reaction, cysteine-nitrosothiol, was prepared and its antibacterial effect was tested on Salmonella strains, Streptococcus faecium, and spores and vegetative cells of Clostridium sporogenes. Cysteine-nitrosothiol showed a higher inhibitory effect than nitrite. Preliminary results on the effect of simultaneous use of nitrite and cysteine on Clostridium sporogenes spores were also presented. PMID:4589130

  18. Stereoselective HDAC inhibition from cysteine-derived zinc-binding groups.

    PubMed

    Butler, Kyle V; He, Rong; McLaughlin, Kathryn; Vistoli, Giulio; Langley, Brett; Kozikowski, Alan P

    2009-08-01

    A series of small-molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10, was determined to have IC(50) values below 1 microM. The compounds were also tested in a cellular assay of oxidative stress-induced neurodegeneration. Many of the inhibitors gave near-complete protection against cell death at 10 microM without the neurotoxicity seen with hydroxamic acid-based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc-binding groups (ZBGs). Derivatives of L-cysteine were active in the HDAC inhibition assays, while the derivatives of D-cysteine were inactive. Notably, the finding that both the D- and L-cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.

  19. Cysteine Supplementation May be Beneficial in a Subgroup of Mitochondrial Translation Deficiencies.

    PubMed

    Bartsakoulia, Marina; Mϋller, Juliane S; Gomez-Duran, Aurora; Yu-Wai-Man, Patrick; Boczonadi, Veronika; Horvath, Rita

    2016-08-30

    Mitochondrial encephalomyopathies are severe, relentlessly progressive conditions and there are very few effective therapies available to date. We have previously suggested that in two rare forms of reversible mitochondrial disease (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy) supplementation with L-cysteine can improve mitochondrial protein synthesis, since cysteine is required for the 2-thiomodification of mitochondrial tRNAs. We studied whether supplementation with L-cysteine or N-acetyl-cysteine (NAC) results in any improvement of the mitochondrial function in vitro in fibroblasts of patients with different genetic forms of abnormal mitochondrial translation. We studied in vitro in fibroblasts of patients carrying the common m.3243A>G and m.8344A>G mutations or autosomal recessive mutations in genes affecting mitochondrial translation, whether L-cysteine or N-acetyl-cysteine supplementation have an effect on mitochondrial respiratory chain function. Here we show that supplementation with L-cysteine, but not with N-acetyl-cysteine partially rescues the mitochondrial translation defect in vitro in fibroblasts of patients carrying the m.3243A>G and m.8344A>G mutations. In contrast, N-acetyl-cysteine had a beneficial effect on mitochondrial translation in TRMU and MTO1 deficient fibroblasts. Our results suggest that L-cysteine or N-acetyl-cysteine supplementation may be a potential treatment for selected subgroups of patients with mitochondrial translation deficiencies. Further studies are needed to explore the full potential of cysteine supplementation as a treatment for patients with mitochondrial disease.

  20. Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, N.; Yun, P.; Nadkarni, M.A.

    Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel {beta}-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions ofmore » K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.« less

  1. Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

    PubMed

    Aich, Pulakesh; Biswas, Sampa

    2018-06-01

    Pro-domain of a cysteine cathepsin contains a highly conserved Ex 2 Rx 2 Fx 2 Nx 3 Ix 3 N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

  2. Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lovrinovic, Marina; Niemeyer, Christof M.

    2005-09-30

    We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

  3. Transmembrane Domains of Attraction on the TSH Receptor

    PubMed Central

    Ali, M. Rejwan; Mezei, Mihaly; Davies, Terry F.

    2015-01-01

    The TSH receptor (TSHR) has the propensity to form dimers and oligomers. Our data using ectodomain-truncated TSHRs indicated that the predominant interfaces for oligomerization reside in the transmembrane (TM) domain. To map the potentially interacting residues, we first performed in silico studies of the TSHR transmembrane domain using a homology model and using Brownian dynamics (BD). The cluster of dimer conformations obtained from BD analysis indicated that TM1 made contact with TM4 and two residues in TM2 made contact with TM5. To confirm the proximity of these contact residues, we then generated cysteine mutants at all six contact residues predicted by the BD analysis and performed cysteine cross-linking studies. These results showed that the predicted helices in the protomer were indeed involved in proximity interactions. Furthermore, an alternative experimental approach, receptor truncation experiments and LH receptor sequence substitution experiments, identified TM1 harboring a major region involved in TSHR oligomerization, in agreement with the conclusion from the cross-linking studies. Point mutations of the predicted interacting residues did not yield a substantial decrease in oligomerization, unlike the truncation of the TM1, so we concluded that constitutive oligomerization must involve interfaces forming domains of attraction in a cooperative manner that is not dominated by interactions between specific residues. PMID:25406938

  4. A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans*

    PubMed Central

    Molitor, Bastian; Stassen, Marc; Modi, Anuja; El-Mashtoly, Samir F.; Laurich, Christoph; Lubitz, Wolfgang; Dawson, John H.; Rother, Michael; Frankenberg-Dinkel, Nicole

    2013-01-01

    Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor). PMID:23661702

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

    PubMed Central

    Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process. PMID:26764487

  6. Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages

    PubMed Central

    Fan, Xiaoteng; Pan, Yang; Lv, Shan; Pan, Chuanying; Lei, Anmin

    2017-01-01

    The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry. PMID:28700739

  7. Inhibition of trypanosomal cysteine proteinases by their propeptides.

    PubMed

    Lalmanach, G; Lecaille, F; Chagas, J R; Authié, E; Scharfstein, J; Juliano, M A; Gauthier, F

    1998-09-25

    The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

  8. L-cysteine suppresses ghrelin and reduces appetite in rodents and humans.

    PubMed

    McGavigan, A K; O'Hara, H C; Amin, A; Kinsey-Jones, J; Spreckley, E; Alamshah, A; Agahi, A; Banks, K; France, R; Hyberg, G; Wong, C; Bewick, G A; Gardiner, J V; Lehmann, A; Martin, N M; Ghatei, M A; Bloom, S R; Murphy, K G

    2015-03-01

    High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety.

  9. l-cysteine suppresses ghrelin and reduces appetite in rodents and humans

    PubMed Central

    McGavigan, A K; O'Hara, H C; Amin, A; Kinsey-Jones, J; Spreckley, E; Alamshah, A; Agahi, A; Banks, K; France, R; Hyberg, G; Wong, C; Bewick, G A; Gardiner, J V; Lehmann, A; Martin, N M; Ghatei, M A; Bloom, S R; Murphy, K G

    2015-01-01

    Background: High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. Methods: We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. Results: l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. Conclusions: Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety. PMID:25219528

  10. EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

    PubMed

    Pearson, Jaclyn S; Giogha, Cristina; Mühlen, Sabrina; Nachbur, Ueli; Pham, Chi L L; Zhang, Ying; Hildebrand, Joanne M; Oates, Clare V; Lung, Tania Wong Fok; Ingle, Danielle; Dagley, Laura F; Bankovacki, Aleksandra; Petrie, Emma J; Schroeder, Gunnar N; Crepin, Valerie F; Frankel, Gad; Masters, Seth L; Vince, James; Murphy, James M; Sunde, Margaret; Webb, Andrew I; Silke, John; Hartland, Elizabeth L

    2017-01-13

    Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling 1-3 . RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis 4,5 . Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

  11. An N-terminal glycine to cysteine mutation in the collagen COL1A1 gene produces moderately severe osteogenesis imperfecta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilcox, W.; Scott, L.; Cohn, D.

    Osteogenesis imperfecta (OI) is usually due to mutations in the type I procollagen genes COL1A1 and COL1A2. Point mutations close to the N-terminus are generally milder than those near the C-terminus of the molecule (the gradient hypothesis of collagen mutations). We describe a patient with moderately severe OI due to a mutation in the N-terminal portion of the triple helical domain of the {alpha}1(I) chain. Electrophoretic analysis of collagen isolated from fibroblast cultures suggested the abnormal presence of a cysteine in the N-terminal portion of the {alpha}1(I) chain. Five overlapping DNA fragments amplified from fibroblast RNA were screened for mutationsmore » using single strand conformational polymorphism (SSCP) and heteroduplex analyses. Direct DNA sequence analysis of the single positive fragment demonstrated a G to T transversion, corresponding to a glycine to cysteine substitution at position 226 of the triple helical domain of the {alpha}1(I) chain. The mutation was confirmed by restriction enzyme analysis of amplified genomic DNA. The mutation was not present in fibroblasts from either phenotypically normal parent. Combining this mutation with other reported mutations, glycine to cysteine substitutions at positions 205, 211, 223, and 226 produce a moderately severe phenotype whereas flanking mutations at positions 175 and 382 produce a mild phenotype. This data supports a regional rather than a gradient model of the relationship between the nature and location of type I collagen mutations and OI phenotype.« less

  12. Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella enterica and Escherichia coli

    PubMed Central

    Loddeke, Melissa; Schneider, Barbara; Oguri, Tamiko; Mehta, Iti; Xuan, Zhenyu

    2017-01-01

    ABSTRACT Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR. Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica ΔcyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and

  13. Prediction of redox-sensitive cysteines using sequential distance and other sequence-based features.

    PubMed

    Sun, Ming-An; Zhang, Qing; Wang, Yejun; Ge, Wei; Guo, Dianjing

    2016-08-24

    Reactive oxygen species can modify the structure and function of proteins and may also act as important signaling molecules in various cellular processes. Cysteine thiol groups of proteins are particularly susceptible to oxidation. Meanwhile, their reversible oxidation is of critical roles for redox regulation and signaling. Recently, several computational tools have been developed for predicting redox-sensitive cysteines; however, those methods either only focus on catalytic redox-sensitive cysteines in thiol oxidoreductases, or heavily depend on protein structural data, thus cannot be widely used. In this study, we analyzed various sequence-based features potentially related to cysteine redox-sensitivity, and identified three types of features for efficient computational prediction of redox-sensitive cysteines. These features are: sequential distance to the nearby cysteines, PSSM profile and predicted secondary structure of flanking residues. After further feature selection using SVM-RFE, we developed Redox-Sensitive Cysteine Predictor (RSCP), a SVM based classifier for redox-sensitive cysteine prediction using primary sequence only. Using 10-fold cross-validation on RSC758 dataset, the accuracy, sensitivity, specificity, MCC and AUC were estimated as 0.679, 0.602, 0.756, 0.362 and 0.727, respectively. When evaluated using 10-fold cross-validation with BALOSCTdb dataset which has structure information, the model achieved performance comparable to current structure-based method. Further validation using an independent dataset indicates it is robust and of relatively better accuracy for predicting redox-sensitive cysteines from non-enzyme proteins. In this study, we developed a sequence-based classifier for predicting redox-sensitive cysteines. The major advantage of this method is that it does not rely on protein structure data, which ensures more extensive application compared to other current implementations. Accurate prediction of redox-sensitive cysteines not

  14. Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload

    PubMed Central

    Deshpande, Anup Arunrao; Bhatia, Muskan; Laxman, Sunil; Bachhawat, Anand Kumar

    2017-01-01

    Cysteine is an essential requirement in living organisms. However, due to its reactive thiol side chain, elevated levels of intracellular cysteine can be toxic and therefore need to be rapidly eliminated from the cellular milieu. In mammals and many other organisms, excess cysteine is believed to be primarily eliminated by the cysteine dioxygenase dependent oxidative degradation of cysteine, followed by the removal of the oxidative products. However, other mechanisms of tackling excess cysteine are also likely to exist, but have not thus far been explored. In this study, we use Saccharomyces cerevisiae, which naturally lacks a cysteine dioxygenase, to investigate mechanisms for tackling cysteine overload. Overexpressing the high affinity cysteine transporter, YCT1, enabled yeast cells to rapidly accumulate high levels of intracellular cysteine. Using targeted metabolite analysis, we observe that cysteine is initially rapidly interconverted to non-reactive cystine in vivo. A time course revealed that cells systematically convert excess cysteine to inert thiol forms; initially to cystine, and subsequently to cystathionine, S-Adenosyl-L-homocysteine (SAH) and S-Adenosyl L-methionine (SAM), in addition to eventually accumulating glutathione (GSH) and polyamines. Microarray based gene expression studies revealed the upregulation of arginine/ornithine biosynthesis a few hours after the cysteine overload, and suggest that the non-toxic, non-reactive thiol based metabolic products are eventually utilized for amino acid and polyamine biogenesis, thereby enabling cell growth. Thus, cells can handle potentially toxic amounts of cysteine by a combination of thiol trapping, metabolic redistribution to non-reactive thiols and subsequent consumption for anabolism. PMID:28435838

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

    PubMed Central

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

    2013-01-01

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

  16. Targeting cysteine proteases in trypanosomatid disease drug discovery.

    PubMed

    Ferreira, Leonardo G; Andricopulo, Adriano D

    2017-12-01

    Chagas disease and human African trypanosomiasis are endemic conditions in Latin America and Africa, respectively, for which no effective and safe therapy is available. Efforts in drug discovery have focused on several enzymes from these protozoans, among which cysteine proteases have been validated as molecular targets for pharmacological intervention. These enzymes are expressed during the entire life cycle of trypanosomatid parasites and are essential to many biological processes, including infectivity to the human host. As a result of advances in the knowledge of the structural aspects of cysteine proteases and their role in disease physiopathology, inhibition of these enzymes by small molecules has been demonstrated to be a worthwhile approach to trypanosomatid drug research. This review provides an update on drug discovery strategies targeting the cysteine peptidases cruzain from Trypanosoma cruzi and rhodesain and cathepsin B from Trypanosoma brucei. Given that current chemotherapy for Chagas disease and human African trypanosomiasis has several drawbacks, cysteine proteases will continue to be actively pursued as valuable molecular targets in trypanosomatid disease drug discovery efforts. Copyright © 2017. Published by Elsevier Inc.

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

    PubMed

    Mason, Alexander F; Thordarson, Pall

    2016-07-20

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

  18. Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction.

    PubMed

    García-Santamarina, Sarela; Boronat, Susanna; Hidalgo, Elena

    2014-04-29

    Activation of redox cascades through hydrogen peroxide-mediated reversible cysteine oxidation is a major mechanism for intracellular signaling. Understanding why some cysteine residues are specifically oxidized, in competition with other proximal cysteine residues and in the presence of strong redox buffers, is therefore crucial for understanding redox signaling. In this review, we explore the recent advances in thiol-redox chemistry linked to signaling. We describe the last findings in the field of redox sensors, those that are naturally present in different model organisms as well as those that have been engineered to quantify intracellular hydrogen peroxide concentrations. Finally, we provide a summary of the newest approaches developed to study reversible cysteine oxidation at the proteomic level.

  19. Mapping of the local environmental changes in proteins by cysteine scanning

    PubMed Central

    Yamazaki, Yoichi; Nagata, Tomoko; Terakita, Akihisa; Kandori, Hideki; Shichida, Yoshinori; Imamoto, Yasushi

    2014-01-01

    Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes. PMID:27493492

  20. A 170kDa multi-domain cystatin of Fasciola gigantica is active in the male reproductive system.

    PubMed

    Geadkaew, Amornrat; Kosa, Nanthawat; Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

    2014-09-01

    Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often <20% and at only 26.8% between the highest matching domains 6 and 10. Several domains contain degenerated QVVAG core motifs and/or lack other important residues of active type 2 cystatins. Domain-specific antisera detected multiple forms of FgMDC ranging from <10 to >120kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. The role of the Fe-S cluster in the sensory domain of nitrogenase transcriptional activator VnfA from Azotobacter vinelandii.

    PubMed

    Nakajima, Hiroshi; Takatani, Nobuyuki; Yoshimitsu, Kyohei; Itoh, Mitsuko; Aono, Shigetoshi; Takahashi, Yasuhiro; Watanabe, Yoshihito

    2010-02-01

    Transcriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobacter vinelandii. In the present study, we have purified full-length VnfA produced in E. coli as recombinant proteins (Strep-tag attached and tag-less proteins), enabling detailed characterization of VnfA for the first time. The EPR spectra of whole cells producing tag-less VnfA (VnfA) show distinctive signals assignable to a 3Fe-4S cluster in the oxidized form ([Fe(3)S(4)](+)). Although aerobically purified VnfA shows no vestiges of any Fe-S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe(3)S(4)](+) dominantly in the protein. Additional spectroscopic evidence of [Fe(3)S(4)](+)in vitro is provided by anaerobically purified Strep-tag attached VnfA. Thus, spectroscopic studies both in vivo and in vitro indicate the involvement of [Fe(3)S(4)](+) as a prosthetic group in VnfA. Molecular mass analyses reveal that VnfA is a tetramer both in the presence and absence of the Fe-S cluster. Quantitative data of iron and acid-labile sulfur in reconstituted VnfA are fitted with four 3Fe-4S clusters per a tetramer, suggesting that one subunit bears one cluster. In vivobeta-gal assays reveal that the Fe-S cluster which is presumably anchored in the GAF domain by the N-terminal cysteine residues is essential for VnfA to exert its transcription activity on the target nitrogenase genes. Unlike the NifAL system of A. vinelandii, O(2) shows no effect on the transcriptional activity of VnfA but reactive oxygen species is reactive to cause disassembly of the Fe-S cluster and turns active VnfA inactive.

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

    PubMed Central

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

    2015-01-01

    Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation. PMID:26642319

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

    PubMed

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

    2009-08-01

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

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

    PubMed

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

    2012-11-01

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

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

    PubMed Central

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

    2017-01-01

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

  6. Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation

    PubMed Central

    Ymer, Susie I.; Greenall, Sameer A.; Cvrljevic, Anna; Cao, Diana X.; Donoghue, Jacqui F.; Epa, V. Chandana; Scott, Andrew M.; Adams, Timothy E.; Johns, Terrance G.

    2011-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation. PMID:24212795

  7. Differences in conformational stability of the two alpha domains of the disease-associated and non-disease-associated subtypes of HLA-B27.

    PubMed

    Rana, Manish Kumar; Luthra-Guptasarma, Manni

    2017-01-01

    The MHC Class I molecule, HLA-B27, is strongly linked with development of the inflammatory arthritic disease, ankylosing spondylitis (AS); whereas the B*2705 subtype shows strong association, B*2709 is not associated with disease, even though the two subtypes differ in only a single residue at position 116. Currently, attention is focused on the misfolding propensities of these two subtypes, including studies of disulfide-linked dimers and non-covalently formed high molecular weight (HMW) aggregates. Using mutants retaining only a single cysteine at positions C67 or C164, and using a cysteine-reactive, environment-sensitive, fluorescence probe (acrylodan), we find that within the same overall population of identical single-cysteine HLA-B27 molecules, there exist sub-populations which (a) possess free cysteines which react with acrylodan, (b) form disulfide-linked dimers, and (c) form HMW aggregates. Further, using acrylodan fluorescence, we find (d) that the α1 and α2 domains unfold independently of each other in HMW aggregates, (e) that these two domains of B*2709 are less stable to chemical and thermal denaturation than the corresponding domains of B*2705, suggesting easier clearance of misfolded molecules in the former, and (f) C67 is much more exposed in B*2705 than in B*2709, which could potentially explain how B*2705 more easily forms C67-mediated disulfide-bonded dimers. Copyright © 2016. Published by Elsevier B.V.

  8. Conotoxin Φ-MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti-Apoptotic Activity.

    PubMed

    Jin, Ai-Hua; Dekan, Zoltan; Smout, Michael J; Wilson, David; Dutertre, Sébastien; Vetter, Irina; Lewis, Richard J; Loukas, Alex; Daly, Norelle L; Alewood, Paul F

    2017-11-20

    Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC 50 17.85 μm) while inhibiting apoptosis (EC 50 2.2 μm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    Li, R; Kast, J

    2017-01-01

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

  10. Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

    PubMed

    Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

    2016-01-01

    Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC 50 >1 mM) on the activities of five major isoforms of human CYP in vitro.

  11. Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

    PubMed

    Beltrán González, Andrea N; Vicentini, Florencia; Calvo, Daniel J

    2018-01-01

    l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABA A ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABA A ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABA A ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABA A ρ1 receptors. © 2017 International Society for Neurochemistry.

  12. A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.

    PubMed

    Seal, R P; Amara, S G

    1998-12-01

    To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.

  13. S-Nitroso-N-acetyl-L-cysteine ethyl ester (SNACET) and N-acetyl-L-cysteine ethyl ester (NACET)-Cysteine-based drug candidates with unique pharmacological profiles for oral use as NO, H2S and GSH suppliers and as antioxidants: Results and overview.

    PubMed

    Tsikas, Dimitrios; Schwedhelm, Kathrin S; Surdacki, Andrzej; Giustarini, Daniela; Rossi, Ranieri; Kukoc-Modun, Lea; Kedia, George; Ückert, Stefan

    2018-02-01

    S -Nitrosothiols or thionitrites with the general formula RSNO are formally composed of the nitrosyl cation (NO + ) and a thiolate (RS - ), the base of the corresponding acids RSH. The smallest S -nitrosothiol is HSNO and derives from hydrogen sulfide (HSH, H 2 S). The most common physiological S -nitrosothiols are derived from the amino acid L-cysteine (CysSH). Thus, the simplest S -nitrosothiol is S -nitroso-L-cysteine (CysSNO). CysSNO is a spontaneous potent donor of nitric oxide (NO) which activates soluble guanylyl cyclase to form cyclic guanosine monophosphate (cGMP). This activation is associated with multiple biological actions that include relaxation of smooth muscle cells and inhibition of platelet aggregation. Like NO, CysSNO is a short-lived species and occurs physiologically at concentrations around 1 nM in human blood. CysSNO can be formed from CysSH and higher oxides of NO including nitrous acid (HONO) and its anhydride (N 2 O 3 ). The most characteristic feature of RSNO is the S-transnitrosation reaction by which the NO + group is reversibly transferred to another thiolate. By this way numerous RSNO can be formed such as the low-molecular-mass S -nitroso- N -acetyl-L-cysteine (SNAC) and S -nitroso-glutathione (GSNO), and the high-molecular-mass S -nitrosol-L-cysteine hemoglobin (HbCysSNO) present in erythrocytes and S -nitrosol-L-cysteine albumin (AlbCysSNO) present in plasma at concentrations of the order of 200 nM. All above mentioned RSNO exert NO-related biological activity, but they must be administered intravenously. This important drawback can be overcome by lipophilic charge-free RSNO. Thus, we prepared the ethyl ester of SNAC, the S -nitroso- N -acetyl-L-cysteine ethyl ester (SNACET), from synthetic N -acetyl-L-cysteine ethyl ester (NACET). Both NACET and SNACET have improved pharmacological features over N -acetyl-L-cysteine (NAC) and S -nitroso- N -acetyl-L-cysteine (SNAC), respectively, including higher oral bioavailability. SNACET

  14. Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines

    PubMed Central

    Chandel, Avinash; Das, Krishna K.; Bachhawat, Anand K.

    2016-01-01

    Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeast. We show here that glutathione depletion in yeast leads to the activation of two cytoplasmically inward-facing channels: the plasma membrane, Cch1p, and the vacuolar calcium channel, Yvc1p. Deletion of these channels partially rescues cells from glutathione depletion–induced cell death. Subsequent investigations on the Yvc1p channel, a homologue of the mammalian TRP channels, revealed that the channel is activated by glutathionylation. Yvc1p has nine cysteine residues, of which eight are located in the cytoplasmic regions and one on the transmembrane domain. We show that three of these cysteines, Cys-17, Cys-79, and Cys-191, are specifically glutathionylated. Mutation of these cysteines to alanine leads to a loss in glutathionylation and a concomitant loss in calcium channel activity. We further investigated the mechanism of glutathionylation and demonstrate a role for the yeast glutathione S-transferase Gtt1p in glutathionylation. Yvc1p is also deglutathionylated, and this was found to be mediated by the yeast thioredoxin, Trx2p. A model for redox activation and deactivation of the yeast Yvc1p channel is presented. PMID:27708136

  15. Transcription factor DecR (YbaO) controls detoxification of L-cysteine in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Tanaka, Kan; Ishihama, Akira

    2016-09-01

    YbaO is an uncharacterized AsnC-family transcription factor of Escherichia coli. In both Salmonella enterica and Pantoea ananatis, YbaO homologues were identified to regulate the adjacent gene encoding cysteine desulfhydrase for detoxification of cysteine. Using the genomic SELEX (systematic evolution of ligands by exponential enrichment) screening system, we identified the yhaOM operon, located far from the ybaO gene on the E. coli genome, as a single regulatory target of YbaO. In both gel shift assay in vitro and reporter and Northern blot assays in vivo, YbaO was found to regulate the yhaOM promoter. The growth of mutants lacking either ybaO or its targets yhaOM was delayed in the presence of cysteine, indicating involvement of these genes in cysteine detoxification. In the major pathway of cysteine degradation, hydrogen sulfide is produced in wild-type E. coli, but its production was not observed in each of the ybaO, yhaO and yhaM mutants. The yhaOM promoter was activated in the presence of cysteine, implying the role of cysteine in activation of YbaO. Taken together, we propose that YbaO is the cysteine-sensing transcriptional activator of the yhaOM operon, which is involved in the detoxification of cysteine. We then propose the naming of ybaO as decR (regulator of detoxification of cysteine).

  16. Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Svintradze, David V.; Virginia Commonwealth University, Richmond, VA 23219-1540; Peterson, Darrell L.

    Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces,more » which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.« less

  17. Cysteine Inhibits Mercury Methylation by Geobacter sulfurreducens PCA Mutant Δ omcBESTZ

    DOE PAGES

    Lin, Hui; Lu, Xia; Liang, Liyuan; ...

    2015-04-21

    For cysteine enhances Hg uptake and methylation by Geobacter sulfurreducens PCA wild type (WT) strain in short-term assays. The prevalence of this enhancement in other strains remains poorly understood. We examined the influence of cysteine concentration on time-dependent Hg(II) reduction, sorption and methylation by PCA-WT and its c-type cytochrome-deficient mutant ( omcBESTZ) in phosphate buffered saline. Without cysteine, the mutant methylated twice as much Hg(II) as the PCA-WT, whereas addition of cysteine inhibited Hg methylation, regardless of the reaction time. PCA-WT, but, exhibited both time-dependent and cysteine concentration-dependent methylation. In 144 hour assay, nearly complete sorption of the Hg(II) bymore » PCA-WT occurred in the presence of 1 mM cysteine, resulting in our highest observed methylmercury production. Moreover, the chemical speciation modeling and experimental data suggest that uncharged Hg(II) species are more readily taken up, and that this uptake is kinetic limiting, thereby affecting Hg methylation by both mutant and WT.« less

  18. Cysteine cathepsin S processes leptin, inactivating its biological activity.

    PubMed

    Oliveira, Marcela; Assis, Diego M; Paschoalin, Thaysa; Miranda, Antonio; Ribeiro, Eliane B; Juliano, Maria A; Brömme, Dieter; Christoffolete, Marcelo Augusto; Barros, Nilana M T; Carmona, Adriana K

    2012-08-01

    Leptin is a 16  kDa hormone mainly produced by adipocytes that plays an important role in many biological events including the regulation of appetite and energy balance, atherosclerosis, osteogenesis, angiogenesis, the immune response, and inflammation. The search for proteolytic enzymes capable of processing leptin prompted us to investigate the action of cysteine cathepsins on human leptin degradation. In this study, we observed high cysteine peptidase expression and hydrolytic activity in white adipose tissue (WAT), which was capable of degrading leptin. Considering these results, we investigated whether recombinant human cysteine cathepsins B, K, L, and S were able to degrade human leptin. Mass spectrometry analysis revealed that among the tested enzymes, cathepsin S exhibited the highest catalytic activity on leptin. Furthermore, using a Matrigel assay, we observed that the leptin fragments generated by cathepsin S digestion did not exhibit angiogenic action on endothelial cells and were unable to inhibit food intake in Wistar rats after intracerebroventricular administration. Taken together, these results suggest that cysteine cathepsins may be putative leptin activity regulators in WAT.

  19. Desulfurization of Cysteine-Containing Peptides Resulting from Sample Preparation for Protein Characterization by MS

    PubMed Central

    Wang, Zhouxi; Rejtar, Tomas; Zhou, Zhaohui Sunny; Karger, Barry L.

    2010-01-01

    In this paper, we have examined two cysteine modifications resulting from sample preparation for protein characterization by MS: (1) a previously observed conversion of cysteine to dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine to alanine. Using model peptides, the conversion of cysteine to dehydroalanine via β-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 °C, pH 7.0– 9.0) without disulfide reduction and alkylation.. Furthermore, the surprising conversion of cysteine to alanine was shown to occur by heating cysteine containing peptides in the presence of a phosphine (TCEP). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical-based desulfurization mechanism unrelated to β-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. PMID:20049891

  20. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi

    Purification and crystallization of ginkbilobin-2 and its selenomethionine derivative allowed the collection of complete data to 2.38 Å resolution and multiwavelength anomalous diffraction data sets, respectively. The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 Å resolution and from SeMet-rGnk2 at 2.79 Å resolution using amore » synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2{sub 1}3, with unit-cell parameters a = b = c = 143.2 Å.« less

  1. The phenotypic spectrum in patients with arginine to cysteine mutations in the COL2A1 gene

    PubMed Central

    Hoornaert, K P; Dewinter, C; Vereecke, I; Beemer, F A; Courtens, W; Fryer, A; Fryssira, H; Lees, M; Müllner‐Eidenböck, A; Rimoin, D L; Siderius, L; Superti‐Furga, A; Temple, K; Willems, P J; Zankl, A; Zweier, C; De Paepe, A; Coucke, P; Mortier, G R

    2006-01-01

    Background The majority of COL2A1 missense mutations are substitutions of obligatory glycine residues in the triple helical domain. Only a few non‐glycine missense mutations have been reported and among these, the arginine to cysteine substitutions predominate. Objective To investigate in more detail the phenotype resulting from arginine to cysteine mutations in the COL2A1 gene. Methods The clinical and radiographic phenotype of all patients in whom an arginine to cysteine mutation in the COL2A1 gene was identified in our laboratory, was studied and correlated with the abnormal genotype. The COL2A1 genotyping involved DHPLC analysis with subsequent sequencing of the abnormal fragments. Results Six different mutations (R75C, R365C, R519C, R704C, R789C, R1076C) were found in 11 unrelated probands. Each mutation resulted in a rather constant and site‐specific phenotype, but a perinatally lethal disorder was never observed. Spondyloarthropathy with normal stature and no ocular involvement were features of patients with the R75C, R519C, or R1076C mutation. Short third and/or fourth toes was a distinguishing feature of the R75C mutation and brachydactyly with enlarged finger joints a key feature of the R1076C substitution. Stickler dysplasia with brachydactyly was observed in patients with the R704C mutation. The R365C and R789C mutations resulted in classic Stickler dysplasia and spondyloepiphyseal dysplasia congenita (SEDC), respectively. Conclusions Arginine to cysteine mutations are rather infrequent COL2A1 mutations which cause a spectrum of phenotypes including classic SEDC and Stickler dysplasia, but also some unusual entities that have not yet been recognised and described as type II collagenopathies. PMID:16155195

  2. N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes.

    PubMed

    Alicigüzel, Y; Aslan, M

    2004-09-01

    In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species.

  3. Effect of L-cysteine on the oxidation of cyclohexane catalyzed by manganeseporphyrin.

    PubMed

    Zhou, Wei-You; Tian, Peng; Chen, Yong; He, Ming-Yang; Chen, Qun; Chen, Zai Xin

    2015-06-01

    Effect of L-cysteine as the cocatalyst on the oxidation of cyclohexane by tert-butylhydroperoxide (TBHP) catalyzed by manganese tetraphenylporphyrin (MnTPP) has been investigated. The results showed that L-cysteine could moderately improve the catalytic activity of MnTPP and significantly increase the selectivity of cyclohexanol. Different from imidazole and pyridine, the L-cysteine may perform dual roles in the catalytic oxidation of cyclohexane. Besides as the axial ligand for MnTPP, the L-cysteine could also react with cyclohexyl peroxide formed as the intermediate to produce alcohol as the main product. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

    PubMed Central

    1996-01-01

    Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

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

    PubMed

    Habich, Markus; Riemer, Jan

    2017-01-01

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

  6. Cysteine-functionalized silica-coated magnetite nanoparticles as potential nanoadsorbents

    NASA Astrophysics Data System (ADS)

    Enache, Daniela F.; Vasile, Eugenia; Simonescu, Claudia M.; Răzvan, Anca; Nicolescu, Alina; Nechifor, Aurelia-Cristina; Oprea, Ovidiu; Pătescu, Rodica-Elena; Onose, Cristian; Dumitru, Florina

    2017-09-01

    Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@ICPTES-cysteine MNPs have been prepared by the deposition of silica onto magnetite nanoparticles via controlled hydrolysis of TEOS. The new formed silica surface has been functionalized by grafting 3-(triethoxysilyl) propyl isocyanate (ICPTES) and, subsequently, by condensation of isocyanate moiety with cysteine. The morphology of magnetic silica nanoparticles has been investigated by FTIR, PXRD, TEM-HRTEM/SEM/EDX as well as TG experiments. HRTEM microscopy revealed that the Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2@ICPTES-cysteine nanoparticles are all of spherical shape with particle of ca. 10-30 nm diameters and the silica-coated magnetites have a core-shell structure. Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@ICPTES-cysteine MNPs have been tested for their sorption capacity of Pb(II) from synthetic aqueous solutions and the influence of pH solution, contact time, initial heavy metal ion concentrations, and adsorption isotherms on the sorption behavior were also studied. The kinetic studies revealed that the Pb(II) sorption process is mainly controlled by chemical mechanisms. Fe3O4@SiO2@ICPTES-cysteine, with a sorption capacity of 81.8 mg Pb(II)/g, has the potential to be an efficient Pb(II) adsorbent.

  7. Proteomic Approaches to Quantify Cysteine Reversible Modifications in Aging and Neurodegenerative Diseases

    PubMed Central

    Gu, Liqing; Robinson, Renã A. S.

    2016-01-01

    Cysteine is a highly reactive amino acid and is subject to a variety of reversible post-translational modifications (PTMs), including nitrosylation, glutathionylation, palmitoylation, as well as formation of sulfenic acid and disulfides. These modifications are not only involved in normal biological activities, such as enzymatic catalysis, redox signaling and cellular homeostasis, but can also be the result of oxidative damage. Especially in aging and neurodegenerative diseases, oxidative stress leads to aberrant cysteine oxidations that affect protein structure and function leading to neurodegeneration as well as other detrimental effects. Methods that can identify cysteine modifications by type, including the site of modification, as well as the relative stoichiometry of the modification can be very helpful for understanding the role of the thiol proteome and redox homeostasis in the context of disease. Cysteine reversible modifications however, are challenging to investigate as they are low abundant, diverse, and labile especially under endogenous conditions. Thanks to the development of redox proteomic approaches, large-scale quantification of cysteine reversible modifications is possible. These approaches cover a range of strategies to enrich, identify, and quantify cysteine reversible modifications from biological samples. This review will focus on nongel-based redox proteomics workflows that give quantitative information about cysteine PTMs and highlight how these strategies have been useful for investigating the redox thiol proteome in aging and neurodegenerative diseases. PMID:27666938

  8. A nitric oxide/cysteine interaction mediates the activation of soluble guanylate cyclase

    PubMed Central

    Fernhoff, Nathaniel B.; Derbyshire, Emily R.; Marletta, Michael A.

    2009-01-01

    Nitric oxide (NO) regulates a number of essential physiological processes by activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. The mechanism of NO sensing was previously thought to result exclusively from NO binding to the sGC heme; however, recent studies indicate that heme-bound NO only partially activates sGC and additional NO is involved in the mechanism of maximal NO activation. Furthermore, thiol oxidation of sGC cysteines results in the loss of enzyme activity. Herein the role of cysteines in NO-stimulated sGC activity investigated. We find that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking a non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway. PMID:20007374

  9. In vivo functional mapping of the conserved protein domains within murine Themis1.

    PubMed

    Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal; Barr, Valarie; Akpan, Itoro; Samelson, Lawrence; Love, Paul E

    2014-09-01

    Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

  10. The Cysteine Dioxgenase Knockout Mouse: Altered Cysteine Metabolism in Nonhepatic Tissues Leads to Excess H2S/HS− Production and Evidence of Pancreatic and Lung Toxicity

    PubMed Central

    Roman, Heather B.; Hirschberger, Lawrence L.; Krijt, Jakub; Valli, Alessandro; Kožich, Viktor

    2013-01-01

    Abstract Aims: To define the consequences of loss of cysteine dioxygenase (CDO) on cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS− (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO−/− mice that were fed either a taurine-free or taurine-supplemented diet. Results: CDO−/− mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of cysteine, consistent with the loss of CDO. CDO−/− mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS−. Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthionine in pancreas of CDO−/− mice were observed, suggesting a unique cysteine metabolism in the pancreas. Innovation: The CDO−/− mouse model provides new insights into tissue-specific cysteine metabolism, particularly the role of pancreas in metabolism of excess cysteine by CBS-catalyzed reactions, and will be a useful model for studying the effects of excess endogenous production of H2S/HS−. Conclusion: The CDO−/− mouse clearly demonstrates that H2S/HS− production in tissues can exceed the capacity of the animal to oxidize sulfide to sulfate and demonstrates that pancreas and lung are more susceptible to toxicity from endogenous H2S/HS−production than are liver and kidney. Antioxid. Redox Signal. 19, 1321–1336. PMID:23350603

  11. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  12. Analysis of Structures, Functions, and Epitopes of Cysteine Protease from Spirometra erinaceieuropaei Spargana

    PubMed Central

    Liu, Li Na; Cui, Jing; Zhang, Xi; Wei, Tong; Jiang, Peng; Wang, Zhong Quan

    2013-01-01

    Spirometra erinaceieuropaei cysteine protease (SeCP) in sparganum ES proteins recognized by early infection sera was identified by MALDI-TOF/TOF-MS. The aim of this study was to predict the structures and functions of SeCP protein by using the full length cDNA sequence of SeCP gene with online sites and software programs. The SeCP gene sequence was of 1 053 bp length with a 1011 bp biggest ORF encoding 336-amino acid protein with a complete cathepsin propeptide inhibitor domain and a peptidase C1A conserved domain. The predicted molecular weight and isoelectric point of SeCP were 37.87 kDa and 6.47, respectively. The SeCP has a signal peptide site and no transmembrane domain, located outside the membrane. The secondary structure of SeCP contained 8 α-helixes, 7 β-strands, and 20 coils. The SeCP had 15 potential antigenic epitopes and 19 HLA-I restricted epitopes. Based on the phylogenetic analysis of SeCP, S. erinaceieuropaei has the closest evolutionary status with S. mansonoides. SeCP was a kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights on the diagnostic antigens and target molecular of antisparganum drugs. PMID:24392448

  13. Cysteine oxidation impairs systemic glucocorticoid responsiveness in children with difficult-to-treat asthma.

    PubMed

    Stephenson, Susan T; Brown, Lou Ann S; Helms, My N; Qu, Hongyan; Brown, Sheena D; Brown, Milton R; Fitzpatrick, Anne M

    2015-08-01

    The mechanisms underlying glucocorticoid responsiveness are largely unknown. Although redox regulation of the glucocorticoid receptor (GR) has been reported, it has not been studied in asthmatic patients. We characterized systemic cysteine oxidation and its association with inflammatory and clinical features in healthy children and children with difficult-to-treat asthma. We hypothesized that cysteine oxidation would be associated with increased markers of oxidative stress and inflammation, increased features of asthma severity, decreased clinically defined glucocorticoid responsiveness, and impaired GR function. PBMCs were collected from healthy children (n = 16) and children with asthma (n = 118) aged 6 to 17 years. Children with difficult-to-treat asthma underwent glucocorticoid responsiveness testing with intramuscular triamcinolone. Cysteine, cystine, and inflammatory chemokines and reactive oxygen species generation were quantified, and expression and activity of the GR were assessed. Cysteine oxidation was present in children with difficult-to-treat asthma and accompanied by increased reactive oxygen species generation and increased CCL3 and CXCL1 mRNA expression. Children with the greatest extent of cysteine oxidation had more features of asthma severity, including poorer symptom control, greater medication use, and less glucocorticoid responsiveness despite inhaled glucocorticoid therapy. Cysteine oxidation also modified the GR protein by decreasing available sulfhydryl groups and decreasing nuclear GR expression and activity. A highly oxidized cysteine redox state promotes a posttranslational modification of the GR that might inhibit its function. Given that cysteine oxidation is prevalent in children with difficult-to-treat asthma, the cysteine redox state might represent a potential therapeutic target for restoration of glucocorticoid responsiveness in this population. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by

  14. Cysteine Racemization on IgG Heavy and Light Chains

    PubMed Central

    Zhang, Qingchun; Flynn, Gregory C.

    2013-01-01

    Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of d-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide. PMID:24142697

  15. Cysteine Cathepsin Activity Regulation by Glycosaminoglycans

    PubMed Central

    Lenarčič, Brigita

    2014-01-01

    Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail. PMID:25587532

  16. Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function

    USDA-ARS?s Scientific Manuscript database

    This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess six-cysteine-containing chitin-binding domains (CBDs) related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of...

  17. Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro.

    PubMed

    Wollenweber, Frank Arne; Hanecker, Patrizia; Bayer-Karpinska, Anna; Malik, Rainer; Bäzner, Hansjörg; Moreton, Fiona; Muir, Keith W; Müller, Susanna; Giese, Armin; Opherk, Christian; Dichgans, Martin; Haffner, Christof; Duering, Marco

    2015-03-01

    Mutations in NOTCH3 cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common monogenic cause of stroke and vascular dementia. Misfolding and aggregation of NOTCH3 proteins triggered by cysteine-affecting mutations are considered to be the key disease mechanisms. However, the significance of cysteine-sparing mutations is still debated. We studied a family with inherited small vessel disease by standardized medical history, clinical examination, MRI, ultrastructural analysis of skin biopsies, and Sanger sequencing of all NOTCH3 exons. In addition, we performed in vitro characterization of NOTCH3 variants using recombinant protein fragments and a single-particle aggregation assay. We identified a novel cysteine-sparing NOTCH3 mutation (D80G) in 4 family members, which was absent in a healthy sibling. All mutation carriers exhibited a CADASIL typical brain imaging and clinical phenotype, whereas skin biopsy showed inconsistent results. In vitro aggregation behavior of the D80G mutant was similar compared with cysteine-affecting mutations. This was reproduced with cysteine-sparing mutations from previously reported families having a phenotype consistent with CADASIL. Our findings support the view that cysteine-sparing mutations, such as D80G, might cause CADASIL with a phenotype largely indistinguishable from cysteine mutations. The in vitro aggregation analysis of atypical NOTCH3 mutations offers novel insights into pathomechanisms and might represent a tool for estimating their clinical significance. © 2015 American Heart Association, Inc.

  18. Effects of cysteine introduction into three homologous cytochromes C.

    PubMed

    Kobayashi, Yoshiko; Sonoyama, Takafumi; Takeda, Taku; Sambongi, Yoshihiro

    2009-05-01

    A cysteine residue was systematically introduced into three homologous cytochromes c from Hydrogenobacter thermophilus, Hydrogenophilus thermoluteolus, and Pseudomonas aeruginosa at a conserved position. The H. thermoluteolus variant showed the most decreased thermal stability as compared with the wild type, which might have been due in part to crosslinked polymer formation. The effects of cysteine introduction differed even at the conserved position in these homologous proteins.

  19. Effects of L-cysteine on lead acetate induced neurotoxicity in albino mice.

    PubMed

    Mahmoud, Y I; Sayed, S S

    2016-07-01

    Lead is a toxic heavy metal that adversely affects nervous tissues; it often occurs as an environmental pollutant. We investigated histological changes in the cerebral cortex, hippocampus and cerebellum of adult albino mice following exposure to lead acetate. We also studied the possible ameliorative effect of the chelating agent, L-cysteine, on lead-induced neurotoxicity. We divided albino mice into six groups: 1) vehicle-only control, 2) L-cysteine control, 3 and 4) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, and 5 and 6) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, followed by 50 mg/kg L-cysteine for 7 days. Lead acetate administration caused disorganization of cell layers, neuronal loss and degeneration, and neuropil vacuolization. Brain sections from lead-intoxicated mice treated with L-cysteine showed fewer pathological changes; the neuropil showed less vacuolization and the neurons appeared less damaged. L-cysteine at the dose we used only marginally alleviated lead-induced toxicity.

  20. Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

    PubMed

    Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

    2016-11-01

    Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways. Copyright © 2016 Biophysical Society. Published by Elsevier

  1. Synthesis of the sulfur amino acids: cysteine and methionine.

    PubMed

    Wirtz, Markus; Droux, Michel

    2005-12-01

    This review will assess new features reported for the molecular and biochemical aspects of cysteine and methionine biosynthesis in Arabidopsis thaliana with regards to early published data from other taxa including crop plants and bacteria (Escherichia coli as a model). By contrast to bacteria and fungi, plant cells present a complex organization, in which the sulfur network takes place in multiple sites. Particularly, the impact of sulfur amino-acid biosynthesis compartmentalization will be addressed in respect to localization of sulfur reduction. To this end, the review will focus on regulation of sulfate reduction by synthesis of cysteine through the cysteine synthase complex and the synthesis of methionine and its derivatives. Finally, regulatory aspects of sulfur amino-acid biosynthesis will be explored with regards to interlacing processes such as photosynthesis, carbon and nitrogen assimilation.

  2. Facile and green synthesis of highly stable L-cysteine functionalized copper nanoparticles

    NASA Astrophysics Data System (ADS)

    Kumar, Nikhil; Upadhyay, Lata Sheo Bachan

    2016-11-01

    A simple eco-friendly method for L-cysteine capped copper nanoparticles (CCNPs) synthesis in aqueous solution has been developed. Glucose and L-cysteine were used as reducing agent and capping/functionalizing agent, respectively. Different parameters such as capping agent concentration, pH, reaction temperature, and reducing agent concentration were optimized during the synthesis. The L-cysteine capped copper nanoparticle were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, Particle size and zeta potential analyser, and high resolution transmission electron microscopy. Spherical shaped cysteine functionalized/capped copper nanoparticles with an average size of 40 nm were found to be highly stable at room temperature (RT) for a period of 1 month

  3. Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

    PubMed

    Lipton, Stuart A; Choi, Yun-Beom; Takahashi, Hiroto; Zhang, Dongxian; Li, Weizhong; Godzik, Adam; Bankston, Laurie A

    2002-09-01

    Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

  4. Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

    PubMed

    Nozaki, T; Asai, T; Sanchez, L B; Kobayashi, S; Nakazawa, M; Takeuchi, T

    1999-11-05

    The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

  5. Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

    PubMed Central

    Sun, Meihong; Liu, Shiyang; Qi, Baoxiu; Li, Xinzheng

    2013-01-01

    Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family. PMID:24155879

  6. Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains.

    PubMed

    Muradov, Khakim G; Granovsky, Alexey E; Schey, Kevin L; Artemyev, Nikolai O

    2002-03-26

    Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

  7. L-cysteine desulfidase: an [4Fe-4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia, and sulfide.

    PubMed

    Tchong, Shih-I; Xu, Huimin; White, Robert H

    2005-02-08

    A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being

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

    PubMed

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

    2012-11-06

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

  9. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  10. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  11. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  12. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  13. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  14. Cysteine proteases and cell differentiation: excystment of the ciliated protist Sterkiella histriomuscorum.

    PubMed

    Villalobo, Eduardo; Moch, Clara; Fryd-Versavel, Ghislaine; Fleury-Aubusson, Anne; Morin, Loïc

    2003-12-01

    The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.

  15. Neuronal growth on L- and D-cysteine self-assembled monolayers reveals neuronal chiral sensitivity.

    PubMed

    Baranes, Koby; Moshe, Hagay; Alon, Noa; Schwartz, Shmulik; Shefi, Orit

    2014-05-21

    Studying the interaction between neuronal cells and chiral molecules is fundamental for the design of novel biomaterials and drugs. Chirality influences all biological processes that involve intermolecular interaction. One common method used to study cellular interactions with different enantiomeric targets is the use of chiral surfaces. Based on previous studies that demonstrated the importance of cysteine in the nervous system, we studied the effect of L- and D-cysteine on single neuronal growth. L-Cysteine, which normally functions as a neuromodulator or a neuroprotective antioxidant, causes damage at elevated levels, which may occur post trauma. In this study, we grew adult neurons in culture enriched with L- and D-cysteine as free compounds or as self-assembled monolayers of chiral surfaces and examined the effect on the neuronal morphology and adhesion. Notably, we have found that exposure to the L-cysteine enantiomer inhibited, and even prevented, neuronal attachment more severely than exposure to the D-cysteine enantiomer. Atop the L-cysteine surfaces, neuronal growth was reduced and degenerated. Since the cysteine molecules were attached to the surface via the thiol groups, the neuronal membrane was exposed to the molecular chiral site. Thus, our results have demonstrated high neuronal chiral sensitivity, revealing chiral surfaces as indirect regulators of neuronal cells and providing a reference for studying chiral drugs.

  16. Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example

    PubMed Central

    Kim, Yong Joon; Kaluz, Stefan; Mehta, Anil; Weinert, Emily; Rivera, Shannon; Van Meir, Erwin G.

    2017-01-01

    The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating zinc during expression and streamlining the purification time, resulting in a high yield of optimally folded protein (120 mg per 4 L expression media) that is suitable for structural NMR studies. The structural integrity of the final recombinant p300-CH1 has been verified to be optimal using onedimensional 1H NMR spectroscopy and circular dichroism. This protocol is applicable for the purification of other zinc finger containing proteins. PMID:28966947

  17. Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice

    PubMed Central

    Niewiadomski, Julie; Zhou, James Q.; Roman, Heather B.; Liu, Xiaojing; Hirschberger, Lawrence L.; Locasale, Jason W.; Stipanuk, Martha H.

    2016-01-01

    To gain further insights into the effect of elevated cysteine levels on energy metabolism and the possible mechanisms by which cysteine may have these effects, we conducted studies in cysteine dioxygenase (Cdo1)–null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated cysteine levels with greater weight gain and with elevated hepatic Scd1 expression holds in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggested impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated cysteine levels with excess H2S production and impairments in energy metabolism suggest that H2S signaling could be involved. PMID:26995761

  18. Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

    NASA Astrophysics Data System (ADS)

    Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

    2013-10-01

    Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

  19. Analysis of S-nitrosothiols via Copper Cysteine (2C) and Copper Cysteine - Carbon Monoxide (3C) Methods

    PubMed Central

    Rogers, Stephen C.; Gibbons, Lindsey B.; Griffin, Sherraine; Doctor, Allan

    2012-01-01

    This chapter summarizes the principles of RSNO measurement in the gas phase, utilizing ozone-based chemiluminescence and the copper cysteine (2C) ± carbon monoxide (3C) reagent. Although an indirect method for quantifying RSNOs, this assay represents one of the most robust methodologies available. It exploits the NO• detection sensitivity of ozone based chemiluminscence, which is within the range required to detect physiological concentrations of RSNO metabolites. Additionally, the specificity of the copper cysteine (2C and 3C) reagent for RSNOs negates the need for sample pretreatment, thereby minimizing the likelihood of sample contamination (false positive results), NO species inter-conversion, or the loss of certain highly labile RSNO species. Herein, we outline the principles of this methodology, summarizing key issues, potential pitfalls and corresponding solutions. PMID:23116707

  20. A site-directed mutagenesis analysis of tNOX functional domains

    NASA Technical Reports Server (NTRS)

    Chueh, Pin-Ju; Morre, Dorothy M.; Morre, D. James

    2002-01-01

    Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

  1. Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors

    PubMed Central

    Nga, Bui T. T.; Takeshita, Yuki; Yamamoto, Misa; Yamamoto, Yoshimi

    2014-01-01

    Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed. PMID:25045530

  2. Oral Administration of (S)-Allyl-l-Cysteine and Aged Garlic Extract to Rats: Determination of Metabolites and Their Pharmacokinetics.

    PubMed

    Park, Taehoon; Oh, Ju-Hee; Lee, Joo Hyun; Park, Sang Cheol; Jang, Young Pyo; Lee, Young-Joo

    2017-11-01

    ( S )-Allyl-l-cysteine is the major bioactive compound in garlic. ( S )-Allyl-l-cysteine is metabolized to ( S )-allyl-l-cysteine sulfoxide, N -acetyl-( S )-allyl-l-cysteine, and N -acetyl-( S )-allyl-l-cysteine sulfoxide after oral administration. An accurate LC-MS/MS method was developed and validated for the simultaneous quantification of ( S )-allyl-l-cysteine and its metabolites in rat plasma, and the feasibility of using it in pharmacokinetic studies was tested. The analytes were quantified by multiple reaction monitoring using an atmospheric pressure ionization mass spectrometer. Because significant quantitative interference was observed between ( S )-allyl-l-cysteine and N -acetyl-( S )-allyl-l-cysteine as a result of the decomposition of N -acetyl-( S )-allyl-l-cysteine at the detector source, chromatographic separation was required to discriminate ( S )-allyl-l-cysteine and its metabolites on a reversed-phase C 18 analytical column with a gradient mobile phase consisting of 0.1% formic acid and acetonitrile. The calibration curves of ( S )-allyl-l-cysteine, ( S )-allyl-l-cysteine sulfoxide, N -acetyl-( S )-allyl-l-cysteine, and N -acetyl-( S )-allyl-l-cysteine sulfoxide were linear over each concentration range, and the lower limits of quantification were 0.1 µg/mL [( S )-allyl-l-cysteine and N -acetyl-( S )-allyl-l-cysteine] and 0.25 µg/mL [( S )-allyl-l-cysteine sulfoxide and N -acetyl-( S )-allyl-l-cysteine sulfoxide]. Acceptable intraday and inter-day precisions and accuracies were obtained at three concentration levels. The method satisfied the regulatory requirements for matrix effects, recovery, and stability. The validated LC-MS/MS method was successfully used to determine the concentration of ( S )-allyl-l-cysteine and its metabolites in rat plasma samples after the administration of ( S )-allyl-l-cysteine or aged garlic extract. Georg Thieme Verlag KG Stuttgart · New York.

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

    PubMed Central

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

    2015-01-01

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

  4. Influence of cysteine doping on photoluminescence intensity from semiconducting single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Kurnosov, N. V.; Leontiev, V. S.; Linnik, A. S.; Karachevtsev, V. A.

    2015-03-01

    Photoluminescence (PL) from semiconducting single-walled carbon nanotubes can be applied for detection of cysteine. It is shown that cysteine doping (from 10-8 to 10-3 M) into aqueous suspension of nanotubes with adsorbed DNA leads to increase of PL intensity. The PL intensity was enhanced by 27% at 10-3 M cysteine concentration in suspension. Most likely, the PL intensity increases due to the passivation of p-defects on the nanotube by the cysteine containing reactive thiol group. The effect of doping with other amino acids without this group (methionine, serine, aspartic acid, lysine, proline) on the PL intensity is essentially weaker.

  5. Formation of Hg(II) tetrathiolate complexes with cysteine at neutral pH

    DOE PAGES

    Warner, Thomas; Jalilehvand, Farideh

    2016-01-04

    Mercury(II) ions precipitate from aqueous cysteine (H 2Cys) solutions containing H 2Cys/Hg(II) mole ratio ≥ 2.0 as Hg( S-HCys) 2. In absence of additional cysteine, the precipitate dissolves at pH ~12 with the [Hg( S, N-Cys) 2] 2- complex dominating. With excess cysteine (H 2Cys/Hg(II) mole ratio ≥ 4.0), higher complexes form and the precipitate dissolves at lower pH values. Previously, we found that tetrathiolate [Hg( S-Cys) 4] 6- complexes form at pH = 11.0; in this work we extend the investigation to pH values of physiological interest. We examined two series of Hg(II)-cysteine solutions in which C Hg(II) variedmore » between 8 – 9 mM and 80 – 100 mM, respectively, with H 2Cys/Hg(II) mole ratios from 4 to ~20. The solutions were prepared in the pH range 7.1 – 8.8, at the pH at which the initial Hg( S-HCys) 2 precipitate dissolved. The variations in the Hg(II) speciation were followed by 199Hg NMR, X-ray absorption and Raman spectroscopic techniques. Our results show that in the dilute solutions (C Hg(II) = 8 – 9 mM), mixtures of di-, tri- (major) and tetrathiolate complexes exist at moderate cysteine excess (C H2Cys ~ 0.16 M) at pH 7.1. In the more concentrated solutions (C Hg(II) = 80 – 100 mM) with high cysteine excess (C H2Cys > 0.9 M), tetrathiolate [Hg( S-cysteinate) 4] m-6 ( m = 0 – 4) complexes dominate in the pH range 7.3 – 7.8, with lower charge than for the [Hg( S-Cys) 4] 6- complex due to protonation of some ( m) of the amino groups of the coordinated cysteine ligands. In conclusion, the results of this investigation could provide a key to the mechanism of biosorption and accumulation of Hg(II) ions in biological / environmental systems.« less

  6. L-Cysteine inhibits root elongation through auxin/PLETHORA and SCR/SHR pathway in Arabidopsis thaliana.

    PubMed

    Wang, Zhen; Mao, Jie-Li; Zhao, Ying-Jun; Li, Chuan-You; Xiang, Cheng-Bin

    2015-02-01

    L-Cysteine plays a prominent role in sulfur metabolism of plants. However, its role in root development is largely unknown. Here, we report that L-cysteine reduces primary root growth in a dosage-dependent manner. Elevating cellular L-cysteine level by exposing Arabidopsis thaliana seedlings to high L-cysteine, buthionine sulphoximine, or O-acetylserine leads to altered auxin maximum in root tips, the expression of quiescent center cell marker as well as the decrease of the auxin carriers PIN1, PIN2, PIN3, and PIN7 of primary roots. We also show that high L-cysteine significantly reduces the protein level of two sets of stem cell specific transcription factors PLETHORA1/2 and SCR/SHR. However, L-cysteine does not downregulate the transcript level of PINs, PLTs, or SCR/SHR, suggesting that an uncharacterized post-transcriptional mechanism may regulate the accumulation of PIN, PLT, and SCR/SHR proteins and auxin transport in the root tips. These results suggest that endogenous L-cysteine level acts to maintain root stem cell niche by regulating basal- and auxin-induced expression of PLT1/2 and SCR/SHR. L-Cysteine may serve as a link between sulfate assimilation and auxin in regulating root growth. © 2014 Institute of Botany, Chinese Academy of Sciences.

  7. Cysteine reacts to form blue-green pigments with thiosulfinates obtained from garlic (Allium sativum L.).

    PubMed

    Shin, Young Keum; Kyung, Kyu Hang

    2014-01-01

    Cysteine was found to form pigments with garlic thiosulfinates in this investigation, in contrast to previous reports. Pigments were formed only when the molar concentration ratios of cysteine to total thiosulfinates were smaller than 2:1. Cysteine does not form pigments with thiosulfinates in the same manner as other pigment-forming amino compounds because it has a sulfhydryl (SH) group. A colour reaction of cysteine with thiosulfinates is proposed where colourless disulphide-type S-alk(en)yl mercaptocysteines (SAMCs) are formed first by the SH-involved reaction between cysteine and thiosulfinates, and then SAMCs react with residual thiosulfinates to form pigments. When the cysteine to total thiosulfinate molar concentration ratio was 2:1 or greater, total thiosulfinates were consumed to form SAMCs without leaving any thiosulfinates remaining available for the following colour reactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Efficient Site-Specific Labeling of Proteins via Cysteines

    PubMed Central

    Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

    2011-01-01

    Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

  9. Efficient site-specific labeling of proteins via cysteines.

    PubMed

    Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

    2008-03-01

    Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

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

    PubMed

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

    2015-09-24

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

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

    PubMed Central

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

    2015-01-01

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. PMID:26400108

  12. Dealing with the sulfur part of cysteine: four enzymatic steps degrade l-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.

    PubMed

    Höfler, Saskia; Lorenz, Christin; Busch, Tjorven; Brinkkötter, Mascha; Tohge, Takayuki; Fernie, Alisdair R; Braun, Hans-Peter; Hildebrandt, Tatjana M

    2016-07-01

    Amino acid catabolism is essential for adjusting pool sizes of free amino acids and takes part in energy production as well as nutrient remobilization. The carbon skeletons are generally converted to precursors or intermediates of the tricarboxylic acid cycle. In the case of cysteine, the reduced sulfur derived from the thiol group also has to be oxidized in order to prevent accumulation to toxic concentrations. Here we present a mitochondrial sulfur catabolic pathway catalyzing the complete oxidation of l-cysteine to pyruvate and thiosulfate. After transamination to 3-mercaptopyruvate, the sulfhydryl group from l-cysteine is transferred to glutathione by sulfurtransferase 1 and oxidized to sulfite by the sulfur dioxygenase ETHE1. Sulfite is then converted to thiosulfate by addition of a second persulfide group by sulfurtransferase 1. This pathway is most relevant during early embryo development and for vegetative growth under light-limiting conditions. Characterization of a double mutant produced from Arabidopsis thaliana T-DNA insertion lines for ETHE1 and sulfurtransferase 1 revealed that an intermediate of the ETHE1 dependent pathway, most likely a persulfide, interferes with amino acid catabolism and induces early senescence. © 2016 Scandinavian Plant Physiology Society.

  13. Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore

    PubMed Central

    Seyedmohammad, Saeed; Fuentealba, Natalia Alveal; Marriott, Robert A.J.; Goetze, Tom A.; Edwardson, J. Michael; Barrera, Nelson P.; Venter, Henrietta

    2016-01-01

    Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel. PMID:26934982

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

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

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

  15. Cysteine Cathepsins Activate ELR Chemokines and Inactivate Non-ELR Chemokines*

    PubMed Central

    Repnik, Urska; Starr, Amanda E.; Overall, Christopher M.; Turk, Boris

    2015-01-01

    Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9–12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca2+ mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9–12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation. PMID:25833952

  16. Crystal structure and activity studies of the C11 cysteine peptidase from Parabacteroides merdae in the human gut microbiome

    DOE PAGES

    McLuskey, Karen; Grewal, Jaspreet S.; Das, Debanu; ...

    2016-03-03

    Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other familiesmore » in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys 147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys 147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca 2+ for activity. Altogether, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.« less

  17. Enhancement of L-cysteine production by disruption of yciW in Escherichia coli.

    PubMed

    Kawano, Yusuke; Ohtsu, Iwao; Takumi, Kazuhiro; Tamakoshi, Ai; Nonaka, Gen; Funahashi, Eri; Ihara, Masaki; Takagi, Hiroshi

    2015-02-01

    Using in silico analysis, the yciW gene of Escherichia coli was identified as a novel L-cysteine regulon that may be regulated by the transcriptional activator CysB for sulfur metabolic genes. We found that overexpression of yciW conferred tolerance to L-cysteine, but disruption of yciW increased L-cysteine production in E. coli. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

    PubMed

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

    2003-08-29

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

  19. Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice.

    PubMed

    Niewiadomski, Julie; Zhou, James Q; Roman, Heather B; Liu, Xiaojing; Hirschberger, Lawrence L; Locasale, Jason W; Stipanuk, Martha H

    2016-01-01

    To gain further insights into the effects of elevated cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in cysteine dioxygenase (Cdo1)-null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated cysteine levels with greater weight gain and with elevated hepatic Scd1 expression are also seen in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggests impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated cysteine levels with excess H2 S production and impairments in energy metabolism suggest that H2 S signaling could be involved. © 2016 New York Academy of Sciences.

  20. [Plant signaling peptides. Cysteine-rich peptides].

    PubMed

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation.

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

    PubMed

    Takemoto, Yumi

    2014-12-01

    The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

  3. Dietary L-cysteine improves the antioxidative potential and lipid metabolism in rats fed a normal diet.

    PubMed

    Lee, Seulki; Han, Kyu-Ho; Nakamura, Yumi; Kawakami, Sakura; Shimada, Ken-ichiro; Hayakawa, Touru; Onoue, Hirotake; Fukushima, Michihiro

    2013-01-01

    L-cysteine works as a precursor of the antioxidant, glutathione. We investigated the effects of L-cysteine (1% and 2%) on lipid metabolism and the antioxidative system in rats fed a normal diet. Administering L-cysteine dependently decreased the food intake, fat mass weight and body weight dose. Dietary L-cysteine also decreased the triglyceride levels in the serum and liver. However, there were no significant differences in the hepatic TBARS and glutathione (GSH) levels among the groups. The activities of catalase and glutathione reductase in the rats receiving 2% L-cysteine were significantly higher (p<0.05) than in the control rats. These results suggest that dietary L-cysteine dose-dependently affected the antioxidative enzyme activities, and the lipid levels in the serum and liver which might be related to the reduced food intake.

  4. Acetylation of aromatic cysteine conjugates by recombinant human N-acetyltransferase 8.

    PubMed

    Deol, Reema; Josephy, P David

    2017-03-01

    1. The mercapturic acid (MA) pathway is a metabolic route for the processing of glutathione conjugates to MA (N-acetylcysteine conjugates). An N-acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a MA, which is excreted in the urine. We expressed human NAT8 in HEK293T cells and developed an HPLC-MS method for the quantitation of the S-aryl-substituted cysteine conjugates and their MA. 2. We measured the activity of the enzyme for acetylation of benzyl-, 4-nitrobenzyl-, and 1-menaphthylcysteine substrates. 3. NAT8 catalyzed the acetylation of all three cysteine conjugates with similar Michaelis-Menten kinetics.

  5. Beyond cysteine: recent developments in the area of targeted covalent inhibition.

    PubMed

    Mukherjee, Herschel; Grimster, Neil P

    2018-05-29

    Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion.

    PubMed

    Santos, André L S; d'Avila-Levy, Claudia M; Dias, Felipe A; Ribeiro, Rachel O; Pereira, Fernanda M; Elias, Camila G R; Souto-Padrón, Thaïs; Lopes, Angela H C S; Alviano, Celuta S; Branquinha, Marta H; Soares, Rosangela M A

    2006-01-01

    In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

  7. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    NASA Astrophysics Data System (ADS)

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

    2017-05-01

    The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

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

    PubMed

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

    2008-08-01

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

  9. Cd–cysteine precursor nanowire templated microwave-assisted transformation route to CdS nanotubes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Xiao-Lin, E-mail: liu_x_l@sina.cn; Zhu, Ying-Jie; Zhang, Qian

    2012-12-15

    Graphical abstract: Cadmium sulfide polycrystalline nanotubes have been successfully synthesized by microwave-assisted transformation method using Cd–cysteine precursor nanowires as the source material and template in ethylene glycol at 160 °C or ethanol at 60 °C. Display Omitted Highlights: ► Cd–cysteine precursor nanowires were successfully synthesized in alkaline solution. ► CdS nanotubes were prepared by templated microwave-assisted transformation method. ► CdS nanotubes can well duplicate the size and morphology of precursor nanowires. ► This method has the advantages of the simplicity and low cost. -- Abstract: We report the Cd–cysteine precursor nanowire templated microwave-assisted transformation route to CdS nanotubes. In thismore » method, the Cd–cysteine precursor nanowires are synthesized using CdCl{sub 2}·2.5H{sub 2}O, L-cysteine and ethanolamine in water at room temperature. The Cd–cysteine precursor nanowires are used as the source material and template for the subsequent preparation of CdS nanotubes by a microwave-assisted transformation method using ethylene glycol or ethanol as the solvent. This method has the advantages of the simplicity and low cost, and may be extended to the synthesis of nanotubes of other compounds. The products are characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM).« less

  10. Aged garlic extract and S-allyl cysteine prevent formation of advanced glycation endproducts.

    PubMed

    Ahmad, Muhammad Saeed; Pischetsrieder, Monika; Ahmed, Nessar

    2007-04-30

    Hyperglycaemia causes increased protein glycation and the formation of advanced glycation endproducts which underlie the complications of diabetes and ageing. Glycation is accompanied by metal-catalysed oxidation of glucose and Amadori products to form free radicals capable of protein fragmentation. Aged garlic extract is a potent antioxidant with established lipid-lowering effects attributed largely to a key ingredient called S-allyl cysteine. This study investigated the ability of aged garlic extract and S-allyl cysteine to inhibit advanced glycation in vitro. Bovine serum albumin (BSA) was glycated in the presence of Cu(2+) ions and different concentrations of aged garlic extract and protein fragmentation was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Lysozyme was glycated by glucose or methylglyoxal in the presence of different concentrations of aged garlic extract or S-allyl cysteine with subsequent analysis of glycation-derived crosslinking using SDS-PAGE. Amadori-rich protein was prepared by dialysing lysozyme that had been glycated by ribose for 24 h. This ribated lysozyme was reincubated and the effects of aged garlic extract, S-allyl cysteine and pyridoxamine on glycation-induced crosslinking was monitored. Aged garlic extract inhibited metal-catalysed protein fragmentation. Both aged garlic extract and S-allyl cysteine inhibited formation of glucose and methylglyoxal derived advanced glycation endproducts and showed potent Amadorin activity when compared to pyridoxamine. S-allyl cysteine inhibited formation of carboxymethyllysine (CML), a non-crosslinked advanced glycation endproduct derived from oxidative processes. Further studies are required to assess whether aged garlic extract and S-allyl cysteine can protect against the harmful effects of glycation and free radicals in diabetes and ageing.

  11. The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase*

    PubMed Central

    Tchesnokov, Egor P.; Fellner, Matthias; Siakkou, Eleni; Kleffmann, Torsten; Martin, Lois W.; Aloi, Sekotilani; Lamont, Iain L.; Wilbanks, Sigurd M.; Jameson, Guy N. L.

    2015-01-01

    Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site. PMID:26272617

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2017-08-01

    l-Cysteine is deliberately added to various flour types since l-Cysteine has enabled favorable baking conditions such as low viscosity, increased elasticity and rise during baking. In Turkey, usage of l-Cysteine as a food additive isn't allowed in wheat flour according to the Turkish Food Codex Regulation on food additives. There is an urgent need for effective methods to detect l-Cysteine in wheat flour. In this study, for the first time, a new, rapid, effective, non-destructive and cost-effective method was developed for detection of l-Cysteine in wheat flour using Raman microscopy. Detection of l-Cysteine in wheat flour was accomplished successfully using Raman microscopy combined chemometrics of PCA (Principal Component Analysis) and HCA (Hierarchical Cluster Analysis). In this work, 500-2000cm -1 spectral range (fingerprint region) was determined to perform PCA and HCA analysis. l-Cysteine and l-Cystine were determined with detection limit of 0.125% (w/w) in different wheat flour samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2011-04-25

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

  16. Interdependence of the rad50 hook and globular domain functions.

    PubMed

    Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

    2015-02-05

    Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2017-05-01

    Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34 S-labeled L-cysteine from O-acetyl-L-serine and 34 S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur ( 34 S) and nitrogen ( 15 N) atoms was also achieved by performing enzyme reactions with 15 N-labeled L-serine, acetyl-CoA, and 34 S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34 S-labeled N-acetyl-L-cysteine (NAC) by incubating 34 S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34 S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology. Copyright © 2017 Elsevier Inc

  18. Optical Absorption and Electric Resistivity of an l-Cysteine Film

    NASA Astrophysics Data System (ADS)

    Kamada, Masao; Hideshima, Takuya; Azuma, Junpei; Yamamoto, Isamu; Imamura, Masaki; Takahashi, Kazutoshi

    2016-12-01

    The optical and electric properties of an l-cysteine film have been investigated to understand its applicability to bioelectronics. The fundamental absorption is the allowed transition having the threshold at 5.8 eV and the absorption is due to the charge-transfer type transition from sulfur-3sp to oxygen-2p and/or carbon-2p states, while absorptions more than 9 eV can be explained with intra-atomic transitions in the functional groups. The electric resistivity is 2.0 × 104 Ω m at room temperature and increases as the sample temperature decreases. The results indicate that the l-cysteine film is a p-type semiconductor showing the hole conduction caused by the sulfur-3sp occupied states and unknown impurity or defect states as acceptors. The electron affinity of the l-cysteine film is derived as ≦-0.3 eV.

  19. Electronic structure of transition metal-cysteine complexes from X-ray absorption spectroscopy.

    PubMed

    Leung, Bonnie O; Jalilehvand, Farideh; Szilagyi, Robert K

    2008-04-17

    The electronic structures of HgII, NiII, CrIII, and MoV complexes with cysteine were investigated by sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy and density functional theory. The covalency in the metal-sulfur bond was determined by analyzing the intensities of the electric-dipole allowed pre-edge features appearing in the XANES spectra below the ionization threshold. Because of the well-defined structures of the selected cysteine complexes, the current work provides a reference set for further sulfur K-edge XAS studies of bioinorganic active sites with transition metal-sulfur bonds from cysteine residues as well as more complex coordination compounds with thiolate ligands.

  20. L-Cysteine Production in Escherichia coli Based on Rational Metabolic Engineering and Modular Strategy.

    PubMed

    Liu, Han; Fang, Guochen; Wu, Hui; Li, Zhimin; Ye, Qin

    2018-05-01

    L-cysteine is an amino acid with important physiological functions and has a wide range of applications in medicine, food, animal feed, and cosmetics industry. In this study, the L-cysteine synthesis in Escherichia coliEscherichia coli is divided into four modules: the transport module, sulfur module, precursor module, and degradation module. The engineered strain LH03 (overexpression of the feedback-insensitive cysE and the exporter ydeD in JM109) accumulated 45.8 mg L -1 of L-cysteine in 48 hr with yield of 0.4% g/g glucose. Further modifications of strains and culture conditions which based on the rational metabolic engineering and modular strategy improved the L-cysteine biosynthesis significantly. The engineered strain LH06 (with additional overexpression of serA, serC, and serB and double mutant of tnaA and sdaA in LH03) produced 620.9 mg L -1 of L-cysteine with yield of 6.0% g/g glucose, which increased the production by 12 times and the yield by 14 times more than those of LH03 in the original condition. In fed-batch fermentation performed in a 5-L reactor, the concentration of L-cysteine achieved 5.1 g L -1 in 32 hr. This work demonstrates that the combination of rational metabolic engineering and module strategy is a promising approach for increasing the L-cysteine production in E. coli. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Evolutionary acquisition of cysteines determines FOXO paralog-specific redox signaling.

    PubMed

    Putker, Marrit; Vos, Harmjan R; van Dorenmalen, Kim; de Ruiter, Hesther; Duran, Ana G; Snel, Berend; Burgering, Boudewijn M T; Vermeulen, Michiel; Dansen, Tobias B

    2015-01-01

    Reduction-oxidation (redox) signaling, the translation of an oxidative intracellular environment into a cellular response, is mediated by the reversible oxidation of specific cysteine thiols. The latter can result in disulfide formation between protein hetero- or homodimers that alter protein function until the local cellular redox environment has returned to the basal state. We have previously shown that this mechanism promotes the nuclear localization and activity of the Forkhead Box O4 (FOXO4) transcription factor. In this study, we sought to investigate whether redox signaling differentially controls the human FOXO3 and FOXO4 paralogs. We present evidence that FOXO3 and FOXO4 have acquired paralog-specific cysteines throughout vertebrate evolution. Using a proteome-wide screen, we identified previously unknown redox-dependent FOXO3 interaction partners. The nuclear import receptors Importin-7 (IPO7) and Importin-8 (IPO8) form a disulfide-dependent heterodimer with FOXO3, which is required for its reactive oxygen species-induced nuclear translocation. FOXO4 does not interact with IPO7 or IPO8. IPO7 and IPO8 control the nuclear import of FOXO3, but not FOXO4, in a redox-sensitive and disulfide-dependent manner. Our findings suggest that evolutionary acquisition of cysteines has contributed to regulatory divergence of FOXO paralogs, and that phylogenetic analysis can aid in the identification of cysteines involved in redox signaling.

  2. Human Lamin B Contains a Farnesylated Cysteine Residue*

    PubMed Central

    Farnsworth, Christopher C.; Wolda, Sharon L.; Gelb, Michael H.; Glomset, John A.

    2012-01-01

    We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of link-age to the mevalonic acid derivative, and established the derivative’s structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H] mevalonic acid or [35S] cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B. PMID:2684976

  3. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    DOE PAGES

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; ...

    2017-05-17

    Here, the Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combiningmore » the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.« less

  4. [Protective Effect of S-isopentenyl-L-cysteine against DNA Damage in Irradiated Mice].

    PubMed

    Zheng, Qi-sheng; Yu, Guang-yun; He, Xin; Jiang, Ming; Chu, Xiao-fei; Zhao, Shu-yi; Fan, Sai-jun; Liu, Pei-xun

    2015-10-01

    To evaluate the protective effect of S-isopentenyl-L-cysteine,a new cysteine derivative,on DNA damage induced by radiation by using acute radiation injury animal models. Forty ICR mice were randomly divided into five groups:the control group,1.0Gy gamma irradiation group,1.0Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,7.2Gy gamma irradiation group,and 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,with 8 mice in each group.The comet assay and bone marrow polychromatic micronucleus experiments were performed to evaluate the double-strand DNA breaks in ICR mice exposed to 1.0 and 7.2Gy gamma-ray, respectively. The tail DNA percentage,tail length,tail moment,and olive tail moment of peripheral blood lymphocytes in 7.2Gy gamma irradiation group were significantly higher than that of the control group (P<0.01).And it was also observed that above experimental indexes of 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group was significantly less than that of 7.2Gy gamma irradiation group (P<0.05). In addition,the micronucleus rate of 1.0Gy gamma irradiation group and 7.2Gy gamma irradiation group were both significantly higher than in the control group (P<0.01). In addition,in mice given S-isopentenyl-L-cysteine before irradiation,the micronucleus rate of ICR mice exposed to 1.0 and 7.2Gy gamma-ray decreased from (39.5000 ± 3.3141)‰ to (28.1667±4.1345)‰ (P=0.033) and from (76.5000 ± 4.6242)‰ to (22.8333 ± 3.6553)‰(P=0.000),respectively. The bone marrow polychromatic micronucleus experiment indicated that the value of polychromatic erythrocyte (PCE)/normochromatic erythrocyte(NCE) of ICR mice exposed to 1.0 and 7.2Gy gamma-ray was less than the control group(P<0.05). Meanwhile,after irradiating by certain dose,the value of PCE/NCE in mice given S-isopentenyl-L-cysteine before irradiation was significantly higher than the corresponding groups (P<0.05). S-isopentenyl-L-cysteine has a good protective

  5. Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases.

    PubMed

    Hossain, Md Israil; Iwasaki, Hirohide; Okochi, Yoshifumi; Chahine, Mohamed; Higashijima, Shinichi; Nagayama, Kuniaki; Okamura, Yasushi

    2008-06-27

    The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

  6. Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase.

    PubMed

    Xu, Lisheng; Wang, Zhiyuan; Mao, Pingting; Liu, Junzhong; Zhang, Hongjuan; Liu, Qian; Jiao, Qing-Cai

    2013-04-01

    An economical method for production of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater (KHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative KHW utilization strategy to synthesize S-phenyl-L-cysteine. Tryptophan synthase could efficiently convert L-serine contained in KHW to S-phenyl-L-cysteine at pH 9.0, 40°C and Trion X-100 of 0.02%. In a scale up study, L-serine conversion rate reach 97.1% with a final S-phenyl-L-cysteine concentration of 38.6 g l(-1). Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    PubMed

    Qi, Peng; Zhang, Dun; Wan, Yi

    2014-11-01

    Sulfate-reducing bacteria (SRB) have been extensively studied in corrosion and environmental science. However, fast enumeration of SRB population is still a difficult task. This work presents a novel specific SRB detection method based on inhibition of cysteine protease activity. The hydrolytic activity of cysteine protease was inhibited by taking advantage of sulfide, the characteristic metabolic product of SRB, to attack active cysteine thiol group in cysteine protease catalytic sites. The active thiol S-sulfhydration process could be used for SRB detection, since the amount of sulfide accumulated in culture medium was highly related with initial bacterial concentration. The working conditions of cysteine protease have been optimized to obtain better detection capability, and the SRB detection performances have been evaluated in this work. The proposed SRB detection method based on inhibition of cysteine protease activity avoided the use of biological recognition elements. In addition, compared with the widely used most probable number (MPN) method which would take up to at least 15days to accomplish whole detection process, the method based on inhibition of papain activity could detect SRB in 2 days, with a detection limit of 5.21×10(2) cfu mL(-1). The detection time for SRB population quantitative analysis was greatly shortened. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome.

    PubMed

    Song, Jiangning; Wang, Minglei; Burrage, Kevin

    2006-07-21

    High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.

  9. Guided Act and Feel Indonesia (GAF-ID) - Internet-based behavioral activation intervention for depression in Indonesia: study protocol for a randomized controlled trial.

    PubMed

    Arjadi, Retha; Nauta, Maaike H; Scholte, Willem F; Hollon, Steven D; Chowdhary, Neerja; Suryani, Angela O; Bockting, Claudi L H

    2016-09-17

    Depression is a leading cause of disease burden across the world. However, in low-middle income countries (LMICs), access to mental health services is severely limited because of the insufficient number of mental health professionals available. The WHO initiated the Mental Health Gap Action Program (mhGAP) aiming to provide a coherent strategy for closing the gap between what is urgently needed and what is available in LMICs. Internet-based treatment is a promising strategy that can be made available to a large number of people now that Internet access is increasing rapidly throughout the world. The present study will investigate whether such an Internet-based treatment for depression is effective in Indonesia. An Internet-based behavioral activation treatment, with support by lay counselors who will provide online feedback on the assignments and supportive phone contact to encourage participants to work in the program (Guided Act and Feel Indonesia/GAF-ID), is compared to an online-delivered minimal psychoeducation without any support (psychoeducation/PE). Initial assessment for inclusion is based on a Patient Health Questionnaire-9 (PHQ-9) score of at least 10 and meeting criteria for major depressive disorder or persistent depressive disorder as assessed using the Structured Clinical Interview for DSM-5 (SCID-5). Participants with depression (N = 312) will be recruited and randomly assigned to GAF-ID or PE. Overall assessments will be done at baseline, post intervention (10 weeks from baseline) and follow-ups (3 months and 6 months from baseline). The primary outcome is the reduction of depression symptoms as measured by the PHQ-9 after 10 weeks from baseline. To our knowledge, this is the first study in Indonesia that examines the effectiveness of an Internet-based intervention for depression in a randomized controlled trial. The hope is that it can serve as a starting point for bridging the mental health gap in Indonesia and other LMICs. Nederlands

  10. A novel electrochemical sensor based on metal-organic framework for electro-catalytic oxidation of L-cysteine.

    PubMed

    Hosseini, Hadi; Ahmar, Hamid; Dehghani, Ali; Bagheri, Akbar; Tadjarodi, Azadeh; Fakhari, Ali Reza

    2013-04-15

    A novel electrochemical sensor based on Au-SH-SiO₂ nanoparticles supported on metal-organic framework (Au-SH-SiO₂@Cu-MOF) has been developed for electrocatalytic oxidation and determination of L-cysteine. The Au-SH-SiO₂@Cu-MOF was characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction and cyclic voltammetry. The electrochemical behavior of L-cysteine at the Au-SH-SiO₂@Cu-MOF was investigated by cyclic voltammetry. The Au-SH-SiO₂@Cu-MOF showed a very efficient electrocatalytic activity for the oxidation of L-cysteine in 0.1 M phosphate buffer solution (pH 5.0). The oxidation overpotentials of L-cysteine decreased significantly and their oxidation peak currents increased dramatically at Au-SH-SiO₂@Cu-MOF. The potential utility of the sensor was demonstrated by applying it to the analytical determination of L-cysteine concentration. The results showed that the electrocatalytic current increased linearly with the L-cysteine concentration in the range of 0.02-300 μM and the detection limit was 0.008 μM. Finally, the sensor was applied to determine L-cysteine in water and biological samples. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed Central

    Hernández, Hilda M.; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis. PMID:25348828

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

    PubMed Central

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

    2015-01-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

  13. Pressor response to L-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons.

    PubMed

    Takemoto, Yumi

    2013-03-01

    The sulfur-containing non-essential amino acid L-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to L-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected D-cysteine produced no cardiovascular changes, while L-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of L-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of L-cysteine-injected rats than those injected with D-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of L-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of L-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.

  14. Crystallization and preliminary X-ray diffraction studies of the cysteine protease ervatamin A from Ervatamia coronaria

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chakraborty, Sibani; Biswas, Sampa; Chakrabarti, Chandana

    2005-06-01

    Ervatamin A is a papain-family cysteine protease with high activity and stability. It has been isolated and purified from the latex of the medicinal flowering plant E. coronaria and crystallized by the vapour-diffusion technique. Crystals diffracted to 2.1 Å and the structure was solved by molecular replacement. The ervatamins are highly stable cysteine proteases that are present in the latex of the medicinal plant Ervatamia coronaria and belong to the papain family, members of which share similar amino-acid sequences and also a similar fold comprising two domains. Ervatamin A from this family, a highly active protease compared with others frommore » the same source, has been purified to homogeneity by ion-exchange chromatography and crystallized by the vapour-diffusion method. Needle-shaped crystals of ervatamin A diffract to 2.1 Å resolution and belong to space group C222{sub 1}, with unit-cell parameters a = 31.10, b = 144.17, c = 108.61 Å. The solvent content using an ervatamin A molecular weight of 27.6 kDa is 43.9%, with a V{sub M} value of 2.19 Å{sup 3} Da{sup −1} assuming one protein molecule in the asymmetric unit. A molecular-replacement solution has been found using the structure of ervatamin C as a search model.« less

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

    PubMed Central

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

    2015-01-01

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

  16. DNA sequence transfer between two high-cysteine chorion gene families in the silkmoth Bombyx mori.

    PubMed Central

    Iatrou, K; Tsitilou, S G; Kafatos, F C

    1984-01-01

    We have previously shown that one type of high-cysteine silkmoth chorion protein (Hc-A) has evolved from the A family of chorion proteins by radical modifications of the NH2-terminal and COOH-terminal polypeptide arms: most of the arm sequences have been deleted, while short cysteine- and glycine-containing repeats have expanded into long arrays. Strikingly similar modifications of the arms have led to the evolution of a second type of high-cysteine protein (Hc-B) from the B family of chorion proteins. It appears that the parallel evolution of these high-cysteine-encoding gene families has not been entirely independent: examination of 3' untranslated regions shows evidence of information transfer between the two families. PMID:6589605

  17. L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

    PubMed

    Nakatsu, Daiki; Horiuchi, Yuta; Kano, Fumi; Noguchi, Yoshiyuki; Sugawara, Taichi; Takamoto, Iseki; Kubota, Naoto; Kadowaki, Takashi; Murata, Masayuki

    2015-03-10

    Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

  18. A high-performance electrochemical sensor for biologically meaningful l-cysteine based on a new nanostructured l-cysteine electrocatalyst.

    PubMed

    Cao, Fei; Huang, Yikun; Wang, Fei; Kwak, Dongwook; Dong, Qiuchen; Song, Donghui; Zeng, Jie; Lei, Yu

    2018-08-17

    As a new class of l-cysteine electrocatalyst explored in this study, Au/CeO 2 composite nanofibers (CNFs) were employed to modify the screen printed carbon electrode (SPCE) to fabricate a novel l-cysteine (CySH) electrochemical sensor with high performance. Its electrochemical behavior and the roles of Au and CeO 2 in the composite toward electro-oxidation of CySH were elucidated and demonstrated using cyclic voltammetry and amperometry techniques for the first time through the comparison with pure CeO 2 NFs. More specifically, the Au/CeO 2 CNFs modified SPCE possessed greatly enhanced electrocatalytic activity toward CySH oxidation. An ultra high sensitivity of 321 μA mM -1 cm -2 was obtained, which is almost 2.7 times higher than that of pure CeO 2 NFs, revealing that the presence of Au imposed an important influence on the electrocatalytic activity toward CySH. The detailed reasons on such high performance were also discussed. In addition, the as-prepared sensor showed a low detection limit of 10 nM (signal to noise ratio of 3), a wide linear range up to 200 μM for the determination of CySH, an outstanding reproducibility and good long-term stability, as well as an excellent selectivity against common interferents such as tryptophan, tyrosine, methionine, ascorbic acid and uric acid. All these features indicate that the Au/CeO 2 composite nanofiber is a promising candidate as a new class of l-cysteine electrocatalyst in the development of highly sensitive and selective CySH electrochemical sensor. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    PubMed Central

    2012-01-01

    Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role

  20. Molecular and bioinformatical characterization of a novel superfamily of cysteine-rich peptides from arthropods.

    PubMed

    Zeng, Xian-Chun; Nie, Yao; Luo, Xuesong; Wu, Shifen; Shi, Wanxia; Zhang, Lei; Liu, Yichen; Cao, Hanjun; Yang, Ye; Zhou, Jianping

    2013-03-01

    The full-length cDNA sequences of two novel cysteine-rich peptides (referred to as HsVx1 and MmKTx1) were obtained from scorpions. The two peptides represent a novel class of cysteine-rich peptides with a unique cysteine pattern. The genomic sequence of HsVx1 is composed of three exons interrupted by two introns that are localized in the mature peptide encoding region and inserted in phase 1 and phase 2, respectively. Such a genomic organization markedly differs from those of other peptides from scorpions described previously. Genome-wide search for the orthologs of HsVx1 identified 59 novel cysteine-rich peptides from arthropods. These peptides share a consistent cysteine pattern with HsVx1. Genomic comparison revealed extensive intron length differences and intronic number and position polymorphisms among the genes of these peptides. Further analysis identified 30 cases of intron sliding, 1 case of intron gain and 22 cases of intron loss occurred with the genes of the HsVx1 and HsVx1-like peptides. It is interesting to see that three HsVx1-like peptides XP_001658928, XP_001658929 and XP_001658930 were derived from a single gene (XP gene): the former two were generated from alternative splicing; the third one was encoded by a DNA region in the reverse complementary strand of the third intron of the XP gene. These findings strongly suggest that the genes of these cysteine-rich peptides were evolved by intron sliding, intron gain/loss, gene recombination and alternative splicing events in response to selective forces without changing their cysteine pattern. The evolution of these genes is dominated by intron sliding and intron loss. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine.

    PubMed

    Basic, Amina; Blomqvist, Madeleine; Dahlén, Gunnar; Svensäter, Gunnel

    2017-03-14

    Hydrogen sulfide (H 2 S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H 2 S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H 2 S-producing enzymes; Sulfide from H 2 S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H 2 S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H 2 S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. Numerous enzymes, identified as cysteine synthase, were involved in the production of H 2 S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H 2 S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin.

  2. Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis.

    PubMed

    Levdikov, Vladimir M; Blagova, Elena; Young, Vicki L; Belitsky, Boris R; Lebedev, Andrey; Sonenshein, Abraham L; Wilkinson, Anthony J

    2017-02-17

    CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (c G MP-stimulated phosphodiesterases, a denylate cyclases, F hlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

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

    2006-12-01

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

  4. Photo-fermentative bacteria aggregation triggered by L-cysteine during hydrogen production

    PubMed Central

    2013-01-01

    Background Hydrogen recovered from organic wastes and solar energy by photo-fermentative bacteria (PFB) has been suggested as a promising bioenergy strategy. However, the use of PFB for hydrogen production generally suffers from a serious biomass washout from photobioreactor, due to poor flocculation of PFB. In the continuous operation, PFB cells cannot be efficiently separated from supernatant and rush out with effluent from reactor continuously, which increased the effluent turbidity, meanwhile led to increases in pollutants. Moreover, to replenish the biomass washout, substrate was continuously utilized for cell growth rather than hydrogen production. Consequently, the poor flocculability not only deteriorated the effluent quality, but also decreased the potential yield of hydrogen from substrate. Therefore, enhancing the flocculability of PFB is urgent necessary to further develop photo-fermentative process. Results Here, we demonstrated that L-cysteine could improve hydrogen production of Rhodopseudomonas faecalis RLD-53, and more importantly, simultaneously trigger remarkable aggregation of PFB. Experiments showed that L-cysteine greatly promoted the production of extracellular polymeric substances, especially secretion of protein containing more disulfide bonds, and help for enhancement stability of floc of PFB. Through formation of disulfide bonds, L-cysteine not only promoted production of EPS, in particular the secretion of protein, but also stabilized the final confirmation of protein in EPS. In addition, the cell surface elements and functional groups, especially surface charged groups, have also been changed by L-cysteine. Consequently, absolute zeta potential reached a minimum value at 1.0 g/l of L-cysteine, which obviously decreased electrostatic repulsion interaction energy based on DLVO theory. Total interaction energy barrier decreased from 389.77 KT at 0.0 g/l of L-cysteine to 127.21 kT at 1.0 g/l. Conclusions Thus, the strain RLD-53 overcame the

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

    PubMed

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

    2016-09-01

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

  6. Chromatographic methods for determination of S-substituted cysteine derivatives--a comparative study.

    PubMed

    Kubec, Roman; Dadáková, Eva

    2009-10-09

    A novel HPLC method for determination of a wide variety of S-substituted cysteine derivatives in Allium species has been developed and validated. This method allows simultaneous separation and quantification of S-alk(en)ylcysteine S-oxides, gamma-glutamyl-S-alk(en)ylcysteines and gamma-glutamyl-S-alk(en)ylcysteine S-oxides in a single run. The procedure is based on extraction of these amino acids and dipeptides by methanol, their derivatization by dansyl chloride and subsequent separation by reversed phase HPLC. The main advantages of the new method are simplicity, excellent stability of derivatives, high sensitivity, specificity and the ability to simultaneously analyze the whole range of S-substituted cysteine derivatives. This method was critically compared with other chromatographic procedures used for quantification of S-substituted cysteine derivatives, namely with two other HPLC methods (derivatization by o-phthaldialdehyde/tert-butylthiol and fluorenylmethyl chloroformate), and with determination by gas chromatography or capillary electrophoresis. Major advantages and drawbacks of these analytical procedures are discussed. Employing these various chromatographic methods, the content and relative proportions of individual S-substituted cysteine derivatives were determined in four most frequently consumed alliaceous vegetables (garlic, onion, shallot, and leek).

  7. L-Cysteine Capped CdSe Quantum Dots Synthesized by Photochemical Route.

    PubMed

    Singh, Avinash; Kunwar, Amit; Rath, M C

    2018-05-01

    L-cysteine capped CdSe quantum dots were synthesized via photochemical route in aqueous solution under UV photo-irradiation. The as grown CdSe quantum dots exhibit broad fluorescence at room temperature. The CdSe quantum dots were found to be formed only through the reactions of the precursors, i.e., Cd(NH3)2+4 and SeSO2-3 with the photochemically generated 1-hydroxy-2-propyl radicals, (CH3)2COH radicals, which are formed through the process of H atom abstraction by the photoexcited acetone from 2-propanol. L-Cysteine was found to act as a suitable capping agent for the CdSe quantum dots and increases their biocompatability. Cytotoxicty effects of these quantum dots were evaluated in Chinese Hamster Ovary (CHO) epithelial cells, indicated a significant lower level for the L-cysteine capped CdSe quantum dots as compare to the bare ones.

  8. The basics of thiols and cysteines in redox biology and chemistry.

    PubMed

    Poole, Leslie B

    2015-03-01

    Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic, or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts to functional sites their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low-molecular-weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes, including signal transduction. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. GAMETOPHYTE DEFECTIVE 1, a putative subunit of RNases P/MRP, is essential for female gametogenesis and male competence in Arabidopsis.

    PubMed

    Wang, Si-Qi; Shi, Dong-Qiao; Long, Yan-Ping; Liu, Jie; Yang, Wei-Cai

    2012-01-01

    RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing) are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1), a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants.

  10. Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.

    PubMed

    Lee, Chia-Fang; Paull, Tanya T; Person, Maria D

    2013-10-04

    Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.

  11. A novel reaction-based fluorescent probe for the detection of cysteine in milk and water samples.

    PubMed

    Wang, Jialin; Wang, Hao; Hao, Yanfeng; Yang, Shaoxiang; Tian, Hongyu; Sun, Baoguo; Liu, Yongguo

    2018-10-01

    A novel fluorescent probe 3'-hydroxy-3-oxo-3H-spiro [isobenzofuran-1,9'-xanthene]-6'-yl-2,4-dinitrobenzenesulfonate (probe 1) was designed and synthesized as a visual sensor for the detection of cysteine levels in milk and water samples. The addition of cysteine to the solution of probe 1 resulted in an increase in fluorescence intensity and color change, from light yellow to yellow-green. The distinct color response indicated that probe 1 could be used as a visual sensor for cysteine. Cysteine can be detected quantitatively at concentrations between 0 and 400 μM and the detection limit of the fluorescence response to the probe was 6.5 μM. This suggests that probe 1 could be used as a signaling tool to determine the cysteine levels in samples, such as milk and water. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Comparison of Visuospatial and Verbal Abilities in First Psychotic Episode of Schizophrenia Spectrum Disorder: Impact on Global Functioning and Quality of Life

    PubMed Central

    Rodriguez, Mabel; Spaniel, Filip; Konradova, Lucie; Sedlakova, Katerina; Dvorska, Karolina; Prajsova, Jitka; Kratochvilova, Zuzana; Levcik, David; Vlcek, Kamil; Fajnerova, Iveta

    2015-01-01

    Objectives: Deficit in visuospatial functions can influence both simple and complex daily life activities. Despite the fact that visuospatial deficit was reported in schizophrenia, research on visuospatial functions as an independent entity is limited. Our study aims to elucidate the impact of visuospatial deficit in comparison with verbal deficit on global functioning and quality of life in the first psychotic episode of schizophrenia spectrum disorder (FES). The significance of clinical symptoms and antipsychotic medication was also studied. Methods: Thirty-six FES patients and a matched group of healthy controls (HC group) were assessed with a neuropsychological battery focused on visuospatial (VIS) and verbal (VERB) functions. Using multiple regression analysis, we evaluated the cumulative effect of VERB and VIS functions, psychiatric symptoms (PANSS) and antipsychotic medication on global functioning (GAF) and quality of life (WHOQOL-BREF) in the FES group. Results: The FES group demonstrated significant impairment both in VIS and VERB cognitive abilities compared to the HC group. Antipsychotic medication did not significantly affect either VIS or VERB functioning. PANSS was not related to cognitive functioning, apart from the Trail Making Test B. In the FES group, the GAF score was significantly affected by the severity of positive symptoms and VERB functioning, explaining together 60% of GAF variability. The severity of negative and positive symptoms affected only the Physical health domain of WHOQOL-BREF. The degree of VERB deficit was associated with both Physical and Psychological health. Although we did not find any relation between VIS functioning, GAF, and WHOQOL-BREF, a paradoxical finding emerged in the Environment quality domain, where a worse quality of the environment was associated with better VIS functioning. Conclusions: Our results suggest that the deficit in VIS functions is an integral part of cognitive deficit in schizophrenia spectrum

  13. Evaluation of the inhibitory effect of N-acetyl-L-cysteine on Babesia and Theileria parasites.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Yokoyama, Naoaki; Igarashi, Ikuo

    2017-08-01

    N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

    PubMed

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

    2015-08-01

    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

  15. Controlling the Chiral Inversion Reaction of the Metallopeptide Ni-Asparagine-Cysteine-Cysteine with Dioxygen

    PubMed Central

    Glass, Amanda M.; Krause, Mary E.; Laurence, Jennifer S.; Jackson, Timothy A.

    2014-01-01

    Synthetically generated metallopeptides have the potential to serve a variety of roles in biotechnology applications, but the use of such systems is often hampered by the inability to control secondary reactions. We have previously reported that the NiII complex of the tripeptide LLL-asparagine-cysteine-cysteine, LLL-NiII-NCC, undergoes metal-facilitated chiral inversion to DLD-NiII-NCC, which increases the observed superoxide scavenging activity. However, the mechanism for this process remained unexplored. Electronic absorption and circular dichroism studies of the chiral inversion reaction of NiII-NCC reveal a unique dependence on dioxygen. Specifically, in the absence of dioxygen, the chiral inversion is not observed, even at elevated pH, whereas the addition of O2 initiates this reactivity and concomitantly generates superoxide. Scavenging experiments using acetaldehyde are indicative of the formation of carbanion intermediates, demonstrating that inversion takes place by deprotonation of the alpha carbons of Asn1 and Cys3. Together, these data are consistent with the chiral inversion being dependent on the formation of a NiIII-NCC intermediate from NiII-NCC and O2. The data further suggest that the anionic thiolate and amide ligands in NiII-NCC inhibit Cα–H deprotonation for the NiII oxidation state, leading to a stable complex in the absence of O2. Together, these results offer insights into the factors controlling reactivity in synthetic metallopeptides. PMID:22928993

  16. A practical synthesis of (+)-biotin from L-cysteine.

    PubMed

    Seki, Masahiko; Hatsuda, Masanori; Mori, Yoshikazu; Yoshida, Shin-ichi; Yamada, Shin-ichi; Shimizu, Toshiaki

    2004-11-19

    Alpha-amino aldehyde 4, which is readily derived from L-cysteine through cyclization and elaboration of the carboxy group, was subjected to the Strecker reaction, which, via sodium bisulfite adduct 16, afforded alpha-amino nitrile 5 with high diastereoselectivity (syn/anti=11:1) and in high yield. Amide 6, derived from 5, was converted to thiolactone 8, a key intermediate in the synthesis of (+)-biotin (1), by a novel S,N-carbonyl migration and cyclization reaction. The Fukuyama coupling reaction of 8 with the zinc reagent 21, which has an ester group, in the presence of a heterogeneous Pd/C catalyst allowed the efficient installation of the 4-carboxybutyl chain to provide 9. Compound 9 was hydrogenated and the protecting groups removed to furnish 1 in 10 steps and in 34 % overall yield from L-cysteine.

  17. The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

    PubMed Central

    Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

  18. The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

    PubMed

    Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-11-13

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

  19. Technical Note: Response time evolution of XR-QA2 GafChromic™ film models.

    PubMed

    Aldelaijan, Saad; Tomic, Nada; Papaconstadopoulos, Pavlos; Schneider, James; Seuntjens, Jan; Shih, Shelley; Lewis, David; Devic, Slobodan

    2018-01-01

    To evaluate the response of the newest XR-QA2 GafChromic™ film model in terms of postexposure signal growth and energy response in comparison with the older XR-QA (Version 2) model. Pieces of film were irradiated to air kerma in air values up to 12 cGy with several beam qualities (5.3-8.25 mm Al) commonly used for CT scanning. Film response was scored in terms of net reflectance from scanned film images at various points in time postirradiation ranging from 1 to 7 days and 5 months postexposure. To reconstruct the measurement signal changes with postirradiation delay, we irradiated one film piece and then scanned it at different point times starting from 2" min and up to 3 days postexposure. For all beam qualities and dose range investigated, it appears that the XR-QA2 film signal completely saturated after 15 h. Compared to 15 h postirradiation scanning time, the observed variation in net reflectance were 3%, 2%, and 1% for film scanned 2" min, 20 min, and 3 h after exposure, respectively, which is well within the measurement uncertainty of the XR-QA2 based reference radiochromic film dosimetry system. A comparison between the XR-QA (Version 2) and the XR-QA2 film response after several months (relative to their responses after 24 h) show differences in up to 8% and 1% for each film model respectively. The replacement of cesium bromide in the older XR-QA (Version 2) film model with bismuth oxide in the newer XR-QA2 film, while keeping the same single sensitive layer structure, lead to a significantly more stable postexposure response. © 2017 American Association of Physicists in Medicine.

  20. Novel Cysteine-Centered Sulfur Metabolic Pathway in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

    PubMed Central

    Oh, Doo-Byoung; Kwon, Ohsuk; Lee, Sang Yup; Sibirny, Andriy A.; Kang, Hyun Ah

    2014-01-01

    In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species. PMID:24959887

  1. Biosynthesis of Se-methyl-seleno-l-cysteine in Basidiomycetes fungus Lentinula edodes (Berk.) Pegler.

    PubMed

    Klimaszewska, M; Górska, S; Dawidowski, M; Podsadni, P; Turło, J

    2016-01-01

    The aim of the current study was to investigate whether the Basidiomycetes fungus Lentinula edodes can biosynthesize Se-methyl-seleno-l-cysteine, a seleno-amino acid with strong anticancer activity, and to optimize the culture conditions for its biosynthesis. We hypothesize that preparations obtained from Se-methyl-seleno-l-cysteine-enriched mycelia from this medicinal mushroom would possess stronger cancer-preventive properties than current preparations. By optimizing the concentration of selenium in the culture medium, we increased the mycelial concentration of Se-methyl-seleno-l-cysteine from essentially non-detectable levels to 120 µg/g dry weight. Significantly elevated levels of this amino acid also correlated with significant (twofold) inhibition of mycelial growth. Increases in the concentration of mycelial Se-methyl-seleno-l-cysteine appeared to be highly correlated with the enhanced biosynthesis of selenomethionine and total selenium content in mycelium. We have demonstrated that in L. edodes, enhanced biosynthesis of this non-protein amino acid eliminates excess selenium.

  2. Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

    PubMed

    Villapakkam, Anuradha C; Handke, Luke D; Belitsky, Boris R; Levdikov, Vladimir M; Wilkinson, Anthony J; Sonenshein, Abraham L

    2009-11-01

    Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

  3. Direct detection of cysteine using functionalized BaTiO3 nanoparticles film based self-powered biosensor.

    PubMed

    Selvarajan, Sophia; Alluri, Nagamalleswara Rao; Chandrasekhar, Arunkumar; Kim, Sang-Jae

    2017-05-15

    Simple, novel, and direct detection of clinically important biomolecules have continuous demand among scientific community as well as in market. Here, we report the first direct detection and facile fabrication of a cysteine-responsive, film-based, self-powered device. NH 2 functionalized BaTiO 3 nanoparticles (BT-NH 2 NPs) suspended in a three-dimensional matrix of an agarose (Ag) film, were used for cysteine detection. BaTiO 3 nanoparticles (BT NPs) semiconducting as well as piezoelectric properties were harnessed in this study. The changes in surface charge properties of the film with respect to cysteine concentrations were determined using a current-voltage (I-V) technique. The current response increased with cysteine concentration (linear concentration range=10µM-1mM). Based on the properties of the composite (BT/Ag), we created a self-powered cysteine sensor in which the output voltage from a piezoelectric nanogenerator was used to drive the sensor. The potential drop across the sensor was measured as a function of cysteine concentrations. Real-time analysis of sensor performance was carried out on urine samples by non-invasive method. This novel sensor demonstrated good selectivity, linear concentration range and detection limit of 10µM; acceptable for routine analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Cysteine as a green corrosion inhibitor for Cu37Zn brass in neutral and weakly alkaline sulphate solutions.

    PubMed

    Radovanović, Milan B; Petrović, Marija B; Simonović, Ana T; Milić, Snežana M; Antonijević, Milan M

    2013-07-01

    The aim of this study was to investigate electrochemical properties of brass in neutral and weakly alkaline solutions in the presence of cysteine as a nontoxic and ecological corrosion inhibitor. Potentiodynamic measurements, open circuit potential measurements, as well as chronoamperometric measurements were the methods used during investigation of the inhibitory effect of cysteine on the corrosion behaviour of brass. Potentiodynamic measurements showed that cysteine behaves as a mixed-type inhibitor in the investigated media. Based on polarization curves for brass in a weakly alkaline solution of sodium sulphate at varying cysteine concentrations, an interaction occurs between Cu(+) ions and the inhibitor, resulting in the formation of a protective complex on the electrode surface. The results of chronoamperometric measurements confirm the results obtained by potentiodynamic measurements. Optical microphotography of the brass surface also confirms the formation of a protective film in the presence of a 1 × 10(-4) mol/dm(3) cysteine. Adsorption of cysteine on the brass surface proceeds according to the Langmuir adsorption isotherm.

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

    PubMed

    Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

    2013-11-01

    Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

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

    PubMed

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

    2017-10-19

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

  7. Synthesis and characterization of arsenic-doped cysteine-capped thoria-based nanoparticles

    NASA Astrophysics Data System (ADS)

    Pereira, F. J.; Díez, M. T.; Aller, A. J.

    2013-09-01

    Thoria materials have been largely used in the nuclear industry. Nonetheless, fluorescent thoria-based nanoparticles provide additional properties to be applied in other fields. Thoria-based nanoparticles, with and without arsenic and cysteine, were prepared in 1,2-ethanediol aqueous solutions by a simple precipitation procedure. The synthesized thoria-based nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (ED-XRS), Raman spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and fluorescence microscopy. The presence of arsenic and cysteine, as well as the use of a thermal treatment facilitated fluorescence emission of the thoria-based nanoparticles. Arsenic-doped and cysteine-capped thoria-based nanoparticles prepared in 2.5 M 1,2-ethanediol solutions and treated at 348 K showed small crystallite sizes and strong fluorescence. However, thoria nanoparticles subjected to a thermal treatment at 873 K also produced strong fluorescence with a very narrow size distribution and much smaller crystallite sizes, 5 nm being the average size as shown by XRD and TEM. The XRD data indicated that, even after doping of arsenic in the crystal lattice of ThO2, the samples treated at 873 K were phase pure with the fluorite cubic structure. The Raman and FT-IR spectra shown the most characteristics vibrational peaks of cysteine together with other peaks related to the bonds of this molecule to thoria and arsenic when present.

  8. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

    PubMed

    Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

    2014-01-01

    The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

  9. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

    PubMed Central

    Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

    2014-01-01

    The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

  10. Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume

    PubMed Central

    Tabe, Linda; Wirtz, Markus; Molvig, Lisa; Droux, Michel; Hell, Ruediger

    2010-01-01

    There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosyntheis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F2 seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein. PMID:19939888

  11. Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume.

    PubMed

    Tabe, Linda; Wirtz, Markus; Molvig, Lisa; Droux, Michel; Hell, Ruediger

    2010-03-01

    There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosynthesis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F(2) seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein.

  12. Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.

    PubMed

    Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E V; Hansson, Gunnar C

    2013-06-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.

  13. Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bradshaw, William J.; Public Health England, Porton Down, Salisbury SP4 0JG; Roberts, April K.

    2015-02-19

    Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket. In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely tomore » be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Jihun; Blaber, Michael; FSU)

    2010-11-09

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

  15. Effects of pH, temperature, and chemical structure on the stability of S-(purin-6-yl)-L-cysteine: evidence for a novel molecular rearrangement mechanism to yield N-(purin-6-yl)-L-cysteine.

    PubMed

    Elfarra, A A; Hwang, I Y

    1996-01-01

    The stability of S-(purin-6-yl)-L-cysteine (SPC), a kidney-selective prodrug of 6-mercaptopurine and a putative metabolite of 6-chloropurine, was investigated under various pH and temperature conditions. At room temperature, the half-life (t 1/2) of SPC at either highly acidic (pH 3.6) or basic conditions (pH 9.6) was longer than at neutral or slightly acidic or basic conditions (pH 5.7-8.75). The primary degradation product, N-(purin-6-yl)-L-cysteine (NPC), was isolated using Sephadex LH-20 chromatography and characterized by 1H NMR and FAB/MS after derivatization with 2-iodoacetic acid. These results reveal novel stability requirements and implicate the cysteinyl amino group and the purinyl N-1 nitrogen in the mechanism of SPC rearrangement to NPC. Further evidence for this hypothesis was provided by the findings that the stability of SPC in phosphate buffer (pH 7.4) at 37 degrees C was similar to that of S-(guanin-6-yl)-L-cysteine, whereas S-(purin-6-yl)-N-acetyl-L-cysteine and S-(purin-6-yl)glutathione which have their cysteine amino groups blocked were much more stable than SPC. S-(Purin-6-yl)-L-homocysteine (SPHC) was also more stable than SPC, possibly because the formation of a 6-membered ring transition state as would be expected with SPHC is kinetically less favored than the formation of a 5-membered ring transition state as would be expected with SPC. These results may explain previous in vivo metabolism results of SPC and its analogs and may contribute to a better understanding of stability of structurally related cysteine S-conjugates.

  16. Differential expression of cysteine desulfurases in soybean

    PubMed Central

    2011-01-01

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

  17. Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses

    PubMed Central

    Hernandez, Hilda; Niehauser, Sarah; Boltz, Stacey A.; Gawandi, Vijay; Phillips, Robert S.; Amster, I. Jonathan

    2006-01-01

    A method for improving the identification of peptides in a shotgun proteome analysis using accurate mass measurement has been developed. The improvement is based upon the derivatization of cysteine residues with a novel reagent, 2,4-dibromo-(2′-iodo)acetanilide. The derivitization changes the mass defect of cysteine-containing proteolytic peptides in a manner that increases their identification specificity. Peptide masses were measured using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron mass spectrometry. Reactions with protein standards show that the derivatization of cysteine is rapid and quantitative, and the data suggest that the derivatized peptides are more easily ionized or detected than unlabeled cysteine-containing peptides. The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. This procedure permits the identification of proteins from the M. maripaludis proteome that are not usually observed by the standard protocol and shows that better protein coverage is obtained with this methodology. PMID:16689545

  18. Salt Effect Accelerates Site-Selective Cysteine Bioconjugation

    PubMed Central

    2016-01-01

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

  19. Comparison of dose response functions for EBT3 model GafChromic™ film dosimetry system.

    PubMed

    Aldelaijan, Saad; Devic, Slobodan

    2018-05-01

    Different dose response functions of EBT3 model GafChromic™ film dosimetry system have been compared in terms of sensitivity as well as uncertainty vs. error analysis. We also made an assessment of the necessity of scanning film pieces before and after irradiation. Pieces of EBT3 film model were irradiated to different dose values in Solid Water (SW) phantom. Based on images scanned in both reflection and transmission mode before and after irradiation, twelve different response functions were calculated. For every response function, a reference radiochromic film dosimetry system was established by generating calibration curve and by performing the error vs. uncertainty analysis. Response functions using pixel values from the green channel demonstrated the highest sensitivity in both transmission and reflection mode. All functions were successfully fitted with rational functional form, and provided an overall one-sigma uncertainty of better than 2% for doses above 2 Gy. Use of pre-scanned images to calculate response functions resulted in negligible improvement in dose measurement accuracy. Although reflection scanning mode provides higher sensitivity and could lead to a more widespread use of radiochromic film dosimetry, it has fairly limited dose range and slightly increased uncertainty when compared to transmission scan based response functions. Double-scanning technique, either in transmission or reflection mode, shows negligible improvement in dose accuracy as well as a negligible increase in dose uncertainty. Normalized pixel value of the images scanned in transmission mode shows linear response in a dose range of up to 11 Gy. Copyright © 2018 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

  20. Interactions between N-acetyl-L-cysteine protected CdTe quantum dots and doxorubicin through spectroscopic method

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Xiupei, E-mail: xiupeiyang@163.com; College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637000; Lin, Jia

    2015-06-15

    Highlights: • CdTe quantum dots with the diameter of 3–5 nm were synthesized in aqueous solution. • The modified CdTe quantum dots showed well fluorescence properties. • The interaction between the CdTe quantum dots and doxorubicin (DR) was investigated. - Abstract: N-acetyl-L-cysteine protected cadmium telluride quantum dots with a diameter of 3–5 nm were synthesized in aqueous solution. The interaction between N-acetyl-L-cysteine/cadmium telluride quantum dots and doxorubicin was investigated by ultraviolet–visible absorption and fluorescence spectroscopy at physiological conditions (pH 7.2, 37 °C). The results indicate that electron transfer has occurred between N-acetyl-L-cysteine/cadmium telluride quantum dots and doxorubicin under light illumination.more » The quantum dots react readily with doxorubicin to form a N-acetyl-L-cysteine/cadmium telluride-quantum dots/doxorubicin complex via electrostatic attraction between the −NH{sub 3}{sup +} moiety of doxorubicin and the −COO{sup −} moiety of N-acetyl-L-cysteine/cadmium telluride quantum dots. The interaction of N-acetyl-L-cysteine/cadmium telluride-quantum dots/doxorubicin complex with bovine serum albumin was studied as well, showing that the complex might induce the conformation change of bovine serum due to changes in microenvironment of bovine serum.« less

  1. Structural influence in the interaction of cysteine with five coordinated copper complexes: Theoretical and experimental studies

    NASA Astrophysics Data System (ADS)

    Huerta-Aguilar, Carlos Alberto; Thangarasu, Pandiyan; Mora, Jesús Gracia

    2018-04-01

    Copper complexes of N,N,N‧,N‧-tetrakis(pyridyl-2-ylmethyl)-1,2-diaminoethane (L1) and N,N,N‧,N‧-tetrakis(pyridyl-2-ylmethyl)-1,3-diaminopropane (L2) prepared were characterized completely by different analytical methods. The X-structure of the complexes shows that Cu(II) presents in trigonal bi-pyramidal (TBP) geometry, consisting with the electronic spectra where two visible bands corresponding to five coordinated structure were observed. Thus TD-DFT was used to analyze the orbital contribution to the electronic transitions for the visible bands. Furthermore, the interaction of cysteine with the complexes was spectrally studied, and the results were explained through DFT analysis, observing that the geometrical parameters and oxidation state of metal ions play a vital role in the binding of cysteine with copper ion. It appears that the TBP structure is being changed into octahedral geometry during the addition of cysteine to the complexes as two bands (from complex) is turned to a broad band in visible region, signifying the occupation of cysteine molecule at sixth position of octahedral geometry. In the molecular orbital analysis, the existence of a strong overlapping of HOMOs (from cysteine) with LUMOs of Cu ion was observed. The total energy of the systems calculated by DFT shows that cysteine binds favorably with copper (I) than that with Cu(II).

  2. Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Xiaojing; Kuk, Jane; Moffat, Keith

    2008-11-12

    Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 {angstrom} resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an 'arm' structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common andmore » may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.« less

  3. Global analysis of gene expression in response to L-Cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica

    PubMed Central

    2011-01-01

    Background Entamoeba histolytica, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. E. histolytica completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in E. histolytica, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism. Results In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (≥3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion. Conclusions To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of

  4. Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin.

    PubMed

    Kamikubo, Yuichi; De Guzman, Roberto; Kroon, Gerard; Curriden, Scott; Neels, Jaap G; Churchill, Michael J; Dawson, Philip; Ołdziej, Stanisław; Jagielska, Anna; Scheraga, Harold A; Loskutoff, David J; Dyson, H Jane

    2004-06-01

    The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is

  5. Inhibitory effect of CT domain of CCN3/NOV on proliferation and differentiation of osteogenic mesenchymal stem cells, Kusa-A1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Katsuki, Yuko; Oral Pathology, Graduate School of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549; Sakamoto, Kei

    CCN3/NOV activates the Notch signal through the carboxyl terminal cysteine-rich (CT) domain. CCN3 transfection to Kusa-A1 inhibited osteogenic differentiation and cell proliferation, which is accompanied by upregulation of Hes/Hey, Notch downstream targets, and p21, a CDK inhibitor. Upregulation of Hes/Hey and p21 was abrogated by the deletion of CT domain. Anti-proliferative activity of CCN3 was also abrogated by CT domain deletion whereas anti-osteogenic activity was not completely abrogated. We found that CT domain-deleted CCN3 still possesses antagonistic effect on BMP-2. These results suggest that CCN3 employs Notch and BMP pathways in anti-osteogenic activity while it inhibits cell proliferation uniquely bymore » Notch/p21 pathway.« less

  6. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl H2O... 21 Food and Drugs 3 2011-04-01 2011-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184.1272 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

  7. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl H2O... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine monohydrochloride. 184.1272 Section 184.1272 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

  8. Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

    PubMed

    Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

    1992-05-15

    Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

  9. Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

    PubMed Central

    Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

    1992-01-01

    Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

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

    PubMed

    Takemoto, Yumi

    2014-04-01

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

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

    PubMed

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

    2017-10-01

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

  12. Ga2O3 photocatalyzed on-line tagging of cysteine to facilitate peptide mass fingerprinting.

    PubMed

    Qiao, Liang; Su, Fangzheng; Bi, Hongyan; Girault, Hubert H; Liu, Baohong

    2011-09-01

    β-Ga(2)O(3) is a wide-band-gap semiconductor having strong oxidation ability under light irradiation. Herein, the steel target plates modified with β-Ga(2)O(3) nanoparticles have been developed to carry out in-source photo-catalytic oxidative reactions for online peptide tagging during laser desorption/ionization mass spectrometry (LDI-MS) analysis. Under UV laser irradiation, β-Ga(2)O(3) can catalyze the photo-oxidation of 2-methoxyhydroquinone added to a sample mixture to 2-methoxy benzoquinone that can further react with the thiol groups of cysteine residues by Michael addition reaction. The tagging process leads to appearance of pairs of peaks with an m/z shift of 138.1Th. This online labelling strategy is demonstrated to be sensitive and efficient with a detection-limit at femtomole level. Using the strategy, the information on cysteine content in peptides can be obtained together with peptide mass, therefore constraining the database searching for an advanced identification of cysteine-containing proteins from protein mixtures. The current peptide online tagging method can be important for specific analysis of cysteine-containing proteins especially the low-abundant ones that cannot be completely isolated from other high-abundant non-cysteine-proteins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Low-quality birds do not display high-quality signals: The cysteine-pheomelanin mechanism of honesty

    PubMed Central

    Galván, Ismael; Wakamatsu, Kazumasa; Camarero, Pablo R; Mateo, Rafael; Alonso-Alvarez, Carlos

    2015-01-01

    The mechanisms that make that the costs of producing high-quality signals are unaffordable to low-quality signalers are a current issue in animal communication. The size of the melanin-based bib of male house sparrows Passer domesticus honestly signals quality. We induced the development of new bibs while treating males with buthionine-sulfoximine (BSO), a substance that depletes the levels of the antioxidant glutathione (GSH) and the amino acid cysteine, two elements that switch melanogenesis from eumelanin to pheomelanin. Final bib size is negatively related to pheomelanin levels in the bib feathers. BSO reduced cysteine and GSH levels in all birds, but improved phenotypes (bibs larger than controls) were only expressed by high-quality birds (BSO birds with largest bibs initially). Negative associations between final bib size and cysteine levels in erythrocytes, and between pheomelanin and cysteine levels, were observed in high-quality birds only. These findings suggest that a mechanism uncoupling pheomelanin and cysteine levels may have evolved in low-quality birds to avoid producing bibs of size not corresponding to their quality and greater relative costs. Indeed, greater oxidative stress in cells was not observed in low-quality birds. This may represent the first mechanism maintaining signal honesty without producing greater relative costs on low-quality signalers. PMID:25330349

  14. Synthesis, characterization, mucoadhesion and biocompatibility of thiolated carboxymethyl dextran-cysteine conjugate.

    PubMed

    Shahnaz, G; Perera, G; Sakloetsakun, D; Rahmat, D; Bernkop-Schnürch, A

    2010-05-21

    This study was aimed at improving the mucoadhesive properties of carboxymethyl dextran by the covalent attachment of cysteine. Mediated by a carbodiimide, l-cysteine was covalently attached to the polymer. The resulting CMD-cysteine conjugate (CMD-(273) conjugate) displayed 273+/-20 micromol thiol groups per gram of polymer (mean+/-S.D.; n=3). Within 2h the viscosity of an aqueous mucus/CMD-(273) conjugate mixture pH 7.4 increased at 37 degrees C by more than 85% compared to a mucus/carboxymethyl dextran mixture indicating enlarged interactions between the mucus and the thiolated polymer. Due to the immobilization of cysteine, the swelling velocity of the polymer was significantly accelerated (p<0.05). In aqueous solutions the CMD-(273) conjugate was capable of forming inter- and/or intramolecular disulfide bonds. Because of this crosslinking process within the polymeric network, the cohesive properties of the conjugate were also improved. Tablets comprising the unmodified polymer disintegrated within 15 min, whereas tablets of the CMD-(273) conjugate remained stable for 160 min (means+/-S.D.; n=3). Results from LDH and MTT assays on Caco-2 cells revealed 4.96+/-0.98% cytotoxicity and 94.1+/-0.9% cell viability for the CMD-(273) conjugate, respectively. Controlled release of model compound from CMD-(273) conjugate tablets was observed over 6h. These findings suggest that CMD-(273) conjugate is a promising novel polymer for drug delivery systems providing improved mucoadhesive and cohesive properties, greater stability and biocompatibility. Copyright 2010 Elsevier B.V. All rights reserved.

  15. The relationship between the INTERMED patient complexity instrument and Level of Care Utilisation System (LOCUS).

    PubMed

    Thurber, Steven; Wilson, Ann; Realmuto, George; Specker, Sheila

    2018-03-01

    To investigate the concurrent and criterion validity of two independently developed measurement instruments, INTERMED and LOCUS, designed to improve the treatment and clinical management of patients with complex symptom manifestations. Participants (N = 66) were selected from hospital records based on the complexity of presenting symptoms, with tripartite diagnoses across biological, psychiatric and addiction domains. Biopsychosocial information from hospital records were submitted to INTERMED and LOCUS grids. In addition, Global Assessment of Functioning (GAF) ratings were gathered for statistical analyses. The product moment correlation between INTERMED and LOCUS was 0.609 (p = .01). Inverse zero-order correlations for INTERMED and LOCUS total score and GAF were obtained. However, only the beta weight for LOCUS and GAF was significant. An exploratory principal components analysis further illuminated areas of convergence between the instruments. INTERMED and LOCUS demonstrated shared variance. INTERMED appeared more sensitive to complex medical conditions and severe physiological reactions, whereas LOCUS findings are more strongly related to psychiatric symptoms. Implications are discussed.

  16. Molecular switch-modulated fluorescent copper nanoclusters for selective and sensitive detection of histidine and cysteine.

    PubMed

    Gu, Zefeng; Cao, Zhijuan

    2018-06-07

    A novel assay for histidine and cysteine has been constructed based on modulation of fluorescent copper nanoclusters (CuNCs) by molecular switches. In our previous work, a dumbbell DNA template with a poly-T (thymine) loop has been developed as an excellent template for the formation of strongly fluorescent CuNCs. Herein, for the first time, we established this biosensor for sensing two amino acids by using dumbbell DNA-templated CuNCs as the single probe. Among 20 natural amino acids, only histidine and cysteine can selectively quench fluorescence emission of CuNCs, because of the specific interaction of these compounds with copper ions. Furthermore, by using nickel ions (Ni 2+ ) and N-ethylmaleimide as the masking agents for histidine and cysteine respectively, an integrated logic gate system was designed by coupling with the fluorescent CuNCs and demonstrated selective and sensitive detection of cysteine and histidine. Under optimal conditions, cysteine can be detected in the concentration ranges of 0.01-10.0 μM with the detection limit (DL) of as low as 98 pM, while histidine can be detected in the ranges of 0.05-40.0 μM with DL of 1.6 nM. In addition, histidine and cysteine can be observed with the naked eye under a hand-held UV lamp (DL, 50 nM), which can be easily adapted to automated high-throughput screening. Finally, the strategy has been successfully utilized for biological fluids. The proposed system can be conducted in homogeneous solution, eliminating the need for organic cosolvents, separation processes of nanomaterials, or any chemical modifications. Overall, the assay provides an alternative method for simultaneous detection of cysteine and histidine by taking the advantages of high speed, no label and enzyme requirement, and good sensitivity and specificity, and will satisfy the great demand for determination of amino acids in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. Graphical abstract.

  17. Position-dependent interactions between cysteine residues and the helix dipole

    PubMed Central

    Miranda, JJ L.

    2003-01-01

    A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site-directed mutagenesis was used to introduce cysteine residues at the N-terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pKa between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge-dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position-dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family. PMID:12493830

  18. A new simple phthalimide-based fluorescent probe for highly selective cysteine and bioimaging for living cells

    NASA Astrophysics Data System (ADS)

    Shen, Youming; Zhang, Xiangyang; Zhang, Youyu; Zhang, Chunxiang; Jin, Junling; Li, Haitao

    2017-10-01

    A new turn-on phthalimide fluorescent probe has designed and synthesized for sensing cysteine (Cys) based on excited state intramolecular proton transfer (ESIPT) process. It is consisted of a 3-hydroxyphthalimide derivative moiety as the fluorophore and an acrylic ester group as a recognition receptor. The acrylic ester acts as an ESIPT blocking agent. Upon addition of cystein, intermolecular nucleophilic attack of cysteine on acrylic ester releases the fluorescent 3-hydroxyphthalimide derivative, thereby enabling the ESIPT process and leading to enhancement of fluorescence. The probe displays high sensitivity, excellent selectivity and with large Stokes shift toward cysteine. The linear interval range of the fluorescence titration ranged from 0 to 1.0 × 10- 5 M and detection limit is low (6 × 10- 8 M). In addition, the probe could be used for bio-imaging in living cells.

  19. Cysteine-grafted nonwoven geotextile: a new and efficient material for heavy metals sorption--Part B.

    PubMed

    Vandenbossche, M; Vezin, H; Touati, N; Jimenez, M; Casetta, M; Traisnel, M

    2014-10-01

    The development of a new material designed to trap heavy metals from sediments or wastewater, based on a polypropylene non-woven covalently grafted with cysteine, has been reported in a previous paper (Part A). The non-woven was first functionalized with acrylic acid (AA) which is used as spacer, and then cysteine was immobilized on the substrate through covalent coupling in order to obtain the so-called PP-g-AA-cysteine. Some preliminary heavy metals adsorption tests gave interesting results: at 20 °C for 24 h and in a 1000 mg/L heavy metals solution, PP-g-AA-cysteine adsorbs 95 mg Cu/g PP (CuSO4 solution), 104 mg Cu/g PP (Cu(NO3)2 solution), 135 mg Pb/g PP (Pb(NO3)2 solution) and 21 mg Cr/g PP (Cr(NO3)3 solution). In this second part of the work, heavy metals sorption tests were carried out with Cu (II), Pb (II), and Cr (III) separately, in order to determine the sorption capacity of this new sorbent as a function of (i) the heavy metals concentration in the solution, (ii) the contact time with the solution, (iii) the pH and (iv) the ionic strength of the solution containing heavy metals. Moreover, the sorption capacity of PP-g-AA-Cysteine was studied using a polluted solution consisting of a mixture of these different heavy metals. An Electron Paramagnetic Resonance study was finally carried out in order to determine the coordination geometry in the environment of the copper trapped by the PP-g-AA-cysteine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. The Cysteine-Rich Domain of the Macrophage Mannose Receptor Is a Multispecific Lectin That Recognizes Chondroitin Sulfates a and B and Sulfated Oligosaccharides of Blood Group Lewisa and Lewisx Types in Addition to the Sulfated N-Glycans of Lutropin

    PubMed Central

    Leteux, Christine; Chai, Wengang; Loveless, R. Wendy; Yuen, Chun-Ting; Uhlin-Hansen, Lars; Combarnous, Yves; Jankovic, Mila; Maric, Svetlana C.; Misulovin, Ziva; Nussenzweig, Michel C.; Ten Feizi

    2000-01-01

    The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells. PMID:10748230

  1. L-Cysteine Administration Attenuates Pancreatic Fibrosis Induced by TNBS in Rats by Inhibiting the Activation of Pancreatic Stellate Cell

    PubMed Central

    Hu, GuoYong; Shen, Jie; Wang, Feng; Xu, Ling; Dai, WeiQi; Xiong, Jie; Ni, JianBo; Guo, ChuanYong; Wan, Rong; Wang, XingPeng

    2012-01-01

    Background and Aims Recent studies have shown that activated pancreatic stellate cells (PSCs) play a major role in pancreatic fibrogenesis. We aimed to study the effect of L-cysteine administration on fibrosis in chronic pancreatitis (CP) induced by trinitrobenzene sulfonic acid (TNBS) in rats and on the function of cultured PSCs. Methods CP was induced by TNBS infusion into rat pancreatic ducts. L-cysteine was administrated for the duration of the experiment. Histological analysis and the contents of hydroxyproline were used to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis of α-SMA in the pancreas was performed to detect the activation of PSCs in vivo. The collagen deposition related proteins and cytokines were determined by western blot analysis. DNA synthesis of cultured PSCs was evaluated by BrdU incorporation. We also evaluated the effect of L-cysteine on the cell cycle and cell activation by flow cytometry and immunocytochemistry. The expression of PDGFRβ, TGFβRII, collagen 1α1 and α-SMA of PSCs treated with different concentrations of L-cysteine was determined by western blot. Parameters of oxidant stress were evaluated in vitro and in vivo. Nrf2, NQO1, HO-1, IL-1β expression were evaluated in pancreas tissues by qRT-PCR. Results The inhibition of pancreatic fibrosis by L-cysteine was confirmed by histological observation and hydroxyproline assay. α-SMA, TIMP1, IL-1β and TGF-β1 production decreased compared with the untreated group along with an increase in MMP2 production. L-cysteine suppressed the proliferation and extracellular matrix production of PSCs through down-regulating of PDGFRβ and TGFβRII. Concentrations of MDA+4-HNE were decreased by L-cysteine administration along with an increase in GSH levels both in tissues and cells. In addition, L-cysteine increased the mRNA expression of Nrf2, NQO1 and HO-1 and reduced the expression of IL-1β in L-cysteine treated group when compared with control group. Conclusion L-cysteine

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

    USDA-ARS?s Scientific Manuscript database

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

  3. L-cysteine efflux in erythrocytes as a function of human age: correlation with reduced glutathione and total anti-oxidant potential.

    PubMed

    Kumar, Prabhanshu; Maurya, Pawan Kumar

    2013-06-01

    Thiol compounds such as cysteine (Cys) and reduced glutathione (GSH) play an important role in human aging and age-related diseases. In erythrocytes, GSH is synthesized by glutamic acid, cysteine, and glycine, but the rate of GSH synthesis is determined only by the availability of L-cysteine. Cysteine supplementation has been shown to ameliorate several parameters that are known to degenerate during human aging. We have studied L-cysteine efflux in vitro in human erythrocytes as a function of age by suspending cells in solution containing 10 mM L-cysteine for uptake; later cells were re-suspended in phosphate-buffered saline (PBS)-glucose to allow efflux. Change in the free sulfhydryl (-SH) concentration was then measured to calculate the rate of efflux. The GSH/oxidized glutathione (GSSG) ratio was taken as a control to study the oxidation/reduction state of the erythrocyte. The total anti-oxidant potential of plasma was measured in terms of ferric reducing ability of plasma (FRAP) values. We have shown a significant (p<0.0001) decline in the efflux of L-cysteine in erythrocytes during human aging, and the GSH/GSSG ratio decreases as a function of human age. The decline in L-cysteine efflux during aging correlates with the decrease in GSH and the FRAP value. This finding may help to explain the shift in the redox status and low GSH concentration that might determine the rate of L-cysteine efflux observed in erythrocytes and an important factor in the development of oxidative stress in erythrocytes during aging.

  4. Metabolic Design of Corynebacterium glutamicum for Production of l-Cysteine with Consideration of Sulfur-Supplemented Animal Feed.

    PubMed

    Joo, Young-Chul; Hyeon, Jeong Eun; Han, Sung Ok

    2017-06-14

    l-Cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. However, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. In this study, we constructed recombinant Corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. The aims of this work were to investigate the effect of the combined overexpression of serine acetyltransferase (CysE), O-acetylserine sulfhydrylase (CysK), and the transcriptional regulator CysR on l-cysteine production. The CysR-overexpressing strain accumulated approximately 2.7-fold more intracellular sulfide than the control strain (empty pMT-tac vector). Moreover, in the resulting CysEKR recombinant strain, combinatorial overexpression of genes involved in l-cysteine production successfully enhanced its production by approximately 3.0-fold relative to that in the control strain. This study demonstrates a biotechnological model for the production of animal feed supplements such as l-cysteine using metabolically engineered C. glutamicum.

  5. Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

    PubMed

    De Oliveira Neto, Osmundo Brilhante; Batista, João Aguiar Nogueira; Rigden, Daniel John; Franco, Octávio Luiz; Fragoso, Rodrigo Rocha; Monteiro, Ana Carolina Santos; Monnerat, Rose Gomes; Grossi-De-Sa, Maria Fátima

    2004-06-01

    The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.

  6. Cysteine reversal of the novel neuromuscular blocking drug CW002 in dogs: pharmacodynamics, acute cardiovascular effects, and preliminary toxicology.

    PubMed

    Sunaga, Hiroshi; Malhotra, Jaideep K; Yoon, Edward; Savarese, John J; Heerdt, Paul M

    2010-04-01

    CW002 is a neuromuscular blocking drug that is inactivated by endogenous L-cysteine. This study determined the exogenous L-cysteine dose-response relationship for CW002 reversal along with acute cardiovascular effects and organ toxicity in dogs. Six dogs were each studied four times during isoflurane-nitrous oxide anesthesia and recording of muscle twitch, arterial pressure, and heart rate. CW002 (0.08 mg/kg or 9 x ED95) was injected, and the time to spontaneous muscle recovery was determined. CW002 was then administered again followed 1 min later by 10, 20, 50, or 100 mg/kg L-cysteine (1 dose/experiment). After twitch recovery, CW002 was given a third time to determine whether residual L-cysteine influenced duration. Preliminary toxicology was performed in an additional group of dogs that received CW002 followed by vehicle (n = 8) or 200 mg/kg L-cysteine (n = 8). Animals were awakened and observed for 2 or 14 days before sacrificing and anatomic, biochemical, and histopathologic analyses. L-cysteine at all doses accelerated recovery from CW002, with both 50 and 100 mg/kg decreasing median duration from more than 70 min to less than 5 min. After reversal, duration of a subsequent CW002 dose was also decreased in a dose-dependent manner. Over the studied dose range, L-cysteine had less than 10% effect on blood pressure and heart rate. Animals receiving a single 200-mg/kg dose of L-cysteine showed no clinical, anatomic, biochemical, or histologic evidence of organ toxicity. The optimal L-cysteine dose for rapidly reversing the neuromuscular blockade produced by a large dose of CW002 in dogs is approximately 50 mg/kg, which has no concomitant hemodynamic effect. A dose of 200 mg/kg had no evident organ toxicity.

  7. Differential actions of L-cysteine on responses to nitric oxide, nitroxyl anions and EDRF in the rat aorta

    PubMed Central

    Ellis, Anthie; Guang Li, Chun; Rand, Michael J

    2000-01-01

    The effects of L-cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO•), nitroxyl (NO−) derived from Angeli's salt and endothelium-derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 μM or less of L-cysteine had no effect on responses. Relaxations produced by exogenous NO• (0.25–2.5 μM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001–0.3 μM) were enhanced by 1 and 3 mM L-cysteine. The enhancements by L-cysteine of responses to NO• and sodium nitroprusside may be attributed to the formation of S-nitrosocysteine. Relaxations mediated by the nitroxyl anion (0.3 μM) donated from Angeli's salt were more prolonged than those produced by NO•, and nitroxyl-induced relaxations were reduced by L-cysteine (1 and 3 mM). EDRF-mediated relaxations produced by acetylcholine (0.01–10 μM), ATP (3–100 μM) and the calcium ionophore A23187 (0.1 μM) were significantly reduced by 3 mM L-cysteine. The similarity between the inhibitory effects of L-cysteine on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion. PMID:10694238

  8. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

    PubMed

    Semashko, Tatiana A; Vorotnikova, Elena A; Sharikova, Valeriya F; Vinokurov, Konstantin S; Smirnova, Yulia A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N; Filippova, Irina Y

    2014-03-15

    This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. Published by Elsevier Inc.

  9. Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein.

    PubMed

    Hu, A; Norrby, E

    1994-09-01

    The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not

  10. Genetic selection for a highly functional cysteine-less membrane protein using site-saturation mutagenesis

    PubMed Central

    Arendt, Cassandra S.; Ri, Keirei; Yates, Phillip A.; Ullman, Buddy

    2007-01-01

    We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiol-specific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. PMID:17481563

  11. Cysteine proteases and wheat (Triticum aestivum L) under drought: A still greatly unexplored association.

    PubMed

    Botha, Anna-Maria; Kunert, Karl J; Cullis, Christopher A

    2017-09-01

    Bread wheat (Triticum aestivum L.) provides about 19% of global dietary energy. Environmental stress, such as drought, affects wheat growth causing premature plant senescence and ultimately plant death. A plant response to drought is an increase in protease-mediated proteolysis with rapid degradation of proteins required for metabolic processes. Among the plant proteases that are increased in their activity following stress, cysteine proteases are the best characterized. Very little is known about particular wheat cysteine protease sequences, their expression and also localization. The current knowledge on wheat cysteine proteases belonging to the five clans (CA, CD, CE, CF and CP) is outlined, in particular their expression and possible function under drought. The first successes in establishing an annotated wheat genome database are further highlighted which has allowed more detailed mining of cysteine proteases. We also share our thoughts on future research directions considering the growing availability of genomic resources of this very important food crop. Finally, we also outline future application of developed knowledge in transgenic wheat plants for environmental stress protection and also as senescence markers to monitor wheat growth under environmental stress conditions. © 2017 John Wiley & Sons Ltd.

  12. Effect of cysteine and humic acids on bioavailability of Ag from Ag nanoparticles to a freshwater snail

    USGS Publications Warehouse

    Luoma, Samuel N.; Tasha Stoiber,; Croteau, Marie-Noele; Isabelle Romer,; Ruth Merrifeild,; Lead, Jamie

    2016-01-01

    Metal-based engineered nanoparticles (NPs) will undergo transformations that will affect their bioavailability, toxicity and ecological risk when released to the environment, including interactions with dissolved organic material. The purpose of this paper is to determine how interactions with two different types of organic material affect the bioavailability of silver nanoparticles (AgNPs). Silver uptake rates by the pond snail Lymnaea stagnalis were determined after exposure to 25 nmol l-1 of Ag as PVP AgNPs, PEG AgNPs or AgNO3, in the presence of either Suwannee River humic acid or cysteine, a high-affinity thiol-rich organic ligand. Total uptake rate of Ag from the two NPs was either increased or not strongly affected in the presence of 1 – 10 mg 1-1 humic acid. Humic substances contain relatively few strong ligands for Ag explaining their limited effects on Ag uptake rate. In contrast, Ag uptake rate was substantially reduced by cysteine. Three components of uptake from the AgNPs were quantified in the presence of cysteine using a biodynamic modeling approach: uptake of dissolved Ag released by the AgNPs, uptake of a polymer or large (>3kD) Ag-cysteine complex and uptake of the nanoparticle itself. Addition of 1:1 Ag:cysteine reduced concentrations of dissolved Ag, which contributed to, but did not fully explain the reductions in uptake. A bioavailable Ag-cysteine complex (> 3kD) appeared to be the dominant avenue of uptake from both PVP AgNPs and PEG AgNPs in the presence of cysteine. Quantifying the different avenues of uptake sets the stage for studies to assess toxicity unique to NPs.

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

    EPA Science Inventory

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

  14. N-Acetyl cysteine and clomiphene citrate for induction of ovulation in polycystic ovary syndrome: a cross-over trial.

    PubMed

    Badawy, Ahmed; State, Omnia; Abdelgawad, Soma

    2007-01-01

    To compare clomiphene citrate plus N-acetyl cysteine versus clomiphene citrate for inducing ovulation in patients with polycystic ovary syndrome. Prospective cross-over trial. University teaching hospital and a private practice setting. Five hundred and seventy-three patients were treated with clomiphene citrate for one menstrual cycle among which 470 patients were treated with clomiphene citrate plus N-acetyl cysteine for another cycle. All women suffered from polycystic ovary syndrome. Patients had clomiphene citrate 50-mg tablets twice daily alone or with N-acetyl cysteine 1,200 mg/day orally for 5 days starting on day 3 of the menstrual cycle. Primary outcomes were number of mature follicles, serum E2, serum progesterone, and endometrial thickness. Secondary outcome was the occurrence of pregnancy. Ovulation rate improved significantly after the addition of N-acetyl cysteine (17.9% versus 52.1%). Although the number of mature follicles was more in the N-acetyl cysteine group (2.1+/-0.88 versus 3.2+/-0.93), the difference was not statistically significant. The mean E2 levels (pg/ml) at the time of human chorionic gonadotropine injection, serum progesterone levels (ng/ml) on days 21-23 of the cycle, and the endometrial thickness were significantly improved in the N-acetyl cysteine group. The overall pregnancy rate was 11.5% in the N-acetyl cysteine group. Insulin resistance occurred in 260 patients (55.4%). There was no significant difference between the insulin resistance group (n = 260) and non-insulin resistance group (n = 210) as regards ovulation rate, number of follicles, serum E2 (pg/ml), serum progesterone (ng/ml), endometrial thickness (mm), or pregnancy rate. N-Acetyl cysteine is proved effective in inducing or augmenting ovulation in polycystic ovary patients.

  15. Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms

    PubMed Central

    Lohoefer, Fabian; Reeps, Christian; Lipp, Christina; Rudelius, Martina; Haertl, Felix; Matevossian, Edouard; Zernecke, Alma; Eckstein, Hans-Henning; Pelisek, Jaroslav

    2014-01-01

    Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT–PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT–PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA. PMID:24833013

  16. Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

    PubMed Central

    Kaeoket, Kampon; Chanapiwat, Panida; Tummaruk, Padet; Techakumphu, Mongkol

    2010-01-01

    Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L−1 (group I, control), 5 mmol L−1 (group II), 10 mmol L−1 (group III) and 15 mmol L−1 (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P < 0.01) percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups (group II and group III) compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group III. In conclusion, 5 or 10 mmol L−1 was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen–thawed boar semen. PMID:20601963

  17. Visible‐Light‐Mediated Selective Arylation of Cysteine in Batch and Flow

    PubMed Central

    Bottecchia, Cecilia; Rubens, Maarten; Gunnoo, Smita B.; Hessel, Volker; Madder, Annemieke

    2017-01-01

    Abstract A mild visible‐light‐mediated strategy for cysteine arylation is presented. The method relies on the use of eosin Y as a metal‐free photocatalyst and aryldiazonium salts as arylating agents. The reaction can be significantly accelerated in a microflow reactor, whilst allowing the in situ formation of the required diazonium salts. The batch and flow protocol described herein can be applied to obtain a broad series of arylated cysteine derivatives and arylated cysteine‐containing dipeptides. Moreover, the method was applied to the chemoselective arylation of a model peptide in biocompatible reaction conditions (room temperature, phosphate‐buffered saline (PBS) buffer) within a short reaction time. PMID:28805276

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    2015-06-24

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

  19. Metabolism, excretion, and pharmacokinetics of S-allyl-L-cysteine in rats and dogs.

    PubMed

    Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji; Kodera, Yukihiro

    2015-05-01

    The metabolism, excretion, and pharmacokinetics of S-allyl-l-cysteine (SAC), an active key component of garlic supplements, were examined in rats and dogs. A single dose of SAC was administered orally or i.v. to rats (5 mg/kg) and dogs (2 mg/kg). SAC was well absorbed (bioavailability >90%) and its four metabolites-N-acetyl-S-allyl-l-cysteine (NAc-SAC), N-acetyl-S-allyl-l-cysteine sulfoxide (NAc-SACS), S-allyl-l-cysteine sulfoxide (SACS), and l-γ-glutamyl-S-allyl-l-cysteine-were identified in the plasma and/or urine. Renal clearance values (<0.01 l/h/kg) of SAC indicated its extensive renal reabsorption, which contributed to the long elimination half-life of SAC, especially in dogs (12 hours). The metabolism of SAC to NAc-SAC, principal metabolite of SAC, was studied in vitro and in vivo. Liver and kidney S9 fractions of rats and dogs catalyzed both N-acetylation of SAC and deacetylation of NAc-SAC. After i.v. administration of NAc-SAC, SAC appeared in the plasma and its concentration declined in parallel with that of NAc-SAC. These results suggest that the rate and extent of the formation of NAc-SAC are determined by the N-acetylation and deacetylation activities of liver and kidney. Also, NAc-SACS was detected in the plasma after i.v. administration of either NAc-SAC or SACS, suggesting that NAc-SACS could be formed via both N-acetylation of SACS and S-oxidation of NAc-SAC. In conclusion, this study demonstrated that the pharmacokinetics of SAC in rats and dogs is characterized by its high oral bioavailability, N-acetylation and S-oxidation metabolism, and extensive renal reabsorption, indicating the critical roles of liver and kidney in the elimination of SAC. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Cardiovascular actions of L-cysteine and L-cysteine sulfinic acid in the nucleus tractus solitarius of the rat.

    PubMed

    Takemoto, Yumi

    2014-07-01

    The sulfur-containing excitatory amino acid (EAA) L-cysteine sulfinic acid (CSA), a neurotransmitter candidate, is endogenously synthesized from L-cysteine (Cys). Exogenous Cys administration into the brain produces cardiovascular effects; these effects likely occur via synaptic stimulation of central nervous system (CNS) neurons that regulate peripheral cardiovascular function. However, the cardiovascular responses produced by CNS Cys administration could result from CSA biosynthesized in synapse. The present study examined the role of CSA in Cys-induced cardiovascular responses within the nucleus tractus solitarius (NTS) of anesthetized rats. The NTS receives input from various visceral afferents that gate autonomic reflexes, including cardiovascular reflexes. Within the NTS, both Cys and CSA microinjections produced decrease responses in arterial blood pressure and heart rate that were similar to those produced by L-glutamate. Co-injection of the ionotropic EAA receptor antagonist kynurenic acid abolished Cys-, but not CSA-, induced cardiovascular responses. This finding suggests that only Cys-induced cardiovascular responses are mediated by kynurenate-sensitive receptors. This study provides the first demonstration that Cys- and CSA-induced cardiovascular responses occur via different mechanisms in the NTS of rats. Further, this study also indicates that Cys-induced cardiovascular responses do not occur via CSA. Thus, within the NTS, endogenous Cys and/or CSA might be involved in cardiovascular regulation.

  1. Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

    PubMed

    Abegg, Daniel; Frei, Reto; Cerato, Luca; Prasad Hari, Durga; Wang, Chao; Waser, Jerome; Adibekian, Alexander

    2015-09-07

    In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Reduction of mercury from mackerel fillet using combined solution of cysteine, EDTA, and sodium chloride.

    PubMed

    Hajeb, P; Jinap, S

    2012-06-13

    An acidic solution containing mercury chelating agents to eliminate mercury in raw fish (mackerel) fillet was developed. The solution contained hydrochloric acid, sodium hydroxide, cysteine, EDTA, and NaCl. The optimum conditions for mercury reduction were achieved using response surface methodology (RSM) at cysteine concentration of 1.25%, EDTA of 275 mg/L, NaCl of 0.5%, pH of 3.75, and exposure time of 18 min. The optimized conditions produced a solution which can remove up to 91% mercury from raw fish fillet. Cysteine and EDTA were identified as potential chelating agents with the greatest potential for use. The solution can be employed in fish industries to reduce mercury in highly contaminated fish.

  3. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

    PubMed

    Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

    2009-12-01

    The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis. (c) 2009 European Peptide Society and John Wiley & Sons, Ltd.

  4. Structural studies on Pax-8 Prd domain/DNA complex.

    PubMed

    Campagnolo, M; Pesaresi, A; Zelezetsky, I; Geremia, S; Randaccio, L; Bisca, A; Tell, G

    2007-04-01

    Pax-8 is a member of the Pax family of transcription factors and is essential in the development of thyroid follicular cells. Pax-8 has two DNA-binding domains: the paired domain and the homeo domain. In this study, a preliminary X-ray diffraction analysis of the mammalian Pax-8 paired domain in complex with the C-site of the thyroglobulin promoter was achieved. The Pax-8 paired domain was crystallized by the hanging-drop vapor-diffusion method in complex with both a blunt-ended 26 bp DNA fragment and with a sticky-ended 24 bp DNA fragment with two additional overhanging bases. Crystallization experiments make clear that the growth of transparent crystals with large dimensions and regular shape is particularly influenced by ionic strength. The crystals of Pax-8 complex with blunt-ended and sticky-ended DNA, diffracted synchrotron radiation to 6.0 and 8.0 A resolution and belongs both to the C centered monoclinic system with cell dimensions: a = 89.88 A, b = 80.05 A, c = 67.73 A, and beta = 124.3 degrees and a = 256.56, b = 69.07, c = 99.32 A, and beta = 98.1 degrees , respectively. Fluorescence experiments suggest that the crystalline disorder, deduced by the poor diffraction, can be attributed to the low homogeneity of the protein-DNA sample. The theoretical comparative model of the Pax-8 paired domain complexed with the C-site of the thyroglobulin promoter shows the probable presence of some specific protein-DNA interactions already observed in other Pax proteins and the important role of the cysteine residues of PAI subdomain in the redox control of the DNA recognition.

  5. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

    PubMed

    Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

    2016-12-01

    Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a K m of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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

    PubMed

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

    2015-03-01

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

  7. Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen

    PubMed Central

    Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

    2009-01-01

    The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L−1 of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L−1 of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50°C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P < 0.05), and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk (group IV) improved both progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The use of DHA-enriched hen egg yolk alone (group II) did not enhance any of the post-thawed semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity. PMID:19633681

  8. Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen.

    PubMed

    Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

    2009-09-01

    The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50 degrees C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P < 0.05), and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk (group IV) improved both progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The use of DHA-enriched hen egg yolk alone (group II) did not enhance any of the post-thawed semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

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

    DOE PAGES

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

    2015-01-01

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

  10. Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

    2010-01-01

    Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

  11. Investigation of Electro-Kinetic Behavior of Cysteine on Electrodeposition of Ni Through the AC and DC Techniques

    NASA Astrophysics Data System (ADS)

    Ebadi, Mehdi; Basirun, Wan J.; Sim, Yoke-L.; Mahmoudian, Mohammad R.

    2013-11-01

    Electrodeposition of nickel was studied by the AC (as a novel technique) and DC techniques in nickel chloride aqueous solutions, mixed with various amounts of cysteine (0 to 6 mM). Cyclic voltammetry and chronoamperometry data have shown that the electrodeposition of Ni in the presence of cysteine is not diffusion controlled, but is closer to instantaneous nucleation. However, the current distribution decreased with the addition of further cysteine. The nucleation sites were decreased from 1.72 × 106 to 0.190 × 106 (cm-2) when the concentration of cysteine was increased from 0 to 4 mM. AC impedance during electrodeposition shows that the charge transfer resistance is increased from 0.645 to 5.26 Ω cm2 when the concentration of cysteine is increased from 0.5 to 4 mM. The electro-corrosion tests were done to investigate the corrosion behavior of the electrodeposited layers. X-ray diffraction and scanning electron microscopy containing Energy dispersive X-ray were used to estimate the grain size of the electrodeposited layers and capture the micrograph images and roughness of the Ni-electrodeposited surface.

  12. Neuroprotective effect of S-allyl-l-cysteine derivatives against endoplasmic reticulum stress-induced cytotoxicity is independent of calpain inhibition.

    PubMed

    Imai, Toru; Kosuge, Yasuhiro; Saito, Hiroaki; Uchiyama, Taketo; Wada, Taira; Shimba, Shigeki; Ishige, Kumiko; Miyairi, Shinichi; Makishima, Makoto; Ito, Yoshihisa

    2016-03-01

    S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on μ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC. Copyright © 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  13. One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine

    PubMed Central

    Kanie, Shusei; Nishikawa, Toshio; Ojika, Makoto; Oba, Yuichi

    2016-01-01

    Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine. PMID:27098929

  14. Spectrophotometric determination of L-cysteine by using polyvinylpyrrolidone-stabilized silver nanoparticles in the presence of barium ions

    NASA Astrophysics Data System (ADS)

    Bamdad, Farzad; Khorram, Fateme; Samet, Maryam; Bamdad, Kourosh; Sangi, Mohammad Reza; Allahbakhshi, Fateme

    2016-05-01

    In this article a simple and selective colorimetric probe for cysteine determination using silver nano particles (AgNPS) is described. The determination process was based upon the surface plasmon resonance properties of polyvinylpyrrolidone-stabilized AgNPS. Interaction of AgNPS with cysteine molecules in the presence of barium ions induced a red shift in the surface plasmon resonance (SPR) maximum of AgNPs, as a result of nanoparticle aggregation. Consequently, yellow color of AgNP solution was changed to pink. The linear range for the determination of cysteine was 3.2-8.2 μM (R = 0.9965) with a limit of detection equal to 2.8 μM (3σ). The proposed method was successfully applied to the determination of cysteine in human plasma samples. Acceptable recovery results of the spiked samples confirmed the validity of the proposed method.

  15. One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine.

    PubMed

    Kanie, Shusei; Nishikawa, Toshio; Ojika, Makoto; Oba, Yuichi

    2016-04-21

    Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine.

  16. The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle.

    PubMed

    Cong, Xiaofei; Doering, Jonathan; Mazala, Davi A G; Chin, Eva R; Grange, Robert W; Jiang, Honglin

    2016-01-01

    The SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice. We disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems. RT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. This study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.

  17. Analysis of Cysteine Redox Post-Translational Modifications in Cell Biology and Drug Pharmacology.

    PubMed

    Wani, Revati; Murray, Brion W

    2017-01-01

    Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

  18. Isolation and characterization of a cysteine protease of freesia corms.

    PubMed

    Uchikoba, Tetsuya; Okubo, Michiko; Arima, Kazunari; Yonezawa, Hiroo

    2002-02-01

    A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.

  19. Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content

    PubMed Central

    Crawford, Matthew A.; Tapscott, Timothy; Fitzsimmons, Liam F.; Liu, Lin; Reyes, Aníbal M.; Libby, Stephen J.; Trujillo, Madia; Fang, Ferric C.; Radi, Rafael

    2016-01-01

    ABSTRACT The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. PMID:27094335

  20. l-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2

    PubMed Central

    Nakatsu, Daiki; Horiuchi, Yuta; Kano, Fumi; Noguchi, Yoshiyuki; Sugawara, Taichi; Takamoto, Iseki; Kubota, Naoto; Kadowaki, Takashi; Murata, Masayuki

    2015-01-01

    Increase in the concentration of plasma l-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged l-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged l-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued l-cysteine–induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. l-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in l-cysteine–treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to l-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D. PMID:25713368

  1. Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

    PubMed

    Lorenz, Eric; Schmacht, Maximilian; Senz, Martin

    2016-11-01

    Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29±0.29% for cysteine to 3.65±0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

    Abstract Motivation: Cysteine-rich proteins cover many important families in nature but there are currently no methods specifically designed for modeling the structure of these proteins. The accuracy of disulfide connectivity pattern prediction, particularly for the proteins of higher-order connections, e.g. >3 bonds, is too low to effectively assist structure assembly simulations. Results: We propose a new hierarchical order reduction protocol called Cyscon for disulfide-bonding prediction. The most confident disulfide bonds are first identified and bonding prediction is then focused on the remaining cysteine residues based on SVR training. Compared with purely machine learning-based approaches, Cyscon improved the average accuracy of connectivity pattern prediction by 21.9%. For proteins with more than 5 disulfide bonds, Cyscon improved the accuracy by 585% on the benchmark set of PDBCYS. When applied to 158 non-redundant cysteine-rich proteins, Cyscon predictions helped increase (or decrease) the TM-score (or RMSD) of the ab initio QUARK modeling by 12.1% (or 14.4%). This result demonstrates a new avenue to improve the ab initio structure modeling for cysteine-rich proteins. Availability and implementation: http://www.csbio.sjtu.edu.cn/bioinf/Cyscon/ Contact: zhng@umich.edu or hbshen@sjtu.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26254435

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

    PubMed Central

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

    2017-01-01

    Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae. Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses. PMID:27852951

  4. Divergent unprotected peptide macrocyclisation by palladium-mediated cysteine arylation.

    PubMed

    Rojas, Anthony J; Zhang, Chi; Vinogradova, Ekaterina V; Buchwald, Nathan H; Reilly, John; Pentelute, Bradley L; Buchwald, Stephen L

    2017-06-01

    Macrocyclic peptides are important therapeutic candidates due to their improved physicochemical properties in comparison to their linear counterparts. Here we detail a method for a divergent macrocyclisation of unprotected peptides by crosslinking two cysteine residues with bis-palladium organometallic reagents. These synthetic intermediates are prepared in a single step from commercially available aryl bis-halides. Two bioactive linear peptides with cysteine residues at i , i + 4 and i , i + 7 positions, respectively, were cyclised to introduce a diverse array of aryl and bi-aryl linkers. These two series of macrocyclic peptides displayed similar linker-dependent lipophilicity, phospholipid affinity, and unique volume of distributions. Additionally, one of the bioactive peptides showed target binding affinity that was predominantly affected by the length of the linker. Collectively, this divergent strategy allowed rapid and convenient access to various aryl linkers, enabling the systematic evaluation of the effect of appending unit on the medicinal properties of macrocyclic peptides.

  5. Spectrophotometric determination of L-cysteine by using polyvinylpyrrolidone-stabilized silver nanoparticles in the presence of barium ions.

    PubMed

    Bamdad, Farzad; Khorram, Fateme; Samet, Maryam; Bamdad, Kourosh; Sangi, Mohammad Reza; Allahbakhshi, Fateme

    2016-05-15

    In this article a simple and selective colorimetric probe for cysteine determination using silver nano particles (AgNPS) is described. The determination process was based upon the surface plasmon resonance properties of polyvinylpyrrolidone-stabilized AgNPS. Interaction of AgNPS with cysteine molecules in the presence of barium ions induced a red shift in the surface plasmon resonance (SPR) maximum of AgNPs, as a result of nanoparticle aggregation. Consequently, yellow color of AgNP solution was changed to pink. The linear range for the determination of cysteine was 3.2-8.2 μM (R=0.9965) with a limit of detection equal to 2.8 μM (3σ). The proposed method was successfully applied to the determination of cysteine in human plasma samples. Acceptable recovery results of the spiked samples confirmed the validity of the proposed method. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2007-01-01

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

  7. The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

    PubMed Central

    Slatter, David A.; Bihan, Dominique G.; Jarvis, Gavin E.; Stone, Rachael; Pugh, Nicholas; Giddu, Sumana; Farndale, Richard W.

    2012-01-01

    Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRPcys, chemical cross-linking using heterobifunctional reagents generates CRPcys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRPcys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze–thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays. PMID:22555281

  8. The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues.

    PubMed

    Slatter, David A; Bihan, Dominique G; Jarvis, Gavin E; Stone, Rachael; Pugh, Nicholas; Giddu, Sumana; Farndale, Richard W

    2012-07-01

    Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

  11. Identification of cysteine protease inhibitors as new drug leads against Naegleria fowleri.

    PubMed

    Zyserman, Ingrid; Mondal, Deboprosad; Sarabia, Francisco; McKerrow, James H; Roush, William R; Debnath, Anjan

    2018-05-01

    Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. PAM occurs principally in healthy children of less than 13 years old with a history of recent exposure to warm fresh water. While as yet not a reportable disease, the Centers for Disease Control and Prevention (CDC) documents a total of 143 cases in the United States. Only four patients have survived. Infection results from water containing N. fowleri entering the nose, followed by migration of the amebae to the brain. Within the brain, N. fowleri infection results in extensive necrosis, leading to death in 3-7 days. Mortality among patients with PAM is greater than 95%. The drugs of choice in treating PAM are the antifungal amphotericin B, and the antileishmanial, miltefosine. However neither drug is FDA-approved for this indication and the use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with amphotericin B have survived PAM. Therefore, development of new, safe and effective drugs is a critical unmet need to avert future deaths of children. The molecular mechanisms underlying the pathogenesis of PAM are poorly understood but it is known that cysteine proteases of N. fowleri play a role in the progression of PAM. We therefore assessed the in vitro activity of the synthetic vinyl sulfone cysteine protease inhibitor, K11777, and 33 analogs with valine, phenylalanine or pyridylalanine at P2 position, against cysteine protease activity in the lysate of N. fowleri. Inhibitors with phenylalanine or pyridylalanine at P2 position were particularly effective in inhibiting the cysteine protease activity of N. fowleri cell lysate with IC 50 ranging between 3 nM and 6.6 μM. Three of the 34 inhibitors also showed inhibitory activity against N. fowleri in a cell viability assay and were 1.6- to 2.5-fold more potent than the standard of care drug miltefosine. Our study provides the first evidence

  12. The toxic effects of l-Cysteine-capped cadmium sulfide nanoparticles on the aquatic plant Spirodela polyrrhiza

    NASA Astrophysics Data System (ADS)

    Khataee, Alireza; Movafeghi, Ali; Nazari, Fatemeh; Vafaei, Fatemeh; Dadpour, Mohammad Reza; Hanifehpour, Younes; Joo, Sang Woo

    2014-12-01

    Plants play an important role in the fate of nanoparticles in the environment through their uptake, bioaccumulation, and transfer to trophic chains. However, the impacts of nanoparticles on plants as essential components of all ecosystems are not well documented. In the present study, the toxic effects of l-Cysteine-capped CdS nanoparticles on Spirodela polyrrhiza as an aquatic higher plant species were studied. l-Cysteine-capped CdS nanoparticles were synthesized using hydrothermal method and their characteristics were determined by XRD, SEM, HR-TEM, and FT-IR techniques. The diameter of majority of synthesized nanoparticles was about 15-20 nm. Subsequently, the uptake of l-Cysteine-capped CdS nanoparticles by the plant species was confirmed using epifluorescence microscopy. The activity of peroxidase and superoxide dismutase as antioxidant enzymes was assayed and the relative frond number was calculated in the presence of different concentrations of l-Cysteine-capped CdS nanoparticles. The obtained results revealed the toxic effects of the synthesized nanoparticles on S. polyrrhiza, leading to growth reduction and significant changes in antioxidant enzymes' activity.

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

    PubMed Central

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

    2014-01-01

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

  14. Cysteine could change the transport mechanism of PVP-coated silver nanoparticles in porous media

    NASA Astrophysics Data System (ADS)

    Yang, X.; Lin, S.; Wiesner, M.

    2012-12-01

    Silver nanoparticles (AgNPs) can hardly be removed by wastewater treatment plant and have big potential to enter groundwater, jeopardizing the water quality & aquatic ecosystem. Most AgNPs have surface coatings such as polyvinylpyrrolidone (PVP) which dominate their transport in porous media. Our previous study shows that PVP may promote the deposition of AgNPs on silica surface by a bridging mechanism. This study further explored how cysteine, a natural organic matter type, may influence the role of the PVP coating on AgNP translocation. Dynamic Light Scattering (DLS) measurement (Figure 1A) shows that the PVP coating rendered the AgNP dispersion high stability during the measuring period (3hrs). Addition of 100 ppm cysteine to the dispersion resulted in a rapid decrease in particle size from 100nm to 52nm within one hour, following which no further decline in particle size occurred. Column experiment results (Figure 1B) show that corresponding to the particle size change was a substantial decrease in particle deposition rates: introduction of 100 ppm cysteine into the particle dispersion resulted in a decrease in AgNP attenuation by the porous medium from 67% to 26%. The decline in particle size suggested that cysteine may have displaced the macromolecular PVP from the particle surface. Desorption of PVP resulted in a weakening or vanish of polymer bridging effect which in turn lowered the deposition rates substantially. This study demonstrated an implication of environmental transformation of coated AgNPs to their mobility in saturated sand aquifers. Acknowledgment Xinyao Yang appreciates the Natural Science Foundation of China (Grant No.:41101475) for covering the registration fee and traveling costs.igure 1 Particle size measurement (A) and breakthrough curves (B) of PVP-coated silver nanoparticle in the absence and presence of cysteine: pH=7.0, ionic strength=1mM, flow rate=1ml/min.

  15. Ultrasensitive Detection of Cu2+ Using a Microcantilever Sensor Modified with L-Cysteine Self-Assembled Monolayer.

    PubMed

    Xu, Xiaohe; Zhang, Na; Brown, Gilbert M; Thundat, Thomas G; Ji, Hai-Feng

    2017-10-01

    A microcantilever was modified with a self-assembled monolayer (SAM) of L-cysteine for the sensitively and selectively response to Cu(II) ions in aqueous solution. The microcantilever undergoes bending due to sorption of Cu(II) ions. The interaction of Cu(II) ions with the L-cysteine on the cantilever is diffusion controlled and does not follow a simple Langmuir adsorption model. A concentration of 10 -10  M Cu(II) was detected in a fluid cell using this technology. Other cations, such as Ni 2+ , Zn 2+ , Pb 2+ , Cd 2+ , Ca 2+ , K + , and Na + , did not respond with a significant deflection, indicating that this L-cysteine-modified cantilever responded selectively and sensitively to Cu(II).

  16. Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

    PubMed

    Trejo, Sebastián A; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L; Canals, Francesc; Avilés, Francesc X

    2009-07-01

    Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

  17. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structuremore » was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.« less

  18. DNA/nickel oxide nanoparticles/osmium(III)-complex modified electrode toward selective oxidation of l-cysteine and simultaneous detection of l-cysteine and homocysteine.

    PubMed

    Sharifi, Ensiyeh; Salimi, Abdollah; Shams, Esmaeil

    2012-08-01

    The modification of glassy carbon (GC) electrode with electrodeposited nickel oxide nanoparticles (NiOxNPs) and deoxyribonucleic acid (DNA) is utilized as a new efficient platform for entrapment of osmium (III) complex. Surface morphology and electrochemical properties of the prepared nanocomposite modified electrode (GC/DNA/NiOxNPs/Os(III)-complex) were investigated by FESEM, cyclic voltammetry and electrochemical impedance spectroscopy techniques. Cyclic voltammetric results indicated the excellent electrocatalytic activity of the resulting electrode toward oxidation of l-cysteine (CySH) at reduced overpotential (0.1 V vs. Ag/AgCl). Using chronoamperometry to CySH detection, the sensitivity and detection limit of the biosensor are obtained as 44 μA mM(-1) and 0.07 μM with a concentration range up to 1000 μM. The electrocatalytic activity of the modified electrode not only for oxidation of low molecular-mass biothiols derivatives such as, glutathione, l-cystine, l-methionine and electroactive biological species ( dopamine, uric acid, glucose) is negligible but also for very similar biothiol compound (homocysteine) no recognizable response is observed at the applied potential window. Furthermore, the simultaneous voltammetric determination of l-cysteine and homocysteine compounds without any separation or pretreatment process was reported for the first time in this work. Finally, the applicability of sensor for the analysis of CySH concentration in complex serum samples was successfully demonstrated. Highly selectivity, excellent electrocatalytic activity and stability, remarkable antifouling property toward thiols and their oxidation products, as well as the ability for simultaneous detection of l-cysteine and homocysteine are remarkably advantageous of the proposed DNA based biosensor. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Effect of L-cysteine on remote organ injury in rats with severe acute pancreatitis induced by bile-pancreatic duct obstruction.

    PubMed

    Yang, Li-Juan; Wan, Rong; Shen, Jia-Qing; Shen, Jie; Wang, Xing-Peng

    2013-08-01

    Remote organ failure occurs in cases of acute pancreatitis (AP); however, the reports on AP induced by pancreatic duct obstruction are rare. In this study we determined the effect of L-cysteine on pancreaticobiliary inflammation and remote organ damage in rats after pancreaticobiliary duct ligation (PBDL). AP was induced by PBDL in rats with 5/0 silk. Sixty rats were randomly divided into 4 groups. Groups A and B were sham-operated groups that received injections of saline or L-cysteine (10 mg/kg) intraperitoneally (15 rats in each group). Groups C and D were PBDL groups that received injections of saline or L-cysteine (10 mg/kg) intraperitoneally (15 rats in each group). The tissue samples of the pancreas and remote organs such as the lung, liver, intestine and kidney were subsequently examined for pathological changes under a light microscope. The samples were also stored for the determination of malondialdehyde and glutathione levels. Blood urea nitrogen (BUN), plasma amylase, ALT and AST levels were determined spectrophotometrically using an automated analyzer. Also, we evaluated the effect of L-cysteine on remote organ injury in rats with AP induced by retrograde infusion of 3.5% sodium taurocholate (NaTc) into the bile-pancreatic duct. Varying degrees of injury in the pancreas, lung, liver, intestine and kidney were observed in the rats 24 hours after PBDL. The severity of injury to the lung, liver and intestine was attenuated, while injury status was not changed significantly in the pancreas and kidney after L-cysteine treatment. Oxidative stress was also affected by L-cysteine in PBDL-treated rats. The concentration of tissue malondialdehyde decreased in the pancreas and remote organs of PBDL and L-cysteine administrated rats, and the concentration of glutathione increased more significantly than that of the model control group. However, L-cysteine administration reduced the severity of injury in remote organs but not in the pancreas in rats with Na

  20. A substitutional mutation in the DNA binding domain of the androgen receptor causes complete androgen insensitivity syndrome.

    PubMed

    Komori, S; Sakata, K; Kasumi, H; Tsuji, Y; Hamada, K; Koyama, K

    1999-10-01

    DNA analysis of the androgen receptor gene in a patient with complete androgen insensitivity syndrome identified a substitutional mutation (tyrosine converted to cysteine at position 571) in the DNA binding domain. In vitro transfection experiments with the patients' androgen receptor gene, indicated normal expression of the androgen receptor in transfected COS-7 cells compared to the wild type gene. There was also no evidence of impaired thermal stability of the 5 alpha-dihydrotestosterone-androgen receptor complex. However, the capacity of the androgen receptor to activate target gene transcription was found to be completely disrupted in a luciferase assay. These results confirmed that only one substitutional mutation in the DNA binding domain was related to the pathogenesis of the complete androgen insensitivity syndrome.

  1. Unusual specific heat of almost dry L-cysteine and L-cystine amino acids.

    PubMed

    Ishikawa, M S; Lima, T A; Ferreira, F F; Martinho, H S

    2015-03-01

    A detailed quantitative analysis of the specific heat in the 0.5- to 200-K temperature range for almost dry L-cysteine and its dimer, L-cystine, amino acids is presented. We report the occurrence of a sharp first-order transition at ∼76 K for L-cysteine associated with the thiol group ordering which was successfully modeled with the two-dimensional Ising model. We demonstrated that quantum rotors, two-level systems (TLS), Einstein oscillators, and acoustic phonons (the Debye model) are essential ingredients to correctly describe the overall experimental data. Our analysis pointed out the absence of the TLS contribution to the low temperature specific heat of L-cysteine. This result was similar to that found in other noncrystalline amorphous materials, e.g., amorphous silicon, low density amorphous water, and ultrastable glasses. L-cystine presented an unusual nonlinear acoustic dispersion relation ω(q)=vq0.95 and a Maxwell-Boltzmann-type distribution of tunneling barriers. The presence of Einstein oscillators with ΘE∼70 K was common in both systems and adequately modeled the boson peak contributions.

  2. Cysteine-mediated aggregation of Au nanoparticles: the development of a H2O2 sensor and oxidase-based biosensors.

    PubMed

    Wang, Fuan; Liu, Xiaoqing; Lu, Chun-Hua; Willner, Itamar

    2013-08-27

    The cysteine-stimulated aggregation of Au nanoparticles (Au NPs) is used as an auxiliary reporting system for the optical detection of H2O2, for optical probing of the glucose oxidase (GOx) and the catalyzed oxidation of glucose, for probing the biocatalytic cascade composed of acetylcholine esterase/choline oxidase (AChE/ChOx), and for following the inhibition of AChE. The analytical paradigm is based on the I(-)-catalyzed oxidation of cysteine by H2O2 to cystine, a process that prohibits the cysteine-triggered aggregation of the Au NPs. The system enabled the analysis of H2O2 with a detection limit of 2 μM. As the GOx-biocatalyzed oxidation of glucose yields H2O2, and the AChE/ChOx cascade leads to the formation of H2O2, the two biocatalytic processes could be probed by the cysteine-stimulated aggregation of the Au NPs. Since AChE is inhibited by 1,5-bis(4-allyldimethylammonium phenyl)pentane-3-one dibromide, the biocatalytic AChE/ChOx cascade is inhibited by the inhibitor, thus leading to the enhanced cysteine-mediated aggregation of the NPs. The results suggest the potential implementation of the cysteine-mediated aggregation of Au NPs in the presence of AChE/ChOx as a sensing platform for the optical detection of chemical warfare agents.

  3. High-performance liquid chromatography assay of cysteine and homocysteine using fluorosurfactant-functionalized gold nanoparticles as postcolumn resonance light scattering reagents.

    PubMed

    Xiao, Qunyan; Gao, Huiling; Yuan, Qipeng; Lu, Chao; Lin, Jin-Ming

    2013-01-25

    Herein, a new postcolumn resonance light scattering (RLS) detection approach coupled with high-performance liquid chromatography (HPLC) was developed to detect cysteine and homocysteine. In the established system, the fluorosurfactant-capped gold nanoparticles (AuNPs) were first employed as postcolumn RLS reagents. The detection principle was based on the enhancement of RLS intensity of AuNPs upon the addition of cysteine/homocysteine. The RLS signals were detected by a common fluorescence detector at λ(EX)=λ(EM)=560 nm. The linear ranges for both cysteine and homocysteine were in the range of 5.0-50 μM. The detection limits were 5.9 pmol for cysteine and 12 pmol for homocysteine at a signal-to-noise ratio of 3. HPLC separation and RLS detection conditions were optimized in detail. The applicability of the proposed method has been validated by detecting cysteine and homocysteine in human urine samples. Recoveries from spiked urine samples were 95.0-103.0%. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2017-08-11

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

  5. Lead(II) Complex Formation with L-cysteine in Aqueous Solution

    PubMed Central

    Jalilehvand, Farideh; Sisombath, Natalie S.; Schell, Adam C.; Facey, Glenn A.

    2015-01-01

    The lead(II) complexes formed with the multidentate chelator L-cysteine (H2Cys) in alkaline aqueous solution were studied using 207Pb, 13C and 1H NMR, Pb LIII-edge X-ray absorption and UV-vis. spectroscopic techniques, complemented by electro-spray ion mass spectrometry (ESI-MS). The H2Cys/Pb(II) mole ratios were varied from 2.1 to 10.0 for two sets of solutions with CPb(II) = 0.01 and 0.1 M, respectively, prepared at pH values (9.1 – 10.4) for which precipitates of Pb(II)-cysteine dissolved. At low H2Cys/Pb(II) mole ratios (2.1 – 3.0) a mixture of the dithiolate [Pb(S,N-Cys)2]2− and [Pb(S,N,O-Cys)(S-HCys)]− complexes with the average Pb-(N/O) and Pb-S distances 2.42 ± 0.04 Å and 2.64 ± 0.04 Å, respectively, was found to dominate. At high concentration of free cysteinate (> 0.7 M) a significant amount converts to the trithiolate [Pb(S,N-Cys)(S-HCys)2]2−, including a minor amount of a PbS3 coordinated [Pb(S-HCys)3]− complex. The coordination mode was evaluated by fitting linear combinations of EXAFS oscillations to the experimental spectra, and by the 207Pb NMR signals in the chemical shift range δPb = 2006 – 2507 ppm, which became increasingly deshielded with increasing free cysteinate concentration. One-pulse magic angle spinning (MAS) 207Pb NMR spectra of crystalline Pb(aet)2 (Haet = 2-aminoethanethiol or cysteamine) with PbS2N2 coordination were measured for comparison (δiso = 2105 ppm). The UV-vis. spectra displayed absorption maxima at 298 – 300 nm (S− → PbII charge transfer) for the dithiolate PbS2N(N/O) species; with increasing ligand excess a shoulder appeared at ∼ 330 nm for the trithiolate PbS3N and PbS3 (minor) complexes. The results provide spectroscopic fingerprints for structural models for Pb(II) coordination modes to proteins and enzymes. PMID:25695880

  6. Lead(II) complex formation with l-cysteine in aqueous solution

    DOE PAGES

    Jalilehvand, Farideh; Sisombath, Natalie S.; Schell, Adam C.; ...

    2015-02-19

    The lead(II) complexes formed with the multidentate chelator l-cysteine (H 2Cys) in an alkaline aqueous solution were studied using 207Pb, 13C, and 1H NMR, Pb L III-edge X-ray absorption, and UV–vis spectroscopic techniques, complemented by electrospray ion mass spectrometry (ESI-MS). The H 2Cys/Pb II mole ratios were varied from 2.1 to 10.0 for two sets of solutions with C PbII = 0.01 and 0.1 M, respectively, prepared at pH values (9.1–10.4) for which precipitates of lead(II) cysteine dissolved. At low H 2Cys/Pb II mole ratios (2.1–3.0), a mixture of the dithiolate [Pb(S,N-Cys) 2] 2– and [Pb(S,N,O-Cys)(S-HCys)] – complexes with averagemore » Pb–(N/O) and Pb–S distances of 2.42 ± 0.04 and 2.64 ± 0.04 Å, respectively, was found to dominate. At high concentration of free cysteinate (>0.7 M), a significant amount converts to the trithiolate [Pb(S,N-Cys)(S-HCys) 2] 2–, including a minor amount of a PbS 3-coordinated [Pb(S-HCys) 3] – complex. The coordination mode was evaluated by fitting linear combinations of EXAFS oscillations to the experimental spectra and by examining the 207Pb NMR signals in the chemical shift range δ Pb = 2006–2507 ppm, which became increasingly deshielded with increasing free cysteinate concentration. One-pulse magic-angle-spinning (MAS) 207Pb NMR spectra of crystalline Pb(aet) 2 (Haet = 2-aminoethanethiol or cysteamine) with PbS 2N 2 coordination were measured for comparison (δ iso = 2105 ppm). The UV–vis spectra displayed absorption maxima at 298–300 nm (S – → Pb II charge transfer) for the dithiolate PbS 2N(N/O) species; with increasing ligand excess, a shoulder appeared at ~330 nm for the trithiolate PbS 3N and PbS 3 (minor) complexes. Finally, the results provide spectroscopic fingerprints for structural models for lead(II) coordination modes to proteins and enzymes.« less

  7. Strongly enhanced Fenton degradation of organic pollutants by cysteine: An aliphatic amino acid accelerator outweighs hydroquinone analogues.

    PubMed

    Li, Tuo; Zhao, Zhenwen; Wang, Quan; Xie, Pengfei; Ma, Jiahai

    2016-11-15

    Quinone-hydroquinone analogues have been proven to be efficient promoters of Fenton reactions by accelerating the Fe(III)/Fe(II) redox cycle along with self-destruction. However, so far there is little information on non-quinone-hydroquinone cocatalyst for Fenton reactions. This study found that cysteine, a common aliphatic amino acid, can strongly enhance Fenton degradation of organic pollutants by accelerating Fe(III)/Fe(II) redox cycle, as quinone-hydroquinone analogues do. Further, cysteine is superior to quinone-hydroquinone analogues in catalytic activity, H 2 O 2 utilization and atmospheric limits. The cocatalysis mechanism based on the cycle of cysteine/cystine was proposed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Detection of Acetaldehyde in the Esophageal Tissue among Healthy Male Subjects after Ethanol Drinking and Subsequent L-Cysteine Intake.

    PubMed

    Okata, Hideki; Hatta, Waku; Iijima, Katsunori; Asanuma, Kiyotaka; Tsuruya, Atsuki; Asano, Naoki; Koike, Tomoyuki; Hamada, Shin; Nakayama, Toru; Masamune, Atsushi; Shimosegawa, Tooru

    2018-04-01

    Ethanol is oxidized by alcohol dehydrogenase to acetaldehyde, a recognized carcinogen for the esophagus. However, no previous study has measured the acetaldehyde levels in the esophageal tissue. L-cysteine has been shown to reduce the acetaldehyde levels in the saliva; however, it is unknown whether L-cysteine intake affects the acetaldehyde concentration in the esophageal tissue. The aim of this study was to measure the acetaldehyde concentration in the esophageal tissue after ethanol drinking and evaluate the effect of L-cysteine intake on the acetaldehyde levels in the esophagus. We enrolled 10 male subjects with active acetaldehyde dehydrogenase-2*1/*1 (ALDH2*1/*1) genotype and 10 male subjects with the inactive acetaldehyde dehydrogenase-2*1/*2 (ALDH2*1/*2) genotype, the mean ages of whom were 25.6 and 27.9 years, respectively. In this prospective, single-blind, placebo-controlled study using L-cysteine and placebo lozenges (first and second examination), saliva and blood were collected before and after ethanol drinking. Esophageal tissue was obtained by endoscopic biopsy at 60 minutes after drinking, and the acetaldehyde and ethanol concentrations were measured. The acetaldehyde concentration of the saliva was significantly lower in those taking L-cysteine than in those taking the placebo. Acetaldehyde in the esophageal tissue was detected only in those taking L-cysteine lozenges. There were no correlations between the acetaldehyde concentrations in the esophageal tissue and saliva or blood. In conclusion, we detected acetaldehyde in the human esophageal tissue after ethanol drinking. Unexpectedly, intake of L-cysteine lozenges appears to contribute to detection of acetaldehyde in the esophageal tissue.

  9. Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

    PubMed

    Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A L

    2015-08-01

    Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. © 2015 American Society of Plant Biologists. All Rights Reserved.

  10. Optimization of Maillard Reaction in Model System of Glucosamine and Cysteine Using Response Surface Methodology

    PubMed Central

    Arachchi, Shanika Jeewantha Thewarapperuma; Kim, Ye-Joo; Kim, Dae-Wook; Oh, Sang-Chul; Lee, Yang-Bong

    2017-01-01

    Sulfur-containing amino acids play important roles in good flavor generation in Maillard reaction of non-enzymatic browning, so aqueous model systems of glucosamine and cysteine were studied to investigate the effects of reaction temperature, initial pH, reaction time, and concentration ratio of glucosamine and cysteine. Response surface methodology was applied to optimize the independent reaction parameters of cysteine and glucosamine in Maillard reaction. Box-Behnken factorial design was used with 30 runs of 16 factorial levels, 8 axial levels and 6 central levels. The degree of Maillard reaction was determined by reading absorption at 425 nm in a spectrophotometer and Hunter’s L, a, and b values. ΔE was consequently set as the fifth response factor. In the statistical analyses, determination coefficients (R2) for their absorbance, Hunter’s L, a, b values, and ΔE were 0.94, 0.79, 0.73, 0.96, and 0.79, respectively, showing that the absorbance and Hunter’s b value were good dependent variables for this model system. The optimum processing parameters were determined to yield glucosamine-cysteine Maillard reaction product with higher absorbance and higher colour change. The optimum estimated absorbance was achieved at the condition of initial pH 8.0, 111°C reaction temperature, 2.47 h reaction time, and 1.30 concentration ratio. The optimum condition for colour change measured by Hunter’s b value was 2.41 h reaction time, 114°C reaction temperature, initial pH 8.3, and 1.26 concentration ratio. These results can provide the basic information for Maillard reaction of aqueous model system between glucosamine and cysteine. PMID:28401086

  11. Optimization of Maillard Reaction in Model System of Glucosamine and Cysteine Using Response Surface Methodology.

    PubMed

    Arachchi, Shanika Jeewantha Thewarapperuma; Kim, Ye-Joo; Kim, Dae-Wook; Oh, Sang-Chul; Lee, Yang-Bong

    2017-03-01

    Sulfur-containing amino acids play important roles in good flavor generation in Maillard reaction of non-enzymatic browning, so aqueous model systems of glucosamine and cysteine were studied to investigate the effects of reaction temperature, initial pH, reaction time, and concentration ratio of glucosamine and cysteine. Response surface methodology was applied to optimize the independent reaction parameters of cysteine and glucosamine in Maillard reaction. Box-Behnken factorial design was used with 30 runs of 16 factorial levels, 8 axial levels and 6 central levels. The degree of Maillard reaction was determined by reading absorption at 425 nm in a spectrophotometer and Hunter's L, a, and b values. ΔE was consequently set as the fifth response factor. In the statistical analyses, determination coefficients (R 2 ) for their absorbance, Hunter's L, a, b values, and ΔE were 0.94, 0.79, 0.73, 0.96, and 0.79, respectively, showing that the absorbance and Hunter's b value were good dependent variables for this model system. The optimum processing parameters were determined to yield glucosamine-cysteine Maillard reaction product with higher absorbance and higher colour change. The optimum estimated absorbance was achieved at the condition of initial pH 8.0, 111°C reaction temperature, 2.47 h reaction time, and 1.30 concentration ratio. The optimum condition for colour change measured by Hunter's b value was 2.41 h reaction time, 114°C reaction temperature, initial pH 8.3, and 1.26 concentration ratio. These results can provide the basic information for Maillard reaction of aqueous model system between glucosamine and cysteine.

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

    EPA Science Inventory

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

  13. Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (Cyprinus carpio).

    PubMed

    Öğretmen, Fatih; İnanan, Burak Evren; Kutluyer, Filiz; Kayim, Murathan

    2015-06-01

    Amino acids have an important biological role for prevention of cell damage during cryopreservation. The objective of this study is to determine the effects of cysteine on postthaw sperm motility, duration of sperm motility, DNA damage, and fertility in the common carp (Cyprinus carpio). Sperm collected from 10 individuals was cryopreserved in extenders containing different cysteine concentrations (2.5, 5, 10, and 20 mM). Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution, the semen was aspirated into 0.25-mL straws; the straws were placed on the tray, frozen in nitrogen vapor, and plunged into liquid nitrogen. DNA damage was evaluated by comet assay after cryopreservation. Our results indicated that an increase in the concentration of cysteine caused a significant increase in the motility rate and duration of sperm in the common carp (C carpio; P < 0.05). Comparing all concentrations of cysteine, the best concentration of cysteine was 20 mM. Higher postthaw motility (76.00 ± 1.00%) and fertilization (97.00 ± 1.73%) rates were obtained with the extender at the concentration of 20 mM. Supplementation of the extender with cysteine was increased the fertilization and hatching rate and decreased DNA damage. Consequently, cysteine affected the motility, fertilization, and DNA damage positively, and extenders could be supplemented with cysteine. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Calibration of GafChromic EBT3 for absorbed dose measurements in 5 MeV proton beam and (60)Co γ-rays.

    PubMed

    Vadrucci, M; Esposito, G; Ronsivalle, C; Cherubini, R; Marracino, F; Montereali, R M; Picardi, L; Piccinini, M; Pimpinella, M; Vincenti, M A; De Angelis, C

    2015-08-01

    To study EBT3 GafChromic film in low-energy protons, and for comparison purposes, in a reference (60)Co beam in order to use it as a calibrated dosimetry system in the proton irradiation facility under construction within the framework of the Oncological Therapy with Protons (TOP)-Intensity Modulated Proton Linear Accelerator for RadioTherapy (IMPLART) Project at ENEA-Frascati, Italy. EBT3 film samples were irradiated at the Istituto Nazionale di Fisica Nucleare-Laboratori Nazionali di Legnaro, Italy, with a 5 MeV proton beam generated by a 7 MV Van de Graaff CN accelerator. The nominal dose rates used were 2.1 Gy/min and 40 Gy/min. The delivered dose was determined by measuring the particle fluence and the energy spectrum in air with silicon surface barrier detector monitors. A preliminary study of the EBT3 film beam quality dependence in low-energy protons was conducted by passively degrading the beam energy. EBT3 films were also irradiated at ENEA-National Institute of Ionizing Radiation Metrology with gamma radiation produced by a (60)Co source characterized by an absorbed dose to water rate of 0.26 Gy/min as measured by a calibrated Farmer type ionization chamber. EBT3 film calibration curves were determined by means of a set of 40 film pieces irradiated to various doses ranging from 0.5 Gy to 30 Gy absorbed dose to water. An EPSON Expression 11000XL color scanner in transmission mode was used for film analysis. Scanner response stability, intrafilm uniformity, and interfilm reproducibility were verified. Optical absorption spectra measurements were performed on unirradiated and irradiated EBT3 films to choose the most sensitive color channel to the dose range used. EBT3 GafChromic films show an under response up to about 33% for low-energy protons with respect to (60)Co gamma radiation, which is consistent with the linear energy transfer dependence already observed with higher energy protons, and a negligible dose-rate dependence in the 2-40 Gy/min range

  15. Prospective study of serum cysteine and cysteinylglycine and cancer of the head and neck, esophagus, and stomach in a cohort of male smokers

    USDA-ARS?s Scientific Manuscript database

    Background: The nonessential amino acid cysteine is known to be involved in many antioxidant and anticarcinogenic pathways. Cysteinylglycine is a pro-oxidant metabolite of glutathione and a precursor of cysteine. Objective: To examine the relation between serum cysteine and cysteinylglycine and ris...

  16. The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain

    PubMed Central

    Husain, S. S.; Lowe, G.

    1970-01-01

    Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin. PMID:5420046

  17. Structure of the substrate-binding b′ domain of the Protein disulfide isomerase-like protein of the testis

    PubMed Central

    Bastos-Aristizabal, Sara; Kozlov, Guennadi; Gehring, Kalle

    2014-01-01

    Protein Disulfide Isomerase-Like protein of the Testis (PDILT) is a testis-specific member of the PDI family. PDILT displays similar domain architecture to PDIA1, the founding member of this protein family, but lacks catalytic cysteines needed for oxidoreduction reactions. This suggests special importance of chaperone activity of PDILT, but how it recognizes misfolded protein substrates is unknown. Here, we report the high-resolution crystal structure of the b′ domain of human PDILT. The structure reveals a conserved hydrophobic pocket, which is likely a principal substrate-binding site in PDILT. In the crystal, this pocket is occupied by side chains of tyrosine and tryptophan residues from another PDILT molecule, suggesting a preference for binding exposed aromatic residues in protein substrates. The lack of interaction of the b′ domain with the P-domains of calreticulin-3 and calmegin hints at a novel way of interaction between testis-specific lectin chaperones and PDILT. Further studies of this recently discovered PDI member would help to understand the important role that PDILT plays in the differentiation and maturation of spermatozoids. PMID:24662985

  18. Chemical fingerprints of cold physical plasmas - an experimental and computational study using cysteine as tracer compound.

    PubMed

    Lackmann, J-W; Wende, K; Verlackt, C; Golda, J; Volzke, J; Kogelheide, F; Held, J; Bekeschus, S; Bogaerts, A; Schulz-von der Gathen, V; Stapelmann, K

    2018-05-16

    Reactive oxygen and nitrogen species released by cold physical plasma are being proposed as effectors in various clinical conditions connected to inflammatory processes. As these plasmas can be tailored in a wide range, models to compare and control their biochemical footprint are desired to infer on the molecular mechanisms underlying the observed effects and to enable the discrimination between different plasma sources. Here, an improved model to trace short-lived reactive species is presented. Using FTIR, high-resolution mass spectrometry, and molecular dynamics computational simulation, covalent modifications of cysteine treated with different plasmas were deciphered and the respective product pattern used to generate a fingerprint of each plasma source. Such, our experimental model allows a fast and reliable grading of the chemical potential of plasmas used for medical purposes. Major reaction products were identified to be cysteine sulfonic acid, cystine, and cysteine fragments. Less-abundant products, such as oxidized cystine derivatives or S-nitrosylated cysteines, were unique to different plasma sources or operating conditions. The data collected point at hydroxyl radicals, atomic O, and singlet oxygen as major contributing species that enable an impact on cellular thiol groups when applying cold plasma in vitro or in vivo.

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

    PubMed

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

    2015-05-01

    Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems. © 2015 American Society of Plant Biologists. All Rights Reserved.

  20. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    NASA Astrophysics Data System (ADS)

    Pandey, Chandra Mouli; Sumana, Gajjala; Tiwari, Ida

    2014-09-01

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38 × 10-4 cm s-1. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.

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

    PubMed

    Liu, Xuehong; Dawson, David C

    2011-11-29

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

  2. Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.

    PubMed

    Yoshida, Akihiro; Yoshimura, Mamiko; Ohara, Naoya; Yoshimura, Shigeru; Nagashima, Shiori; Takehara, Tadamichi; Nakayama, Koji

    2009-11-01

    Hydrogen sulfide is one of the predominant volatile sulfur compounds (VSCs) produced by oral bacteria. This study developed and evaluated a system for detecting hydrogen sulfide production by oral bacteria. L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (METase) and beta carbon-sulfur (beta C-S) lyase were used to degrade homocysteine and cysteine, respectively, to produce hydrogen sulfide. Enzymatic reactions resulting in hydrogen sulfide production were assayed by reaction with bismuth trichloride, which forms a black precipitate when mixed with hydrogen sulfide. The enzymatic activities of various oral bacteria that result in hydrogen sulfide production and the capacity of bacteria from periodontal sites to form hydrogen sulfide in reaction mixtures containing L-cysteine or DL-homocysteine were assayed. With L-cysteine as the substrate, Streptococcus anginosus FW73 produced the most hydrogen sulfide, whereas Porphyromonas gingivalis American Type Culture Collection (ATCC) 33277 and W83 and Fusobacterium nucleatum ATCC 10953 produced approximately 35% of the amount produced by the P. gingivalis strains. Finally, the hydrogen sulfide found in subgingival plaque was analyzed. Using bismuth trichloride, the hydrogen sulfide produced by oral bacteria was visually detectable as a black precipitate. Hydrogen sulfide production by oral bacteria was easily analyzed using bismuth trichloride. However, further innovation is required for practical use.

  3. Cysteine, histidine and glycine exhibit anti-inflammatory effects in human coronary arterial endothelial cells.

    PubMed

    Hasegawa, S; Ichiyama, T; Sonaka, I; Ohsaki, A; Okada, S; Wakiguchi, H; Kudo, K; Kittaka, S; Hara, M; Furukawa, S

    2012-02-01

    The activation of nuclear factor-kappa B (NF-κB) in vascular endothelial cells may be involved in vascular pathogeneses such as vasculitis or atherosclerosis. Recently, it has been reported that some amino acids exhibit anti-inflammatory effects. We investigated the inhibitory effects of a panel of amino acids on cytokine production or expression of adhesion molecules that are involved in inflammatory diseases in various cell types. The activation of NF-κB was determined in human coronary arterial endothelial cells (HCAECs) because NF-κB modulates the production of many cytokines and the expression of adhesion molecules. We examined the inhibitory effects of the amino acids cysteine, histidine and glycine on the induction of NF-κB activation, expression of CD62E (E-selectin) and the production of interleukin (IL)-6 in HCAECs stimulated with tumour necrosis factor (TNF)-α. Cysteine, histidine and glycine significantly reduced NF-κB activation and inhibitor κBα (IκBα) degradation in HCAECs stimulated with TNF-α. Additionally, all the amino acids inhibited the expression of E-selectin and the production of IL-6 in HCAECs, and the effects of cysteine were the most significant. Our results show that glycine, histidine and cysteine can inhibit NF-κB activation, IκBα degradation, CD62E expression and IL-6 production in HCAECs, suggesting that these amino acids may exhibit anti-inflammatory effects during endothelial inflammation. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

  4. The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

    PubMed Central

    Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

  5. The second transmembrane domain of the human type 1 angiotensin II receptor participates in the formation of the ligand binding pocket and undergoes integral pivoting movement during the process of receptor activation.

    PubMed

    Domazet, Ivana; Holleran, Brian J; Martin, Stéphane S; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-05-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT(1) receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT(1), L81C-AT(1), A85C-AT(1), T88C-AT(1), and A89C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant D74C-N111G-AT(1) became insensitive to MTSEA, whereas mutant L81C-N111G-AT(1) lost some sensitivity and mutant V86C-N111G-AT(1) became sensitive to MTSEA. Our results suggest that constitutive activation of the AT(1) receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.

  6. l-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

    PubMed

    Iqbal, S; Riaz, A; Andrabi, S M H; Shahzad, Q; Durrani, A Z; Ahmad, N

    2016-11-01

    The effects of l-cysteine in extender on antioxidant enzymes profile during cryopreservation, post-thaw quality parameters and in vivo fertility of Nili-Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of l-cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm) and frozen in 0.5-ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre-freezing and post-thawing in extender containing 2.0 mm l-cysteine as compared to other groups. Post-thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curvilinear velocity (μm s -1 ), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l-cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l-cysteine than in the control. In conclusion, the addition of 2.0 mm l-cysteine in extender improved the antioxidant enzymes profile, post-thaw quality and in vivo fertility of Nili-Ravi buffalo bull spermatozoa. © 2016 Blackwell Verlag GmbH.

  7. A single-cysteine mutant and chimeras of essential Leishmania Erv can complement the loss of Erv1 but not of Mia40 in yeast.

    PubMed

    Specht, Sandra; Liedgens, Linda; Duarte, Margarida; Stiegler, Alexandra; Wirth, Ulrike; Eberhardt, Maike; Tomás, Ana; Hell, Kai; Deponte, Marcel

    2018-05-01

    Mia40/CHCHD4 and Erv1/ALR are essential for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals. In contrast, many protists, including important apicomplexan and kinetoplastid parasites, lack Mia40. Furthermore, the Erv homolog of the model parasite Leishmania tarentolae (LtErv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 (ScMia40). Here we addressed structure-function relationships of ScErv1 and LtErv as well as their compatibility with the oxidative protein folding system in yeast using chimeric, truncated, and mutant Erv constructs. Chimeras between the N-terminal arm of ScErv1 and a variety of truncated LtErv constructs were able to rescue yeast cells that lack ScErv1. Yeast cells were also viable when only a single cysteine residue was replaced in LtErv C17S . Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of LtErv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered LtErv incompatible with ScMia40. The question whether parasite Erv homologs might also exert the function of Mia40 was addressed in another set of complementation assays. However, neither the KISS domain nor other truncated or mutant LtErv constructs were able to rescue yeast cells that lack ScMia40. The general relevance of Erv and its candidate substrate small Tim1 was analyzed for the related parasite L. infantum. Repeated unsuccessful knockout attempts suggest that both genes are essential in this human pathogen and underline the potential of mitochondrial protein import pathways for future intervention strategies. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  8. L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

    PubMed Central

    Cooper, Arthur J L; Krasnikov, Boris F; Okuno, Etsuo; Jeitner, Thomas M

    2003-01-01

    Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent

  9. Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains.

    PubMed

    Bot, Simonetta; Andreuzzi, Eva; Capuano, Alessandra; Schiavinato, Alvise; Colombatti, Alfonso; Doliana, Roberto

    2015-01-01

    EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues. Copyright © 2014. Published by Elsevier B.V.

  10. Molecularly imprinted polymer based electrochemical detection of L-cysteine at carbon paste electrode.

    PubMed

    Aswini, K K; Vinu Mohan, A M; Biju, V M

    2014-04-01

    A methacrylic acid (MAA) based molecularly imprinted polymer (MIP) modified carbon paste electrode (CPE) was developed for electrochemical detection of L-cysteine (Cys). Characterisation of MIP was done with FTIR and the modified electrode with cyclic voltammetry (CV) and differential pulse voltammetry (DPV). CV, DPV and impedance analysis demonstrated that the modified electrode is responsive towards the target molecule. The optimum percentage composition of MIP for MIP/CPE and the effect of pH towards the electrode response for Cys were studied. The detection of Cys in the range of 2×10(-8) to 18×10(-8)M at MIP/CPE was monitored by DPV with a limit of detection of 9.6nM and R(2) of 0.9974. Also, various physiological interferents such as ascorbic acid, L-tryptophan, D-glucose, D-cysteine and L-cysteine were found to have little effect on DPV response at MIP/CPE. The utility of the electrode was proved by the effective detection of Cys from tap water and human blood plasma samples with reproducible results. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. The extraction and purification of a cysteine transfer ribonucleic acid from baker's yeast.

    PubMed Central

    Holness, N J; Atfield, G

    1976-01-01

    1. A modification of the RPC 1 system of A.D. Kelmers, G.D. Novelli & M.P. Stulberg (1965) (J. Biol. Chem. 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane. 2. By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA. 3. This preparation accepted at least 60% of the theoretical quantity of [3-14C]cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine. 4. A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M. Connelly & B.P. Doctor (1965) (J. Biol. Chem. 241, 715-719). 5. Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine. The base compositions of the two were similar. PMID:776175

  12. Potential protective effect of L-cysteine against the toxicity of acrylamide and furan in exposed Xenopus laevis embryos: an interaction study.

    PubMed

    Williams, John Russell; Rayburn, James R; Cline, George R; Sauterer, Roger; Friedman, Mendel

    2014-08-06

    The embryo toxicities of two food-processing-induced toxic compounds, acrylamide and furan, with and without added L-cysteine were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus (FETAX). The following measures of developmental toxicity were used: (a) 96 h LC50, the median concentration causing 50% embryo lethality; (b) 96 h EC50, the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic index (96 h LC50/96 h EC50), an estimate of teratogenic risk. Calculations of toxic units (TU) were used to assess possible antagonism, synergism, or response addition of several mixtures. The evaluated compounds demonstrated counterintuitive effects. Furan had lower than expected toxicity in Xenopus embryos and, unlike acrylamide, does not seem to be teratogenic. However, the short duration of the tests may not show the full effects of furan if it is truly primarily genotoxic and carcinogenic. L-Cysteine showed unexpected properties in the delay of hatching of the embryos. The results from the interaction studies between combination of two or three components (acrylamide plus L-cysteine; furan plus L-cysteine; acrylamide plus furan; acrylamide plus furan and L-cysteine) show that furan and acrylamide seem to have less than response addition at 1:1 toxic unit ratio in lethality. Acrylamide and L-cysteine show severe antagonism even at low 19 acrylamide/1 L-cysteine TU ratios. Data from the mixture of acrylamide, furan, and L-cysteine show a slight antagonism, less than would have been expected from binary mixture exposures. Bioalkylation mechanisms and their prevention are discussed. There is a need to study the toxicological properties of mixtures of acrylamide and furan concurrently formed in heat-processed food.

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

    PubMed

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

    2010-08-01

    Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system. (c) 2010 Elsevier Inc. All rights reserved.

  14. The s48/45 six-cysteine proteins: mediators of interaction throughout the Plasmodium life cycle.

    PubMed

    Arredondo, Silvia A; Kappe, Stefan H I

    2017-06-01

    During their life cycle Plasmodium parasites rely upon an arsenal of proteins that establish key interactions with the host and vector, and between the parasite sexual stages, with the purpose of ensuring infection, reproduction and proliferation. Among these is a group of secreted or membrane-anchored proteins known as the six-cysteine (6-cys) family. This is a small but important family with only 14 members thus far identified, each stage-specifically expressed during the parasite life cycle. 6-cys proteins often localise at the parasite surface or interface with the host and vector, and are conserved in different Plasmodium species. The unifying feature of the family is the s48/45 domain, presumably involved in adhesion and structurally related to Ephrins, the ligands of Eph receptors. The most prominent s48/45 members are currently under functional investigation and are being pursued as vaccine candidates. In this review, we examine what is known about the 6-cys family, their structure and function, and discuss future research directions. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  15. Human ESP1/CRP2, a member of the LIM domain protein family: Characterization of the cDNA and assignment of the gene locus to chromosome 14q32.3

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Karim, Mohammad Azharul; Ohta, Kohji; Matsuda, Ichiro

    1996-01-15

    The LIM domain is present in a wide variety of proteins with diverse functions and exhibits characteristic arrangements of Cys and His residues with a novel zinc-binding motif. LIM domain proteins have been implicated in development, cell regulation, and cell structure. A LIM domain protein was identified by screening a human cDNA library with rat cysteine-rich intestinal protein (CRIP) as a probe, under conditions of low stringency. Comparison of the predicted amino acid sequence with several LIM domain proteins revealed 93% of the residues to be identical to rat LIM domain protein, termed ESP1 or CRP2. Thus, the protein ismore » hereafter referred to as human ESP1/CRP2. The cDNA encompasses a 1171-base region, including 26, 624, and 521 bases in the 5{prime}-noncoding region, coding region, and 3{prime}-noncoding regions, respectively, and encodes the entire ESP1/CRP2 protein has two LIM domains, and each shares 35.1% and 77 or 79% identical residues with human cysteine-rich protein (CRP) and rat CRIP, respectively. Northern blot analysis of ESP1/CRP2 in various human tissues showed distinct tissue distributions compared with CRP and CRIP, suggesting that each might serve related but specific roles in tissue organization or function. Using a panel of human-rodent somatic cell hybrids, the ESP1/CRP2 locus was assigned to chromosome 14. Fluorescence in situ hybridization, using cDNA and a genome DNA fragment of the ESP1/CRP2 as probes, confirms this assignment and relegates regional localization to band 14q32.3 47 refs., 7 figs.« less

  16. Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis.

    PubMed

    Chen, Wenjun; Wang, Xiaoyun; Lv, Xiaoli; Tian, Yanli; Xu, Yanquan; Mao, Qiang; Shang, Mei; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2014-09-01

    Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P < 0.01) and EPG (P < 0.01) in CsCB2 and CsCB3 groups were significantly lower than in control group. In conclusion, we profiled secreted cathepsin B cysteine proteases family for the first time and demonstrated that all CsCB family were C. sinensis excretory/secretory products that may regulate host immune responses.

  17. l-Cysteine suppresses hypoxia-ischemia injury in neonatal mice by reducing glial activation, promoting autophagic flux and mediating synaptic modification via H2S formation.

    PubMed

    Xin, Danqing; Chu, Xili; Bai, Xuemei; Ma, Weiwei; Yuan, Hongtao; Qiu, Jie; Liu, Changxing; Li, Tong; Zhou, Xin; Chen, Wenqiang; Liu, Dexiang; Wang, Zhen

    2018-05-08

    We previously reported that l-Cysteine, an H 2 S donor, significantly alleviated brain injury after hypoxia-ischemic (HI) injury in neonatal mice. However, the mechanisms underlying this neuroprotective effect of l-Cysteine against HI insult remain unknown. In the present study, we tested the hypothesis that the protective effects of l-Cysteine are associated with glial responses and autophagy, and l-Cysteine attenuates synaptic injury as well as behavioral deficits resulting from HI. Consistent with our previous findings, we found that treatment with l-Cysteine after HI reduced early brain injury, improved behavioral deficits and synaptic damage, effects which were associated with an up-regulation of synaptophysin and postsynaptic density protein 95 expression in the lesioned cortex. l-Cysteine attenuated the accumulation of CD11b + /CD45 high cells, activation of microglia and astrocytes and diminished HI-induced increases in reactive oxygen species and malondialdehyde within the lesioned cortex. In addition, l-Cysteine increased microtubule associated protein 1 light chain 3-II and Beclin1 expression, decreased p62 expression and phosphor-mammalian target of rapamycin and phosphor-signal transducer and activator of transcription 3. Further support for a critical role of l-Cysteine was revealed from results demonstrating that treatment with an inhibitor of the H 2 S-producing enzyme, amino-oxyacetic acid, reversed the beneficial effects of l-Cysteine described above. These results demonstrate that l-Cysteine effectively alleviates HI injury and improves behavioral outcomes by inhibiting reactive glial responses and synaptic damage and an accompanying triggering of autophagic flux. Accordingly, l-Cysteine may provide a new a therapeutic approach for the treatment of HI via the formation of H 2 S. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Cell entry by a novel European filovirus requires host endosomal cysteine proteases and Niemann-Pick C1

    PubMed Central

    Ng, Melinda; Ndungo, Esther; Jangra, Rohit K.; Cai, Yingyun; Postnikova, Elena; Radoshitzky, Sheli R.; Dye, John M.; de Arellano, Eva Ramírez; Negredo, Ana; Palacios, Gustavo; Kuhn, Jens H.; Chandran, Kartik

    2014-01-01

    Lloviu virus (LLOV), a phylogenetically divergent filovirus, is the proposed etiologic agent of die-offs of Schreiber’s long-fingered bats (Miniopterus schreibersii) in western Europe. Studies of LLOV remain limited because the infectious agent has not yet been isolated. Here, we generated a recombinant vesicular stomatitis virus expressing the LLOV spike glycoprotein (GP) and used it to show that LLOV GP resembles other filovirus GP proteins in structure and function. LLOV GP must be cleaved by endosomal cysteine proteases during entry, but is much more protease-sensitive than EBOV GP. The EBOV/MARV receptor, Niemann Pick C1 (NPC1), is also required for LLOV entry, and its second luminal domain is recognized with high affinity by a cleaved form of LLOV GP, suggesting that receptor binding would not impose a barrier to LLOV infection of humans and non-human primates. The use of NPC1 as an intracellular entry receptor may be a universal property of filoviruses. PMID:25310500

  19. A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain.

    PubMed

    Alexandre, Maxime T A; Arents, Jos C; van Grondelle, Rienk; Hellingwerf, Klaas J; Kennis, John T M

    2007-03-20

    Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the C-terminal part of Avena sativa phototropin 1. The N-terminal part contains two light, oxygen, or voltage (LOV) sensing domains, LOV1 and LOV2; each binds a flavin mononucleotide (FMN) chromophore (lambdamax = 447 nm, termed D447) and forms the light-sensitive domains, of which LOV2 is the principal component. Blue-light absorption produces a covalent adduct between a very conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary by several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well-characterized LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by 3 orders of magnitude in the submolar concentration range, via a base-catalyzed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cysteine. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethyl pyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)-1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contibute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)-1, leading to a

  20. Permeability and toxicity characteristics of L-cysteine and 2-methyl-thiazolidine-4-carboxylic acid in Caco-2 cells.

    PubMed

    Kartal-Hodzic, Alma; Marvola, Tuuli; Schmitt, Mechthild; Harju, Kirsi; Peltoniemi, Marikki; Sivén, Mia

    2013-01-01

    Acetaldehyde is a known mutagenic substance and has been classified as a group-one carcinogen by the WHO. It is possible to bind acetaldehyde locally in the gastrointestinal (GI) tract with the semi-essential amino acid l-cysteine, which reacts covalently with acetaldehyde and forms compound 2-methyl-thiozolidine-4-carboxylic acid (MTCA). The Caco-2 cell line was used to determine the permeation of l-cysteine and MTCA, as well as the possible cell toxicity of both substances. Neither of the substances permeated through the Caco-2 cells at the concentrations used in this study, and only the highest concentration of MTCA affected the viability of the cells in the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) test. These results showed that when l-cysteine is administered in formulations releasing it locally in the lower parts of GI tract, it is not absorbed but can react with acetaldehyde, and that neither l-cysteine nor MTCA is harmful to the cells when present locally in the upper parts of GI tract. This study also shows that MTCA is sensitive at a lower pH of 5.5. Since stable MTCA is desired in different parts of the GI tract, this observation raises concern over the influence of lower pH on l-cysteine-containing product ability to bind and eliminate carcinogenic acetaldehyde.

  1. FRET ratiometric probes reveal the chiral-sensitive cysteine-dependent H2S production and regulation in living cells

    NASA Astrophysics Data System (ADS)

    Wei, Lv; Yi, Long; Song, Fanbo; Wei, Chao; Wang, Bai-Fan; Xi, Zhen

    2014-04-01

    Hydrogen sulfide (H2S) is an endogenously produced gaseous signalling molecule with multiple biological functions. In order to visualize and quantify the endogenous in situ production of H2S in living cells, here we developed two new sulphide ratiometric probes (SR400 and SR550) based on fluorescence resonance energy transfer (FRET) strategy for live capture of H2S. The FRET-based probes show excellent selectivity toward H2S in a high thiol background under physiological buffer. The probe can be used to in situ visualize cysteine-dependent H2S production in a chiral-sensitive manner in living cells. The ratiometric imaging studies indicated that D-Cys induces more H2S production than that of L-Cys in mitochondria of human embryonic kidney 293 cells (HEK293). The cysteine mimics propargylglycine (PPG) has also been found to inhibit the cysteine-dependent endogenous H2S production in a chiral-sensitive manner in living cells. D-PPG inhibited D-Cys-dependent H2S production more efficiently than L-PPG, while, L-PPG inhibited L-Cys-dependent H2S production more efficiently than D-PPG. Our bioimaging studies support Kimura's discovery of H2S production from D-cysteine in mammalian cells and further highlight the potential of D-cysteine and its derivatives as an alternative strategy for classical H2S-releasing drugs.

  2. Cysteine-containing peptides having antioxidant properties

    DOEpatents

    Bielicki, John K [Castro Valley, CA

    2009-10-13

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

  3. Cysteine-containing peptides having antioxidant properties

    DOEpatents

    Bielicki, John K [Castro Valley, CA

    2008-10-21

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

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

    PubMed

    Majcher, Małgorzata A; Jeleń, Henryk H

    2007-07-11

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

  5. Nafion/lead nitroprusside nanoparticles modified carbon ceramic electrode as a novel amperometric sensor for L-cysteine.

    PubMed

    Razmi, H; Heidari, H

    2009-05-01

    This work describes the electrochemical and electrocatalytic properties of carbon ceramic electrode (CCE) modified with lead nitroprusside (PbNP) nanoparticles as a new electrocatalyst material. The structure of deposited film on the CCE was characterized by energy dispersive X-ray (EDX), Fourier transform infrared (FTIR), and scanning electron microscopy (SEM). The cyclic voltammogram (CV) of the PbNP modified CCE showed two well-defined redox couples due to [Fe(CN)5NO](3-)/[Fe(CN)5NO](2-) and Pb(IV)/Pb(II) redox reactions. The modified electrode showed electrocatalytic activity toward the oxidation of L-cysteine and was used as an amperometric sensor. Also, to reduce the fouling effect of L-cysteine and its oxidation products on the modified electrode, a thin film of Nafion was coated on the electrode surface. The sensor response was linearly changed with L-cysteine concentration in the range of 1 x 10(-6) to 6.72 x 10(-5)mol L(-1) with a detection limit (signal/noise ratio [S/N]=3) of 0.46 microM. The sensor sensitivity was 0.17 microA (microM)(-1), and some important advantages such as simple preparation, fast response, good stability, interference-free signals, antifouling properties, and reproducibility of the sensor for amperometric determination of L-cysteine were achieved.

  6. Comparison of volatile sulphur compound production by cheese-ripening yeasts from methionine and methionine-cysteine mixtures.

    PubMed

    López Del Castillo-Lozano, M; Delile, A; Spinnler, H E; Bonnarme, P; Landaud, S

    2007-07-01

    Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with L-methionine or L-methionine/L-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with L-methionine or L-methionine/L-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when L-cysteine was supplemented, even at a low concentration (0.2 g l(-1)). This effect was due mainly to a significant decrease in L-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by L-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.

  7. Density functional theory study on the interactions of l-cysteine with graphene: adsorption stability and magnetism

    NASA Astrophysics Data System (ADS)

    Luo, Huijuan; Li, Hejun; Fu, Qiangang; Chu, Yanhui; Cao, Xiaoyu; Sun, Can; Yuan, Xiaoyan; Liu, Lei

    2013-12-01

    Understanding the interactions between graphene and biomolecules is of fundamental relevance to the area of nanobiotechnology. Herein, we take l-cysteine as the probe biomolecule and investigate its adsorption on pristine graphene and B-, N-, Al-, Ni-, Ga-, Pd-doped graphene using density functional theory calculations. Three kinds of upright adsorption configurations, via unprotonated functional groups (-SH, -NH2, -COOH), are considered. The calculations reveal pristine graphene physically adsorbs l-cysteine. N-doped graphene shows physisorption towards the S-end and N-end l-cysteine, and chemisorption towards the O-end radical. Strong chemisorption, with site-specific preference, occurs on Al-, Ni-, Ga- and Pd-doped graphene, accompanied by severe structural changes. Spin polarization with an unusual mirror symmetry on Ni- and Pd-doped graphene is induced by chemisorption of unprotonated l-cysteine, except for O-end adsorption on Pd-doped graphene. The magnetization arises mainly from spin polarization of the C 2pz orbital, with a minor magnetism located on Ni or Pd. The influence of van der Waals forces is also evaluated. A thorough analysis of the adsorption stability and magnetism of these systems would be beneficial to facilitate applications in graphene-based biosensing, biomolecule immobilization, magnetic bio-separation and other fields in bionanotechnology.

  8. Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway

    PubMed Central

    Allen, James W. A.; Ginger, Michael L.; Ferguson, Stuart J.

    2004-01-01

    The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif. Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH). There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms. The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1. In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus. Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli. However, a CXXCH variant was matured by the E. coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T. brucei cytochrome c can accommodate a second cysteine in a CXXCH motif. The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space. Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system. PMID:15500440

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

    PubMed

    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.

  10. A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

    NASA Technical Reports Server (NTRS)

    Porter Moore, C.; Zhang, J. Z.; Hamilton, S. L.

    1999-01-01

    Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

  11. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

    PubMed Central

    Lavergne, Vincent; Harliwong, Ivon; Jones, Alun; Miller, David; Taft, Ryan J.; Alewood, Paul F.

    2015-01-01

    Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions. PMID:26150494

  12. Parameters optimization defined by statistical analysis for cysteine-dextran radiolabeling with technetium tricarbonyl core.

    PubMed

    Núñez, Eutimio Gustavo Fernández; Faintuch, Bluma Linkowski; Teodoro, Rodrigo; Wiecek, Danielle Pereira; da Silva, Natanael Gomes; Papadopoulos, Minas; Pelecanou, Maria; Pirmettis, Ioannis; de Oliveira Filho, Renato Santos; Duatti, Adriano; Pasqualini, Roberto

    2011-04-01

    The objective of this study was the development of a statistical approach for radiolabeling optimization of cysteine-dextran conjugates with Tc-99m tricarbonyl core. This strategy has been applied to the labeling of 2-propylene-S-cysteine-dextran in the attempt to prepare a new class of tracers for sentinel lymph node detection, and can be extended to other radiopharmaceuticals for different targets. The statistical routine was based on three-level factorial design. Best labeling conditions were achieved. The specific activity reached was 5 MBq/μg. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  13. Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.

    PubMed

    Chung, Heaseung Sophia; Murray, Christopher I; Venkatraman, Vidya; Crowgey, Erin L; Rainer, Peter P; Cole, Robert N; Bomgarden, Ryan D; Rogers, John C; Balkan, Wayne; Hare, Joshua M; Kass, David A; Van Eyk, Jennifer E

    2015-10-23

    S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. © 2015 American Heart Association, Inc.

  14. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pandey, Chandra Mouli; Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005; Sumana, Gajjala, E-mail: sumanagajjala@gmail.com

    2014-09-08

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constantmore » of 3.38 × 10{sup −4 }cm s{sup −1}. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.« less

  15. Kinetics and thermodynamics of oxidation mediated reaction in L-cysteine and its methyl and ethyl esters in dimethyl sulfoxide-d6 by NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Dougherty, Ryan J.; Singh, Jaideep; Krishnan, V. V.

    2017-03-01

    L-Cysteine (L-Cys), L-Cysteine methyl ester (L-CysME) or L-Cysteine ethyl ester (L-CysEE), when dissolved in dimethyl sulfoxide, undergoes an oxidation process. This process is slow enough and leads to nuclear magnetic resonance (NMR) spectral changes that could be monitored in real time. The oxidation mediated transition is modeled as a pseudo-first order kinetics and the thermodynamic parameters are estimated using the Eyring's formulation. L-Cysteine and their esters are often used as biological models due to the remarkable thiol group that can be found in different oxidation states. This oxidation mediated transition is due to the combination of thiol oxidation to a disulfide followed by solvent-induced effects may be relevant in designing cysteine-based molecular models.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biterova, Ekaterina I.; Barycki, Joseph J.; UNL)

    2009-12-01

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

  17. Tryptophan and Cysteine Oxidation Products Dominate in α-Lactalbumin-Derived Peptides Analyzed with LC-MSn.

    PubMed

    Koivumäki, Tuuli P; Gürbüz, Göker; Heinonen, I Marina

    2017-09-01

    α-Lactalbumin (α-La), a major milk whey protein, is comprised of several amino acids prone to metal-catalyzed oxidation (MCO) typical in processing and during storage of foods. New tools are needed for the detection of characteristic oxidation products especially from tryptophan and cysteine that often remain unrecognized when using the traditional methods of carbonyl formation monitoring. In this study, the oxidative changes in α-La were investigated through tryptic digestion and collection of 3 descriptive peptides fitted into a metal-catalyzed oxidation (Fenton reaction) model. The peptide samples were oxidized at +37 °C for 14 d and explored with liquid chromatography-quadrupole ion trap-mass spectrometer (LC-MS n ). The fractionated α-La peptides were valyl-glycyl-isoleucyl-asparaginyl-tyrosyl-tryptophyl-leucyl-alanyl-histidyl-lysine (VGINYWLAHK), leucyl-aspartyl-glutaminyl-tryptophyl-leucyl-cysteinyl-glutamyl-lysine (LDQWLCEK), and tryptophyl +16 -leucyl-alanyl-histidyl-lysyl-alanyl-leucyl-cysteine (W +16 LAHKALC). Oxidation of several amino acids, such as cysteine, histidine, lysine, and tryptophan was observed. In the peptide LDQWLCEK, cysteine was rapidly trioxidized to sulfonic acid, followed by other amino acid side chains as secondary oxidation sites. Tryptophan oxidation was more pronounced in the peptides W +16 LAHKALC and VGINYWLAHK, and also formation of the harmful N-formylkynurenine was observed. As a conclusion, several stable and promising oxidation markers are proposed for α-La, which could be implemented in the evaluation of quality and safety of dairy protein-containing products. © 2017 Institute of Food Technologists®.

  18. Effects of randomized supplementation of methionine or alanine on cysteine and glutathione production during the early phase of treatment of children with edematous malnutrition123

    PubMed Central

    Green, Curtis O; Hsu, Jean W; Taylor-Bryan, Carolyn; Reid, Marvin; Forrester, Terrence; Jahoor, Farook

    2014-01-01

    Background: We have shown that a low glutathione concentration and synthesis rate in erythrocytes are associated with a shortage of protein-derived cysteine in children with edematous severe acute malnutrition (SAM). Objective: We tested the hypothesis that methionine supplementation may increase protein-derived cysteine and upregulate cysteine synthesis, thereby improving glutathione synthesis during the early treatment of edematous SAM. Design: The cysteine flux, its de novo synthesis and release from protein breakdown, and erythrocyte glutathione synthesis rate were measured in 12 children with edematous SAM in the fed state by using stable isotope tracers at 3 clinical phases as follows: 3 ± 1 d (±SE) [clinical phase 1 (CP1)], 8 ± 1 d [clinical phase 2 (CP2)], and 14 ± 2 d (clinical phase 3) after admission. Subjects were randomly assigned to receive equimolar supplements (0.5 mmol ⋅ kg−1 ⋅ d−1) of methionine or alanine (control) immediately after CP1. Results: In the methionine compared with the alanine group, cysteine flux derived from protein breakdown was faster at CP2 than CP1 (P < 0.05), and the change in plasma cysteine concentration from CP1 to CP2 was greater (P < 0.05). However, there was no evidence of a difference in cysteine de novo synthesis and its total flux or erythrocyte glutathione synthesis rate and concentration between groups. Conclusions: Methionine supplementation increased cysteine flux from body protein but had no significant effect on glutathione synthesis rates. Although cysteine is made from methionine, increased dietary cysteine may be necessary to partially fulfill its demand in edematous SAM because glutathione synthesis rates and concentrations were less than previous values shown at full recovery. This study was registered at clinicaltrials.gov as NCT00473031. PMID:24598154

  19. L-cysteine protected copper nanoparticles as colorimetric sensor for mercuric ions.

    PubMed

    Soomro, Razium A; Nafady, Ayman; Sirajuddin; Memon, Najma; Sherazi, Tufail H; Kalwar, Nazar H

    2014-12-01

    This report demonstrates a novel, simple and efficient protocol for the synthesis of copper nanoparticles in aqueous solution using L-cysteine as capping or protecting agent. UV-visible (UV-vis) spectroscopy was employed to monitor the LSPR band of L-cysteine functionalized copper nanoparticles (Cyst-Cu NPs) based on optimizing various reaction parameters. Fourier Transform Infrared (FTIR) spectroscopy provided information about the surface interaction between L-cysteine and Cu NPs. Transmission Electron Microscopy (TEM) confirmed the formation of fine spherical, uniformly distributed Cyst-Cu NPs with average size of 34 ± 2.1 nm. X-ray diffractometry (XRD) illustrated the formation of pure metallic phase crystalline Cyst-Cu NPs. As prepared Cyst-Cu NPs were tested as colorimetric sensor for determining mercuric (Hg(2+)) ions in an aqueous system. Cyst-Cu NPs demonstrated very sensitive and selective colorimetric detection of Hg(2+) ions in the range of 0.5 × 10(-6)-3.5 × 10(-6) mol L(-1) based on decrease in LSPR intensity as monitored by a UV-vis spectrophotometer. The developed sensor is simple, economic compared to those based on precious metal nanoparticles and sensitive to detect Hg(2+) ions with detection limit down to 4.3 × 10(-8) mol L(-1). The sensor developed in this work has a high potential for rapid and on-site detection of Hg(2+) ions. The sensor was successfully applied for assessment of Hg(2+) ions in real water samples collected from various locations of the Sindh River. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

    PubMed Central

    Broer, Rene; Boson, Bertrand; Spaan, Willy; Cosset, François-Loïc; Corver, Jeroen

    2006-01-01

    The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity. PMID:16415007

  1. The effects of L-cysteine and N-acetyl-L-cysteine on homocysteine metabolism and haemostatic markers, and on cardiac and aortic histology in subchronically methionine-treated Wistar male rats.

    PubMed

    Kostić, Sanja; Mićovic, Žarko; Andrejević, Lazar; Cvetković, Saša; Stamenković, Aleksandra; Stanković, Sanja; Obrenović, Radmila; Labudović-Borović, Milica; Hrnčić, Dragan; Jakovljević, Vladimir; Djurić, Dragan

    2018-06-23

    Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways; methionine-rich diets were used to induce hyperhomocysteinemia, and cardiovascular pathology was often observed. Other sulfur amino acids interfere with this metabolism, i.e., L-cysteine (Cys) and N-aceyl-L-cysteine (NAC), and probably also affect cardiovascular system. Their effects are controversial due to their ability to act both as anti- or pro-oxidant. Thus, this study aimed to elucidate their influence on levels of homocysteine, folate and vitamin B12, levels of different haemostatic parameters (fibrinogen, D-dimer, vWF Ag, vWF Ac) in rat serum or plasma as well as their effects on cardiac and aortic tissue histology in subchronically methionine-treated rats. Wistar albino rats were divided into 4 experimental groups: (a) control group (0.9% sodium chloride 0.1-0.2 mL/day) (n = 10) (K); (b) DL-methionine (0.8 mmol/kg/bw/day) (n = 10) (M); (c) DL-methionine (0.8 mmol/kg/bw/day) + L-cysteine (7 mg/kg/bw/day) (n = 8) (C); (d) DL-methionine (0.8 mmol/ kg/bw/day) + N-acetyl-L-cysteine (50 mg/kg/bw/day) (n = 8) (N). All substances were applied i.p., treatment duration 3 weeks. Lower levels of vitamin B12 in all the groups were found. Folate was reduced only in N group. Decreased fibrinogen was noted in C and N groups and increased D-dimer only in C. VWF activity was reduced in M and C groups. Deleterious effects in heart were observed, especially after Cys and NAC application. Aortic tissue remained unchanged. In conclusion, it could be said that sulfur amino acids have the significant impact on cardiovascular system in subchronically methionine-treated rats. This study points out the relevance of their complex interactions and deleterious effects mediated by either direct influence or procoagulant properties.

  2. Engineered Human Antibody Constant Domains with Increased Stability*S⃞

    PubMed Central

    Gong, Rui; Vu, Bang K.; Feng, Yang; Prieto, DaRue A.; Dyba, Marzena A.; Walsh, Joseph D.; Prabakaran, Ponraj; Veenstra, Timothy D.; Tarasov, Sergey G.; Ishima, Rieko; Dimitrov, Dimiter S.

    2009-01-01

    The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (Tm = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (Tm = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. PMID:19307178

  3. Liposome-coated mesoporous silica nanoparticles loaded with L-cysteine for photoelectrochemical immunoassay of aflatoxin B1.

    PubMed

    Lin, Youxiu; Zhou, Qian; Zeng, Yongyi; Tang, Dianping

    2018-06-02

    The authors describe a photoelectrochemical (PEC) immunoassay for determination of aflatoxin B 1 (AFB 1 ) in foodstuff. The competitive immunoreaction is carried out on a microplate coated with a capture antibody against AFB 1 using AFB 1 -bovine serum albumin (BSA)-liposome-coated mesoporous silica nanoparticles (MSN) loaded with L-cysteine as a support. The photocurrent is produced by a photoactive material consisting of cerium-doped Bi 2 MoO 6 . Initially, L-cysteine acting as the electron donor is gated in the pores by interaction between mesoporous silica and liposome. Thereafter, AFB 1 -BSA conjugates are covalently bound to the liposomes. Upon introduction of the analyte (AFB 1 ), the labeled AFB 1 -BSA complex competes with the analyte for the antibody deposited on the microplate. Accompanying with the immunocomplex, the liposomes on the MSNs are lysed upon addition of Triton X-100. This results in the opening of the pores and in a release of L-cysteine. Free cysteine then induces the electron-hole scavenger of the photoactive nanosheets to increase the photocurrent. The photocurrent (relative to background signal) increases with increasing AFB 1 concentration. Under optimum conditions, the photoactive nanosheets display good photoelectrochemical responses, and allow the detection of AFB 1 at a concentration as low as 0.1 pg·mL -1 within a linear response in the 0.3 pg·mL -1 to 10 ng·mL -1 concentration range. Accuracy was evaluated by analyzing naturally contaminated and spiked peanut samples by using a commercial AFB 1 ELISA kit as the reference, and well-matching results were obtained. Graphical abstract Schematic presentation of a photoelectrochemical immunoassay for AFB 1 . It is based on the use of Ce-doped Bi 2 MoO 6 nanosheets and of liposome-coated mesoporous silica nanoparticles loaded with L-cysteine.

  4. Sequence analysis of DBL2β domain of vargene of Indonesian Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Sulistyaningsih, E.; Romadhon, B. D.; Palupi, I.; Hidayah, F.; Dewi, R.; Prasetyo, A.

    2018-03-01

    Malaria is a major health problem in tropical countries including Indonesia. The most deadly agent is Plasmodium falciparum. In P. falciparum infection, PfEMP1 is supposed to play an important role in the pathogenesis of malaria. PfEMP1 is encoded by var gene family, it is a polymorphic protein where the extra-cellular portion contains of three distinct binding domains: Duffy binding-like (DBL), Cysteine-rich interdomain regions (CIDR) and C2. PfEMP1 varies in domain composition and binding specificity. The study explored the characteristic of Indonesian DBL2β-var genes and investigated its role to the malaria outcome. Twenty blood samples from clinically mild to severe malaria patients in Jember, East Java were collected for DNA extraction. Diagnosis was confirmed by Giemsa-stained thick blood smear. PCR was conducted using specific primer targeting on the full-length of DBL2ß and resulted approximately single band of 1,7 kb in a sample. This band was observed only from severe malaria sample. Sequence analysis directly from PCR product showed 74-99% similarities with previous sequences in Gene Bank. In conclusion, the DBL2β domain of vargene of Indonesian isolates was 1603 nucleotides in length and there was a possible association of the existence of DBL2β domain with the severity of malaria outcome.

  5. Preparation of CuO/ZnO nanocomposite and its application as a cysteine/homocysteine colorimetric and fluorescence detector.

    PubMed

    Šimšíková, Michaela; Čechal, Jan; Zorkovská, Anna; Antalík, Marián; Šikola, Tomáš

    2014-11-01

    Cysteine and homocysteine play a crucial role in many biological functions but abnormal levels of these amino acids may lead to various forms of pathogenesis. Therefore, selective and easy-to-use methods for the detection of cysteine and homocysteine are essential for the early diagnosis of developing diseases. In this paper we report on a rapid, straightforward and highly selective method for the detection of cysteine (Cys) and homocysteine (Hcy) which uses a CuO/ZnO nanocomposite as a dual colorimetric and fluorometric assay. The presence of Cys and Hcy in a solution of these nanorods (NRs) induces a change in its color from light blue to dark grey which is visible to the naked eye. This is accompanied by a blue shift in the absorption spectra from 725 nm to 650 nm and a decrease in the intensity of CuO/ZnO nanocomposite emission. These changes are ascribed to the reduction of Cu(II) to Cu(0), and the oxidation of cysteine (homocysteine) and subsequent formation of the disulfide bond. This novel assay method does not respond to any other amino-acid which is present in living organisms; therefore the selective determination of cysteine (homocysteine) with a lower analyte limit of 40 μM (4.8 μg mL(-1)) can be carried out in aqueous solutions without the need for any sophisticated instrumentation, fluorophore molecules or complicated procedures. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Luciferin-Regenerating Enzyme Mediates Firefly Luciferase Activation Through Direct Effects of D-Cysteine on Luciferase Structure and Activity.

    PubMed

    Hemmati, Roohullah; Hosseinkhani, Saman; Sajedi, Reza H; Azad, Taha; Tashakor, Amin; Bakhtiari, Nuredin; Ataei, Farangis

    2015-01-01

    Luciferin-regenerating enzyme (LRE) contributes to in vitro recycling of D-luciferin. In this study, reinvestigation of the luciferase-based LRE assay is reported. Here, using quick change site-directed mutagenesis seven T-LRE (Lampyris turkestanicusLRE) mutants were constructed and the most functional mutant of T-LRE (T(69)R) was selected for this research and the effects of D- and L-cysteine on T(69)R T-LRE-luciferase-coupled assay are examined. Our results demonstrate that bioluminescent signal of T(69)R T-LRE-luciferase-coupled assay increases and then reach equilibrium state in the presence of 5 mm D-cysteine. In addition, results reveal that 5 mm D- and L-cysteine in the absence of T(69)R T-LRE cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far-UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D-cysteine increases the activity of luciferase due to weak redox potential, antiaggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time-course originate from the direct effects of D-cysteine on structure and activity of firefly luciferase. © 2015 The American Society of Photobiology.

  7. Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

    1988-06-01

    E-64, a specific inhibitor of cysteine proteinases, was incorporated into artificial seeds at low levels (0.01-0.25% by weight). It prolonged developmental time and increased mortality of the larval cowpea weevil, Callosobruchus maculatus (F.), in direct proportion to its concentration in the artificial seeds. The fecundity of females emerging from the artificial seeds was significantly decreased by E-64 concentrations of 0.06% and higher. These observations are compatible with the hypothesis that the midgut cysteine proteinase in C. maculatus is essential for normal growth and development.

  8. Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N-terminal fungalysin-thermolysin-propeptide domain.

    PubMed

    Nickerson, Nicholas N; Joag, Vineet; McGavin, Martin J

    2008-09-01

    The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.

  9. Insights into Strand Exchange in BTB Domain Dimers from the Crystal Structures of FAZF and Miz1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stogios, Peter J.; Cuesta-Seijo, Jose Antonio; Chen, Lu

    2010-09-22

    The BTB domain is a widely distributed protein-protein interaction motif that is often found at the N-terminus of zinc finger transcription factors. Previous crystal structures of BTB domains have revealed tightly interwound homodimers, with the N-terminus from one chain forming a two-stranded anti-parallel {beta}-sheet with a strand from the other chain. We have solved the crystal structures of the BTB domains from Fanconi anemia zinc finger (FAZF) and Miz1 (Myc-interacting zinc finger 1) to resolutions of 2.0 {angstrom} and 2.6 {angstrom}, respectively. Unlike previous examples of BTB domain structures, the FAZF BTB domain is a nonswapped dimer, with each N-terminalmore » {beta}-strand associated with its own chain. As a result, the dimerization interface in the FAZF BTB domain is about half as large as in the domain-swapped dimers. The Miz1 BTB domain resembles a typical swapped BTB dimer, although it has a shorter N-terminus that is not able to form the interchain sheet. Using cysteine cross-linking, we confirmed that the promyelocytic leukemia zinc finger (PLZF) BTB dimer is strand exchanged in solution, while the FAZF BTB dimer is not. A phylogenic tree of the BTB fold based on both sequence and structural features shows that the common ancestor of the BTB domain in BTB-ZF (bric a brac, tramtrack, broad-complex zinc finger) proteins was a domain-swapped dimer. The differences in the N-termini seen in the FAZF and Miz1 BTB domains appear to be more recent developments in the structural evolution of the domain.« less

  10. MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

    PubMed

    Amiri, Azam; Bandani, Ali Reza; Alizadeh, Houshang

    2016-04-01

    Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting. © 2015 Wiley Periodicals, Inc.

  11. Alleviative effects of s-allyl cysteine and s-ethyl cysteine on MCD diet-induced hepatotoxicity in mice.

    PubMed

    Lin, Chun-che; Yin, Mei-chin; Liu, Wen-hu

    2008-11-01

    Alleviative effects of s-allyl cysteine (SAC) and s-ethyl cysteine (SEC) upon methionine and choline deficient (MCD) diet-induced hepatotoxicity in mice were examined. SAC or SEC at 1g/L was added into drinking water for 7 weeks with MCD diet. MCD feeding significantly increased hepatic triglyceride and cholesterol levels, and elevated the activity of glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (P < 0.05). However, the intake of SAC or SEC significantly decreased hepatic triglyceride accumulation, and reduced G6PDH and FAS activities (P < 0.05). MCD feeding significantly lowered serum and hepatic glutathione (GSH) levels, increased malondialdehyde (MDA) and oxidized glutathione (GSSG) formation, and suppressed the activity and mRNA expression of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (P < 0.05). The intake of SAC or SEC significantly increased serum and hepatic GSH levels, decreased MDA and GSSG formation, restored the activity and mRNA expression of GPX, SOD and catalase (P < 0.05). MCD feeding significantly enhanced the mRNA expression of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, matrix metalloproteinases-9 (MMP-9) and collagen-alpha1 (P < 0.05). The intake of SAC and SEC significantly blunted the mRNA expression of IL-1beta, IL-6, TNF-alpha, TGF-beta1 and collagen-alpha1 (P < 0.05). SEC was greater than SAC in suppressing IL-6 and TNF-alpha expression (P < 0.05), but SAC was greater than SEC in suppressing collagen-alpha1 and TGF-beta1 expression (P < 0.05). These data suggest that SAC and SEC are potent agents against MCD-induced hepatotoxicity.

  12. Cysteine optical sensing with an up-conversion host and two chemosensors derived from rhodamine: Construction, characterization and performance

    NASA Astrophysics Data System (ADS)

    Lin, Chen; Zhigang, Fang

    2017-03-01

    This paper focused on two rhodamine chemosensors for cysteine optical sensing. To minimize their photobleaching caused by excitation light, up-conversion NaYF4:Yb3 +/Er3 + nanocrystals were prepared and used as excitation host. Photophysical measurement on this host and the two chemosensors suggested that chemosensor absorption matched well with host emission. An efficient energy transfer between them was discussed and confirmed by their spectral analysis and emission lifetime comparison. Job's plot suggested that our chemosensors followed a simple recognition mechanism towards cysteine with binding stoichiometry of 1:1. Both chemosensors showed emission "off-on" effect triggered by cysteine and good photostability. Linear working curves with maximum sensitivity of 2.61 were obtained. S substituent was positive to improve selectivity.

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

    PubMed Central

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

    2014-01-01

    Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30–Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130–Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33–Cys130′ and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33–Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1. PMID:24625320

  14. An in situ XPS study of L-cysteine co-adsorbed with water on polycrystalline copper and gold

    NASA Astrophysics Data System (ADS)

    Jürgensen, Astrid; Raschke, Hannes; Esser, Norbert; Hergenröder, Roland

    2018-03-01

    The interactions of biomolecules with metal surfaces are important because an adsorbed layer of such molecules introduces complex reactive functionality to the substrate. However, studying these interactions is challenging: they usually take place in an aqueous environment, and the structure of the first few monolayers on the surface is of particular interest, as these layers determine most interfacial properties. Ideally, this requires surface sensitive analysis methods that are operated under ambient conditions, for example ambient pressure x-ray photoelectron spectroscopy (AP-XPS). This paper focuses on an AP-XPS study of the interaction of water vapour and l-Cysteine on polycrystalline copper and gold surfaces. Thin films of l-Cysteine were characterized with XPS in UHV and in a water vapour atmosphere (P ≤ 1 mbar): the structure of the adsorbed l-Cysteine layer depended on substrate material and deposition method, and exposure of the surface to water vapour led to the formation of hydrogen bonds between H2O molecules and the COO- and NH2 groups of adsorbed l-Cysteine zwitterions and neutral molecules, respectively. This study also proved that it is possible to investigate monolayers of biomolecules in a gas atmosphere with AP-XPS using a conventional laboratory Al-Kα x-ray source.

  15. Fluorescent coumarin-based probe for cysteine and homocysteine with live cell application

    NASA Astrophysics Data System (ADS)

    Wei, Ling-Fang; Thirumalaivasan, Natesan; Liao, Yu-Cheng; Wu, Shu-Pao

    2017-08-01

    Cysteine (Cys) and homocysteine (Hcy) are two of important biological thiols and function as important roles in several biological processes. The development of Cys and Hcy probes will help to explore the functions of biothiols in biological systems. In this work, a new coumarin-based probe AC, containing an acryloyl moiety, was developed for Cys and Hcy detection in cells. Cys and Hcy undergo a nucleophilic addition and subsequent cyclization reaction to remove to the acryloyl group and yield a fluorescent product, 7-hydroxylcomuarin. The probe AC showed good selectivity for cysteine and homocysteine over glutathione and other amino acids and had low detection limits of 65 nM for Cys and 79 nM for Hcy, respectively. Additionally, confocal imaging experiments demonstrated that the probe AC can be applied to visualize Cys and Hcy in living cells.

  16. Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis; Implications for Vaccine Development

    PubMed Central

    van de Waterbeemd, Bas; Zomer, Gijsbert; van den IJssel, Jan; van Keulen, Lonneke; Eppink, Michel H.; van der Ley, Peter; van der Pol, Leo A.

    2013-01-01

    Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis. PMID:23372704

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

    PubMed Central

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

    2013-01-01

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

  18. Homology modeling, molecular docking and MD simulation studies to investigate role of cysteine protease from Xanthomonas campestris in degradation of Aβ peptide.

    PubMed

    Dhanavade, Maruti J; Jalkute, Chidambar B; Barage, Sagar H; Sonawane, Kailas D

    2013-12-01

    Cysteine protease is known to degrade amyloid beta peptide which is a causative agent of Alzheimer's disease. This cleavage mechanism has not been studied in detail at the atomic level. Hence, a three-dimensional structure of cysteine protease from Xanthomonas campestris was constructed by homology modeling using Geno3D, SWISS-MODEL, and MODELLER 9v7. All the predicted models were analyzed by PROCHECK and PROSA. Three-dimensional model of cysteine protease built by MODELLER 9v7 shows similarity with human cathepsin B crystal structure. This model was then used further for docking and simulation studies. The molecular docking study revealed that Cys17, His87, and Gln88 residues of cysteine protease form an active site pocket similar to human cathepsin B. Then the docked complex was refined by molecular dynamic simulation to confirm its stable behavior over the entire simulation period. The molecular docking and MD simulation studies showed that the sulfhydryl hydrogen atom of Cys17 of cysteine protease interacts with carboxylic oxygen of Lys16 of Aβ peptide indicating the cleavage site. Thus, the cysteine protease model from X. campestris having similarity with human cathepsin B crystal structure may be used as an alternate approach to cleave Aβ peptide a causative agent of Alzheimer's disease. © 2013 Elsevier Ltd. All rights reserved.

  19. An automated and efficient conformation search of L-cysteine and L,L-cystine using the scaled hypersphere search method

    NASA Astrophysics Data System (ADS)

    Kishimoto, Naoki; Waizumi, Hiroki

    2017-10-01

    Stable conformers of L-cysteine and L,L-cystine were explored using an automated and efficient conformational searching method. The Gibbs energies of the stable conformers of L-cysteine and L,L-cystine were calculated with G4 and MP2 methods, respectively, at 450, 298.15, and 150 K. By assuming thermodynamic equilibrium and the barrier energies for the conformational isomerization pathways, the estimated ratios of the stable conformers of L-cysteine were compared with those determined by microwave spectroscopy in a previous study. Equilibrium structures of 1:1 and 2:1 cystine-Fe complexes were also calculated, and the energy of insertion of Fe into the disulfide bond was obtained.

  20. Domain Requirements for DNA Unwinding by Mycobacterial UvrD2, an Essential DNA Helicase†

    PubMed Central

    Sinha, Krishna Murari; Stephanou, Nicolas C.; Unciuleac, Mihaela-Carmen; Glickman, Michael S.; Shuman, Stewart

    2008-01-01

    Mycobacterial UvrD2 is a DNA-dependent ATPase with 3′ to 5′ helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis. PMID:18702526