Sample records for c-type heme-binding proteins

  1. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

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    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.


    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  2. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

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    Wojtowicz, Halina [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Wojaczynski, Jacek [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Mariusz [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Kroliczewski, Jaroslaw [Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, 50-148 Wroclaw (Poland); Latos-Grazynski, Lechoslaw [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Teresa, E-mail: [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland)


    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and {sup 1}H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  3. SCMHBP: prediction and analysis of heme binding proteins using propensity scores of dipeptides. (United States)

    Liou, Yi-Fan; Charoenkwan, Phasit; Srinivasulu, Yerukala; Vasylenko, Tamara; Lai, Shih-Chung; Lee, Hua-Chin; Chen, Yi-Hsiung; Huang, Hui-Ling; Ho, Shinn-Ying


    Heme binding proteins (HBPs) are metalloproteins that contain a heme ligand (an iron-porphyrin complex) as the prosthetic group. Several computational methods have been proposed to predict heme binding residues and thereby to understand the interactions between heme and its host proteins. However, few in silico methods for identifying HBPs have been proposed. This work proposes a scoring card method (SCM) based method (named SCMHBP) for predicting and analyzing HBPs from sequences. A balanced dataset of 747 HBPs (selected using a Gene Ontology term GO:0020037) and 747 non-HBPs (selected from 91,414 putative non-HBPs) with an identity of 25% was firstly established. Consequently, a set of scores that quantified the propensity of amino acids and dipeptides to be HBPs is estimated using SCM to maximize the predictive accuracy of SCMHBP. Finally, the informative physicochemical properties of 20 amino acids are identified by utilizing the estimated propensity scores to be used to categorize HBPs. The training and mean test accuracies of SCMHBP applied to three independent test datasets are 85.90% and 71.57%, respectively. SCMHBP performs well relative to comparison with such methods as support vector machine (SVM), decision tree J48, and Bayes classifiers. The putative non-HBPs with high sequence propensity scores are potential HBPs, which can be further validated by experimental confirmation. The propensity scores of individual amino acids and dipeptides are examined to elucidate the interactions between heme and its host proteins. The following characteristics of HBPs are derived from the propensity scores: 1) aromatic side chains are important to the effectiveness of specific HBP functions; 2) a hydrophobic environment is important in the interaction between heme and binding sites; and 3) the whole HBP has low flexibility whereas the heme binding residues are relatively flexible. SCMHBP yields knowledge that improves our understanding of HBPs rather than merely

  4. Modulation of function in a minimalist heme-binding membrane protein. (United States)

    Shinde, Sandip; Cordova, Jeanine M; Woodrum, Brian W; Ghirlanda, Giovanna


    De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512-518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of -128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K(d,app) = 6.5 × 10(-8) M), and lowers the redox potential of the cofactor to -172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices. © SBIC 2012

  5. Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes (United States)

    Hofbauer, Stefan; Hagmüller, Andreas; Schaffner, Irene; Mlynek, Georg; Krutzler, Michael; Stadlmayr, Gerhard; Pirker, Katharina F.; Obinger, Christian; Daims, Holger; Djinović-Carugo, Kristina; Furtmüller, Paul G.


    Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0 Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2 μM (circular dichroism spectroscopy) and 16.8 μM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis. PMID:25602700

  6. Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.

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    Rachel Senturia

    Full Text Available Human DiGeorge Critical Region 8 (DGCR8 is an essential microRNA (miRNA processing factor that is activated via direct interaction with Fe(III heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD. We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star also binds heme. The extinction coefficients (ε of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

  7. Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis.

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    Halina Wójtowicz


    Full Text Available Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-beta fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.

  8. Heme Binding Proteins of Bartonella henselae Are Required when Undergoing Oxidative Stress During Cell and Flea Invasion (United States)

    Liu, MaFeng; Ferrandez, Yann; Bouhsira, Emilie; Monteil, Martine; Franc, Michel; Boulouis, Henri-Jean; Biville, Francis


    Bartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea. PMID:23144761

  9. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

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    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)


    functionally characterized. On the basis of prior work, we predicted that cTHAP4 is composed of a heme-binding nitrobindin domain, making THAP4 the only human THAP protein predicted to bind a cofactor. Nitrobindin, a recently characterized protein from Arabidopsis thaliana, is structurally similar and exhibits nitric oxide (NO)-binding properties that resemble the heme-binding nitrophorins. Nitrophorins use a heme moiety to store, transport, and release NO in a pH-specific manner. Although the exact function of nitrobindin is not fully known, the similarities between the well-characterized nitrophorins imply a role in NO transport, sensing, or metabolism. To better elucidate the possible function of THAP4, we solved the hemebound structure of cTHAP4 to a resolution of 1.79 {angstrom}.

  10. SpyB, a Small Heme-Binding Protein, Affects the Composition of the Cell Wall in Streptococcus pyogenes. (United States)

    Edgar, Rebecca J; Chen, Jing; Kant, Sashi; Rechkina, Elena; Rush, Jeffrey S; Forsberg, Lennart S; Jaehrig, Bernhard; Azadi, Parastoo; Tchesnokova, Veronika; Sokurenko, Evgeni V; Zhu, Haining; Korotkov, Konstantin V; Pancholi, Vijay; Korotkova, Natalia


    Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to β-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

  11. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

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    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  12. The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp

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    Lei Benfang


    Full Text Available Abstract Background The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established. Results The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp. Conclusion We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.

  13. Discovery of Intracellular Heme-binding Protein HrtR, Which Controls Heme Efflux by the Conserved HrtB-HrtA Transporter in Lactococcus lactis* (United States)

    Lechardeur, Delphine; Cesselin, Bénédicte; Liebl, Ursula; Vos, Marten H.; Fernandez, Annabelle; Brun, Célia; Gruss, Alexandra; Gaudu, Philippe


    Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis. PMID:22084241

  14. Discovery of intracellular heme-binding protein HrtR, which controls heme efflux by the conserved HrtB-HrtA transporter in Lactococcus lactis. (United States)

    Lechardeur, Delphine; Cesselin, Bénédicte; Liebl, Ursula; Vos, Marten H; Fernandez, Annabelle; Brun, Célia; Gruss, Alexandra; Gaudu, Philippe


    Most commensal and food bacteria lack heme biosynthesis genes. For several of these, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane hrt (heme-regulated transporter) and a unique transcriptional regulator that we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated, and involves two histidines, His-72 and His-149. HrtR specifically binds a 15-nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis.

  15. HemeBIND: a novel method for heme binding residue prediction by combining structural and sequence information

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    Hu Jianjun


    Full Text Available Abstract Background Accurate prediction of binding residues involved in the interactions between proteins and small ligands is one of the major challenges in structural bioinformatics. Heme is an essential and commonly used ligand that plays critical roles in electron transfer, catalysis, signal transduction and gene expression. Although much effort has been devoted to the development of various generic algorithms for ligand binding site prediction over the last decade, no algorithm has been specifically designed to complement experimental techniques for identification of heme binding residues. Consequently, an urgent need is to develop a computational method for recognizing these important residues. Results Here we introduced an efficient algorithm HemeBIND for predicting heme binding residues by integrating structural and sequence information. We systematically investigated the characteristics of binding interfaces based on a non-redundant dataset of heme-protein complexes. It was found that several sequence and structural attributes such as evolutionary conservation, solvent accessibility, depth and protrusion clearly illustrate the differences between heme binding and non-binding residues. These features can then be separately used or combined to build the structure-based classifiers using support vector machine (SVM. The results showed that the information contained in these features is largely complementary and their combination achieved the best performance. To further improve the performance, an attempt has been made to develop a post-processing procedure to reduce the number of false positives. In addition, we built a sequence-based classifier based on SVM and sequence profile as an alternative when only sequence information can be used. Finally, we employed a voting method to combine the outputs of structure-based and sequence-based classifiers, which demonstrated remarkably better performance than the individual classifier alone

  16. Identification and characterization of the heme-binding proteins SeShp and SeHtsA of Streptococcus equi subspecies equi

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    McClure Michael J


    Full Text Available Abstract Background Heme is a preferred iron source of bacterial pathogens. Streptococcus equi subspecies equi is a bacterial pathogen that causes strangles in horses. Whether S. equi has a heme acquisition transporter is unknown. Results An S. equi genome database was blasted with the heme binding proteins Shp and HtsA of Streptococcus pyogenes, and found that S. equi has the homologue of Shp (designated SeShp and HtsA (designated SeHtsA. Tag-free recombinant SeShp and SeHtsA and 6xHis-tagged SeHtsA (SeHtsAHis were prepared and characterized. Purified holoSeShp and holoSeHtsA bind Fe(II-protoporphyrin IX (heme and Fe(III-protoporphyrin IX (hemin in a 1:1 stoichiometry, respectively, and are designated hemoSeShp and hemiSeHtsA. HemiSeShp and hemiSeHtsAHis can be reconstituted from apoSeShp and apoSeHtsAHis and hemin. HemoSeShp is stable in air and can be oxidized to hemiSeShp by ferricyanide. HemiSeHtsA can be reduced into hemoSeHtsA, which autoxidizes readily. HemoSeShp rapidly transfers its heme to apoSeHtsAHis. In addition, hemoSeShp can also transfer its heme to apoHtsA, and hemoShp is able to donate heme to apoSeHtsAHis. Conclusion The primary structures, optical properties, oxidative stability, and in vitro heme transfer reaction of SeShp and SeHtsA are very similar to those of S. pyogenes Shp and HtsA. The data suggest that the putative cell surface protein SeShp and lipoprotein SeHtsA are part of the machinery to acquire heme in S. equi. The results also imply that the structure, function, and functional mechanism of the heme acquisition machinery are conserved in S. equi and S. pyogenes.

  17. Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding. (United States)

    Watanabe, Yuta; Ishimori, Koichiro; Uchida, Takeshi


    HBP23, a 23-kDa heme-binding protein identified in rats, is a member of the peroxiredoxin (Prx) family, the primary peroxidases involved in hydrogen peroxide catabolism. Although HBP23 has a characteristic Cys-Pro heme-binding motif, the significance of heme binding to Prx family proteins remains to be elucidated. Here, we examined the effect of heme binding to human peroxiredoxin-1 (PRX1), which has 97% amino acid identity to HBP23. PRX1 was expressed in Escherichia coli and purified to homogeneity. Spectroscopic titration demonstrated that PRX1 binds heme with a 1:1 stoichiometry and a dissociation constant of 0.17 μM. UV-vis spectra of heme-PRX1 suggested that Cys52 is the axial ligand of ferric heme. PRX1 peroxidase activity was lost upon heme binding, reflecting the fact that Cys52 is not only the heme-binding site but also the active center of peroxidase activity. Interestingly, heme binding to PRX1 caused a decrease in the toxicity and degradation of heme, significantly suppressing H2O2-dependent heme peroxidase activity and degradation of PRX1-bound heme compared with that of free hemin. By virtue of its cytosolic abundance (∼20 μM), PRX1 thus functions as a scavenger of cytosolic hemin (dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Identification of a copper-repressible C-type heme protein of Methylococcus capsulatus (Bath). A member of a novel group of the bacterial di-heme cytochrome c peroxidase family of proteins. (United States)

    Karlsen, Odd A; Kindingstad, Louise; Angelskår, Solveig M; Bruseth, Live J; Straume, Daniel; Puntervoll, Pål; Fjellbirkeland, Anne; Lillehaug, Johan R; Jensen, Harald B


    Genomic sequencing of the methanotrophic bacterium, Methylococcus capsulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589). Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence similarity to the bacterial di-heme cytochrome c peroxidase (BCCP) family of proteins. Two putative C-type heme-binding motifs were predicted, and confirmed by positive heme staining. Immunospecific recognition and biotinylation of whole cells combined with MS analyses confirmed expression of MCA2590 in M. capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior. Similar to MopE, expression of MCA2590 is regulated by the bioavailability of copper and is most abundant in M. capsulatus cultures grown under low copper conditions, thus indicating an important physiological role under these growth conditions. MCA2590 is distinguished from previously characterized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme-binding motifs in its primary sequence. Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members. Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di-heme cytochrome c peroxidases not previously characterized.

  19. Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum

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    Marc Keppel


    Full Text Available The HrrSA and the ChrSA two-component systems play a central role in the coordination of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum and the prominent pathogen Corynebacterium diphtheriae, both members of the Corynebacteriaceae. In this study, we have performed a comparative analysis of the membrane topology and heme-binding characteristics of the histidine kinases HrrS and ChrS of C. glutamicum. While the cytoplasmic catalytic domains are highly conserved between HrrS and ChrS, the N-terminal sensing parts share only minor sequence similarity. PhoA and LacZ fusions of the N-terminal sensor domains of HrrS and ChrS revealed that both proteins are embedded into the cytoplasmic membrane via six α-helices. Although the overall membrane topology appeared to be conserved, target gene profiling indicated a higher sensitivity of the ChrS system to low heme levels (< 1 μM. In vitro, solubilized and purified full-length proteins bound heme in a 1:1 stoichiometry per monomer. Alanine-scanning of conserved amino acid residues in the N-terminal sensor domain revealed three aromatic residues (Y112, F115, and F118, which apparently contribute to heme binding of HrrS. Exchange of either one or all three residues resulted in an almost abolished heme binding of HrrS in vitro. In contrast, ChrS mutants only displayed a red shift of the soret band from 406 to 418 nm suggesting an altered set of ligands in the triple mutant. In line with target gene profiling, these in vitro studies suggest distinct differences in the heme-protein interface of HrrS and ChrS. Since the membrane topology mapping displayed no extensive loop regions and alanine-scanning revealed potential heme-binding residues in α-helix number four, we propose an intramembrane sensing mechanism for both proteins. Overall, we present a first comparative analysis of the ChrS and HrrS kinases functioning as transient heme sensors in the Corynebacteriaceae.

  20. Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus. (United States)

    Pluym, Mark; Muryoi, Naomi; Heinrichs, David E; Stillman, Martin J


    Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain.

  1. Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins. (United States)

    Wellman-Labadie, Olivier; Lakshminarayanan, Rajamani; Hincke, Maxwell T


    C-type lectin-like proteins are major components of the calcified eggshell of multiple avian species. In this study, two representative avian C-type lectin-like proteins, ovocleidin-17 and ansocalcin, were purified from decalcified chicken and goose eggshell protein extracts and investigated for carbohydrate binding activity as well as antimicrobial activity. Purified ovocleidin-17 and ansocalcin were found to bind bacterial polysaccharides, and were bactericidal against Bacillus subtilis, Staphylococcus aureus and Pseudomona aeruginosa. Bactericidal activity was found to be enhanced in the presence of calcium but was not dependent on its presence. The results suggest that avian C-type lectin-like proteins may play an important antimicrobial role in defence of the avian embryo.

  2. Crystal Structures of Two Novel Dye-Decolorizing Peroxidases Reveal a Beta-Bar Fold With a Conserved Heme-Binding Motif

    Energy Technology Data Exchange (ETDEWEB)

    Zubieta, C.; Krishna, S.S.; Kapoor, M.; Kozbial, P.; McMullan, D.; Axelrod, H.L.; Miller, M.D.; Abdubek, P.; Ambing, E.; Astakhova, T.; Carlton, D.; Chiu, H.J.; Clayton, T.; Deller, M.C.; Duan, L.; Elsliger, M.A.; Feuerhelm, J.; Grzechnik, S.K.; Hale, J.; Hampton, E.; Han, G.W.; /JCSG /SLAC, SSRL /Burnham Inst. Med. Res. /UC, San Diego /Scripps Res. Inst. /Novartis Res. Found.


    BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 Angstroms, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, {alpha}+{beta} ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).

  3. Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: heme binding by an induced fit mechanism. (United States)

    Cobessi, David; Meksem, Ahmed; Brillet, Karl


    Shigella dysentriae and other Gram-negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 A resolution the 3D structure of the TonB-dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C-terminal domain that folds into a 22-stranded transmembrane beta-barrel, which is filled by the N-terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 A apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA-Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N-terminal TonB-box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB-box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB-box. (c) 2009 Wiley-Liss, Inc.

  4. Heme-binding characteristics of the isolated PAS-B domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms. (United States)

    Hayasaka, Koya; Kitanishi, Kenichi; Igarashi, Jotaro; Shimizu, Toru


    Mouse period homolog 2 (mPer2), an important transcriptional regulatory factor associated with circadian rhythms, is composed of two N-terminal PAS (PAS-A and PAS-B) domains and a C-terminal domain. The PAS-A domain of mPer2 binds the heme iron via a Cys axial ligand. A corresponding transcriptional regulatory factor, neuronal PAS 2 protein (NPAS2), also contains PAS-A and PAS-B domains at the N-terminus with heme-binding capability. In particular, the PAS-B domain appears important for protein-protein interactions critical for transcriptional regulation. In the present study, we examined the heme-binding characteristics of the isolated PAS-B domain of mPer2. Our experiments show that the Fe(III) heme binds the isolated PAS-B domain with a heme to protein stoichiometry of 1:1. The Fe(III) protein complex is suggested to consist of an admixture of 6-coordinated His-bound high-spin and low-spin complexes. Marked pH-dependent spectral changes were observed, in contrast to the spectrum of the Fe(III) bound PAS-A domain of mPer2, which appeared pH-resistant. Treatment with diethylpyrocarbonate abolished the heme-binding ability of this protein, supporting the proposal that His is the axial ligand. Heme dissociation was composed of two phases with rate constants of 4.3 × 10⁻⁴ s⁻¹ (50%) and 4.0 × 10⁻³ s⁻¹ (50%), which were markedly higher than that (1.5 × 10⁻⁷ s⁻¹) of the prototype heme protein, myoglobin. The Soret CD band of the H454A PAS-B mutant was significantly different from those of wild-type and other His mutant proteins, strongly suggesting that His454 is one of the axial ligands for the Fe(III) complex. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Artefacts induced on c-type haem proteins by electrode surfaces


    Sousa, Patrícia M. Paes de; Pauleta, Sofia R.; Gonçalves, M. Lurdes Simões; Pettigrew, Graham W.; Moura, Isabel; Moura, José J. G.; Santos, Margarida M. Correia dos


    J Biol Inorg Chem (2011) 16:209–215 DOI 10.1007/s00775-010-0717-z In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control e...

  6. Heme-binding activity of methoxyflavones from Pentzia monodiana Maire (Asteraceae). (United States)

    Ortiz, Sergio; Dali-Yahia, Kamel; Vasquez-Ocmin, Pedro; Grougnet, Raphaël; Grellier, Philippe; Michel, Sylvie; Maciuk, Alexandre; Boutefnouchet, Sabrina


    A heme-binding assay based on mass spectrometry was performed on P. monodiana Maire (Asteraceae) extracts to identify metabolites able to form adducts with heminic part of haemoglobin, as potential antimalarial drugs. Main adducts were characterized and their stability was measured. Isolation of main constituents of P. monodiana Maire lead to identification of the two methoxyflavones 3'-O-methyleupatorin (7) and artemetin (8) involved in the adducts formation. Four seco-tanapartholides (1-4), a guaianolide (5), a germacranolide (6) and two other methoxyflavones (9, 10) were also characterized. Evaluation of isolated compounds on P. falciparum and T. brucei brucei showed a moderate antiprotozoal activity of the two methoxyflavones. Copyright © 2017. Published by Elsevier B.V.

  7. Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation. (United States)

    Rutardottir, Sigurbjörg; Karnaukhova, Elena; Nantasenamat, Chanin; Songtawee, Napat; Prachayasittikul, Virapong; Rajabi, Mohsen; Rosenlöf, Lena Wester; Alayash, Abdu I; Åkerström, Bo


    α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes. (United States)

    Liu, Ke; Qian, Yingjuan; Jung, Yong-Sam; Zhou, Bin; Cao, Ruibing; Shen, Ting; Shao, Donghua; Wei, Jianchao; Ma, Zhiyong; Chen, Puyan; Zhu, Huaimin; Qiu, Yafeng


    Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca(2+) dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and

  9. Protein-Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer. (United States)

    Dodagatta-Marri, Eswari; Mitchell, Daniel A; Pandit, Hrishikesh; Sonawani, Archana; Murugaiah, Valarmathy; Idicula-Thomas, Susan; Nal, Béatrice; Al-Mozaini, Maha M; Kaur, Anuvinder; Madan, Taruna; Kishore, Uday


    Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein-protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.

  10. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N. (NIH)


    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  11. Vixapatin (VP12, a C-Type Lectin-Protein from Vipera xantina palestinae Venom: Characterization as a Novel Anti-angiogenic Compound

    Directory of Open Access Journals (Sweden)

    Philip Lazarovici


    Full Text Available A C-type lectin-like protein (CTL, originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC50 of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC; 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.

  12. A novel C-type lectin, Nattectin-like protein, with a wide range of bacterial agglutination activity in large yellow croaker Larimichthys crocea. (United States)

    Lv, Changhuan; Zhang, Dongling; Wang, Zhiyong


    C-type lectins (CTLs) are generally recognized as a superfamily of Ca(2+)-dependent carbohydrate-binding proteins, which serve as pattern recognition receptors (PRRs) in innate immunity of vertebrates. In this study, the molecular characterization and immune roles of a novel CTL from Larimichthys crocea (designated as LcNTC) were investigated. LcNTC is a novel protein that shared 33%-49% homology with other teleosts CTLs. The full-length cDNA of LcNTC was composed of 859 bp with a 465 bp open reading frame encoding a putative protein of 154 residues. LcNTC contained a single CRD with four conserved disulfide-bonded cysteine residues (Cys(57)-Cys(148), Cys(126)-Cys(140)) and EPN/AND motifs instead of invariant EPN/WND motifs required for carbohydrate-binding specificity and constructing Ca(2+)-binding sites. LcNTC mRNA was detected in all examined tissues with the most abundant in the gill. After challenged with poly I:C and Vibrio parahaemolyticus, the temporal expression of LcNTC was significantly up-regulated in the liver, spleen and head-kidney. LcNTC transcripts were also induced in the gill, skin, spleen and head-kidney post-infection with Cryptocaryon irritans. The recombinant LcNTC (rLcNTC) purified from Escherichia coli BL21 (DE3) exhibited strong agglutination activity against erythrocytes from human, rabbit and large yellow croaker in a Ca(2+)-dependent manner, and the agglutination could be inhibited by D-Mannose, D-Glucose, D-Fructose, α-Lactose, D-Maltose and LPS. Positive microbial agglutination activities of rLcNTC were observed against all tested bacteria in the presence of Ca(2+), including Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus lysoleikticus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). These findings collectively indicated that LcNTC might be involved in the innate immunity of L. crocea as a PRR. Copyright © 2016 Elsevier Ltd. All rights

  13. Allelic mRNA expression imbalance in C-type lectins reveals a frequent regulatory SNP in the human surfactant protein A (SP-A) gene. (United States)

    Azad, A K; Curtis, A; Papp, A; Webb, A; Knoell, D; Sadee, W; Schlesinger, L S


    Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We used allelic mRNA expression imbalance (AEI) technology for surfactant protein (SP)-A1, SP-A2, SP-D, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), macrophage mannose receptor (MRC1) and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker single-nucleotide polymorphisms (SNPs) and was confirmed by next-generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% minor allele frequency); the only variant consistently associated with an approximately twofold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial linkage disequilibrium with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases.

  14. The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase response

    DEFF Research Database (Denmark)

    Thiel, S; Holmskov, U; Hviid, L


    Two ELISAs for estimating mannan-binding protein (MBP) were constructed and the concentration of MBP in plasma was followed in patients undergoing major surgery and in patients having a malarial attack. In both cases increases of MBP in the plasma were observed. The relative increase and the kine...

  15. CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid Cytochrome c Assembly in the Green Alga Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Sara G. Cline


    Full Text Available In bacteria and energy generating organelles, c-type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III. System II was discovered in the green alga Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants (cytochrome csynthesis, which display a block in the apo- to holo- form conversion of cytochrome f and c6, the thylakoid lumen resident c-type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the octatricopeptide-repeat motif (OPR recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in Nicotiana benthamiana. We discuss the possible functions of CCS2 in the heme attachment reaction.

  16. CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid CytochromecAssembly in the Green AlgaChlamydomonas reinhardtii. (United States)

    Cline, Sara G; Laughbaum, Isaac A; Hamel, Patrice P


    In bacteria and energy generating organelles, c -type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III). System II was discovered in the green alga Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants ( c ytochrome c s ynthesis), which display a block in the apo- to holo- form conversion of cytochrome f and c 6 , the thylakoid lumen resident c -type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the o ctatrico p eptide- r epeat motif (OPR) recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in Nicotiana benthamiana . We discuss the possible functions of CCS2 in the heme attachment reaction.

  17. The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A. (United States)

    Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel


    Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional

  18. An alternative function of C-type lectin comprising low-density lipoprotein receptor domain from Fenneropenaeus merguiensis to act as a binding receptor for viral protein and vitellogenin. (United States)

    Kwankaew, Pattamaporn; Praparatana, Rachanida; Runsaeng, Phanthipha; Utarabhand, Prapaporn


    A diversity of C-type lectins (CTLs) was coming reported and they are known to participate in invertebrate innate immunity by act as pattern recognition receptor (PRR). In the present study, a unique CTL containing low-density lipoprotein receptor (LDLR) domain from Fenneropenaeus merguiensis (designated as FmLdlr) was cloned. Its sequence contained a single LDLR domain and one carbohydrate recognition domain (CRD) with a QAP motif putative for galactose-specific binding. The expression of FmLdlr was detected only in hemocytes of healthy shrimp. Its expression was significantly up-regulated by Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenge. The knockdown by FmLdlr dsRNA resulted in severe gene down-regulation. The gene silencing with pathogenic co-inoculation led to reduction of the median lethal time and increasing in the cumulative mortality including the remained WSSV in WSSV co-challenge group. Recombinant proteins of FmLdlr and two domains could agglutinate various bacterial strains which LDLR domain revealed the lowest activity. Only FmLdlr and CRD could enhance phagocytosis and encapsulation by hemocytes. Both FmLdlr and CRD except LDLR domain exhibited the antibacterial activity by inhibiting the growth of pathogenic V. parahaemolyticus in cultured medium and disk diffusion assay. Only FmLdlr and CRD could bind to WSSV proteins, envelope VP28, tegument VP39A and also capsid VP15, which FmLdlr had the higher binding affinity than that of CRD. Altogether, we concluded that FmLdlr contributed in shrimp immune defense through the main action of CRD in capable of bacterial agglutination, enhancing the phagocytosis and encapsulation, antimicrobial activity and binding to viral proteins. Interestingly, ELISA approach revealed that LDLR domain displayed the highest binding affinity to vitellogenin than whole molecule and CRD. We signified a new function of FmLdlr that it might presumably act as a receptor for vitellogenin transportation in

  19. Convulxin, a C-type lectin-like protein, inhibits HCASMCs functions via WAD-motif/integrin-αv interaction and NF-κB-independent gene suppression of GRO and IL-8

    Energy Technology Data Exchange (ETDEWEB)

    Shih, Chun-Ho; Chiang, Tin-Bin [Chang Gung University of Science and Technology, Guishan Dist., Taoyuan City, Taiwan (China); Wang, Wen-Jeng, E-mail: [Chang Gung University of Science and Technology, Guishan Dist., Taoyuan City, Taiwan (China); Department of Neurological Surgery, Chang Gung Memorial Hospital, Guishan Dist., Taoyuan City, Taiwan (China)


    Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of β-chain of CVX (CVX-β) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala–Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenic effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvβ3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-β with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent. - Highlights: • The tetrameric convulxin (CVX) with WAD

  20. Tetranectin, a trimeric plasminogen-binding C-type lectin

    DEFF Research Database (Denmark)

    Holtet, T L; Graversen, Jonas Heilskov; Clemmensen, I


    Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross......-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization....

  1. Primary structure of cytochrome c' of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c'-type cytochromes. (United States)

    Bergmann, D J; Zahn, J A; DiSpirito, A A


    Cytochrome c' of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome C555. This cytochrome is spectrally similar to other cytochromes c' but is larger (16,000 Da) and has a lower midpoint potential (-205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c' from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c', only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c' from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c'-type cytochrome which developed a covalent cross-link between a lysine residue and the c'-heme.

  2. Quantum Torus Algebras and B(C)-Type Toda Systems (United States)

    Wang, Na; Li, Chuanzhong


    In this paper, we construct a new even constrained B(C)-type Toda hierarchy and derive its B(C)-type Block-type additional symmetry. Also we generalize the B(C)-type Toda hierarchy to the N-component B(C)-type Toda hierarchy which is proved to have symmetries of a coupled \\bigotimes ^NQT_+ algebra ( N-fold direct product of the positive half of the quantum torus algebra QT).

  3. Impacts of Shewanella oneidensis c-type cytochromes on aerobic and anaerobic respiration. (United States)

    Gao, Haichun; Barua, Soumitra; Liang, Yili; Wu, Lin; Dong, Yangyang; Reed, Samantha; Chen, Jingrong; Culley, Dave; Kennedy, David; Yang, Yunfeng; He, Zhili; Nealson, Kenneth H; Fredrickson, James K; Tiedje, James M; Romine, Margaret; Zhou, Jizhong


    Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis. © 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  4. Transmission-blocking antibodies against mosquito C-type lectins for dengue prevention.

    Directory of Open Access Journals (Sweden)

    Yang Liu


    Full Text Available C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1, facilitating the attachment of West Nile virus (WNV on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.

  5. The question of histidine content in c-type cytochromes. (United States)

    Cusanovich, M A; Meyer, T; Tedro, S M; Kamen, M D


    Reports that histidine may not occur in heme peptides derived from c-type cytochromes isolated from chloroplasts of Euglena gracilis and Porphyra sp. have not been substantiated in the present investigation, in which the amino acid composition and a partial sequence were determined for a heme peptide derived from the c-type cytochromes of a strain of Euglena closely related to that used in the previous studies. It is concluded that no evidence exists to challenge the generalization that histidine is always present vicinal to the hemebinding site in c-type cytochromes.

  6. Exploring the common dynamics of homologous proteins. Application to the globin family. (United States)

    Maguid, Sandra; Fernandez-Alberti, Sebastian; Ferrelli, Leticia; Echave, Julian


    We present a procedure to explore the global dynamics shared between members of the same protein family. The method allows the comparison of patterns of vibrational motion obtained by Gaussian network model analysis. After the identification of collective coordinates that were conserved during evolution, we quantify the common dynamics within a family. Representative vectors that describe these dynamics are defined using a singular value decomposition approach. As a test case, the globin heme-binding family is considered. The two lowest normal modes are shown to be conserved within this family. Our results encourage the development of models for protein evolution that take into account the conservation of dynamical features.

  7. C-Type Natriuretic Peptide Analog as Therapy for Achondroplasia. (United States)

    Legeai-Mallet, Laurence


    Fibroblast growth factor receptor 3 (FGFR3) is an important regulator of bone formation. Gain-of-function mutations in the FGFR3 gene result in chondrodysplasias which include achondroplasia (ACH), the most common form of dwarfism, in which skull, appendicular and axial skeletons are affected. The skeletal phenotype of patients with ACH showed defective proliferation and differentiation of the chondrocytes in the growth plate cartilage. Both endochondral and membranous ossification processes are disrupted during development. At cellular level, Fgfr3 mutations induce increased phosphorylation of the tyrosine kinase receptor FGFR3, which correlate with an enhanced activation of its downstream signaling pathways. Potential therapeutic strategies have emerged for ACH. Several preclinical studies have been conducted such as the C-type natriuretic peptide (CNP) analog (BMN111), intermittent parathyroid hormone injections, soluble FGFR3 therapy, and meclozine and statin treatments. Among the putative targets to antagonize FGFR3 signaling, CNP (or BMN111) is one of the most promising strategies. BMN111 acts as a key regulator of longitudinal bone growth by downregulating the mitogen-activated protein kinase pathway, which is activated as a result of a FGFR3 gain-of-function mutation. Preclinical studies showed that BMN111 treatment led to a large improvement in skeletal parameters in Fgfr3Y367C/+ mice mimicking ACH. In 2014, a clinical trial (phase 2) of BMN111 in pediatric patients with ACH has started. This first clinical trial marks the first big step towards real treatment for these patients. © 2016 S. Karger AG, Basel.

  8. The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

    Directory of Open Access Journals (Sweden)

    Jinxing Song


    Full Text Available Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11. In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli.

  9. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Konuma, Tsuyoshi [Icahn School of Medicine at Mount Sinai, Department of Structural and Chemical Biology (United States); Harada, Erisa [Suntory Foundation for Life Sciences, Bioorganic Research Institute (Japan); Sugase, Kenji, E-mail:, E-mail: [Kyoto University, Department of Molecular Engineering, Graduate School of Engineering (Japan)


    Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

  10. Signalling through C-type lectin receptors: shaping immune responses

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.


    C-type lectin receptors (CLRs) expressed by dendritic cells are crucial for tailoring immune responses to pathogens. Following pathogen binding, CLRs trigger distinct signalling pathways that induce the expression of specific cytokines which determine T cell polarization fates. Some CLRs can induce

  11. C-type natriuretic peptide and its precursor

    DEFF Research Database (Denmark)

    Lippert, Solvej; Iversen, Peter; Brasso, Klaus


    AIM: Seminal plasma offer a more organ-specific matrix for markers in prostatic disease. We hypothesized that C-type natriuretic peptide (CNP) expression may constitute such a new target. METHODS: Patients with benign prostatic hyperplasia, clinically localized and metastatic prostate cancer were...

  12. C-type lectin receptors orchestrate antifungal immunity

    NARCIS (Netherlands)

    Wevers, Brigitte A.; Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.


    Fungal infections are an emerging threat for human health. A coordinated host immune response is fundamental for successful elimination of an invading fungal microbe. A panel of C-type lectin receptors expressed on antigen-presenting dendritic cells enable innate recognition of fungal cell wall

  13. A novel C-type lectin identified by EST analysis in tissue migratory larvae of Ascaris suum. (United States)

    Yoshida, Ayako; Nagayasu, Eiji; Horii, Yoichiro; Maruyama, Haruhiko


    C-type lectins (CTLs) are a group of proteins which bind to carbohydrate epitopes in the presence of Ca(2+), which have been described in a wide range of species. In this study, a cDNA sequence coding a putative CTL has been identified from the cDNA library constructed from the pig round worm Ascaris suum lung L3 (LL3) larvae, which was designated as A. suum C-type lectin-1 (As-CTL-1). The 510 nucleotide open reading frame of As-CTL-1 cDNA encoded the predicted 169 amino acid protein including a putative signal peptide of 23 residues and C-type lectin/C-type lectin-like domain (CLECT) at residue 26 to 167. As-CTL-1 was most similar to Toxocara canis C-type lectin-1 and 4 (Tc-CTL-1 and 4), and highly homologous to namatode CTLs and mammalian CTLs as well, such as human C-type lectin domain family 4 member G (CLECG4). In addition, As-CTL-1 was strongly expressed in tissue migrating LL3 and the L4 larvae, which were developmental larvae stages within the mammalian host. These results suggest that A. suum larvae might utilize As-CTL-1 to avoid pathogen recognition mechanisms in mammalian hosts due to it is similarity to host immune cell receptors.

  14. Gclust Server: 11424 [Gclust Server

    Lifescience Database Archive (English)


  15. Occurrence and transmission efficiencies of Borrelia burgdorferi ospC types in avian and mammalian wildlife. (United States)

    Vuong, Holly B; Canham, Charles D; Fonseca, Dina M; Brisson, Dustin; Morin, Peter J; Smouse, Peter E; Ostfeld, Richard S


    Borrelia burgdorferi s.s., the bacterium that causes Lyme disease in North America, circulates among a suite of vertebrate hosts and their tick vector. The bacterium can be differentiated at the outer surface protein C (ospC) locus into 25 genotypes. Wildlife hosts can be infected with a suite of ospC types but knowledge on the transmission efficiencies of these naturally infected hosts to ticks is still lacking. To evaluate the occupancy and detection of ospC types in wildlife hosts, we adapted a likelihood-based species patch occupancy model to test for the occurrence probabilities (ψ - "occupancy") and transmission efficiencies (ε - "detection") of each ospC type. We detected differences in ospC occurrence and transmission efficiencies from the null models with HIS (human invasive strains) types A and K having the highest occurrence estimates, but both HIS and non-HIS types having high transmission efficiencies. We also examined ospC frequency patterns with respect to strains known to be invasive in humans across the host species and phylogenetic groups. We found that shrews and to a lesser extent, birds, were important host groups supporting relatively greater frequencies of HIS to non-HIS types. This novel method of simultaneously assessing occurrence and transmission of ospC types provides a powerful tool in assessing disease risk at the genotypic level in naturally infected wildlife hosts and offers the opportunity to examine disease risk at the community level. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Modification of C-type inactivating Shaker potassium channels by chloramine-T. (United States)

    Schlief, T; Schönherr, R; Heinemann, S H


    Shaker potassium channels undergo a slow C-type inactivation which can be hastened dramatically by single-point mutations in or near the pore region. We found that the oxidizing agent chloramine-T (Chl-T) causes an irreversible loss of current for those mutants which show C-type inactivation. For several mutants at position T449, which show a wide spectrum of inactivation time constants, the time constant of current rundown induced by Chl-T correlated with the speed of inactivation. Rundown was accelerated when the channels were in the inactivated state but rundown also occurred when channels were not opened or inactivated. Apparently, only those channels which can undergo C-type inactivation are accessible to Chl-T. In order to gain information about the target amino-acid residue for the action of Chl-T and the structural rearrangements occurring during C-type inactivation, several mutant channel proteins were compared with respect to their response to Chl-T. Since Chl-T can oxidize cysteine and methionine residues, we mutated the possible targets in and close to the pore region, namely C462 to A, and M440 and M448 to I. While the residues M440 and C462 were not important for channel rundown, mutation of M448 to I made the channels more resistant to Chl-T by about one order of magnitude. While inactivation was accelerated upon application of Chl-T in most mutants, mutation of M448 to I abolished this effect on the time course of inactivation, indicating that M448 is one of the target residues for Chl-T.

  17. Chicken lung lectin is a functional C-type lectin and inhibits haemagglutination by influenza A virus.

    NARCIS (Netherlands)

    Hogenkamp, A.|info:eu-repo/dai/nl/243274319; Isohadouten, N.; Reemers, S.S.N.|info:eu-repo/dai/nl/304836508; Romijn, R.A.|info:eu-repo/dai/nl/26228359X; Hemrika, W.|info:eu-repo/dai/nl/121631362; White, M.R.; Tefsen, B.; Vervelde, L.|info:eu-repo/dai/nl/134923391; van Eijk, M.|info:eu-repo/dai/nl/255160216; Veldhuizen, E.J.A.|info:eu-repo/dai/nl/19545264X; Haagsman, H.P.|info:eu-repo/dai/nl/069273278


    Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms.We previously reported the finding of a

  18. C-type natriuretic peptide in prostate cancer

    DEFF Research Database (Denmark)

    Nielsen, Soeren Junge; Iversen, Peter; Rehfeld, Jens F.


    C-type natriuretic peptide (CNP) is expressed in the male reproductive organs in pigs. To examine whether the human prostate also expresses the CNP gene, we measured CNP and N-terminal proCNP in prostate cancer tissue extracts and performed immunohistochemical biopsy staining. Additionally, pro......CNP-derived peptides were quantitated in plasma from patients with prostate cancer. Blood was collected from healthy controls and patients before surgery for localized prostate cancer. Tissue extracts were prepared from tissue biopsies obtained from radical prostatectomy surgery. N-terminal proCNP, proCNP (1...... demonstrated the presence of the peptides in prostatic epithelial cells. The N-terminal proCNP concentrations in plasma were marginally lower in patients with localized prostate cancer compared with control subjects [13.8 pmol/l (11.0-17.2) vs. 15.1 pmol/l (10.4-23.2), p=0.002] but not enough to justify...

  19. [C-type scaphoid fracture in a elite power lifting]. (United States)

    Heckmann, A; Lahoda, L U; Alkandari, Q; Vogt, P M; Knobloch, K


    Power lifting injuries most often involve shoulder injuries with an injury rate of 0.57 to 0.71/1000 hours of power lifting. Wrist injuries are less common in power lifters with 0.05/1000 hours exposure vs. 0.23/1000 h in elite weight lifting men. Often, two contributing factors causing wrist injuries are encountered: a) loss of balance causing the barbell to drift back behind the head of the power lifter, which hyperextends the wrist and b) the maximal weight. We report on an elite power lifting athlete preparing for the World Masters Bench press championships suffering two months of persisting pain during bench press exercise and rest in the snuff-box area following a loss of balance of the bar-bell during bench press with 280 kg load. Following prolonged presentation 2 months after the initial injury with training in the meantime, CT-scan was performed revealing a C-type scaphoid fracture. Surgery was performed as Herbert screw fixation and bone grafting according to the technique of Matti-Russe, followed by an immobilisation of twelve weeks with a plaster. We recommended ending the athletes' power lifting career, however he further exercised with the plaster with consecutive re-operation 3months later and 2nd Matti-Russe and Herbert screw re-do. One year later he became national champion with 240 kg bench pressing. Given the limited scaphoid blood supply and the high complication rate especially among C-type scaphoid fractures, a surgical procedure with bone grafting, Herbert screw fixation and sufficient plaster immobilisation is advocated in scaphoid fractures in elite athletes.

  20. Production, purification, and capsid stability of rhinovirus C types. (United States)

    Griggs, Theodor F; Bochkov, Yury A; Nakagome, Kazuyuki; Palmenberg, Ann C; Gern, James E


    The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori. (United States)

    Shahzad, Toufeeq; Zhan, Ming-Yue; Yang, Pei-Jin; Yu, Xiao-Qiang; Rao, Xiang-Jun


    C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities. © 2017 Wiley Periodicals, Inc.

  2. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C


    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...

  3. C-type Lectin Receptors for Tumor Eradication: Future Directions

    Energy Technology Data Exchange (ETDEWEB)

    Streng-Ouwehand, Ingeborg; Unger, Wendy W. J.; Kooyk, Yvette van, E-mail: [Department of Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam (Netherlands)


    Dendritic cells are key regulators in directing immune responses and therefore are under extensive research for the induction of anti-tumor responses. DCs express a large array of receptors by which they scan their surroundings for recognition and uptake of pathogens. One of the receptor-families is the C-type lectins (CLR), which bind carbohydrate structures and internalize antigens upon recognition. Intracellular routing of antigen through CLR enhances loading and presentation of antigen through MHC class I and II, inducing antigen-specific CD4{sup +} and CD8{sup +} T-cell proliferation and skewing T-helper cells. These characteristics make CLRs very interesting targets for DC-based immunotherapy. Profound research has been done on targeting specific tumor antigens to CLR using either antibodies or the natural ligands such as glycan structures. In this review we will focus on the current data showing the potency of CLR-targeting and discuss improvements that can be achieved to enhance anti-tumor activity in the near future.

  4. Targeting C-type Lectin Receptors for Cancer Immunity

    Directory of Open Access Journals (Sweden)

    Huimin eYan


    Full Text Available C-type lectin receptors (CLRs are a large family of soluble and trans-membrane pattern recognition receptors that are widely and primarily expressed on myeloid cells. CLRs are important for cell-cell communication and host defense against pathogens through the recognition of specific carbohydrate structures. Similar to a family of Toll-like receptors (TLRs, CLRs signaling are involved in the various steps for initiation of innate immune responses and promote secretion of soluble factors such as cytokines and interferons, Moreover, CLRs contribute to endocytosis and antigen-presentation, thereby fine-tune adaptive immune responses. In addition, there may also be a direct activation of acquired immunity. On the other hand, glycans, such as mannose structures, Lewis-type antigens or GalNAc are components of tumor antigens and ligate CLRs, leading to immunoregulation. Therefore agonists or antagonists of CLRs signaling are potential therapeutic reagents for cancer immunotherapy. We aim to overview the current knowledge of CLRs signaling and the application of their ligands on tumor-associating immune response.

  5. Endothelial C-type natriuretic peptide maintains vascular homeostasis. (United States)

    Moyes, Amie J; Khambata, Rayomand S; Villar, Inmaculada; Bubb, Kristen J; Baliga, Reshma S; Lumsden, Natalie G; Xiao, Fang; Gane, Paul J; Rebstock, Anne-Sophie; Worthington, Roberta J; Simone, Michela I; Mota, Filipa; Rivilla, Fernando; Vallejo, Susana; Peiró, Concepción; Sánchez Ferrer, Carlos F; Djordjevic, Snezana; Caulfield, Mark J; MacAllister, Raymond J; Selwood, David L; Ahluwalia, Amrita; Hobbs, Adrian J


    The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor-C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders.

  6. C-type natriuretic peptide in prostate cancer

    DEFF Research Database (Denmark)

    Nielsen, Soeren Junge; Iversen, Peter; Rehfeld, Jens F.


    C-type natriuretic peptide (CNP) is expressed in the male reproductive organs in pigs. To examine whether the human prostate also expresses the CNP gene, we measured CNP and N-terminal proCNP in prostate cancer tissue extracts and performed immunohistochemical biopsy staining. Additionally, pro......CNP-derived peptides were quantitated in plasma from patients with prostate cancer. Blood was collected from healthy controls and patients before surgery for localized prostate cancer. Tissue extracts were prepared from tissue biopsies obtained from radical prostatectomy surgery. N-terminal proCNP, proCNP (1......-50) and CNP were measured in plasma and tissue extracts. Biopsies were stained for CNP-22 and N-terminal proCNP. Tissue extracts from human prostate cancer contained mostly N-terminal proCNP [median 5.3 pmol/g tissue (range 1.0-12.9)] and less CNP [0.14 pmol/g tissue (0.01-1.34)]. Immunohistochemistry...

  7. Endothelial C-type natriuretic peptide maintains vascular homeostasis (United States)

    Moyes, Amie J.; Khambata, Rayomand S.; Villar, Inmaculada; Bubb, Kristen J.; Baliga, Reshma S.; Lumsden, Natalie G.; Xiao, Fang; Gane, Paul J.; Rebstock, Anne-Sophie; Worthington, Roberta J.; Simone, Michela I.; Mota, Filipa; Rivilla, Fernando; Vallejo, Susana; Peiró, Concepción; Sánchez Ferrer, Carlos F.; Djordjevic, Snezana; Caulfield, Mark J.; MacAllister, Raymond J.; Selwood, David L.; Ahluwalia, Amrita; Hobbs, Adrian J.


    The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor–C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders. PMID:25105365

  8. A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense. (United States)

    Xiu, Yunji; Wang, Yinghui; Bi, Jingxiu; Liu, Yuhan; Ning, Mingxiao; Liu, Hui; Li, Shuang; Gu, Wei; Wang, Wen; Meng, Qingguo


    C-type lectins (CTLs) play important roles in invertebrate innate immunity by recognizing and eliminating pathogens. In the present study, a low-density lipoprotein receptor class A (LDLa) domain-containing CTL was identified from the oriental river prawn Macrobrachium nipponense, designated as MnCTLDcp1. The full-length cDNA of MnCTLDcp1 was composed of 1462 bp, with a 999-bp ORF encoding a 332-aa protein. An LDLa and a single C-type lectin-like domain (CTLD) were found. The mRNA transcripts of MnCTLDcp1 was expressed the highest in heart. After the prawns were challenged by Aeromonas hydrophila and Staphylococcus aureus, the expression level of MnCTLDcp1 in heart and hemocytes were all significantly up-regulated. Sugar binding assay revealed that the MnCTLDcp1 could bind to the glycoconjugates of bacteria surface, such as LPS, PGN and they can compete with bacterial as competitors. The recombinant MnCTLDcp1 agglutinates Gram-positive (S. aureus and Bacillus subtilis) and Gram-negative bacteria (A. hydrophila, Vibrio parahaemolyticus, Escherichia coli and Pseudomonas aeruginosa) in the presence of calcium and also could bind to these bacteria. These results clearly suggested that MnCTLDcp1 functions as a pattern-recognition receptor involved in the innate immunity of M. nipponense. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. C-type lectin binds to β-integrin to promote hemocytic phagocytosis in an invertebrate. (United States)

    Wang, Xian-Wei; Zhao, Xiao-Fan; Wang, Jin-Xing


    Phagocytosis is a conserved cellular response among metazoans. Opsonins are some molecules that label targets to increase their susceptibility to phagocytosis. Opsonins are usually captured by receptors on the surface of phagocytes. Our previous study found the C-type lectin FcLec4 from Chinese white shrimp Fenneropenaeus chinensis might function as an opsonin to facilitate bacterial clearance. In the present study we purified the native FcLec4 protein and confirmed its opsonic activity in the near relation, kuruma shrimp Marsupenaeus japonicus. The possible receptor of FcLec4 was identified as β-integrin by panning a T7 phage display library of shrimp hemocytes and then confirmed by co-immunoprecipitation assay. We further proved that the interaction between FcLec4 and β-integrin did not rely on the carbohydrate recognition domain but on the N terminus of FcLec4. In addition, inhibition of FcLec4 expression using RNAi delayed bacterial clearance, and β-integrin knockdown suppressed the opsonic activity of FcLec4. This study is the first to show the direct interaction between an opsonin and its receptor in crustaceans. Our study provides new insights into invertebrate phagocytosis and the functions of C-type lectins.

  10. Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

    DEFF Research Database (Denmark)

    Nielbo, Steen; Thomsen, Jens K; Graversen, Jonas Heilskov


    Tetranectin is a homotrimeric protein containing a C-type lectin-like domain. This domain (TN3) can bind calcium, but in the absence of calcium, the domain binds a number of kringle-type protein ligands. Two of the calcium-coordinating residues are also critical for binding plasminogen kringle 4 (K...

  11. C-type lectin receptors and RIG-I-like receptors: new points on the oncogenomics map

    Directory of Open Access Journals (Sweden)

    Yuzhalin AE


    Full Text Available Anton G Kutikhin, Arseniy E YuzhalinDepartment of Epidemiology, Kemerovo State Medical Academy, Kemerovo, Russian FederationAbstract: The group of pattern recognition receptors includes families of Toll-like receptors, NOD-like receptors, C-type lectin receptors, and RIG-I-like receptors. They are key sensors for a number of infectious agents, some of which are oncogenic, and they launch an immune response against them, normally promoting their eradication. Inherited variations in genes encoding these receptors and proteins and their signaling pathways may affect their function, possibly modulating cancer risk and features of cancer progression. There are numerous studies investigating the association of single nucleotide polymorphisms within or near genes encoding Toll-like receptors and NOD-like receptors, cancer risk, and features of cancer progression. However, there is an almost total absence of articles analyzing the correlation between polymorphisms of genes encoding C-type lectin receptors and RIG-I-like receptors and cancer risk or progression. Nevertheless, there is some evidence supporting the hypothesis that inherited C-type lectin receptor and RIG-I-like receptor variants can be associated with increased cancer risk. Certain C-type lectin receptors and RIG-I-like receptors recognize pathogen-associated molecular patterns of potentially oncogenic infectious agents, and certain polymorphisms of genes encoding C-type lectin receptors and RIG-I-like receptors may have functional consequences at the molecular level that can lead to association of such single nucleotide polymorphisms with risk or progression of some diseases that may modulate cancer risk, so these gene polymorphisms may affect cancer risk indirectly. Polymorphisms of genes encoding C-type lectin receptors and RIG-I-like receptors thereby may be correlated with a risk of lung, oral, esophageal, gastric, colorectal, and liver cancer, as well as nasopharyngeal carcinoma

  12. An EPD/WSD motifs containing C-type lectin from Argopectens irradians recognizes and binds microbes with broad spectrum. (United States)

    Huang, Mengmeng; Zhang, Huan; Jiang, Shuai; Wang, Lingling; Liu, Rui; Yi, Qilin; Song, Linsheng


    C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which play significant roles in nonself-recognition and clearance of invaders. The immune function of a C-type lectin (AiCTL-7) with EPD/WSD motifs from Argopectens irradians was investigated in the present study. The recombinant protein of AiCTL-7 (rAiCTL-7) could bind LPS, PGN, mannan, yeast glucan and poly I:C in vitro, and displayed a broader microbes binding spectrum towards Gram-positive bacteria Staphylococcus aureus, Gram-negative bacteria Escherichia coli, Vibrio anguillarum, as well as fungi Pichia pastoris and Yarrowia lipolytica. Moreover, it could also inhibit the growth of E. coli and significantly (P EPD/WSD motifs containing lectin AiCTL-7 could serve as PRR with wider recognition spectrum, and function both as collectin and selectin participating in the immunity against invaders in scallops. It could be inferred that the diversity and complexity of motifs in Ca(2+) binding site 2 in CRDs endowed C-type lectins with comprehensive recognition spectrum and multiple immune functions against complex living environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

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    Ming Feng Jiang


    Full Text Available Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type milk lysozyme gene (YML, was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75 which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

  14. Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea

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    Jingqun Ao


    Full Text Available The C-type lectin-like receptors (CTLRs play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD, an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1–C6, a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp and WYD (Trp-Tyr-Asp. Ca2+ binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus and guppy (Poecilia retitculata CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca2+-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

  15. High Bacterial Agglutination Activity in a Single-CRD C-Type Lectin from Spodoptera exigua (Lepidoptera: Noctuidae). (United States)

    Gasmi, Leila; Ferré, Juan; Herrero, Salvador


    Lectins are carbohydrate-interacting proteins that play a pivotal role in multiple physiological and developmental aspects of all organisms. They can specifically interact with different bacterial and viral pathogens through carbohydrate-recognition domains (CRD). In addition, lectins are also of biotechnological interest because of their potential use as biosensors for capturing and identifying bacterial species. In this work, three C-type lectins from the Lepidoptera Spodoptera exigua were produced as recombinant proteins and their bacterial agglutination properties were characterized. The lowest protein concentration producing bacterial agglutination against a panel of different Gram+ and Gram- as well as their carbohydrate binding specificities was determined for the three lectins. One of these lectins, BLL2, was able to agglutinate cells from a broad range of bacterial species at an extremely low concentration, becoming a very interesting protein to be used as a biosensor or for other biotechnological applications involving bacterial capture.

  16. Macrophage galactose-type C-type lectin receptor for DC targeting of antitumor glycopeptide vaccines

    DEFF Research Database (Denmark)

    Nuti, M; Zizzari, I; Napoletano, C


    e13528 Background: Dendritic cells (DCs) are the most potent antigen presenting cells and are employed in cancer vaccination. Several receptors are being studied in order to identif strategies to increase DCs activating capacity. The C-type lectin macrophage galactose type C-type lectin (MGL...

  17. C-type lectins do not act as functional receptors for filovirus entry into cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuno, Keita; Nakayama, Eri; Noyori, Osamu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan); Marzi, Andrea; Ebihara, Hideki [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Irimura, Tatsuro [Graduate School of Pharmaceutical Science, University of Tokyo, Tokyo (Japan); Feldmann, Heinz [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Takada, Ayato, E-mail: [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan)


    Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

  18. Differential regulation of C-type lectin expression on tolerogenic dendritic cell subsets

    NARCIS (Netherlands)

    van Vliet, Sandra J.; van Liempt, Ellis; Geijtenbeek, Teunis B. H.; van Kooyk, Yvette


    Antigen presenting cells (APC) express high levels of C-type lectins, which play a major role in cellular interactions as well as pathogen recognition and antigen presentation. The C-type lectin macrophage galactose-type lectin (MGL), expressed by dendritic cells (DC) and macrophages, mediates

  19. Isolation and characterization of two novel C-type lectins from the oriental river prawn, Macrobrachium nipponense. (United States)

    Xiu, Yunji; Hou, Libo; Liu, Xiaoqian; Wang, Yinghui; Gu, Wei; Meng, Qingguo; Wang, Wen


    C-type lectins are a family of calcium-dependent carbohydrate-binding proteins which are believed to play important roles in the innate immunity of invertebrates. This study identified two novel C-type lectins, designated as MnCTLDcp2 and MnCTLDcp3, from the oriental river prawn Macrobrachium nipponense. The full-length cDNA of MnCTLDcp2 was of 1582 bp with an open reading frame (ORF) of 972 bp encoding a polypeptide of 323 amino acids. The complete nucleotide sequence of MnCTLDcp3 cDNA was 583 bp, containing a 555 bp ORF encoding a putative protein of 184 deduced amino acids. The deduced MnCTLDcp2 and MnCTLDcp3 proteins both contained a single C-type lectin-like domain (CTLD). Besides, MnCTLDcp2 contains a signal peptide and an low-density lipoprotein receptor class A (LDLa) domain. Reverse transcription PCR showed that MnCTLDcp2 was expressed in the heart, gill, nerve hepatopancreas and intestine; MnCTLDcp3 was expressed in the hepatopancreas, heart, nerve, gill and muscle. Their expression in the heart tissue was regulated following challenge with bacteria. The microbial agglutination assay showed that both MnCTLDcp2 and MnCTLDcp3 could agglutinated bacteria in the presence of calcium. All these results suggested that MnCTLDcp2 and MnCTLDcp3 functioned as pattern recognition receptors in the immune system of M. nipponense. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Macrophage Inducible C-Type Lectin As a Multifunctional Player in Immunity

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    Emmanuel C. Patin


    Full Text Available The macrophage-inducible C-type lectin (Mincle is an innate immune receptor on myeloid cells sensing diverse entities including pathogens and damaged cells. Mincle was first described as a receptor for the mycobacterial cell wall glycolipid, trehalose-6,6′-dimycolate, or cord factor, and the mammalian necrotic cell-derived alarmin histone deacetylase complex unit Sin3-associated protein 130. Upon engagement by its ligands, Mincle induces secretion of innate cytokines and other immune mediators modulating inflammation and immunity. Since its discovery more than 25 years ago, the understanding of Mincle’s immune function has made significant advances in recent years. In addition to mediating immune responses to infectious agents, Mincle has been linked to promote tumor progression, autoimmunity, and sterile inflammation; however, further studies are required to completely unravel the complex role of Mincle in these distinct host responses. In this review, we discuss recent findings on Mincle’s biology with an emphasis on its diverse functions in immunity.

  1. Association of a hepatopancreas-specific C-type lectin with the antibacterial response of Eriocheir sinensis.

    Directory of Open Access Journals (Sweden)

    Xing-Kun Jin

    Full Text Available Pattern recognition receptors (PPRs are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD. EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS injection. The recombinant EsLecF (rEsLecF protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate.

  2. A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans

    DEFF Research Database (Denmark)

    Stattin, Eva-Lena; Wiklund, Fredrik; Lindblom, Karin


    aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other...... is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo....... proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan...

  3. Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

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    Beier Frank


    Full Text Available Abstract Background Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. Methods Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. Results We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc. In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II and Npr3 (natriuretic peptide decoy receptor genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor, as well as the Npr2 gene (encoding the CNP receptor. Conclusion Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine

  4. Orthogonal plating of Vancouver B1 and C-type periprosthetic femur fracture nonunions. (United States)

    Birch, Christopher E; Blankstein, Michael; Chlebeck, Jesse D; Bartlett Rd, Craig S


    Periprosthetic femoral shaft fractures are a significant complication after total hip arthroplasty (THA). Plate osteosynthesis has been the mainstay of treatment around well-fixed stems. Nonunions are a rare and challenging complication of this fixation method. We report the outcomes of a novel orthogonal plating surgical technique for Vancouver B1 and C-type periprosthetic fractures that previously failed open reduction internal fixation (ORIF). A retrospective review identified all patients with Vancouver B1/C THA periprosthetic femoral nonunions from 2010 to 2015. Exclusion criteria included open fractures and periprosthetic infections. The technique utilised a mechanobiologic strategy of atraumatic exposure, resection of necrotic tissue, bone grafting with adjuvant bone morphogenetic protein (BMP) and revision open reduction internal fixation with orthogonal plate osteosynthesis. 6 Vancouver B1/C periprosthetic femoral nonunions were treated. 5 patients were female with an average age of 80.3 years (range 72-91 years). The fractures occurred at a mean of 5.8 years (range 1-10 years) from their initial arthroplasty procedure. No patients underwent further revision surgery; there were no perioperative complications. All patients had a minimum of 11 months follow-up (mean 18.6, range 11-36 months). All fractures achieved osseous union, defined as solid bridging callus over at least 2 cortices and pain free, independent ambulation, at an average of 24.4 weeks (range 6.1-39.7 weeks). This is the 1st series describing orthogonal locked compression plating using modern implants for periprosthetic femoral nonunions. This technique should be considered in periprosthetic femur fracture nonunions around a well-fixed stem.

  5. c-Type cytochrome-dependent formation of U(IV nanoparticles by Shewanella oneidensis.

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    Matthew J Marshall


    Full Text Available Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI complexes in situ, the biomolecular mechanisms of U(VI reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI and formation of extracellular UO(2 nanoparticles. In particular, the outer membrane (OM decaheme cytochrome MtrC (metal reduction, previously implicated in Mn(IV and Fe(III reduction, directly transferred electrons to U(VI. Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2 nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS. In wild-type cells, this UO(2-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2 nanoparticles with MtrC and OmcA (outer membrane cytochrome. This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2 nanoparticles. In the environment, such association of UO(2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2 or transport in soils and sediments.

  6. Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei. (United States)

    Soni, Awakash; Goyal, Manish; Prakash, Kirtika; Bhardwaj, Jyoti; Siddiqui, Arif Jamal; Puri, Sunil K


    Malaria parasite resides within the host red blood cells, where it degrades vast amount of haemoglobin. During haemoglobin degradation, toxic free heme is liberated which subsequently gets converted into hemozoin. This process is facilitated by action of various proteins viz. heme detoxification protein (HDP), and histidine rich proteins II and III (HRP II & III). Out of these, HDP is the most potent in hemozoin formation and plays indispensible role for parasite survival. Despite this, the detailed study of HDP from rodent and simian parasite has not been performed till date. Here, we have cloned and sequenced hdp gene from different malaria parasites Plasmodium vinckei, Plasmodium yoelii, Plasmodium knowlesi, and Plasmodium cynomolgi. Furthermore, HDP from P. vinckei (PvHDP) was over-expressed and purified for detailed characterization. The PvHDP is cytosolic, expressed throughout the intra erythrocytic stages and its expression is higher in late trophozoite and schizont stages of parasite. The PvHDP interacts with free heme (KD=89 nM) and efficiently converts heme into hemozoin in a time and concentration dependent manner. Moreover, PvHDP showed activity in acidic pH and over a broad range of temperature. Histidine modification of PvHDP using DEPC showed reduction in heme binding and hemozoin formation, thus emphasizing the importance of histidine residues in heme binding and subsequent hemozoin production. Furthermore, applicability of PvHDP to screen anti-plasmodial agents (targeting heme to hemozoin conversion) was also determined using chloroquine, and mefloquine as reference antimalarials. Results showed that these drugs inhibit heme polymerization effectively in a concentration dependent manner. In conclusion, our study identified and biochemically characterized HDP from rodent malaria parasite P. vinckei and this will help to develop a high throughput assay to evaluate new antimalarials targeting hemozoin pathway. Copyright © 2015 Elsevier B.V. All

  7. The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper. (United States)

    Karlsen, O A; Lillehaug, J R; Jensen, H B


    Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

  8. Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

    Energy Technology Data Exchange (ETDEWEB)

    Le Coq, Johanne; Ghosh, Partho (UCSD)


    Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd ({approx}16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10{sup 20} potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

  9. Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

    Directory of Open Access Journals (Sweden)

    Carlo Travaglini-Allocatelli


    Full Text Available Cytochromes c (Cyt c are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i heme translocation and delivery, (ii apoCyt thioreductive pathway, and (iii apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria.

  10. C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice. (United States)

    Kimura, Toru; Nojiri, Takashi; Hino, Jun; Hosoda, Hiroshi; Miura, Koichi; Shintani, Yasushi; Inoue, Masayoshi; Zenitani, Masahiro; Takabatake, Hiroyuki; Miyazato, Mikiya; Okumura, Meinoshin; Kangawa, Kenji


    Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts. C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription. CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22

  11. DMPD: C-type lectin receptors in antifungal immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available nds Microbiol. 2008 Jan;16(1):27-32. Epub 2007 Dec 21. (.png) (.svg) (.html) (.csml) Show C-type lectin rece... 2007 Dec 21. Pathway - PNG File (.png) SVG File (.svg) HTML File (.html) CSML File (.csml) Open .csml file

  12. Structure of the C-type lectin carbohydrate recognition domain of human tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Nielsen, B B; Rasmussen, H


    Tetranectin (TN) is a C-type lectin involved in fibrinolysis, being the only endogenous ligand known to bind specifically to the kringle 4 domain of plasminogen. TN was originally isolated from plasma, but shows a wide tissue distribution. Furthermore, TN has been found in the extracellular matrix...

  13. Dusting the sugar fingerprint: C-type lectin signaling in adaptive immunity

    NARCIS (Netherlands)

    den Dunnen, Jeroen; Gringhuis, Sonja I.; Geijtenbeek, Teunis B. H.


    Pathogen recognition by dendritic cells (DCs) is central to the induction of adaptive immunity. Pattern recognition receptors (PRRs) on DCs interact with pathogens, leading to signaling events that dictate adaptive immune responses. It is becoming clear that C-type lectins are important PRRs that

  14. C-type lectin receptors in the control of T helper cell differentiation

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.


    Pathogen recognition by C-type lectin receptors (CLRs) expressed by dendritic cells is important not only for antigen presentation, but also for the induction of appropriate adaptive immune responses via T helper (TH) cell differentiation. CLRs act either by themselves or in cooperation with other

  15. Modulation of the immune system by ligands of the C-type lectin DC-SIGN

    NARCIS (Netherlands)

    Gijzen, K.


    DC-SIGN is a C-type lectin specifically expressed on dendritic cells that recognize high-mannose and fucose glycans. This carbohydrate specificity of DC-SIGN enables recognition of a large array of ligands which include both pathogen-derived as well as endogenous glycoconjugates. Because of the wide

  16. Innate signaling by the C-type lectin DC-SIGN dictates immune responses

    NARCIS (Netherlands)

    Dunnen, den J.; Gringhuis, S.I.; Geijtenbeek, T.B.H.


    Effective immune responses depend on the recognition of pathogens by dendritic cells (DCs) through pattern recognition receptors (PRRs). These receptors induce specific signaling pathways that lead to the induction of immune responses against the pathogens. It is becoming evident that C-type lectins

  17. C-type lectin interactions with Schistosoma mansoni SEA : Molecular basis and function

    NARCIS (Netherlands)

    Liempt, van P.A.G.


    Outline of this thesis The studies described in this thesis have been performed to gain more insight in the recognition of Schistosoma mansoni glycans by C-type lectins and the consequences for dendritic cell mediated immune responses. As a first approach to understand the molecular interactions

  18. Processing-independent analysis for pro-C-type natriuretic peptide

    DEFF Research Database (Denmark)

    Lippert, Solvej Kølvraa; Rehfeld, Jens F.; Gøtze, Jens Peter


    C-type natriuretic peptide (CNP) is expressed in several human tissues. We designed a specific processing-independent assay for proCNP-derived products and quantitated the concentrations in human seminal plasma from normal and vasectomized men. Antibodies were raised against the N-terminus of human...

  19. Carbohydrate signaling by C-type lectin DC-SIGN affects NF-kappaB activity

    NARCIS (Netherlands)

    Gringhuis, Sonja I.; Geijtenbeek, Teunis B. H.


    Pathogen recognition is central to the induction of adaptive immunity. Dendritic cells (DCs) express different pattern recognition receptors (PRRs), such as Toll-like receptors and C-type lectins, that sense invading pathogens. Pathogens trigger a specific set of PRRs, leading to activation of

  20. Dual function of C-type lectin-like receptors in the immune system.

    NARCIS (Netherlands)

    Cambi, A.; Figdor, C.G.


    Carbohydrate-binding C-type lectin and lectin-like receptors play an important role in the immune system. The large family can be subdivided into subtypes according to their structural similarities and functional differences. The selectins are of major importance in mediating cell adhesion and

  1. Microsomal prostaglandin E synthase type 2 (mPGES2) is a glutathione-dependent heme protein, and dithiothreitol dissociates the bound heme to produce active prostaglandin E2 synthase in vitro. (United States)

    Takusagawa, Fusao


    An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H2 degradation, but not PGE2 formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414-8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE2 synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782-786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe(3+) in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nM) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H2O2 or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE2 synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H2O2 and reduction by dithiothreitol.

  2. Identification of a Macrobrachium nipponense C-type lectin with a close evolutionary relationship to vertebrate lectins. (United States)

    Huang, Xin; Li, Tingting; Jin, Min; Yin, Shaowu; Wang, Wen; Ren, Qian


    C-type lectins (CTLs) are involved in the innate immune defense of vertebrates and invertebrates against invading pathogens. This study cloned and characterized a novel C-type lectin (MnCTL) of the oriental river prawn, Macrobrachium nipponense. The cloned MnCTL cDNA encompasses an open reading frame of 774 nucleotides and encodes polypeptides of 257 residues. The deduced MnCTL protein contains a single carbohydrate recognition domain (CRD) with an EPN (Glu-Pro-Asn) motif in calcium-binding site 2. Phylogenetic analysis indicated that MnCTL has a closer evolutionary relationship with vertebrate lectins than with invertebrate lectins. Tissue expression analysis showed that high levels of MnCTL are ubiquitously distributed in the gills and stomach of M. nipponense. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that MnCTL expression was up-regulated by bacteria or white spot syndrome virus (WSSV) challenge. Knock-down of the MnCTL gene in WSSV-challenged prawns significantly decreased MnALF1 and MnALF2 transcript levels. The recombinant MnCRD (rMnCRD) agglutinated both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio parahaemolyticus) in the presence of calcium. Furthermore, rMnCRD could bind to all the tested bacteria with different activities. The sugar-binding assay showed that rMnCRD was able to bind lipopolysaccharide and peptidoglycan in a concentration-dependent manner. In addition, rMnCRD could accelerate bacterial clearance. On the contrary, MnCTL silencing by dsRNA interference could weaken the bacterial clearance ability. All these findings implicated MnCTL were involved in the antiviral and antibacterial innate immunity of M. nipponense. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Optimal C-type Filter for Harmonics Mitigation and Resonance Damping in Industrial Distribution Systems


    Abdel Aleem, SHE; Zobaa, AF


    Single-tuned passive filters offer reasonable mitigation for harmonic distortion at a specific harmonic frequency with a high filtering percentage, but resonance hazards exist. Traditional damped filters offer high-pass filtering for the high-frequency range, but suffer from extra ohmic losses. C-type filters may operate in a manner similar to the tuned filters with low damping losses and marginal resonance damping capabilities. Also, they can be designed as damped filters with increased reso...

  4. Prenatal diagnosis of methymalonic aciduria and homocystinuria cblC type using DNA analysis

    Directory of Open Access Journals (Sweden)

    Antonietta Zappu


    Full Text Available Methylmalonic aciduria (MMA and homocystinuria, cblC type is the most frequent inborn error of vitamin B12. CblC patients present with a heterogeneous clinical picture.To date, the early prenatal diagnosis of MMA and homocystinuria, cblC type is performed by determination of methylmalonic acid and total homocysteine (Hcy in amniotic fluid supernatant. In this paper we report a case of prenatal diagnosis, using genetic analysis, of MMA and homocystinuria, cblC type in an at risk couple. Direct sequencing analysis of the amplified products of chorionic villi biopsy extracted DNA showed normal sequence in the fetal DNA. Mutation analysis of the MMACHC gene is more cost-effective and less time-consuming than the biochemical approach. Early prenatal treatment may have an impact on the long-term complications associated with cblC disease. Future studies with the aim of determining the long-term benefits of daily parenteral OHCbl started soon after conception in at risk mothers should be considered. In this context early prenatal diagnosis could determine whether therapy needs to be continued.

  5. A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination. (United States)

    Wongpanya, Ratree; Sengprasert, Panjana; Amparyup, Piti; Tassanakajon, Anchalee


    C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Expression of guanylyl cyclase B in the human corpus cavernosum penis and the possible involvement of its ligand C-type natriuretic polypeptide in the induction of penile erection. (United States)

    Küthe, Andrea; Reinecke, Manfred; Uckert, Stefan; Becker, Armin; David, Ivana; Heitland, Aleksandra; Stief, Christian G; Forssmann, Wolf-Georg; Mägert, Hans-Jürgen


    The induction of penile erection depends on the depletion of free intracellular Ca2+ from the cytosol into the sarcoplasmic reticulum of smooth muscle cells of the corpus cavernosum. This process is regulated by a complex system of signal transduction pathways. In this context guanylyl and adenylyl cyclases as well as cyclic nucleotide monophosphate degrading phosphodiesterases have essential roles and represent important target molecules for the development of drugs for erectile dysfunction. Sildenafil, which is an inhibitor of phosphodiesterase 5, is frequently used for this application but, unfortunately, it has undesirable side effects. Therefore, we investigated the suitability of membrane bound guanylyl cyclases as alternative target proteins. We determined mRNA transcripts specific for guanylyl cyclase B, a receptor of the peptide hormone C-type natriuretic polypeptide, in human corpus cavernosum. We performed immunohistochemistry to evaluate the presence of guanylyl cyclase B in corpus cavernosum and helical artery smooth muscle cells. We further investigated whether C-type natriuretic polypeptide increases intracellular cyclic guanosine monophosphate and performed organ bath studies using corpus cavernosum muscle strips and C-type natriuretic polypeptide at concentrations of 1 to 1 microM. mRNA transcripts were detected encoding for guanylyl cyclase-B, a receptor of the peptide hormone C-type natriuretic polypeptide, in human corpus cavernosum. This finding was verified at the protein level by immunohistochemistry that demonstrated guanylyl cyclase B in corpus cavernosum and helical artery smooth muscle cells. We further noted that C-type natriuretic polypeptide increased intracellular cyclic guanosine monophosphate. In organ bath studies with corpus cavernosum muscle strips C-type natriuretic polypeptide at concentrations of 1 to 1 microM. led to smooth muscle relaxation from 5% to 40%. The results indicate a role for C-type natriuretic polypeptide and


    Energy Technology Data Exchange (ETDEWEB)

    Vernazza, P.; Marsset, M.; Groussin, O.; Lamy, P.; Jorda, L.; Mousis, O.; Delsanti, A. [Aix Marseille Univ, CNRS, LAM, Laboratoire d’Astrophysique de Marseille, Marseille (France); Castillo-Rogez, J. [Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Drive, Pasadena, CA 91109 (United States); Beck, P. [UJF-Grenoble 1, CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG), UMR 5274, Grenoble F-38041 (France); Emery, J. [Department of Earth and Planetary Sciences and Planetary Geosciences Institute, University of Tennessee, Knoxville, TN 37996-1410 (United States); Brunetto, R.; Djouadi, Z.; Dionnet, Z. [Institut d’Astrophysique Spatiale, CNRS, UMR-8617, Université Paris-Sud, bâtiment 121, F-91405 Orsay Cedex (France); Delbo, M.; Carry, B. [Laboratoire Lagrange, UNS-CNRS, Observatoire de la Cote d’Azur, Boulevard de l’Observatoire-CS 34229, F-06304 Nice Cedex 4 (France); Marchis, F. [Carl Sagan Center at the SETI Institute, Mountain View, CA 94043 (United States); Zanda, B. [IMCCE, Observatoire de Paris, 77 avenue Denfert-Rochereau, F-75014 Paris Cedex (France); Borondics, F., E-mail: [SMIS Beamline, Soleil Synchrotron, BP48, L’Orme des Merisiers, F-91192 Gif sur Yvette Cedex (France)


    Anhydrous pyroxene-rich interplanetary dust particles (IDPs) have been proposed as surface analogs for about two-thirds of all C-complex asteroids. However, this suggestion appears to be inconsistent with the presence of hydrated silicates on the surfaces of some of these asteroids, including Ceres. Here, we report the presence of enstatite (pyroxene) on the surface of two C-type asteroids (Ceres and Eugenia) based on their spectral properties in the mid-infrared range. The presence of this component is particularly unexpected in the case of Ceres, because most thermal evolution models predict a surface consisting of hydrated compounds only. The most plausible scenario is that Ceres’ surface has been partially contaminated by exogenous enstatite-rich material, possibly coming from the Beagle asteroid family. This scenario questions a similar origin for Ceres and the remaining C-types, and it possibly supports recent results obtained by the Dawn mission (NASA) that Ceres may have formed in the very outer solar system. Concerning the smaller D  ∼ 200 km C-types such as Eugenia, both their derived surface composition (enstatite and amorphous silicates) and low density (<1.5 g cm{sup −3}) suggest that these bodies accreted from the same building blocks, namely chondritic porous, pyroxene-rich IDPs and volatiles (mostly water ice), and that a significant volume fraction of these bodies has remained unaffected by hydrothermal activity likely implying a late accretion. In addition, their current heliocentric distance may best explain the presence or absence of water ice at their surfaces. Finally, we raise the possibility that CI chondrites, Tagish-Lake-like material, or hydrated IDPs may be representative samples of the cores of these bodies.

  8. A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms (United States)

    Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros


    Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

  9. [Surgical treatment for Tile C type pelvis fracture through posterior approach]. (United States)

    Chen, Zhi-wei; Yang, Le-zhong; Liu, Chun-lei


    To study the clinical results of surgical treatment for Tile C type pelvis fractures with internal fixation by posterior approach. From January 2005 to June 2009, 12 patients with Tile C type pelvis fracture were treated by open reduction through posterior approach. There were 8 males and 4 females, with an average age of 39.5 years ranging from 25 to 58 years. The time from injury to operation was ranged from 7 to 10 days with an average of 9.5 days. All the patients were given X-ray, 3-D CT examinations before operation. The fracture were classified by Tile classification: Type C1 in 5 cases, Type C2 in 2 cases, Type C1 and Type C2 in 4 cases, Type C3 in 1 case. All the posterior rings were fixed by re-establishing steel board without anterior ring fixation after stabilization of body condition. All the patients were treated with skin traction for 3 weeks after operation. All 12 patients were followed up for 6 months to 24 months with an average of 12.6 months. All the incisions healed well, and the fractures got union. No pelvic malunion, low back pain or leg length discrepancy was found. According to Majeed criteria for the evaluation of therapeutic effect, 10 patients were excellent, and 2 were good. In the management of the Tile C type pelvis fractures, a stable pelvis can be reconstructed by fixing posterior ring simply through the posterior approach, so that further sequelae can be reduced.

  10. Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation

    DEFF Research Database (Denmark)

    Napoletano, Chiara; Zizzari, Ilaria G; Rughetti, Aurelia


    Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner....... Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for a-N-acetylgalactosamine (Gal...

  11. Regulation of C-Type Lectin Receptor-Mediated Antifungal Immunity

    Directory of Open Access Journals (Sweden)

    Juan Tang


    Full Text Available Of all the pathogen recognition receptor families, C-type lectin receptor (CLR-induced intracellular signal cascades are indispensable for the initiation and regulation of antifungal immunity. Ongoing experiments over the last decade have elicited diverse CLR functions and novel regulatory mechanisms of CLR-mediated-signaling pathways. In this review, we highlight novel insights in antifungal innate and adaptive-protective immunity mediated by CLRs and discuss the potential therapeutic strategies against fungal infection based on targeting the mediators in the host immune system.

  12. Processing-independent analysis for pro-C-type natriuretic peptide

    DEFF Research Database (Denmark)

    Lippert, Solvej Kølvraa; Rehfeld, Jens F.; Gøtze, Jens Peter


    C-type natriuretic peptide (CNP) is expressed in several human tissues. We designed a specific processing-independent assay for proCNP-derived products and quantitated the concentrations in human seminal plasma from normal and vasectomized men. Antibodies were raised against the N-terminus of human......-933 pmol/L, age 18-25 years); gel chromatography suggested the presence of several molecular forms. Parameters associated to male fertility, proCNP concentrations in blood plasma and time of abstinence did not correlate to the seminal proCNP concentrations. Measurement in vasectomized men disclosed seminal...

  13. A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination. (United States)

    Mu, Changkao; Song, Xiaoyan; Zhao, Jianmin; Wang, Lingling; Qiu, Limei; Zhang, Huan; Zhou, Zhi; Wang, Mengqiang; Song, Linsheng; Wang, Chunlin


    C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Crystalline and structural properties of acid-modified lotus rhizome C-type starch. (United States)

    Cai, Jinwen; Cai, Canhui; Man, Jianmin; Yang, Yang; Zhang, Fengmin; Wei, Cunxu


    The crystalline and structural properties of acid-modified C-type starch from lotus rhizomes were investigated using a combination of techniques. The degradation of granule during hydrolysis began from the end distant from the hilum and then propagated into the center of granule, accompanied by loss of birefringence. The crystallinity changed from C-type to A-type via CA-type during hydrolysis. At the early stage of hydrolysis, the amylose content substantially reduced, the peak and conclusion gelatinization temperatures increased, and the enthalpy decreased. During hydrolysis, the double helix content gradually increased and the amorphous component decreased, the lamellar peak intensity firstly increased and then decreased accompanied by hydrolysis of amorphous and crystalline regions. This study elucidated that B-type allomorph was mainly arranged in the distal region of eccentric hilum, A-type allomorph was mainly located in the periphery of hilum end, and the center of granule was a mixed distribution of A- and B-type allomorphs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Mechanistic insights into the role of C-type lectin receptor/CARD9 signaling in human antifungal immunity

    Directory of Open Access Journals (Sweden)

    Rebecca A. Drummond


    Full Text Available Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs. CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS. In the last few years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2 and CARD9-binding partners (MALT1, BCL10 affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation.

  16. Venom of parasitoid, Pteromalus puparum, suppresses host, Pieris rapae, immune promotion by decreasing host C-type lectin gene expression.

    Directory of Open Access Journals (Sweden)

    Qi Fang

    Full Text Available Insect hosts have evolved immunity against invasion by parasitoids, and in co-evolutionary response parasitoids have also developed strategies to overcome host immune systems. The mechanisms through which parasitoid venoms disrupt the promotion of host immunity are still unclear. We report here a new mechanism evolved by parasitoid Pteromalus puparum, whose venom inhibited the promotion of immunity in its host Pieris rapae (cabbage white butterfly.A full-length cDNA encoding a C-type lectin (Pr-CTL was isolated from P. rapae. Quantitative PCR and immunoblotting showed that injection of bacterial and inert beads induced expression of Pr-CTL, with peaks of mRNA and Pr-CTL protein levels at 4 and 8 h post beads challenge, respectively. In contrast, parasitoid venom suppressed Pr-CTL expression when co-injected with beads, in a time and dose-dependent manner. Immunolocalization and immunoblotting results showed that Pr-CTL was first detectable in vesicles present in cytoplasm of granulocytes in host hemolymph, and was then secreted from cells into circulatory fluid. Finally, the secreted Pr-CTL bound to cellular membranes of both granulocytes and plasmatocytes. Injection of double-stranded RNA specific for target gene decreased expression of Pr-CTL, and a few other host immune-related genes. Suppression of Pr-CTL expression also down-regulated antimicrobial and phenoloxidase activities, and reducing phagocytotic and encapsulation rates in host. The inhibitory effect of parasitoid venom on host encapsulation is consistent with its effect in suppressing Pr-CTL expression. Binding assay results showed that recombinant Pr-CTL directly attached to the surface of P. puparum egges. We infer that Pr-CTL may serve as an immune signalling co-effector, first binding to parasitoid eggs, regulating expression of a set of immune-related genes and promoting host immunity.P. puparum venom inhibits promotion of host immune responses by silencing expression of host C-type

  17. Importance of c-Type cytochromes for U(VI reduction by Geobacter sulfurreducens

    Directory of Open Access Journals (Sweden)

    Leang Ching


    Full Text Available Abstract Background In order to study the mechanism of U(VI reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI with acetate serving as the electron donor was investigated. Results The ability of several c-type cytochrome deficient mutants to reduce U(VI was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60% the ability of G. sulfurreducens to reduce U(VI. Involvement in U(VI reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI reduction. A subpopulation of both wild type and U(VI reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III revealed no correlation between the impact of cytochrome deletion on U(VI reduction and reduction of Fe(III hydroxide and chelated Fe(III. Conclusion This study indicates that c-type cytochromes are involved in U(VI reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium

  18. Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K(+) channels. (United States)

    Dean, Terry; Xu, Rong; Joiner, William; Sehgal, Amita; Hoshi, Toshinori


    The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K(+) currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss-of-function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss-null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude.

  19. Identification of the receptor scavenging hemopexin-heme complexes

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Maniecki, Maciej B; Jacobsen, Christian


    Heme released from heme-binding proteins on internal hemorrhage, hemolysis, myolysis, or other cell damage is highly toxic due to oxidative and proinflammatory effects. Complex formation with hemopexin, the high-affinity heme-binding protein in plasma and cerebrospinal fluid, dampens these effects...

  20. Cardiac C-type natriuretic peptide gene expression and plasma concentrations in neonatal piglets

    DEFF Research Database (Denmark)

    Holst Hansen, Lasse; Smith, Julie; Goetze, Jens P


    BACKGROUND: C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family. Cardiac ANP and BNP expressions are firmly established, whereas CNP expression in the mammalian heart remains controversial. In the present report, we used a porcine model of the neonatal period with high...... expressions of cardiac ANP and BNP in order to elucidate the cardiac CNP expression profile. METHODS: Plasma and cardiac tissue were obtained from newborn piglets during the first 72 h of life. The chamber-specific CNP mRNA contents were quantified by real-time PCR analysis. The proCNP concentrations...... in the newborn piglets (n=44) were exceedingly low compared to proANP concentrations (5.3 pmol/L (3.2-8.6) vs. 3438 pmol/L (2790-5418), pANP (130 pmol/g (101-159)) and atrial proANP...

  1. The C-type lectin like fold in natural killer cell receptors

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; Skálová, Tereza; Kolenko, Petr; Kotýnková, Kristýna; Hašek, Jindřich; Man, Petr; Hanč, Pavel; Vaněk, Ondřej; Rozbeský, Daniel; Bezouška, Karel


    Roč. 19, č. 1 (2012), s. 14 ISSN 1211-5894. [Discussions in Structural Molecular Biology /10./. 22.03.2012-24.03.2012, Nové Hrady] R&D Projects: GA AV ČR KJB101120805; GA ČR GA303/09/0477; GA ČR GD305/09/H008; GA ČR GA305/07/1073; GA ČR GAP302/11/0855; GA ČR(CZ) GAP207/10/1040 EU Projects: European Commission(XE) 31220 - SPINE2-COMPLEXES Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Institutional support: RVO:61389013 ; RVO:61388971 Keywords : NK receptor * structure * C-type lectin like Subject RIV: EE - Microbiology, Virology; EB - Genetics ; Molecular Biology (MBU-M)

  2. The role of C-type lectin receptors in immune homeostasis. (United States)

    Yan, Huimin; Ohno, Naohito; Tsuji, Noriko M


    C-type lectin receptors (CLRs) are important pathogen pattern recognition molecules that recognize carbohydrate structures. Upon ligand binding, CLRs induce a variety of cellular responses, such as respiratory burst, production of cytokines and chemokines, and consequently shaping the adaptive immune responses. Recent frontier studies have demonstrated that CLRs play a significant role in development of anti-inflammatory immune responses and maintenance of host immune-homeostasis. In this review we describe the major CLRs involved in production of IL-10 by immune-related cells and functional maturation of regulatory T cells. In addition, we discuss the potential role of CLRs signaling in regulating intestinal immune-homeostasis and inflammation. Further understanding of the precise role for these receptors on immunoregulatory cells will provide unexpected opportunities for developing new therapies for inflammatory diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis. (United States)

    Domínguez-Andrés, Jorge; Arts, Rob J W; Ter Horst, Rob; Gresnigt, Mark S; Smeekens, Sanne P; Ratter, Jacqueline M; Lachmandas, Ekta; Boutens, Lily; van de Veerdonk, Frank L; Joosten, Leo A B; Notebaart, Richard A; Ardavín, Carlos; Netea, Mihai G


    Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.

  4. Modeling the differentiation of A- and C-type baroreceptor firing patterns

    DEFF Research Database (Denmark)

    Sturdy, Jacob; Ottesen, Johnny T.; Olufsen, Mette


    of their axons and their distinct firing patterns elicited in response to specific pressure stimuli. This study has developed a comprehensive model of the afferent baroreceptor discharge built on physiological knowledge of arterial wall mechanics, firing rate responses to controlled pressure stimuli, and ion...... channel dynamics within the baroreceptor neurons. With this model, we were able to predict firing rates observed in previously published experiments in both A- and C-type neurons. These results were obtained by adjusting model parameters determining the maximal ion-channel conductances. The observed...... basal firing, whereas a tenfold higher mechanosensitive conductance is responsible for the greater firing rate observed in A-type neurons. A better understanding of the difference between the two neuron types can potentially be used to gain more insight into the underlying pathophysiology facilitating...

  5. Myeloid C-Type Lectin Receptors in Viral Recognition and Antiviral Immunity. (United States)

    Monteiro, João T; Lepenies, Bernd


    Recognition of viral glycans by pattern recognition receptors (PRRs) in innate immunity contributes to antiviral immune responses. C-type lectin receptors (CLRs) are PRRs capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, antigen processing and presentation, and subsequent T cell activation. The ability of CLRs to elicit and shape adaptive immunity plays a critical role in the inhibition of viral spread within the host. However, certain viruses exploit CLRs for viral entry into host cells to avoid immune recognition. To block CLR interactions with viral glycoproteins, antiviral strategies may involve the use of multivalent glycan carrier systems. In this review, we describe the role of CLRs in antiviral immunity and we highlight their dual function in viral clearance and exploitation by viral pathogens.

  6. Quantitative Expression of C-Type Lectin Receptors in Humans and Mice

    Directory of Open Access Journals (Sweden)

    Hans-Joachim Anders


    Full Text Available C-type lectin receptors and their adaptor molecules are involved in the recognition of glycosylated self-antigens and pathogens. However, little is known about the species- and organ-specific expression profiles of these molecules. We therefore determined the mRNA expression levels of Dectin-1, MR1, MR2, DC-SIGN, Syk, Card-9, Bcl-10, Malt-1, Src, Dec-205, Galectin-1, Tim-3, Trem-1, and DAP-12 in 11 solid organs of human and mice. Mouse organs revealed lower mRNA levels of most molecules compared to spleen. However, Dec-205 and Galectin-1 in thymus, Src in brain, MR2, Card-9, Bcl-10, Src, and Dec-205 in small intestine, MR2, Bcl-10, Src, Galectin-1 in kidney, and Src and Galectin-1 in muscle were at least 2-fold higher expressed compared to spleen. Human lung, liver and heart expressed higher mRNA levels of most genes compared to spleen. Dectin-1, MR1, Syk and Trem-1 mRNA were strongly up-regulated upon ischemia-reperfusion injury in murine kidney. Tim3, DAP-12, Card-9, DC-SIGN and MR2 were further up-regulated during renal fibrosis. Murine kidney showed higher DAP-12, Syk, Card-9 and Dectin-1 mRNA expression during the progression of lupus nephritis. Thus, the organ-, and species-specific expression of C-type lectin receptors is different between mice and humans which must be considered in the interpretation of related studies.

  7. A C-type lectin isolated from the skin of Japanese bullhead shark (Heterodontus japonicus) binds a remarkably broad range of sugars and induces blood coagulation. (United States)

    Tsutsui, Shigeyuki; Dotsuta, Yuma; Ono, Ayaka; Suzuki, Masanari; Tateno, Hiroaki; Hirabayashi, Jun; Nakamura, Osamu


    The aim of this study was to determine the physiological role of skin lectins of the Japanese bullhead shark (Heterodontus japonicus). A skin extract was subjected to affinity chromatography using seven different sugars as ligands. Molecular mass and N-terminal amino acid sequence analyses indicated elution of the same protein by each of the seven respective cognate ligands from sugar affinity columns. The predicted amino acid sequence encoded by the cDNA of this protein [designated as H. japonicus C-type-lectin (HjCL)] identified it as a novel fish subgroup VII C-type lectin evolutionarily related to snake venom lectins. HjCL was predicted to bind to mannose because of the presence of a Glu-Pro-Asn (EPN) motif; however, haemagglutination inhibition assays and glycoconjugate microarray analysis demonstrated its binding to numerous structurally diverse sugars. Competitive sugar-binding assays using affinity chromatography indicated that HjCL bound multiple sugars via a common carbohydrate-recognition domain. The mRNA encoding HjCL was specifically detected in the skin, and immunohistochemical analysis detected its expression in uncharacterized large cells in the epidermis. HjCL agglutinated the bacterial pathogen Edwardsiella tarda and promoted immediate clotting of shark blood, indicating that HjCL is involved in host defence on the skin surface especially when the shark is injured and bleeds. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  8. Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria. (United States)

    Guo, Zhijun; Sevrioukova, Irina F; Denisov, Ilia G; Zhang, Xia; Chiu, Ting-Lan; Thomas, Dafydd G; Hanse, Eric A; Cuellar, Rebecca A D; Grinkova, Yelena V; Langenfeld, Vanessa Wankhede; Swedien, Daniel S; Stamschror, Justin D; Alvarez, Juan; Luna, Fernando; Galván, Adela; Bae, Young Kyung; Wulfkuhle, Julia D; Gallagher, Rosa I; Petricoin, Emanuel F; Norris, Beverly; Flory, Craig M; Schumacher, Robert J; O'Sullivan, M Gerard; Cao, Qing; Chu, Haitao; Lipscomb, John D; Atkins, William M; Gupta, Kalpna; Kelekar, Ameeta; Blair, Ian A; Capdevila, Jorge H; Falck, John R; Sligar, Stephen G; Poulos, Thomas L; Georg, Gunda I; Ambrose, Elizabeth; Potter, David A


    The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER(+) mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria (United States)

    Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.


    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

  10. The Macrophage Galactose-Type C-Type Lectin (MGL Modulates Regulatory T Cell Functions.

    Directory of Open Access Journals (Sweden)

    Ilaria Grazia Zizzari

    Full Text Available Regulatory T cells (Tregs are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments, their presence is related to a poor prognosis, and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study, we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs, restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70, an increase in the Foxp3 methylation status and, ultimately, the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions, suggesting its possible use in the design of anticancer vaccines.

  11. Assessment of C-Type Darrieus Wind Turbine Under Low Wind Speed Condition (United States)

    Misaran, M. S.; Rahman, Md. M.; Muzammil, W. K.; Ismail, M. A.


    Harvesting wind energy in in a low wind speed region is deem un-economical if not daunting task. Study shows that a minimum cut in speed of 3.5 m/s is required to extract a meaningful wind energy for electricity while a mean speed of 6 m/s is preferred. However, in Malaysia the mean speed is at 2 m/s with certain potential areas having 3 m/s mean speed. Thus, this work aims to develop a wind turbine that able to operate at lower cut-in speed and produce meaningful power for electricity generation. A C-type Darrieus blade is selected as it shows good potential to operate in arbitrary wind speed condition. The wind turbine is designed and fabricated in UMS labs while the performance of the wind turbine is evaluated in a simulated wind condition. Test result shows that the wind turbine started to rotate at 1 m/s compared to a NACA 0012 Darrieus turbine that started to rotate at 3 m/s. The performance of the turbine shows that it have good potential to be used in an intermittent arbitrary wind speed condition as well as low mean wind speed condition.

  12. Urinary C-Type Natriuretic Peptide: An Emerging Biomarker for Heart Failure and Renal Remodeling (United States)

    Zakeri, Rosita; Burnett, John C.; Sangaralingham, S. Jeson


    The public health and economic burden of heart failure (HF) is staggering and the need for relevant pathophysiologic and clinical biomarkers to advance the field and improve HF therapy remains high. Renal dysfunction is common among HF patients and is associated with increased HF hospitalization and mortality. It is widely recognized that mechanisms contributing to HF pathogenesis include a complex bidirectional interaction between the kidney and heart, encompassed by the term cardiorenal syndrome (CRS). Among a new wave of urinary biomarkers germane to CRS, C-type natriuretic peptide (CNP) has emerged as an innovative biomarker of renal structural and functional impairment in HF and chronic renal disease states. CNP is a hormone, synthesized in the kidney, and is an important regulator of cell proliferation and organ fibrosis. Hypoxia, cytokines and fibrotic growth factors, which are inherent to both cardiac and renal remodeling processes, are among the recognized stimuli for CNP production and release. In this review we aim to highlight current knowledge regarding the biology and pathophysiologic correlates of urinary CNP, and its potential clinical utility as a diagnostic and prognostic biomarker in HF and renal disease states. PMID:25512164

  13. Role of C-type natriuretic peptide in the function of normal human sperm

    Directory of Open Access Journals (Sweden)

    Hui Xia


    Full Text Available C-type natriuretic peptide (CNP is a newly discovered type of local regulatory factor that mediates its biological effects through the specific, membrane-bound natriuretic peptide receptor-B (NPR-B. Recent studies have established that CNP is closely related to male reproductive function. The aims of this study were to determine the distribution of CNP/NPR-B in human ejaculated spermatozoa through different methods (such as immunolocalization, real time polymerase chain reaction and Western Blot, and then to evaluate the influence of CNP on sperm function i n vitro, such as motility and acrosome reaction. Human semen samples were collected from consenting donors who met the criteria of the World Health Organization for normozoospermia. Our results show that the specific receptor NPR-B of CNP is localized in the acrosomal region of the head and the membrane of the front-end tail of the sperm, and there is no signal of CNP in human sperm. Compared with the control, CNP can induce a significant dose-dependent increase in spermatozoa motility and acrosome reaction. In summary, CNP/NPR-B can affect sperm motility and acrosome reaction, thus regulating the reproductive function of males. CNP may be a new key factor in regulating sperm function.

  14. Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis.

    Directory of Open Access Journals (Sweden)

    Jorge Domínguez-Andrés


    Full Text Available Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.

  15. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. (United States)

    Vatta, M S; Presas, M F; Bianciotti, L G; Rodriguez-Fermepin, M; Ambros, R; Fernandez, B E


    We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of 3H-NE. Experiments were carried out in rat adrenal medulla slices incubated "in vitro." Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities.

  16. Ophioluxin, a convulxin-like C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI. (United States)

    Du, Xiao-Yan; Clemetson, Jeannine M; Navdaev, Alexei; Magnenat, Edith M; Wells, Timothy N C; Clemetson, Kenneth J


    Ophioluxin, a potent platelet agonist, was purified from the venom of Ophiophagus hannah (King cobra). Under nonreducing conditions it has a mass of 85 kDa, similar to convulxin, and on reduction gives two subunits with masses of 16 and 17 kDa, slightly larger than those of convulxin. The N-terminal sequences of both subunits are very similar to those of convulxin and other C-type lectins. Ophioluxin induces a pattern of tyrosine-phosphorylated proteins in platelets like that caused by convulxin, when using appropriate concentrations based on aggregation response, because it is about 2-4 times more powerful as agonist than the latter. Ophioluxin and convulxin induce [Ca(2+)](i) elevation both in platelets and in Dami megakaryocytic cells, and each of these C-type lectins desensitizes responses to the other. Convulxin agglutinates fixed platelets at 2 microg/ml, whereas ophioluxin does not, even at 80 microg/ml. Ophioluxin resembles convulxin more than echicetin or alboaggregin B because polyclonal anti-ophioluxin antibodies recognize both ophioluxin and convulxin, but not echicetin, and platelets adhere to and spread on ophioluxin- or convulxin-precoated surfaces in the same way that is clearly different from their behavior on an alboaggregin B surface. Immobilized ophioluxin was used to isolate the glycoprotein VI-Fcgamma complex from resting platelets, which also contained Fyn, Lyn, Syk, LAT, and SLP76. Ophioluxin is the first multiheterodimeric, convulxin-like snake C-type lectin, as well as the first platelet agonist, to be described from the Elapidae snake family.

  17. The phosphoproteome of a Chlamydomonas reinhardtii eyespot fraction includes key proteins of the light signaling pathway. (United States)

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg


    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.

  18. Evolution of the C-Type Lectin-Like Receptor Genes of the DECTIN-1 Cluster in the NK Gene Complex

    Directory of Open Access Journals (Sweden)

    Susanne Sattler


    Full Text Available Pattern recognition receptors are crucial in initiating and shaping innate and adaptive immune responses and often belong to families of structurally and evolutionarily related proteins. The human C-type lectin-like receptors encoded in the DECTIN-1 cluster within the NK gene complex contain prominent receptors with pattern recognition function, such as DECTIN-1 and LOX-1. All members of this cluster share significant homology and are considered to have arisen from subsequent gene duplications. Recent developments in sequencing and the availability of comprehensive sequence data comprising many species showed that the receptors of the DECTIN-1 cluster are not only homologous to each other but also highly conserved between species. Even in Caenorhabditis elegans, genes displaying homology to the mammalian C-type lectin-like receptors have been detected. In this paper, we conduct a comprehensive phylogenetic survey and give an up-to-date overview of the currently available data on the evolutionary emergence of the DECTIN-1 cluster genes.

  19. Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif. (United States)

    Alenton, Rod Russel R; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo


    C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

  20. C-type natriuretic peptide in complicated pregnancy: increased secretion precedes adverse events. (United States)

    Reid, Rosemary A; Prickett, Timothy C R; Pullar, Barbra E; Darlow, Brian A; Gullam, Joanna E; Espiner, Eric A


    C-type natriuretic peptide (CNP), a vasoactive product of the endothelium, is markedly increased during placentation in ovine pregnancy and is further stimulated by nutrient restriction. Whether CNP products change in human pregnancy is unknown. The objective of the study was to compare serial changes in maternal plasma CNP peptides during normal pregnancy with changes in pregnancy complicated by adverse events and relate these to fetal growth and placental CNP content. This was a prospective observational study undertaken in a tertiary care center. We studied changes in maternal plasma aminoterminal proCNP (NTproCNP) and CNP at monthly intervals, fetal growth, and placental and umbilical plasma CNP peptides in 51 women, 28 of whom experienced an adverse event and 23 were uneventful. Age matched healthy nonpregnant women served as a reference range for NTproCNP. Compared with nonpregnant women, maternal plasma NTproCNP in an uneventful pregnancy was significantly reduced from first sampling (16 wk gestation) until 36 weeks. In contrast, in complicated pregnancy, levels did not decline and were significantly higher (P pregnancy from 20 weeks. Highest values occurred in women later developing hypertension and fetal growth disorders. Placental concentration of NTproCNP was unrelated to maternal NTproCNP but strongly correlated with cord plasma levels. Maternal NTproCNP is significantly raised in women who later exhibit a range of obstetric adverse events. Lack of association with placental concentrations suggests that these changes represent an adaptive response within the maternal circulation to a threatened nutrient supply to the fetus.

  1. Dexamethasone increases production of C-type natriuretic peptide in the sheep brain. (United States)

    Wilson, Michele O; McNeill, Bryony A; Barrell, Graham K; Prickett, Timothy C R; Espiner, Eric A


    Although C-type natriuretic peptide (CNP) has high abundance in brain tissues and cerebrospinal fluid (CSF), the source and possible factors regulating its secretion within the central nervous system (CNS) are unknown. Here we report the dynamic effects of a single IV bolus of dexamethasone or saline solution on plasma, CSF, CNS and pituitary tissue content of CNP products in adult sheep, along with changes in CNP gene expression in selected tissues. Both CNP and NTproCNP (the amino-terminal product of proCNP) in plasma and CSF showed dose-responsive increases lasting 12-16 h after dexamethasone, whereas other natriuretic peptides were unaffected. CNS tissue concentrations of CNP and NTproCNP were increased by dexamethasone in all of the 12 regions examined. Abundance was highest in limbic tissues, pons and medulla oblongata. Relative to controls, CNP gene expression (NPPC) was upregulated by dexamethasone in 5 of 7 brain tissues examined. Patterns of responses differed in pituitary tissue. Whereas the abundance of CNP in both lobes of the pituitary gland greatly exceeded that of brain tissues, neither CNP nor NTproCNP concentration was affected by dexamethasone, despite an increase in NPPC expression. This is the first report of enhanced production and secretion of CNP in brain tissues in response to a corticosteroid. Activation of CNP secretion within CNS tissues by dexamethasone, not exhibited by other natriuretic peptides, suggests an important role for CNP in settings of acute stress. Differential findings in pituitary tissues likely relate to altered processing of proCNP storage and secretion. © 2017 Society for Endocrinology.

  2. Thermal Infrared Imaging Experiments of C-Type Asteroid 162173 Ryugu on Hayabusa2 (United States)

    Okada, Tatsuaki; Fukuhara, Tetsuya; Tanaka, Satoshi; Taguchi, Makoto; Imamura, Takeshi; Arai, Takehiko; Senshu, Hiroki; Ogawa, Yoshiko; Demura, Hirohide; Kitazato, Kohei; Nakamura, Ryosuke; Kouyama, Toru; Sekiguchi, Tomohiko; Hasegawa, Sunao; Matsunaga, Tsuneo; Wada, Takehiko; Takita, Jun; Sakatani, Naoya; Horikawa, Yamato; Endo, Ken; Helbert, Jörn; Müller, Thomas G.; Hagermann, Axel


    The thermal infrared imager TIR onboard Hayabusa2 has been developed to investigate thermo-physical properties of C-type, near-Earth asteroid 162173 Ryugu. TIR is one of the remote science instruments on Hayabusa2 designed to understand the nature of a volatile-rich solar system small body, but it also has significant mission objectives to provide information on surface physical properties and conditions for sampling site selection as well as the assessment of safe landing operations. TIR is based on a two-dimensional uncooled micro-bolometer array inherited from the Longwave Infrared Camera LIR on Akatsuki (Fukuhara et al., 2011). TIR takes images of thermal infrared emission in 8 to 12 μm with a field of view of 16 × 12° and a spatial resolution of 0.05° per pixel. TIR covers the temperature range from 150 to 460 K, including the well calibrated range from 230 to 420 K. Temperature accuracy is within 2 K or better for summed images, and the relative accuracy or noise equivalent temperature difference (NETD) at each of pixels is 0.4 K or lower for the well-calibrated temperature range. TIR takes a couple of images with shutter open and closed, the corresponding dark frame, and provides a true thermal image by dark frame subtraction. Data processing involves summation of multiple images, image processing including the StarPixel compression (Hihara et al., 2014), and transfer to the data recorder in the spacecraft digital electronics (DE). We report the scientific and mission objectives of TIR, the requirements and constraints for the instrument specifications, the designed instrumentation and the pre-flight and in-flight performances of TIR, as well as its observation plan during the Hayabusa2 mission.

  3. B and C types natriuretic peptides modulate norepinephrine uptake and release in the rat hypothalamus. (United States)

    Vatta, M S; Presas, M; Bianciotti, L G; Zarrabeitia, V; Fernández, B E


    We previously reported that atrial natriuretic factor (ANF) regulates catecholamine metabolism in the central nervous system. ANF, B and C types natriuretic peptides (BNP and CNP) also play a regulatory role in body fluid homeostasis, cardiovascular activity and hormonal and neuro-hormonal secretions. The aim of the present work was to investigate BNP and CNP effects on the uptake and release of norepinephrine (NE) in rat hypothalamic slices incubated in vitro. Results showed that BNP (100 nM) and CNP (1, 10 and 100 nM) enhanced total and neuronal [3H]NE uptake but did not modify non-neuronal uptake. BNP (100 nM) and CNP (1 nM) caused a rapid increase in NE uptake (1 min), which was sustained for 60 min. BNP (100 nM) did not modify the intracellular distribution of NE; however, 1 nM CNP increased the granular store and decreased the cytosolic pool of NE. BNP (100 nM) and CNP (1, 10 and 100 nM), diminished spontaneous NE release. In addition, BNP (1, 10, 100 nM) and CNP (1, 10 and 100 pM, as well as 1, 10 and 100 nM) reduced NE output induced by 25 mM KCl. These results suggest that BNP and CNP may be involved in the regulation of several central as well as peripheral physiological functions through the modulation of noradrenergic neurotransmission at the presynaptic neuronal level. Present results provide evidence to consider CNP as the brain natriuretic peptide since physiological concentrations of this peptide (pM) diminished NE evoked release.

  4. MCL and mincle: C-type lectin receptors that sense damaged self and pathogen associated molecular patterns

    Directory of Open Access Journals (Sweden)

    Mark B Richardson


    Full Text Available MCL (macrophage C-type lectin and mincle (macrophage inducible C-type lectin comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage associated and pathogen associated molecular patterns. In this review we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure-activity relationships of activating ligands, particularly trehalose dimycolate (TDM and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies.

  5. SDS-facilitated in vitro formation of a transmembrane B-type cytochrome is mediated by changes in local pH

    DEFF Research Database (Denmark)

    Weber, M.; Schneider, D.; Prodöhl, A.


    cytochrome b(559)', which can be efficiently assembled in vitro from a heme-binding PsbF homo-dimer by combining free heme with the apo-cytochrome b(559)'. Unfolding of the protein dissolved in the mild detergent dodecyl maltoside may be induced by addition of SDS, which at high concentrations leads to dimer...... dissociation. Surprisingly, absorption spectroscopy reveals that heme binding and cytochrome formation at pH 8.0 are optimal at intermediate SDS concentrations. Stopped-flow kinetics revealed that genuine conformational changes are involved in heme binding at these SDS concentrations. GPS (Global Protein...... folding State mapping) NMR measurements showed that optimal heme binding is intimately related to a change in the degree of histidine protonation. In the absence of SDS, the pH curve for heme binding is bell-shaped with an optimum at around pH 6-7. At alkaline pH values, the negative electrostatic...

  6. Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli


    Kim,Jong-Wan; Yoe,Jeehyun; Lee,Gil Ho; Yoe,Sung Moon


    The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by ...

  7. The cartilage-specific lectin C-type lectin domain family 3 member A (CLEC3A) enhances tissue plasminogen activator-mediated plasminogen activation. (United States)

    Lau, Daniela; Elezagic, Dzemal; Hermes, Gabriele; Mörgelin, Matthias; Wohl, Alexander P; Koch, Manuel; Hartmann, Ursula; Höllriegl, Stefan; Wagener, Raimund; Paulsson, Mats; Streichert, Thomas; Klatt, Andreas R


    C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A C-type lectin (MrLec) with high expression in intestine is involved in innate immune response of Macrobrachium rosenbergii. (United States)

    Feng, Jinling; Huang, Xin; Jin, Min; Zhang, Yi; Li, Tingting; Hui, Kaimin; Ren, Qian


    C-type lectins (CTLs) are pattern-recognition proteins that play an important role in innate immunity of vertebrates and invertebrates. In this study, a lectin cDNA named MrLec was cloned and characterized from giant freshwater prawns (Macrobrachiun rosenbergii). The full-length cDNA of MrLec was 1431 bp, which contained an open reading frame of 1041 bp that encoded a protein with 346 amino acids. MrLec was found to contain a typical signal peptide of 18 amino acids and a single carbohydrate-recognition domain with 121 amino acids. The phylogenetic analysis showed that MrLec was grouped with vertebrates and had 57% identity with C-type lectin 3 from Marsupenaeus japonicas. Tissue expression analysis showed that MrLec was ubiquitously distributed at a high level in the intestine, with lower expression levels in the hemocytes, heart, hepatopancreas, gill and stomach. Vibrio parahaemolyticus infection induced the upregulation of MrLec in the gills and intestine. For the white spot syndrome virus (WSSV) challenge, MrLec in gills was upregulated at 24, 36 and 48 h. In intestine, MrLec also went up at 36 and 48 h WSSV challenge. Recombinant MrLec can agglutinate (Ca2+-dependent) and bind both Gram-negative and Gram-positive bacteria. rMrLec could attach to lipopolysaccharide and peptidoglycan in a dose-dependent manner. These results indicated possible MrLec involvement in the immune response of giant freshwater prawns. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. C-type lectin Mermaid inhibits dendritic cell mediated HIV-1 transmission to CD4+ T cells

    NARCIS (Netherlands)

    Nabatov, Alexey A.; de Jong, Marein A. W. P.; de Witte, Lot; Bulgheresi, Silvia; Geijtenbeek, Teunis B. H.


    Dendritic cells (DCs) are important in HIV-1 transmission; DCs capture invading HIV-1 through the interaction of the gp120 oligosaccharides with the C-type lectin DC-SIGN and migrate to the lymphoid tissues where HIV-1 is transmitted to T cells. Thus, the HIV-1 envelope glycoprotein gp120 is an

  10. DC-SIGN, a C-type lectin on dendritic cells that unveils many aspects of dendritic cell biology

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.; Engering, Anneke; van Kooyk, Yvette


    Dendritic cells (DC) are present in essentially every tissue where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T cells. It is becoming clear that not all C-type lectins on DC serve as antigen receptors recognizing

  11. C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous glycoconjugates.

    NARCIS (Netherlands)

    Gijzen, K.; Cambi, A.; Torensma, R.; Figdor, C.G.


    Human C-type lectin receptors (CLRs) characteristically bind glycosylated ligands in a Ca(2+)-dependent way via their carbohydrate recognition domain (CRD). Their carbohydrate preference is dependent on the amino acid sequence in the CRD domain and on the ability and flexibility of the CRD domain to

  12. The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot Fraction Includes Key Proteins of the Light Signaling Pathway1[W (United States)

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg


    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii. PMID:18065559

  13. Genomic organization and reproductive regulation of a large lipid transfer protein in the varroa mite, Varroa destructor (Anderson & Trueman). (United States)

    Cabrera, A R; Shirk, P D; Duehl, A J; Donohue, K V; Grozinger, C M; Evans, J D; Teal, P E A


    The complete genomic region and corresponding transcript of the most abundant protein in phoretic varroa mites, Varroa destructor (Anderson & Trueman), were sequenced and have homology with acarine hemelipoglycoproteins and the large lipid transfer protein (LLTP) super family. The genomic sequence of VdLLTP included 14 introns and the mature transcript coded for a predicted polypeptide of 1575 amino acid residues. VdLLTP shared a minimum of 25% sequence identity with acarine LLTPs. Phylogenetic assessment showed VdLLTP was most closely related to Metaseiulus occidentalis vitellogenin and LLTP proteins of ticks; however, no heme binding by VdLLTP was detected. Analysis of lipids associated with VdLLTP showed that it was a carrier for free and esterified C12 -C22 fatty acids from triglycerides, diacylglycerides and monoacylglycerides. Additionally, cholesterol and β-sitosterol were found as cholesterol esters linked to common fatty acids. Transcript levels of VdLLTP were 42 and 310 times higher in phoretic female mites when compared with males and quiescent deutonymphs, respectively. Coincident with initiation of the reproductive phase, VdLLTP transcript levels declined to a third of those in phoretic female mites. VdLLTP functions as an important lipid transporter and should provide a significant RNA interference target for assessing the control of varroa mites. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  14. C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity. (United States)

    Wiersma, Valerie R; de Bruyn, Marco; Shi, Ce; Gooden, Marloes J M; Wouters, Maartje C A; Samplonius, Douwe F; Hendriks, Djoke; Nijman, Hans W; Wei, Yunwei; Zhou, Jin; Helfrich, Wijnand; Bremer, Edwin


    The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.

  15. The molecular puzzle of C-type lectin like natural killer cell receptors

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; Kolenko, Petr; Skálová, Tereza; Hašek, Jindřich; Vaněk, O.; Rozbeský, D.; Kotýnková, Kristýna; Novák, Petr; Bezouška, K.


    Roč. 18, č. 1 (2011), s. 15 ISSN 1211-5894. [Discussions in Structural Molecular Biology /9./. 24.03.2011-26.03.2011, Nové Hrady] R&D Projects: GA ČR GA303/09/0477; GA ČR GAP207/10/1040; GA ČR GAP302/11/0855 EU Projects: European Commission(XE) 31220 - SPINE2-COMPLEXES Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Keywords : protein structure * X-ray diffraction * natural killer cell Subject RIV: CE - Biochemistry

  16. Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease. (United States)

    Balderas, Miriam A; Nguyen, Chinh T Q; Terwilliger, Austen; Keitel, Wendy A; Iniguez, Angelina; Torres, Rodrigo; Palacios, Frederico; Goulding, Celia W; Maresso, Anthony W


    Bacillus anthracis is a sporulating Gram-positive bacterium that is the causative agent of anthrax and a potential weapon of bioterrorism. The U.S.-licensed anthrax vaccine is made from an incompletely characterized culture supernatant of a nonencapsulated, toxigenic strain (anthrax vaccine absorbed [AVA]) whose primary protective component is thought to be protective antigen (PA). AVA is effective in protecting animals and elicits toxin-neutralizing antibodies in humans, but enthusiasm is dampened by its undefined composition, multishot regimen, recommended boosters, and potential for adverse reactions. Improving next-generation anthrax vaccines is important to safeguard citizens and the military. Here, we report that vaccination with recombinant forms of a conserved domain (near-iron transporter [NEAT]), common in Gram-positive pathogens, elicits protection in a murine model of B. anthracis infection. Protection was observed with both Freund's and alum adjuvants, given subcutaneously and intramuscularly, respectively, with a mixed composite of NEATs. Protection correlated with an antibody response against the NEAT domains and a decrease in the numbers of bacteria in major organs. Anti-NEAT antibodies promote opsonophagocytosis of bacilli by alveolar macrophages. To guide the development of inactive and safe NEAT antigens, we also report the crystal structure of one of the NEAT domains (Hal) and identify critical residues mediating its heme-binding and acquisition activity. These results indicate that we should consider NEAT proteins in the development of an improved antianthrax vaccine. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

    Directory of Open Access Journals (Sweden)

    Chih-Ya Yang

    Full Text Available CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal, N-acetylgalactosamine (GalNAc, and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/- mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5 but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

  18. Molecular characterization, expression and antimicrobial activities of two c-type lysozymes from manila clam Venerupis philippinarum. (United States)

    Yang, Dinglong; Wang, Qing; Cao, Ruiwen; Chen, Lizhu; Liu, Yongliang; Cong, Ming; Wu, Huifeng; Li, Fei; Ji, Chenglong; Zhao, Jianmin


    Lysozymes play an important role in the innate immune responses with which mollusks respond to bacterial invasion through its lytic activity. In the present study, two c-type lysozymes (designed as VpCLYZ-1 and VpCLYZ-2, respectively) were identified and characterized from the manila clam Venerupis philippinarum. The full-length cDNA of VpCLYZ-1 and VpCLYZ-2 was of 629 and 736 bp, encoding a polypeptide of 156 and153 amino acid residues, respectively. The deduced amino acid sequences of VpCLYZs showed high similarity to other known invertebrate c-type lysozymes. Multiple alignments and phylogenetic relationship strongly suggested that VpCLYZ-1 and VpCLYZ-2 belonged to the c-type lysozyme family. Both VpCLYZ-1 and VpCLYZ-2 transcripts were constitutively expressed in a wide range of tissues with different levels. The VpCLYZ-1 transcript was dominantly expressed in hepatopancreas and hemocytes, while VpCLYZ-2 transcript was mainly expressed in the tissues of hepatopancreas and gills. Both the mRNA expression of VpCLYZ-1 and VpCLYZ-2 was significantly up-regulated at 12 h post Vibrio anguillarum challenge. The recombinant VpCLYZ-1 and VpCLYZ-2 (designed as rVpCLYZ-1 and rVpCLYZ-2) exhibited lytic activity against all tested bacteria, and rVpCLYZ-1 showed higher activities than rVpCLYZ-2 in killing Micrococcus luteus and V. anguillarum. Overall, our results suggested that VpCLYZ-1 and VpCLYZ-2 belonged to the c-type lysozyme family, and played important roles in the immune responses of manila clam, especially in the elimination of pathogens. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. In Situ Spectral Kinetics of Cr(VI) Reduction by c-Type Cytochromes in A Suspension of Living Shewanella putrefaciens 200 (United States)

    Liu, Tongxu; Li, Xiaomin; Li, Fangbai; Han, Rui; Wu, Yundang; Yuan, Xiu; Wang, Ying


    Although c-type cytochromes (c-Cyts) mediating metal reduction have been mainly investigated with in vitro purified proteins of dissimilatory metal reducing bacteria, the in vivo behavior of c-Cyts is still unclear given the difficulty in measuring the proteins of intact cells. Here, c-Cyts in living Shewanella putrefaciens 200 (SP200) was successfully quantified using diffuse-transmission UV/Vis spectroscopy due to the strong absorbance of hemes, and the in situ spectral kinetics of Cr(VI) reduction by c-Cyts were examined over time. The reduced product Cr(III) observed on the cell surface may play a role in inhibiting the Cr(VI) reduction and reducing the cell numbers with high concentrations (>200 μM) of Cr(VI) evidenced by the 16S rRNA analysis. A brief kinetic model was established with two predominant reactions, redox transformation of c-Cyts and Cr(VI) reduction by reduced c-Cyts, but the fitting curves were not well-matched with c-Cyts data. The Cr(III)-induced inhibitory effect to the cellular function of redox transformation of c-Cyts was then added to the model, resulting in substantially improved the model fitting. This study provides a case of directly examining the reaction properties of outer-membrane enzyme during microbial metal reduction processes under physiological conditions.

  20. Interaction analysis of grapevine MIKC(c)-type MADS transcription factors and heterologous expression of putative véraison regulators in tomato. (United States)

    Mellway, Robin D; Lund, Steven T


    MIKC(c)-type MADS-domain transcription factors include important regulators of floral development that interact in protein complexes to control the development of floral organs, as described by the ABC model. Members of the SEPALLATA (SEP) and AGAMOUS (AG) MADS clades include proteins involved in stamen and carpel specification and certain members of these families, such as tomato (Solanum lycopersicon) SlRIN and SlTAGL1, have been shown to regulate fruit development and ripening initiation. A number of expression studies have shown that several floral homeotic MADS genes are expressed during grapevine (Vitis vinifera) berry development, including potential homologues of these characterized ripening regulators. To gain insight into the regulation of berry development and ripening in grapevine, we studied the interactions and functions of grapevine floral homeotic MADS genes. Using the yeast 2- and 3-hybrid systems, we determined that the complexes formed during fruit development and ripening may involve several classes of floral homeotic MADS proteins. We found that a heterologously expressed grapevine SEP gene, VviSEP4, is capable of partially complementing the non-ripening phenotype of the tomato rin mutant, indicating that a role for this gene in ripening regulation may be conserved in fleshy fruit ripening. We also found that ectopic expression of a grapevine AG clade gene, VviAG1, in tomato results in the development of fleshy sepals with the chemical characteristics of tomato fruit pericarp. Additionally, we performed 2-hybrid screens on a library prepared from Pinot noir véraison-stage berry and identified proteins that may interact with the MADS factors that are expressed during berry development and that may represent regulatory functions in grape berry development. Copyright © 2013 Elsevier GmbH. All rights reserved.

  1. Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus. (United States)

    Stappers, Mark H T; Clark, Alexandra E; Aimanianda, Vishukumar; Bidula, Stefan; Reid, Delyth M; Asamaphan, Patawee; Hardison, Sarah E; Dambuza, Ivy M; Valsecchi, Isabel; Kerscher, Bernhard; Plato, Anthony; Wallace, Carol A; Yuecel, Raif; Hebecker, Betty; da Glória Teixeira Sousa, Maria; Cunha, Cristina; Liu, Yan; Feizi, Ten; Brakhage, Axel A; Kwon-Chung, Kyung J; Gow, Neil A R; Zanda, Matteo; Piras, Monica; Zanato, Chiara; Jaeger, Martin; Netea, Mihai G; van de Veerdonk, Frank L; Lacerda, João F; Campos, António; Carvalho, Agostinho; Willment, Janet A; Latgé, Jean-Paul; Brown, Gordon D


    Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31 + endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

  2. Identification of AlcR, an AraC-Type Regulator of Alcaligin Siderophore Synthesis in Bordetella bronchiseptica and Bordetella pertussis (United States)

    Pradel, Elizabeth; Guiso, Nicole; Locht, Camille


    A Fur titration assay was used to isolate DNA fragments bearing putative Fur binding sites (FBS) from a partial Bordetella bronchiseptica genomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert was further studied. Successive deletions in the cloned fragment enabled us to map a putative FBS at about 2 kb from one end. Sequence analysis revealed the presence of an FBS upstream from a new gene encoding an AraC-type transcriptional regulator. The deduced protein displays similarity to PchR, an activator of pyochelin siderophore and ferripyochelin receptor synthesis in Pseudomonas aeruginosa. Homologous genes in Bordetella pertussis and Bordetella parapertussis were PCR amplified, and sequence comparisons indicated a very high conservation in the three species. The B. pertussis and B. bronchiseptica chromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions, the mutants did not secrete the alcaligin siderophore and lacked AlcC, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On the basis of its role in regulation of alcaligin biosynthesis, the new gene was designated alcR. Additional sequence determination showed that alcR is located about 2 kb downstream from the alcABC operon and is transcribed in the same orientation. Two tightly linked open reading frames, alcD and alcE, were identified between alcC and alcR. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and colonization in the mouse respiratory infection model are AlcR independent. PMID:9473041

  3. Fast response in-line gas sensor using C-type fiber and Ge-doped ring defect photonic crystal fiber. (United States)

    Kassani, Sahar Hosseinzadeh; Park, Jiyoung; Jung, Yongmin; Kobelke, Jens; Oh, Kyunghwan


    An in-line chemical gas sensor was proposed and experimentally demonstrated using a new C-type fiber and a Ge-doped ring defect photonic crystal fiber (PCF). The C-type fiber segment served as a compact gas inlet/outlet directly spliced to PCF, which overcame previous limitations in packaging and dynamic responses. C-type fiber was prepared by optimizing drawing process for a silica tube with an open slot. Splicing conditions for SMF/C-type fiber and PCF/C-type fiber were experimentally established to provide an all-fiber sensor unit. To enhance the sensitivity and light coupling efficiency we used a special PCF with Ge-doped ring defect to further enhance the sensitivity and gas flow rate. Sensing capability of the proposed sensor was investigated experimentally by detecting acetylene absorption lines.

  4. Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator. (United States)

    Kitatsuji, Chihiro; Izumi, Kozue; Nambu, Shusuke; Kurogochi, Masaki; Uchida, Takeshi; Nishimura, Shin-ichiro; Iwai, Kazuhiro; O'Brian, Mark R; Ikeda-Saito, Masao; Ishimori, Koichiro


    The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.

  5. Scavenger Receptor C-Type Lectin Binds to the Leukocyte Cell Surface Glycan Lewis By a Novel Mechanism

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    Feinberg, H.; Taylor, M.E.; Weis, W.I.; /Stanford U., Med. School /Imperial Coll., London


    The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

  6. Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity. (United States)

    Huang, Xin; Huang, Ying; Shi, Yan-Ru; Ren, Qian; Wang, Wen


    C-type lectins play crucial roles in innate immunity. In the present study, a novel C-type lectin gene, designated as MrCTL, was identified from Macrobrachium rosenbergii. MrCTL contains 2 carbohydrate-recognition domains (CRDs), namely MrCRD1 and MrCRD2. The MrCRD1 contains a QEP motif and MrCRD2 contains a motif of EPD. MrCTL was mainly expressed in the hepatopancreas. The expression level of MrCTL in hepatopancreas was significantly upregulated after a challenge with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). The recombinant MrCTL, MrCRD1 and MrCRD2 have an ability to agglutinate both Gram-negative (V. parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) in a calcium dependent manner. The recombinant MrCTL, MrCRD1 and MrCRD2 bind directly to all tested microorganisms. All these results suggested that MrCTL may have important roles in immune defense against invading pathogens in prawns. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1β in cartilage explants. (United States)

    Peake, Nick J; Pavlov, Anton M; D'Souza, Alveena; Pingguan-Murphy, Belinda; Sukhorukov, Gleb B; Hobbs, Adrian J; Chowdhury, Tina T


    C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1β (IL-1β). SEM showed uniform, 2 to 3 μm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1β and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1β in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

  8. Influence of the ferric uptake regulator (Fur protein on pathogenicity in Pectobacterium carotovorum subsp. brasiliense.

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    Collins Kipngetich Tanui

    Full Text Available Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692, an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs, biofilm formation, exopolysaccharide (EPS production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132, stress response (SodC-superoxide dismutase, plant cell wall degrading enzymes (PrtA and CelV and motility (FlhC and MotA. We conclude that the ferric uptake regulator protein (Fur of Pcb1692 regulates traits that are important to host-pathogens interactions.

  9. Spectroscopic Studies of Abiotic and Biological Nanomaterials: Silver Nanoparticles, Rhodamine 6G Adsorbed on Graphene, and c-Type Cytochromes and Type IV Pili in Geobacter sulfurreducens (United States)

    Thrall, Elizabeth S.

    mechanism, the conductive pili mediate electron transport to extracellular acceptors. The second proposed mechanism is that charge transport proceeds by electron hopping between the heme groups in the many c-type cytochromes produced by G. sulfurreducens. In this picture, the observed conductivity of the pili is due to hopping through associated cytochrome proteins. Our aim is to explore these alternative mechanisms for electron transport in G. sulfurreducens through electrical and optical studies. We report the work we have done thus far to culture and characterize G. sulfurreducens , and we show that preliminary micro-Raman studies of G. sulfurreducens cells confirm that we can detect the spectroscopic signature of c-type cytochrome proteins. Future directions for this ongoing work are briefly discussed.

  10. Shr of Group A Streptococcus is a new type of composite NEAT protein involved in sequestering heme from methemoglobin (United States)

    Ouattara, Mahamoudou; Cunha, Elizabeth Bentley; Li, Xueru; Huang, Ya-Shu; Dixon, Dabney; Eichenbaum, Zehava


    SUMMARY A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in heme acquisition and trafficking across the cell envelope of Gram-positive bacteria. Group A Streptococcus (GAS), a β-hemolytic human pathogen, expresses a NEAT protein, Shr, which binds several hemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane-anchored protein, with a unique N-terminal domain (NTD) and two NEAT domains separated by a central leucine-rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains heme in solution and furthermore reduces the heme iron; this is the first report of heme reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding heme, although they are missing a critical tyrosine residue found in the ligand-binding pocket of other heme-binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester heme directly from methemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in heme uptake found in pyogenic streptococci and Clostridium novyi. PMID:20807204

  11. Differential contribution of sodium channel subtypes to action potential generation in unmyelinated human C-type nerve fibers. (United States)

    Lang, Philip M; Hilmer, Verena B; Grafe, Peter


    Multiple voltage-dependent sodium channels (Na(v)) contribute to action potentials and excitability of primary nociceptive neurons. The aim of the current study was to characterize subtypes of Na(v) that contribute to action potential generation in peripheral unmyelinated human C-type nerve fibers. Registration of C-fiber compound action potentials and determination of membrane threshold was performed by a computerized threshold tracking program. Nerve fibers were stimulated with a 1-ms current pulse either alone or after a small ramp current lasting 300 ms. Compound C-fiber action potentials elicited by supramaximal 1-ms current pulses were rather resistant to application of tetrodotoxin (30-90 nM). However, the same concentrations of tetrodotoxin strongly reduced the peak height and elevated membrane threshold of action potentials evoked at the end of a 300-ms current ramp. A similar effect was observed during application of lidocaine and mexiletine (50 microM each). These data indicate that more than one type of Na(v) contributes to the generation of action potentials in unmyelinated human C-type nerve fibers. The peak height of an action potential produced by a short electrical impulse is dependent on the activation of tetrodotoxin-resistant ion channels. In contrast, membrane threshold and action potential peak height at the end of a slow membrane depolarization are regulated by a subtype of Na(v) with high sensitivity to low concentrations of tetrodotoxin, lidocaine, and mexiletine. The electrophysiologic and pharmacologic characteristics may indicate the functional activity of the Na(v) 1.7 subtype of voltage-dependent sodium channels.

  12. High-molecular-mass multi-c-heme cytochromes from Methylococcus capsulatus bath. (United States)

    Bergmann, D J; Zahn, J A; DiSpirito, A A


    The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6. 0. The heme c concentration was estimated to be 8.2 +/- 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118, 620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94, 000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.

  13. High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath† (United States)

    Bergmann, David J.; Zahn, James A.; DiSpirito, Alan A.


    The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome. PMID:9922265

  14. Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites

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    Michelle S Itano


    Full Text Available Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (~80 nm on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to four hours. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal nanodomain structure, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of < 30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.

  15. Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites (United States)

    Itano, Michelle; Graus, Matthew; Pehlke, Carolyn; Wester, Michael; Liu, Ping; Lidke, Keith; Thompson, Nancy; Jacobson, Ken; Neumann, Aaron


    Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs) that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (~80 nm) on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to four hours. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal nanodomain structure, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of < 30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.

  16. Characterization of the Decaheme c-Type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-Ray Scattering and Neutron Reflectometry (United States)

    Johs, A.; Shi, L.; Droubay, T.; Ankner, J.F.; Liang, L.


    Abstract The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (α-Fe2O3). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 × 90 × 65 Å3. To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by ∼7 Å (the maximum dimension was 96 Å for oxidized OmcA, and 89 Å for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA's role in mediating redox processes. PMID:20550916

  17. An autopsied case of MV2K + C-type sporadic Creutzfeldt-Jakob disease presenting with widespread cerebral cortical involvement and Kuru plaques. (United States)

    Iwasaki, Yasushi; Saito, Yufuko; Aiba, Ikuko; Kobayashi, Atsushi; Mimuro, Maya; Kitamoto, Tetsuyuki; Yoshida, Mari


    MV2-type sporadic Creutzfeldt-Jakob disease (sCJD), which was previously called "Kuru-plaque variant", was gradually revealed to have a wide spectrum and has been classified into three pathological subtypes: MV2K, MV2C and MV2K + C. We herein describe the detailed clinical findings and neuropathologic observations from an autopsied MV2K + C-type Japanese sCJD case with widespread cerebral cortical pathology and Kuru plaques. In the early stages of the disease, the patient exhibited gait disturbance with ataxia and dysarthria as well as gradual appearance of cognitive dysfunction. Diffusion-weighted images (DWI) on MRI revealed extensive cerebral cortical hyperintensity. Pathologic investigation revealed extensive spongiform change in the cerebral cortex, particularly in the deeper layers. Vacuole size varied, and some were confluent. Prion protein (PrP) immunostaining revealed extensive PrP deposition in the cerebral cortex, basal ganglia, thalamus, cerebellum, brainstem and spinal cord. In the cerebral cortex, synaptic-type, Kuru plaque-like, and coarse plaque-type PrP depositions were mainly observed, along with some perivacuolar-type PrP depositions. Kuru plaques and coarse plaque-type PrP depositions also were observed in the cerebellar cortex. PrP gene analysis revealed no mutations, and polymorphic codon 129 exhibited Met/Val heterozygosity. Western blot analysis revealed a mixture of intermediate-type PrPSc and type 2 PrPSc . Based on previous reports regarding MV2-type sCJD and the clinicopathologic findings of the present case, we speculated that it may be possible to clinically distinguish each MV2 subtype. Clinical presentation of the MV2K + C subtype includes predominant cerebral cortical involvement signs with ataxia and DWI hyperintensity of the cerebral cortex on MRI. © 2016 Japanese Society of Neuropathology.

  18. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation. (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd


    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Opposed circulating plasma levels of endothelin-1 and C-type natriuretic peptide in children with Plasmodium falciparum malaria

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    Issifou Saadou


    Full Text Available Abstract Background Molecular mechanisms involved in the pathogenesis of severe Plasmodium falciparum malaria (SM, are not yet fully understood. Both endothelin-1 (ET-1 and C-type natriuretic peptide (CNP are produced by vascular endothelium and act locally as paracrine regulators of vascular tone, ET-1 being a potent vasoconstrictor and CNP having strong vasorelaxant properties. Methods Plasma levels of ET-1 and N-terminal fragments of CNP (NT-proCNP were studied on admission and after 24 hours of treatment, using enzyme-linked-immunosorbent-assay (ELISA technique, in Gabonese children with severe falciparum malaria (SM, n = 50, with uncomplicated malaria (UM, n = 39 and healthy controls (HC, n = 25. Results Compared to HC, malaria patients had significantly higher plasma levels of ET-1 and significantly lower levels of NT-proCNP (p p p = 0.034, whereas UM was not significantly different to HC. In the SM group we found a trend towards lower ET-1 levels compared to UM (p = 0.085. Conclusion In the present study, an imbalance between the vasoconstricitve and vasorelaxant endothelium-derived substances ET-1 and CNP in the plasma of children with falciparum malaria is demonstrated, presumably in favor of vasoconstrictive and pro-inflammatory effects. These results may indicate involvement of ET-1 and CNP in malaria pathogenesis. Furthermore, results of lower ET-1 and CNP levels in SM may reflect endothelial cell damage.

  20. Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development.

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    Shigeto Yoshida


    Full Text Available The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50 of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.

  1. Plasma C-type natriuretic peptide as a predictor for therapeutic response to metoprolol in children with postural tachycardia syndrome.

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    Jing Lin

    Full Text Available POTS is a global public-health disease, but predictor for therapeutic response to metoprolol in children with POTS is lacking. This study was designed to investigate predictive value of plasma C-type natriuretic peptide (CNP in the therapeutic efficacy of metoprolol on postural tachycardia syndrome (POTS in children. Totally 34 children with POTS and 27 healthy children were included in the study. The head-up test or head-up tilt test was used to check heart rate and blood pressure from supine to upright in subjects. A double antibody (competitive sandwich immunoluminometric assay was used to detect plasma CNP. Metoprolol was used to treat children with POTS. The difference in plasma concentrations of CNP between responders and non-responders was compared. An ROC curve was used to analyze plasma CNP to predict efficacy of metoprolol on POTS in children. Plasma CNP in children with POTS was significantly higher than that of healthy children [(51.9 ± 31.4 vs. (25.1 ± 19.1 pg/ml, P 32.55 pg/ml yielded a sensitivity of 95.8% and specificity of 70% in predicting therapeutic efficacy of metoprolol on POTS children. Plasma CNP might serve as a useful predictor for the therapeutic efficacy of metoprolol on POTS in children.

  2. C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia. (United States)

    Behler-Janbeck, Friederike; Takano, Tomotsugu; Maus, Regina; Stolper, Jennifer; Jonigk, Danny; Tort Tarrés, Meritxell; Fuehner, Thomas; Prasse, Antje; Welte, Tobias; Timmer, Mattie S M; Stocker, Bridget L; Nakanishi, Yoichi; Miyamoto, Tomofumi; Yamasaki, Sho; Maus, Ulrich A


    Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

  3. C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia.

    Directory of Open Access Journals (Sweden)

    Friederike Behler-Janbeck


    Full Text Available Among various innate immune receptor families, the role of C-type lectin receptors (CLRs in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG, which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

  4. Thermal Inertia Determination of C-type Asteroid Ryugu from in-situ Surface Brightness Temperature Measurements (United States)

    Hamm, Maximilian; Grott, Matthias; Knollenberg, Jörg; Kührt, Ekkehard; Pelivan, Ivanka


    The Japanese Hayabusa-2 mission is a sample-return mission currently on its way to the C-type asteroid Ryugu. Hayabusa-2 carries the small lander MASCOT (Mobile Asteroid Surface Scout), whose scientific payload includes the infrared radiometer MARA. The primary science goal of MARA is to determine Ryugu's surface brightness temperatures at the landing site for a full asteroid rotation, which will be measured using a long-pass filter, an 8 to 12 µm bandpass, as well as four narrow bandpasses centered at wavelengths between 5 and 15 µm. From these measurements, surface thermal inertia will be derived, but because MARA performs single pixel measurements, heterogeneity in the field of view cannot be resolved. Yet, the surface will likely exhibit different surface textures, and thermal inertia in the field of view could vary from 600 (small rocks) to 50 Jm-2s-0.5K-1 (fine regolith grains). Sub-pixel heterogeneity is a common problem when interpreting radiometer data, since the associated ambiguities cannot be resolved without additional information on surface texture. For MARA, this information will be provided by the MASCOT camera, and in the present paper we have investigated to what extent different thermal inertias can be retrieved from MARA data. To test the applied approach, we generated synthetic MARA data using a thermal model of Ryugu, assuming different thermal inertias for sections of the field of view. We find that sub-pixel heterogeneity systematically deforms the diurnal temperature curve so that it is not possible to fit the data using a single thermal inertia value. However, including the area fractions of the different surface sections enables us to reconstruct the different thermal inertias to within 10% assuming appropriate measurement noise. The presented approach will increase robustness of the Ryugu thermal inertia determination and results will serve as a ground truth for the global measurements performed by the thermal infrared mapper (TIR) on

  5. Shear stress induction of C-type natriuretic peptide (CNP) in endothelial cells is independent of NO autocrine signaling. (United States)

    Zhang, Z; Xiao, Z; Diamond, S L


    C-type natriuretic peptide (CNP) is secreted by endothelial cells and has vasodilatory and antiproliferative activity against smooth muscle cells. Using defined laminar shear stress exposures of cultured bovine aortic endothelial cells, we investigated the regulation of CNP gene by PhosphorImaging the ratio of CNP mRNA to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. A 6 h exposure to arterial shear stress of 25 dyn/cm2 caused a marked elevation (10.5 +/- 6.2-fold: n=10, pshear stress was 2.6 times more potent than a venous level of shear stress of 4 dyn/cm2 in elevating the CNP/GAPDH mRNA ratio. After 6 h, CNP secretion by shear stressed BAEC was elevated over stationary controls by 3.1-fold (n=5, pBAEC. Shear stress elevated CNP mRNA in the presence of L-NAME (400 microM) indicating that autocrine signaling through shear-induced NO production or guanylate cyclase activation was not involved. Similarly, the tyrosine kinase inhibitor genistein (10 microM), which can also block shear-induced NO production, had no effect on CNP mRNA induction by shear stress in BAEC. The intracellular calcium chelator BAPTA/AM (5 microM) attenuated the shear stress-induced CNP mRNA expression by 71%. Interestingly, dexamethasone (1 microM) potentiated by 2-fold the shear stress enhancement of CNP mRNA. Shear stress was a more potent inducer of CNP than either phorbol myristrate acetate or lipopolysaccharide. Hemodynamic shear stress may be an important physiological regulator of CNP expression with consequent effects on vasodilation and regulation of intimal hyperplasia.

  6. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression (United States)

    Scotland, Ramona S.; Cohen, Marc; Foster, Paul; Lovell, Matthew; Mathur, Anthony; Ahluwalia, Amrita; Hobbs, Adrian J.


    The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS-/-) or under acute inflammatory conditions (induced by interleukin-1 or histamine). CNP suppressed basal leukocyte rolling in eNOS-/- mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF4-23. CNP also suppressed leukocyte rolling induced by IL-1 or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders. endothelium | natriuretic peptide receptor type C | atherosclerosis | thrombosis

  7. C-type natriuretic peptide plasma levels are elevated in subjects with achondroplasia, hypochondroplasia, and thanatophoric dysplasia. (United States)

    Olney, Robert C; Prickett, Timothy C R; Espiner, Eric A; Mackenzie, William G; Duker, Angela L; Ditro, Colleen; Zabel, Bernhard; Hasegawa, Tomonobu; Kitoh, Hiroshi; Aylsworth, Arthur S; Bober, Michael B


    C-type natriuretic peptide (CNP) is a crucial regulator of endochondral bone growth. In a previous report of a child with acromesomelic dysplasia, Maroteaux type (AMDM), caused by loss-of-function of the CNP receptor (natriuretic peptide receptor-B [NPR-B]), plasma levels of CNP were elevated. In vitro studies have shown that activation of the MAPK kinase (MEK)/ERK MAPK pathway causes functional inhibition of NPR-B. Achondroplasia, hypochondroplasia, and thanatophoric dysplasia are syndromes of short-limbed dwarfism caused by activating mutations of fibroblast growth factor receptor-3, which result in overactivation of the MEK/ERK MAPK pathway. The purpose of this study was to determine whether these syndromes exhibit evidence of CNP resistance as reflected by increases in plasma CNP and its amino-terminal propeptide (NTproCNP). This was a prospective, observational study. Participants were 63 children and 20 adults with achondroplasia, 6 children with hypochondroplasia, 2 children with thanatophoric dysplasia, and 4 children and 1 adult with AMDM. Plasma levels of CNP and NTproCNP were higher in children with achondroplasia with CNP SD scores (SDSs) of 1.0 (0.3-1.4) (median [interquartile range]) and NTproCNP SDSs of 1.4 (0.4-1.8; P achondroplasia (CNP SDSs of 1.5 [0.7-2.1] and NTproCNP SDSs of 0.5 [0.1-1.0], P < .005). In children with hypochondroplasia, CNP SDSs were 1.3 (0.7-1.5) (P = .08) and NTproCNP SDSs were 1.9 (1.8-2.3) (P < .05). In children with AMDM, CNP SDSs were 1.6 (1.4-3.3) and NTproCNP SDSs were 4.2 (2.7-6.2) (P < .01). In these skeletal dysplasias, elevated plasma levels of proCNP products suggest the presence of tissue resistance to CNP.

  8. QTL involved in the partial restoration of male fertility of C-type cytoplasmic male sterility in maize. (United States)

    Kohls, Susanne; Stamp, Peter; Knaak, Carsten; Messmer, Rainer


    Partial restoration of male fertility limits the use of C-type cytoplasmic male sterility (C-CMS) for the production of hybrid seeds in maize. Nevertheless, the genetic basis of the trait is still unknown. Therefore, the aim to this study was to identify genomic regions that govern partial restoration by means of a QTL analysis carried out in an F(2) population (n = 180). This population was derived from the Corn Belt inbred lines B37C and K55. F(2)BC(1) progenies were phenotyped at three locations in Switzerland. Male fertility was rated according to the quality and number of anthers as well as the anthesis-silking interval. A weak effect of environment on the expression of partial restoration was reflected by high heritabilities of all fertility-related traits. Partial restoration was inherited like an oligogenic trait. Three major QTL regions were found consistently across environments in the chromosomal bins 2.09, 3.06 and 7.03. Therefore, a marker-assisted counter-selection of partial restoration is promising. Minor QTL regions were found on chromosomes 3, 4, 5, 6 and 8. A combination of partial restorer alleles at different QTL can lead to full restoration of fertility. The maternal parent was clearly involved in the partial restoration, because the restorer alleles at QTL in bins 2.09, 6.04 and 7.03 originated from B37. The three major QTL regions collocated with other restorer genes of maize, a phenomenon, which seems to be typical for restorer genes. Therefore, a study of the clusters of restorer genes in maize could lead to a better understanding of their evolution and function. In this respect, the long arm of chromosome 2 is particularly interesting, because it harbors restorer genes for the three major CMS systems (C, T and S) of maize.

  9. Vascular relaxation induced by C-type natriuretic peptide involves the ca2+/NO-synthase/NO pathway.

    Directory of Open Access Journals (Sweden)

    Fernanda A Andrade

    Full Text Available AIMS: C-type natriuretic peptide (CNP and nitric oxide (NO are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3 contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery. MAIN METHODS: Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry. KEY FINDINGS: CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas. SIGNIFICANCE: These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.

  10. lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

    Directory of Open Access Journals (Sweden)

    Nagy Olga


    Full Text Available Abstract Background Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, lemming (lmg mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila melanogaster. Results The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite

  11. New elements in the C-type natriuretic peptide signaling pathway inhibiting swine in vitro oocyte meiotic resumption. (United States)

    Santiquet, Nicolas; Papillon-Dion, Emilie; Djender, Nadjib; Guillemette, Christine; Richard, François J


    C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM. © 2014 by the Society for the Study of Reproduction, Inc.

  12. AraC-Type Regulator Rbf Controls the Staphylococcus epidermidis Biofilm Phenotype by Negatively Regulating the icaADBC Repressor SarR. (United States)

    Rowe, Sarah E; Campbell, Christopher; Lowry, Colm; O'Donnell, Sinead T; Olson, Michael E; Lindgren, Jill K; Waters, Elaine M; Fey, Paul D; O'Gara, James P


    Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating

  13. Enhancement of solubility and yield of a β-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag. (United States)

    Dulal, Hari Prasad; Nagae, Masamichi; Ikeda, Akemi; Morita-Matsumoto, Kana; Adachi, Yoshiyuki; Ohno, Naohito; Yamaguchi, Yoshiki


    Dectin-1 is a C-type lectin-like pattern recognition receptor for β(1-3)-glucans. It plays a crucial role in protecting against fungal invasion through binding to β-glucans which are commonly present on the fungal cell wall. To probe its ligand binding mechanism by NMR, we expressed the recombinant murine Dectin-1 C-type lectin-like domain (CTLD) in E. coli using pCold vector and purified it. However, the high concentration of Dectin-1 CTLD required for NMR analysis could not be attained due to its inherent low solubility and low bacterial expression. In this study, we tried to increase expression and solubility of Dectin-1 CTLD by codon optimization and fusion of a GB1 tag (B1 domain of streptococcal Protein G). GB1 was inserted on either the N-terminal (NT) or C-terminal end as well as both terminal ends of human and mouse Dectin-1 CTLDs. A pure monomeric sample was only obtained with NT-GB1 fused mouse Dectin-1. Expression of mouse Dectin-1 CTLD yielded 0.9 ± 0.2 mg/L culture, codon optimized mouse Dectin-1 CTLD produced 1.4 ± 0.2 mg/L, and the tag-fused domain 7.1 ± 0.3 mg/L. The tag also increased solubility from 0.1 mM to 1.4 mM. The recombinant protein was correctly folded, in a monomeric state, and specifically bound β-glucan laminarin. These results indicate that fusing GB1 to the N-terminus of mouse Dectin-1 domain advantageously increases yield and solubility, allows retention of native structure, and that the site of fusion is critical. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Specific glycan elements determine differential binding of individual egg glycoproteins of the human parasite Schistosoma mansoni by host C-type lectin receptors

    NARCIS (Netherlands)

    Meevissen, M.H.J.; Driessen, N.N.; Smits, H.H.; Versteegh, R.; van Vliet, S.J.; van Kooijk, Y.; Schramm, G.; Deelder, A.M.; de Haas, H.; Yazdanbakhsh, M.; Hokke, C.H.


    During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite's eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the

  15. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x.

    NARCIS (Netherlands)

    Die, van I.M.; Vliet, van SJ; Nyame, AK; Cummings, RD; Bank, CM; Appelmelk, B.J.; Geijtenbeek, T.B.H.; Kooijk, van Y.


    Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies

  16. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

    NARCIS (Netherlands)

    van Die, Irma; van Vliet, Sandra J.; Nyame, A. Kwame; Cummings, Richard D.; Bank, Christine M. C.; Appelmelk, Ben; Geijtenbeek, Teunis B. H.; van Kooyk, Yvette


    Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies

  17. Discordant expression of pro-B-type and pro-C-type natriuretic peptide in newborn infants of mothers with type 1 diabetes

    DEFF Research Database (Denmark)

    Nybo, M.; Nielsen, Lars Bo; Nielsen, S.J.


    Maternal diabetes increases the risk of hypertrophic cardiomyopathy in the fetus. As signaling via the C-type natriuretic peptide (CNP) specific receptor protects against cardiac hypertrophy, we examined whether maternal type 1 diabetes affects the plasma concentrations of proCNP-derived peptides...

  18. Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7.

    Directory of Open Access Journals (Sweden)

    Elad Yunger


    Full Text Available In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks.

  19. Granule structure and distribution of allomorphs in C-type high-amylose rice starch granule modified by antisense RNA inhibition of starch branching enzyme. (United States)

    Wei, Cunxu; Qin, Fengling; Zhou, Weidong; Yu, Huaguang; Xu, Bin; Chen, Chong; Zhu, Lijia; Wang, Youping; Gu, Minghong; Liu, Qiaoquan


    C-type starch, which is a combination of both A-type and B-type crystal starch, is usually found in legumes and rhizomes. We have developed a high-amylose transgenic line of rice (TRS) by antisense RNA inhibition of starch branching enzymes. The starch in the endosperm of this TRS was identified as typical C-type crystalline starch, but its fine granular structure and allomorph distribution remained unclear. In this study, we conducted morphological and spectroscopic studies on this TRS starch during acid hydrolysis to determine the distribution of A- and B-type allomorphs. The morphology of starch granules after various durations of acid hydrolysis was compared by optical microscopy, scanning electron microscopy, and transmission electron microscopy. The results showed that amorphous regions were located at the center part of TRS starch subgranules. During acid hydrolysis, starch was degraded from the interior of the subgranule to the outer surface, while the peripheral part of the subgranules and the surrounding band of the starch granule were highly resistant to acid hydrolysis. The spectroscopic changes detected by X-ray powder diffraction, 13C cross-polarization magic-angle spinning NMR, and attenuated total reflectance Fourier transform infrared showed that the A-type allomorph was hydrolyzed more rapidly than the B-type, and that the X-ray diffraction profile gradually changed from a native C-type to a CB-type with increasing hydrolysis time. Our results showed that, in TRS starch, the A-type allomorph was located around the amorphous region, and was surrounded by the B-type allomorph located in the peripheral region of the subgranules and the surrounding band of the starch granule. Thus, the positions of A- and B-type allomorphs in the TRS C-type starch granule differ markedly from those in C-type legume and rhizome starch.

  20. Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers.

    Directory of Open Access Journals (Sweden)

    Soosan Hadjialirezaei

    Full Text Available Carbohydrate-protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6 pN and 7.1 pN were determined for the MUC1(Tn-MGL and MUC1(STn-MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27 and 37 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST, a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn and MUC1(STn when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.

  1. Lech, M., et al., Quantitative Expression of C-Type Lectin Receptors in Humans and Mice. Int. J. Mol. Sci. 2012, 13, 10113-10131.

    Directory of Open Access Journals (Sweden)

    Hans-Joachim Anders


    Full Text Available The authors wish to add this correction on their paper published in IJMS [1]. Galectin-1 was misclassified as a C-type lectin. Galectin-1 belongs to the family of the S-type lectins, i.e., the galectins. These errors have been amended in an amended version of the manuscript, which is available from the International Journal of Molecular Sciences website. The authors and publisher apologize for the inconvenience. [...

  2. Recombinant Expression, In Vitro Refolding and Characterizing Disulfide Bonds of a Mouse Inhibitory C-Type Lectin-Like Receptor Nkrp1b

    Czech Academy of Sciences Publication Activity Database

    Hernychová, Lucie; Mrázek, Hynek; Ivanova, Ljubina; Kukačka, Zdeněk; Chmelík, Josef; Novák, Petr


    Roč. 64, č. 2015 (2015), s. 85-93 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk LO1509 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : NK cell * C-type lectin -like receptor (CTLR) * Nkrp1 Subject RIV: CE - Biochemistry Impact factor: 1.643, year: 2015

  3. Purification and characterization of a novel C-type hemolytic lectin for clot lysis from the fresh water clam Villorita cyprinoides: a possible natural thrombolytic agent against myocardial infarction. (United States)

    Sudhakar, G R Learnal; Vincent, S G Prakash


    Villorita cyprinoides (black clam) is a fresh water clam that belongs as a bivalve to the group of mollusc. The saline extracts from the muscle reveal high titers of agglutination potency on trypsin-treated rabbit erythrocytes. With the help of affinity chromatography a hemolytic protein with lectin activity which could all be inhibited by D-galactose were isolated. The lectins were separated on DEAE-cellulose and the main component was purified after an additional step of gel filtration on sephadex G-75. The main component is a non-glycosylated protein with a molecular weight of 96,560 Da determined by MALDI-ToF, consisting of a single protein chain and characterized by the lack of polymers and intermediate disulfide bonds. The pure main lectin with clot lytic feature shows two bands at molecular weights 36,360 and 26, 520 Da. Optimal inhibition of the pure lectin is achieved by D-galactose containing oligo- and polysaccharides. The lectin activity decreased above 40 °C and was lost at 62 °C, the stability over the pH range between 7.0 and 8.0 and requires divalent cations for their activity. The novel C-type hemolytic lectin for clot lysis from the clam Villorita cyprinoides was identified and evaluated, the purified hemolytic lectin (0.35 mg/ml and 0.175 mg/ml) enhanced clot lysis activity when compared to the different concentration (5 mg/ml and 1 mg/ml) of commercial streptokinase. In the present study identified hemolytic lectin was a rapid and effective clot lytic molecule and could be developed as new drug molecule in future. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Oxidation of c-Type Cytochromes by the Membrane-Bound Cytochrome Oxidase (Cytochrome aa(3)) of Blue-Green Algae. (United States)

    Kienzl, P F; Peschek, G A


    Respiratory particles containing an aa(3)-type cytochrome oxidase were prepared from Anacystis nidulans, Synechocystis 6714, Synechococcus lividus, Anabaena variabilis, Nostoc sp. strain MAC, Nostoc muscorum, and Mastigocladus laminosus. Oxidation of c-type cytochromes by membrane preparations of the different blue-green algae was observed using purified cytochromes from horse heart, Candida krusei, tuna, Saccharomyces oviformis, Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas palustris, Rhodocyclus purpureus, Paracoccus denitrificans, Anacystis nidulans, Anabaena variabilis, Euglena gracilis, and Scenedesmus obliquus. Rapid oxidations were consistently observed with the mitochondrial c-type cytochromes (horse heart cytochrome c reacts most rapidly) and with cytochromes c(2) from Rhodopseudomonas palustris and Rhodocyclus purpureus; in contrast, the cytochrome c(2) from Rhodospirillum rubrum and the plastidic cytochromes from E. gracilis and Scendesmus obliquus were inactive with all membrane preparations. All reactions were inhibited by low concentrations of KCN, NaN(3), and CO, and they were activated by Tween 80, thus indicating participation of the terminal oxidase. The results are discussed in view of the spectral similarities between the terminal oxidase of blue-green algae and the mitochondrial aa(3)-type cytochrome oxidase of plants and other eukaryotes.

  5. Conformation and location of amorphous and semi-crystalline regions in C-type starch granules revealed by SEM, NMR and XRD. (United States)

    Wang, Shujun; Yu, Jinglin; Yu, Jiugao


    The conformations and locations of amorphous and semi-crystalline regions in C-type starch granules from Chinese yam were evaluated by a combination of morphology and spectroscopy studies during acid hydrolysis. Scanning electron micrographs showed that amorphous or less crystalline areas were essentially located in the centre part of C-type starch granules, whereas the semi-crystalline and amorphous growth rings were found mainly in the outer part of the granules. (13)C cross-polarization magic angle spinning NMR ((13)C CP/MAS NMR) showed that amorphous regions were hydrolyzed faster than the crystalline ones. In addition, B-type polymorphs were shown to be hydrolyzed more rapidly than A-types. Powder X-ray diffraction (XRD) also revealed that the B-polymorph was hydrolyzed more rapidly than the A-type. XRD showed that the amorphous or less crystalline areas were mainly located in the core of starch granules, while the amorphous growth rings are distributed toward the outside of the granules and alternatively arranged with semi-crystalline growth rings. The amorphous or less crystalline areas predominantly consisted of the B-polymorph whereas the outer semi-crystalline and amorphous growth rings were mostly composed of the A-polymorph. Copyright © 2008 Elsevier Ltd. All rights reserved.

  6. Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c{sub 552} and its b-type variant

    Energy Technology Data Exchange (ETDEWEB)

    Tozawa, Kaeko; Ferguson, Stuart J.; Redfield, Christina, E-mail: [University of Oxford, Department of Biochemistry (United Kingdom); Smith, Lorna J., E-mail: [University of Oxford, Department of Chemistry (United Kingdom)


    Cytochrome c{sub 552} from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, {sup 15}N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high {sup 15}N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

  7. Structure of a glycomimetic ligand in the carbohydrate recognition domain of C-type lectin DC-SIGN. Structural requirements for selectivity and ligand design. (United States)

    Thépaut, Michel; Guzzi, Cinzia; Sutkeviciute, Ieva; Sattin, Sara; Ribeiro-Viana, Renato; Varga, Norbert; Chabrol, Eric; Rojo, Javier; Bernardi, Anna; Angulo, Jesus; Nieto, Pedro M; Fieschi, Franck


    In genital mucosa, different fates are described for HIV according to the subtype of dendritic cells (DCs) involved in its recognition. This notably depends on the C-type lectin receptor, langerin or DC-SIGN, involved in gp120 interaction. Langerin blocks HIV transmission by its internalization in specific organelles of Langerhans cells. On the contrary, DC-SIGN enhances HIV trans-infection of T lymphocytes. Thus, approaches aiming to inhibit DC-SIGN, without blocking langerin, represent attractive anti-HIV strategies. We previously demonstrated that dendrons bearing multiple copies of glycomimetic compounds were able to block DC-SIGN-dependent HIV infection in cervical explant models. Optimization of such ligand requires detailed characterization of its binding mode. In the present work, we determined the first high-resolution structure of a glycomimetic/DC-SIGN complex by X-ray crystallography. This glycomimetic, pseudo-1,2-mannobioside, shares shape and conformational properties with Manα1-2Man, its natural counterpart. However, it uses the binding epitope previously described for Lewis X, a ligand specific for DC-SIGN among the C-type lectin family. Thus, selectivity gain for DC-SIGN versus langerin is observed with pseudo-1,2-mannobioside as shown by surface plasmon resonance analysis. In parallel, ligand binding was also analyzed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol. These studies demonstrate that the complex, defined by X-ray crystallography, represents the unique binding mode of this ligand as opposed to the several binding orientations described for the natural ligand. This exclusive binding mode and its selective interaction properties position this glycomimetic as a good lead compound for rational improvement based on a structurally driven approach.

  8. Study of coherent diffractive production reactions of p + C --> [Y$^{0}$K$^{+}$] + C type and observation of the new baryonic states X(2050) --> $\\Sigma$(1385)$^{0}$K$^{+}$ and X(2000) --> $\\Sigma^{0}$K$^{+}$

    CERN Document Server

    Golovkin, S V; Kozhevnikov, A A; Kubarovskii, V P; Kulyavtsev, A I; Kurshetsov, V F; Kushnirenko, A Yu; Landsberg, G L; Molchanov, V V; Mukkin, V A; Solyanik, V I; Vavilov, D V; Victorov, V A; Balats, M Ya; Dzubenko, G B; Kamenskii, A D; Kliger, G K; Kolganov, V Z; Lakaev, V S; Lomkatsi, G S; Nilov, A P; Smolyankin, V T; Vishniakov, V V


    Study of coherent diffractive production reactions of p + C --> [Y$^{0}$K$^{+}$] + C type and observation of the new baryonic states X(2050) --> $\\Sigma$(1385)$^{0}$K$^{+}$ and X(2000) --> $\\Sigma^{0}$K$^{+}$

  9. Developmental regulation of chicken surfactant protein A and its localization in lung

    DEFF Research Database (Denmark)

    Zhang, Weidong; Cuperus, Tryntsje; van Dijk, Albert


    Surfactant Protein A (SP-A) is a collagenous C-type lectin (collectin) that plays an important role in the early stage of the host immune response. In chicken, SP-A (cSP-A) is expressed as a 26 kDa glycosylated protein in the lung. Using immunohistochemistry, cSP-A protein was detected mainly...

  10. Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part II: combination with external radiation improves survival

    Directory of Open Access Journals (Sweden)

    Kushchayev SV


    Full Text Available Sergiy V Kushchayev,1 Tejas Sankar,1 Laura L Eggink,5,6 Yevgeniya S Kushchayeva,5 Philip C Wiener,1,5 J Kenneth Hoober,5,6 Jennifer Eschbacher,3 Ruolan Liu,2 Fu-Dong Shi,2 Mohammed G Abdelwahab,4 Adrienne C Scheck,4 Mark C Preul11Neurosurgery Research Laboratory, 2Neuroimmunology Laboratory, 3Department of Pathology, 4Neurooncology Research, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, 5School of Life Sciences, Arizona State University, Tempe, 6Susavion Biosciences, Inc, Tempe, AZ, USABackground: A peptide mimetic of a ligand for the galactose/N-acetylgalactosamine-specific C-type lectin receptors (GCLR exhibited monocyte-stimulating activity, but did not extend survival when applied alone against a syngeneic murine malignant glioma. In this study, the combined effect of GCLRP with radiation was investigated.Methods: C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells. Animals were grouped based on randomized tumor size by magnetic resonance imaging on day seven. One group that received cranial radiation (4 Gy on days seven and nine only were compared with animals treated with radiation and GCLRP (4 Gy on days seven and nine combined with subcutaneous injection of 1 nmol/g on alternative days beginning on day seven. Magnetic resonance imaging was used to assess tumor growth and correlated with survival rate. Blood and brain tissues were analyzed with regard to tumor and contralateral hemisphere using fluorescence-activated cell sorting analysis, histology, and enzyme-linked immunosorbent assay.Results: GCLRP activated peripheral monocytes and was associated with increased blood precursors of dendritic cells. Mean survival increased (P < 0.001 and tumor size was smaller (P < 0.02 in the GCLRP + radiation group compared to the radiation-only group. Accumulation of dendritic cells in both the tumoral hemisphere (P < 0.005 and contralateral tumor-free hemisphere (P< 0.01 was

  11. P17, an Original Host Defense Peptide from Ant Venom, Promotes Antifungal Activities of Macrophages through the Induction of C-Type Lectin Receptors Dependent on LTB4-Mediated PPARγ Activation

    Directory of Open Access Journals (Sweden)

    Khaddouj Benmoussa


    Full Text Available Despite the growing knowledge with regard to the immunomodulatory properties of host defense peptides, their impact on macrophage differentiation and on its associated microbicidal functions is still poorly understood. Here, we demonstrated that the P17, a new cationic antimicrobial peptide from ant venom, induces an alternative phenotype of human monocyte-derived macrophages (h-MDMs. This phenotype is characterized by a C-type lectin receptors (CLRs signature composed of mannose receptor (MR and Dectin-1 expression. Concomitantly, this activation is associated to an inflammatory profile characterized by reactive oxygen species (ROS, interleukin (IL-1β, and TNF-α release. P17-activated h-MDMs exhibit an improved capacity to recognize and to engulf Candida albicans through the overexpression both of MR and Dectin-1. This upregulation requires arachidonic acid (AA mobilization and the activation of peroxisome proliferator-activated receptor gamma (PPARγ nuclear receptor through the leukotriene B4 (LTB4 production. AA/LTB4/PPARγ/Dectin-1-MR signaling pathway is crucial for P17-mediated anti-fungal activity of h-MDMs, as indicated by the fact that the activation of this axis by P17 triggered ROS production and inflammasome-dependent IL-1β release. Moreover, we showed that the increased anti-fungal immune response of h-MDMs by P17 was dependent on intracellular calcium mobilization triggered by the interaction of P17 with pertussis toxin-sensitive G-protein-coupled receptors on h-MDMs. Finally, we also demonstrated that P17-treated mice infected with C. albicans develop less severe gastrointestinal infection related to a higher efficiency of their macrophages to engulf Candida, to produce ROS and IL-1β and to kill the yeasts. Altogether, these results identify P17 as an original activator of the fungicidal response of macrophages that acts upstream PPARγ/CLRs axis and offer new immunomodulatory therapeutic perspectives in the field of

  12. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    Energy Technology Data Exchange (ETDEWEB)

    Usami, Katsuaki [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Matsuno, Keita; Igarashi, Manabu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Denda-Nagai, Kaori [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Takada, Ayato [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Irimura, Tatsuro, E-mail: [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan)


    Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

  13. C-type starch from high-amylose rice resistant starch granules modified by antisense RNA inhibition of starch branching enzyme. (United States)

    Wei, Cunxu; Xu, Bin; Qin, Fengling; Yu, Huaguang; Chen, Chong; Meng, Xianglen; Zhu, Lijia; Wang, Youping; Gu, Minghong; Liu, Qiaoquan


    High-amylose starch is a source of resistant starch (RS) which has a great benefit on human health. A transgenic rice line (TRS) enriched amylose and RS had been developed by antisense RNA inhibition of starch branching enzymes. In this study, the native starch granules were isolated from TRS grains as well as the wild type, and their crystalline type was carefully investigated before and after acid hydrolysis. In high-amylose TRS rice, the C-type starch, which might result from the combination of both A-type and B-type starch, was observed and subsequently confirmed by multiple physical techniques, including X-ray powder diffraction, solid-state nuclear magnetic resonance, and Fourier transform infrared. Moreover, the change of starch crystalline structure from C- to B-type during acid hydrolysis was also observed in this RS-rich rice. These data could add to our understanding of not only the polymorph structure of cereal starch but also why high-amylose starch is more resistant to digestion.

  14. Gene encoding a c-type cyclin in Mycosphaerella graminicola is involved in aerial mycelium formation, filamentous growth, hyphal swelling, melanin biosynthesis, stress response, and pathogenicity. (United States)

    Choi, Yoon-E; Goodwin, Stephen B


    Mycosphaerella graminicola is an important wheat pathogen causing Septoria tritici blotch. To date, an efficient strategy to control M. graminicola has not been developed. More significantly, we have a limited understanding of the molecular mechanisms of M. graminicola pathogenicity. In this study, we attempted to characterize an MCC1-encoding c-type cyclin, a gene homologous to FCC1 in Fusarium verticillioides. Four independent MCC1 knock-out mutants were generated via Agrobacterium tumefaciens-mediated transformation. All of the MCC1 mutants showed consistent multiple phenotypes. Significant reductions in radial growth on potato dextrose agar (PDA) were observed in all of the MCC1 mutants. In addition, MCC1 gene-deletion mutants produced less aerial mycelium on PDA, showed delayed filamentous growth, had unusual hyphal swellings, produced more melanin, showed an increase in their stress tolerance response, and were reduced significantly in pathogenicity. These results indicate that the MCC1 gene is involved in multiple signaling pathways, including those involved in pathogenicity in M. graminicola.

  15. The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

    Directory of Open Access Journals (Sweden)

    Magdalena eEriksson


    Full Text Available Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern recognition receptors sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs are pattern recognition receptors recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR SIGNR3 in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homologue of the human DC-SIGN receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze if this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium (DSS-induced colitis model was employed. SIGNR3-/- mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3-/- mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

  16. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells* (United States)

    Manzo, Carlo; Torreno-Pina, Juan A.; Joosten, Ben; Reinieren-Beeren, Inge; Gualda, Emilio J.; Loza-Alvarez, Pablo; Figdor, Carl G.; Garcia-Parajo, Maria F.; Cambi, Alessandra


    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection. PMID:23019323

  17. Macrophage-inducible C-type lectin Mincle-expressing dendritic cells contribute to control of splenic Mycobacterium bovis BCG infection in mice. (United States)

    Behler, Friederike; Maus, Regina; Bohling, Jennifer; Knippenberg, Sarah; Kirchhof, Gabriele; Nagata, Masahiro; Jonigk, Danny; Izykowski, Nicole; Mägel, Lavinia; Welte, Tobias; Yamasaki, Sho; Maus, Ulrich A


    The macrophage-inducible C-type lectin Mincle has recently been identified to be a pattern recognition receptor sensing mycobacterial infection via recognition of the mycobacterial cell wall component trehalose-6',6-dimycolate (TDM). However, its role in systemic mycobacterial infections has not been examined so far. Mincle-knockout (KO) mice were infected intravenously with Mycobacterium bovis BCG to mimic the systemic spread of mycobacteria under defined experimental conditions. After intravenous infection with M. bovis BCG, Mincle-KO mice responded with significantly higher numbers of mycobacterial CFU in spleen and liver, while reduced granuloma formation was observed only in the spleen. At the same time, reduced Th1 cytokine production and decreased numbers of gamma interferon-producing T cells were observed in the spleens of Mincle-KO mice relative to the numbers in the spleens of wild-type (WT) mice. The effect of adoptive transfer of defined WT leukocyte subsets generated from bone marrow cells of zDC(+/DTR) mice (which bear the human diphtheria toxin receptor [DTR] under the control of the classical dendritic cell-specific zinc finger transcription factor zDC) to specifically deplete Mincle-expressing classical dendritic cells (cDCs) but not macrophages after diphtheria toxin application on the numbers of splenic and hepatic CFU and T cell subsets was then determined. Adoptive transfer experiments revealed that Mincle-expressing splenic cDCs rather than Mincle-expressing macrophages contributed to the reconstitution of attenuated splenic antimycobacterial immune responses in Mincle-KO mice after intravenous challenge with BCG. Collectively, we show that expression of Mincle, particularly by cDCs, contributes to the control of splenic M. bovis BCG infection in mice. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. The primary structure of cytochrome c-554 from the green photosynthetic bacterium Chloroflexus aurantiacus

    Energy Technology Data Exchange (ETDEWEB)

    Dracheva, S.; Williams, J.C.; Blankenship, R.E. (Arizona State Univ., Tempe (United States)); Van Driessche, G.; Van Beeumen, J.J. (State Univ., Ghent (Belgium))


    The complete nucleotide sequence of the cytochrome c-554 gene from the green photosynthetic bacterium Chloroflexus aurantiacus has been determined. The derived amino acid sequence showed that the cytochrome precursor protein consists of 414 residues and contains 4-Cys-X-X-Cys-His- heme binding motifs. The only regions of the cytochrome c-554 sequence that were found to be significantly similar to the sequences of cytochromes from other organisms were the heme binding sites. The highest similarity was found with the heme binding segments in the four-heme reaction center cytochrome subunit from the purple photosynthetic bacterium Rhodopseudomonas viridis. The importance of this similarity for the evolutionary relationship between Chloroflexus and the purple bacteria is discussed.

  19. Protein Foods (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  20. Prevalence of AmpC type extended spectrum beta lactamases genes in clinical Samples of E.coli Isolated from Poultry and Humans

    Directory of Open Access Journals (Sweden)

    Elham Farrokhnazar


    Full Text Available Emergence of antibiotic resistance among pathogens, particularly in health centers and hospitals, has become a major public health problem. This study identified AmpC-type beta-lactamase against the antibiotic ceftazidime, cefotaxime and cefpodoxime in E.coli isolated from human and poultry and types of producing genes were studied by PCR. In this study, 500 clinical human samples of urine from hospitals of Tehran during 5 months as well as 300 poultry samples were collected and transferred to the microbiology laboratory. Biochemical tests such as TSI, Urea and IMViC were performed on suspected colonies with E.coli. To identify ESBL producing strains, beta-lactamase samples were cultured on Mueller-Hinton agar through antimicrobial susceptibility test by disk agar diffusion based on the standard CLSI for initial screening. PCR reactions were done using specific primers CITM, EBCM, DHAM and MOXM to identify the beta-lactamase AmpC. A number of 200 human and 55 poultry E.coli samples were screened. In human samples, 105 (52.5% were resistant and potential producers of ESBL and AmpC; out of those, 102 (51% produced ESBL and 3 (1.5% potentially produced AmpC. In the study on 55 E.coli isolates from poultry samples based on the results of disk agar diffusion test, 4 (7.2% samples were resistant and potential producers of ESBL. None of the samples were AmpC producers. Through PCR, 2 human samples (1% were CITM positive and one sample (0.5% was DHAM positive. Through the PCR carried out on poultry samples, there were no bands with 4 primers. There was AmpC in human samples; while further studies are required for poultry samples, because poultry significantly contribute in production of food for humans and can be an important source for dissemination of antibiotic resistance. Given the significance of Ampc in providing high levels of beta-lactam antibiotic resistance, particularly third generation cephalosporins which are very common treatments, more

  1. Dissemination of SHV-12 and Characterization of New AmpC-Type Beta-Lactamase Genes among Clinical Isolates of Enterobacter Species in Korea (United States)

    Lee, Sang Hee; Kim, Jae Young; Shin, Sang Heum; An, Young Jun; Choi, Young Wook; Jung, Yeun Chang; Jung, Ha Il; Sohn, Eui Suk; Jeong, Seok Hoon; Lee, Kye Joon


    To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of Enterobacter species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the ampC genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four ampC genes (blaEcloK992004.1, blaEcloK995120.1, blaEcloK99230, and blaEareK9911729) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal ampC gene (blaEcloMHN1) of Enterobacter cloacae MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two ampC genes (blaEcloK9973 and blaEcloK9914325) showed close similarity to the chromosomal ampC gene (blaEcloQ908R) of E. cloacae Q908R (99.7% identity). The results from phylogenetic analysis suggested that six ampC genes could originate from blaEcloMHN1 or blaEcloQ908R. PMID:12791868

  2. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B


    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...... diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract...

  3. Abundance of the multiheme c-type cytochrome OmcB increases in outer biofilm layers of electrode-grown Geobacter sulfurreducens.

    Directory of Open Access Journals (Sweden)

    Camille S Stephen

    Full Text Available When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB and cytoplasmic acetate kinase (AckA were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode. Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10-20 µm distant from the electrode surface compared to inner layers (0-10 µm. In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.

  4. Molecular basis of the differences in binding properties of the highly related C-type lectins DC-SIGN and L-SIGN to Lewis X trisaccharide and Schistosoma mansoni egg antigens

    NARCIS (Netherlands)

    van Liempt, Ellis; Imberty, Anne; Bank, Christine M. C.; van Vliet, Sandra J.; van Kooyk, Yvette; Geijtenbeek, Teunis B. H.; van Die, Irma


    The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal

  5. Molecular basis of the differences in binding properties of the highly related C-type lectins DC-SIGN and L-SIGN to Lewis X trisaccharide and Schistosoma mansoni egg antigens.

    NARCIS (Netherlands)

    Liempt, P.A.G.; Imberty, A; Bank, CM; Vliet, van SJ; Kooijk, van Y.; Geijtenbeek, T.B.H.; Die, van I.M.


    The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal

  6. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente


    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers. The bi...

  7. The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

    Directory of Open Access Journals (Sweden)

    Qiang Lou

    Full Text Available OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

  8. The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway. (United States)

    Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang


    OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

  9. Domain Modeling: NP_055516.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_055516.2 chr15 Crystal structure of Heme Binding protein, an autotransporter hem...oglobine protease from pathogenic Escherichia coli p1wxra_ chr15/NP_055516.2/NP_055516.2_apo_5-1056.pdb swppa 0 ...

  10. Domain Modeling: NP_065829.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_065829.3 chr15 Crystal structure of Heme Binding protein, an autotransporter hem...oglobine protease from pathogenic Escherichia coli p1wxra_ chr15/NP_065829.3/NP_065829.3_apo_819-1906.pdb swppa 0 ...

  11. Genetics and phenomics of hypothyroidism and goiter due to TPO mutations

    NARCIS (Netherlands)

    Ris-Stalpers, Carrie; Bikker, Hennie


    Thyroid peroxidase (TPO) is a heme binding protein localized on the apical membrane of the thyrocyte. TPO enzymatic activity is essential for thyroid hormonogenesis. Inactivating mutations form the molecular basis for a specific subtype of congenital hypothyroidism: thyroid dyshormonogenesis due to

  12. Domain Modeling: NP_060224.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_060224.3 chr6 Crystal structure of Heme Binding protein, an autotransporter hemo...globine protease from pathogenic Escherichia coli p1wxra_ chr6/NP_060224.3/NP_060224.3_apo_387-1439.pdb swppa 0 ...

  13. Membrane-associated c-type cytochromes from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum: purification and characterization of cytochrome c553. (United States)

    Albouy, D; Sturgis, J N; Feiler, U; Nitschke, W; Robert, B


    Tetraheme cytochromes involved in photosynthetic electron transport have previously been described associated with the reaction centers of purple photosynthetic bacteria; however, similar heme proteins have not until now been characterized in the phylogenetically distinct green sulfur bacteria. In this paper we describe the first isolation and characterization of a multitheme, membrane-associated cytochrome from a green sulfur bacterium, Chlorobium limicola forma thiosulfatophilum. We show that this cytochrome contains a single polypeptide of 32 kDa apparent molecular mass on SDS-PAGE and has a characteristic broad alpha-band absorption at 553 nm. By both low-temperature absorption and electron paramagnetic resonance spectroscopy, we demonstrate that there are at least four distinct heme groups.

  14. Increased Antimicrobial Resistance in a Novel CMY-54 AmpC-Type Enzyme with a GluLeu(217-218) Insertion in the Ω-Loop. (United States)

    Pérez-Llarena, Francisco José; Vázquez-Ucha, Juan Carlos; Kerff, Frédéric; Zamorano, Laura; Miró, Elisenda; Cabral, María Póvoa; Fleites, Ana; Lantero, Marta; Martínez-Martínez, Luis; Oliver, Antonio; Galleni, Moreno; Navarro, Ferrán; Beceiro, Alejandro; Bou, Germán


    During a Spanish surveillance study, a natural variant of a CMY-type β-lactamase related to CMY-2 with a GluLeu(217-218) insertion in the Ω-loop (designated CMY-54) was found to increase the minimum inhibitory concentractions to β-lactams in a clinical strain of Escherichia coli. The aim of this study was to characterize CMY-54 by genetic, microbiological, and biochemical analysis. The blaCMY-54 gene is encoded by a plasmid of around 100 kb that hybridizes with K and FIB probes. The genetic context of blaCMY-54 and blaCMY-2 genes was found to be very similar. E. coli expressing CMY-54 under isogenic conditions showed a clear fourfold to eightfold increase in MICs to penicillins, cefotaxime, ceftazidime, and aztreonam compared with CMY-2. The catalytic efficiencies of pure CMY-2 and CMY-54 proteins correlated with their microbiological parameters. CMY-2 protein was more resistant to thermal denaturation than CMY-54, indicating than the Ω-loop of CMY-54 may be wider and more relaxed and probably enables better accommodation of these antimicrobials. Otherwise, the higher stabilization of CMY-2 may induce a slight reduction of the dynamics of this enzyme and primarily affect the hydrolysis of some of the bulkiest antibiotics. In summary, the GluLeu(217-218) insertion observed in CMY-54 compared to CMY-2 produces a β-lactamase with a distinctive catalytic efficacy for β-lactam antimicrobials likely caused by an increased flexibility slightly affecting the active site shape, highlighting the relevance of single mutations on the hydrolytic spectrum in class C β-lactamases.

  15. Molecular and mass spectral identification of the broadly conserved decapod crustacean neuropeptide pQIRYHQCYFNPISCF: the first PISCF-allatostatin (Manduca sexta- or C-type allatostatin) from a non-insect


    Stemmler, Elizabeth A.; Bruns, Emily A.; Cashman, Christopher R.; Dickinson, Patsy S.; Andrew E Christie


    The PISCF-allatostatins (Manduca sexta- or C-type allatostatins) are a family of pentadecapeptides characterized by a pyroglutamine blocked N-terminus, an unamidated –PISCF C-terminus, and a disulfide bridge between two internal Cys residues. Several isoforms of PISCF-AST are known, all from holometabolous insects. Using a combination of transcriptomics and mass spectrometry, we have identified the first PISCF-type peptides from a non-insect species. In silico analysis of crustacean ESTs iden...

  16. Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

    Directory of Open Access Journals (Sweden)

    Norifumi Muraki


    Full Text Available Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

  17. Identification of the major proteins of the organic matrix of emu (Dromaius novaehollandiae) and rhea (Rhea americana) eggshell calcified layer. (United States)

    Mann, K


    1. The eggshell is a composite consisting of 95% calcite and an organic matrix. 2. While many proteins of the chicken eggshell matrix have already been identified, little is known about the matrix of other birds. 3. Isolation of the emu and rhea eggshell matrix and analysis of its major constituents showed that the predominant components were C-type lectin-like proteins related to those of ostrich, chicken and goose. 4. Serum albumin, vitelline membrane outer layer protein I (VMO-I) and the turpentine-induced acute phase serum protein 18-B were identified as minor components of the emu shell matrix. Both eggshell matrices also contained a novel proline- and alanine-rich protein. 5. Like ostrich, and unlike chicken and goose, both emu and rhea eggshell matrix contained two different C-type lectin-like proteins as major components, indicating that the occurrence of two proteins of this family may be widespread among ratites.

  18. Tumor-associated Tn-MUC1 glycoform is internalized through the macrophage galactose-type C-type lectin and delivered to the HLA class I and II compartments in dendritic cells

    DEFF Research Database (Denmark)

    Napoletano, Chiara; Rughetti, Aurelia; Agervig Tarp, Mads P


    by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis......The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes...

  19. Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil

    DEFF Research Database (Denmark)

    Nielsen, B B; Kastrup, J S; Rasmussen, H


    Tetranectin is a plasminogen kringle 4-binding protein. The crystal structure has been determined at 2.8 A resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple alpha-helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected...... to a long alpha-helix. Tetranectin has been classified in a distinct group of the C-type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C-type lectin superfamily, whereas...

  20. Synthesis and structural and electrical investigations of a hexagonal Y(1-x)Gd(x)InO3 (0.0 ≤ x ≤ 1.0) system obtained via metastable C-type intermediates. (United States)

    Shukla, Rakesh; Grover, Vinita; Deshpande, S K; Jain, Dheeraj; Tyagi, Avesh K


    Detailed structural and electrical investigations were carried out on an A-site disordered hexagonal Y(1-x)Gd(x)InO3 (0.0 ≤ x ≤ 1.0) series synthesized by a self-assisted gel-combustion route. The phase relations show profound temperature dependence. The metastable C-type modification could be stabilized for all the compositions, which on further heating get converted to stable hexagonal polymorphs. The conversion temperature (C-type to hexagonal) was found to increase with an increase in Y(3+) content. The system was observed to be single-phasic hexagonal at 1250 °C throughout the composition range. Interestingly, the increase in planar bonds of InO5 polyhedra was found to be twice that of the apical bonds on Gd(3+) substitution. Careful Raman spectroscopic studies highlighted a definitive though subtle structural change from x = 0.7 onward. The same observation is also corroborated by the dielectric studies. Electric field-dependent polarization measurements showed the ferroelectric hysteresis loop for pure YInO3. The system transforms from ferroelectric in YInO3 to almost paraelectric for GdInO3. In the present study, XRD, Raman, and electrical characterizations in conjunction reveal that to tune the electrical properties of the hexagonal rare earth indates, the variation in tilting of InO5 polyhedra has to be influenced, which could not be brought about by isovalent A-site substitution.

  1. Total protein (United States)

    ... page: // Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  2. Protein Structure (United States)

    Asmus, Elaine Garbarino


    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  3. Analysis of two lysozyme genes and antimicrobial functions of their recombinant proteins in Asian seabass.

    Directory of Open Access Journals (Sweden)

    Gui Hong Fu

    Full Text Available Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type and goose-type (g-type lysozymes from Asian seabass (Lates calcarifer. The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50 and Asp(67 and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL domain containing three conserved catalytic residues (Glu(71, Asp(84, Asp(95 essential for catalytic activity. Real time quantitative PCR (qRT-PCR revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

  4. An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

    DEFF Research Database (Denmark)

    Hansen, Soren; Schmidt, Vivi; Steffensen, Maria Abildgaard


    Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of othe...

  5. Nkrp1 Family, from Lectins to Protein Interacting Molecules

    Directory of Open Access Journals (Sweden)

    Daniel Rozbeský


    Full Text Available The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the “sweet story” was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

  6. Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part 1: stimulatory effects on blood monocytes and monocyte-derived cells of the brain

    Directory of Open Access Journals (Sweden)

    Kushchayev SV


    Full Text Available Sergiy V Kushchayev,1 Tejas Sankar,1 Laura L Eggink,4,5 Yevgeniya S Kushchayeva,5 Philip C Wiener,1,5 J Kenneth Hoober,5,6 Jennifer Eschbacher,3 Ruolan Liu,2 Fu-Dong Shi,2 Mohammed G Abdelwahab,4 Adrienne C Scheck,4 Mark C Preul11Neurosurgery Research Laboratory, 2Neuroimmunology Laboratory, 3Department of Pathology, 4Neurooncology Research, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, 5School of Life Sciences, Arizona State University, Tempe, 6Susavion Biosciences, Inc, Tempe, AZ, USAObjectives: Immunotherapy with immunostimulants is an attractive therapy against gliomas. C-type lectin receptors specific for galactose/N-acetylgalactosamine (GCLR regulate cellular differentiation, recognition, and trafficking of monocyte-derived cells. A peptide mimetic of GCLR ligands (GCLRP was used to activate blood monocytes and populations of myeloid-derived cells against a murine glioblastoma.Methods: The ability of GCLRP to stimulate phagocytosis by human microglia and monocyte-derived cells of the brain (MDCB isolated from a human glioblastoma was initially assessed in vitro. Induction of activation markers on blood monocytes was assayed by flow cytometry after administration of GCLRP to naive mice. C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells and were randomized for tumor size by magnetic resonance imaging, which was also used to assess increase in tumor size. Brain tumor tissues were analyzed using flow cytometry, histology, and enzyme-linked immunosorbent assay with respect to tumor, peritumoral area, and contralateral hemisphere regions.Results: GCLRP exhibited strong stimulatory effect on MDCBs and blood monocytes in vitro and in vivo. GCLRP was associated with an increased percentage of precursors of dendritic cells in the blood (P = 0.003, which differentiated into patrolling macrophages in tumoral (P = 0.001 and peritumoral areas (P = 0.04, rather than into dendritic cells

  7. The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

    DEFF Research Database (Denmark)

    Beatson, Richard; Maurstad, Gjertrud; Picco, Gianfranco


    that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC...... carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell......Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through...

  8. Identification of Enterococcus faecium and Enterococcus faecalis as vanC-type Vancomycin-Resistant Enterococci (VRE) from sewage and river water in the provincial city of Miyazaki, Japan. (United States)

    Nishiyama, Masateru; Iguchi, Atsushi; Suzuki, Yoshihiro


    As a first step for assessing the risk to human health posed by vancomycin-resistant enterococci (VRE) in the aquatic environment, we screened sewage and urban river water samples from Miyazaki, Japan for VRE. Because vancomycin-resistant organisms are not as prevalent in sewage and river water as vancomycin-susceptible organisms, the samples were screened by minimum inhibitory concentration test using the vancomycin-supplemented membrane-Enterococcus indoxyl-β-d-glucoside (mEI) agar. The isolates, presumed to be enterococci, were identified using 16S rRNA sequencing analysis. The percentages of VRE isolates screened using 4 μg mL(-1) vancomycin-supplemented mEI agar from sewage and urban river water samples were 12% and 24%, respectively. The vancomycin-resistant genes vanC1 and vanC2/3 were detected in the isolates from both samples by PCR analysis. All enterococci isolates containing vanC1, which is a specific gene for vanC-type of VRE, were identified as Enterococcus casseliflavus/gallinarum. Further, 92% enterococci isolates containing vanC2/3 were identified as E. casseliflavus/gallinarum, the remaining isolates containing vanC2/3 were E. faecium (4%) and E. faecalis (4%). Thereafter, the distribution of E. faecium and E. faecalis, which are the major types of enterococci in humans containing vanC2/3, was observed in the water samples collected.

  9. Combined Methylmalonic Aciduria and Homocystinuria cblC Type of a Taiwanese Infant With c.609G>A and c.567dupT Mutations in the MMACHC Gene

    Directory of Open Access Journals (Sweden)

    Jenn-Tzong Chang


    Full Text Available Combined methylmalonic aciduria and homocystinuria, cobalamin (cblC type (cblC disease, the most common inborn error of vitamin B12, is a rare disorder of intracellular cbl metabolism because of mutations in the MMACHC gene located in chromosome region 1p34.1. It has become possible to establish phenotype–genotype correlations and to observe ethnicity-related trends. This article provides detailed clinical manifestations and outcomes of a Taiwanese infant boy with early-onset cblC disease, heterozygous for c.609G>A and c.567dupT mutations, although there is limited information about cases with c.609G>A or c.567dupT mutation in the literature. He had no significant clinical abnormality during his neonatal period, whereas elevated C3 level was noted at newborn screening. He presented later with life-threatening manifestations and failure to thrive, which resolved through our treatment, although delayed development was still noted at 6 months of age. To date, all reported cblC patients with the c.609G>A mutation have been East Asians. Therefore, we suggest that c.609G>A should be included in the initial mutation screening tests for a cblC patient in East Asian populations.

  10. Whey Protein (United States)

    ... protein daily for 2 years does not improve bone density in postmenopausal women with osteoporosis. Weight loss. Most research suggests that taking whey protein alone, along with diet modifications, or while following an exercise plan does not seem to reduce weight for ...

  11. Protein Extractability

    African Journals Online (AJOL)

    limited to high oleic acid oil and water purification property (Katayon et al., 2006; Foid et al., 2001 and. Folkard et al., 1993), whereas it contains up to. 332.5 g of crude protein per kg of sample (Jose et al., 1999). Studies to characterize the interaction effects of pH and salts on the extraction of. PROTEIN EXTRACTABILITY ...

  12. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc


    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  13. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl


    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  14. Dietary Proteins (United States)

    ... because your body doesn't store it the way it stores fats or carbohydrates. How much you need depends on your age, sex, health, and level of physical activity. Most Americans eat enough protein in their diet.

  15. Protein Crystallization (United States)

    Chernov, Alexander A.


    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  16. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche


    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  17. Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

    Energy Technology Data Exchange (ETDEWEB)

    Westberg, Johan A., E-mail: [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Jiang, Ji, E-mail: [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Andersson, Leif C., E-mail: [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland)


    Highlights: {yields} Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. {yields} Central iron atom of heme and cysteine-114 of STC1 are essential for binding. {yields} STC1 binds Fe{sup 2+} and Fe{sup 3+} heme. {yields} STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys{sup 114} as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H{sub 2}O{sub 2} induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

  18. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H


    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  19. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan


    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  20. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H


    molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine...... membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  1. Interaction entropy for protein-protein binding (United States)

    Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.


    Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

  2. Learning about Proteins (United States)

    ... Videos for Educators Search English Español Learning About Proteins KidsHealth / For Kids / Learning About Proteins What's in ... from the foods you eat. Different Kinds of Protein Protein from animal sources, such as meat and ...

  3. Identification and characterization of a QM protein as a possible peptidoglycan recognition protein (PGRP) from the giant tiger shrimp Penaeus monodon. (United States)

    Udompetcharaporn, Attasit; Junkunlo, Kingkamon; Senapin, Saengchan; Roytrakul, Sittiruk; Flegel, Timothy W; Sritunyalucksana, Kallaya


    In an attempt to identify a peptidoglycan recognition protein (PGRP) in Penaeus (Penaeus) monodon, in vitro pull-down binding assays were used between shrimp proteins and purified peptidoglycan (PG). By gel electrophoresis and mass spectrometry followed by Mascot program analysis, proteins from shrimp hemocyte peripheral membrane proteins showed significant homology to records for a QM protein, actin and prophenoloxidase 2 precursor (proPO2), while proteins from cell-free plasma showed significant homology to records for a vitellogenin, a fibrinogen related protein (FREP) and a C-type lectin. Due to time and resource limitations, specific binding to PG was examined only for recombinant PmQM protein and PmLec that were synthesized based on sequences reported in the Genbank database (accession numbers FJ766846 and DQ078266, respectively). An in vitro assay revealed that hemocytes would bind with and encapsulate agarose beads coated with recombinant PmQM (rPmQM) or rPmLec and that melanization followed 2h post-encapsulation. ELISA tests confirmed specific binding of rPmQM protein to PG. This is the first time that PmQM has been reported as a potential PGRP in shrimp or any other crustacean. The two other potential PGRP identified (FREP and the vitellin-like protein present in male P. monodon, unlike other vitellin subunits) should also be expressed heterologously and tested for their ability to activate shrimp hemocytes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Efficient protein alignment algorithm for protein search. (United States)

    Lu, Zaixin; Zhao, Zhiyu; Fu, Bin


    Proteins show a great variety of 3D conformations, which can be used to infer their evolutionary relationship and to classify them into more general groups; therefore protein structure alignment algorithms are very helpful for protein biologists. However, an accurate alignment algorithm itself may be insufficient for effective discovering of structural relationships among tens of thousands of proteins. Due to the exponentially increasing amount of protein structural data, a fast and accurate structure alignment tool is necessary to access protein classification and protein similarity search; however, the complexity of current alignment algorithms are usually too high to make a fully alignment-based classification and search practical. We have developed an efficient protein pairwise alignment algorithm and applied it to our protein search tool, which aligns a query protein structure in the pairwise manner with all protein structures in the Protein Data Bank (PDB) to output similar protein structures. The algorithm can align hundreds of pairs of protein structures in one second. Given a protein structure, the tool efficiently discovers similar structures from tens of thousands of structures stored in the PDB always in 2 minutes in a single machine and 20 seconds in our cluster of 6 machines. The algorithm has been fully implemented and is accessible online at our webserver, which is supported by a cluster of computers. Our algorithm can work out hundreds of pairs of protein alignments in one second. Therefore, it is very suitable for protein search. Our experimental results show that it is more accurate than other well known protein search systems in finding proteins which are structurally similar at SCOP family and superfamily levels, and its speed is also competitive with those systems. In terms of the pairwise alignment performance, it is as good as some well known alignment algorithms.

  5. Small heat shock proteins, protein degradation and protein aggregation diseases

    NARCIS (Netherlands)

    Vos, Michel J.; Zijlstra, Marianne P.; Carra, Serena; Sibon, Ody C. M.; Kampinga, Harm H.

    Small heat shock proteins have been characterized in vitro as ATP-independent molecular chaperones that can prevent aggregation of un- or misfolded proteins and assist in their refolding with the help of ATP-dependent chaperone machines (e. g., the Hsp70 proteins). Comparison of the functionality of

  6. EDITORIAL: Precision proteins Precision proteins (United States)

    Demming, Anna


    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  7. N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence. (United States)

    Maurer-Stroh, Sebastian; Eisenhaber, Birgit; Eisenhaber, Frank


    biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana ( approximately 0.8 %). Copyright 2002 Elsevier Science Ltd.

  8. Human Antimicrobial Peptides and Proteins

    Directory of Open Access Journals (Sweden)

    Guangshun Wang


    Full Text Available As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32 can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized

  9. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin


    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  10. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.


    Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

  11. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  12. Evolution of protein-protein interactions

    Indian Academy of Sciences (India)

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  13. 24-hour urine protein (United States)

    Urine protein - 24 hour; Chronic kidney disease - urine protein; Kidney failure - urine protein ... Bladder tumor Heart failure High blood pressure during pregnancy ( preeclampsia ) Kidney disease caused by diabetes, high blood pressure, autoimmune disorders, ...

  14. Protein in diet (United States)

    Diet - protein ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a ... to eat animal products to get all the protein you need in your diet. Amino acids are ...

  15. Protein-losing enteropathy (United States)

    ... this page: // Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  16. Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila.

    Directory of Open Access Journals (Sweden)

    Toshio Shibata

    Full Text Available Transglutaminase (TG plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins--two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin-as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.

  17. Nanotechnologies in protein microarrays. (United States)

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene


    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.

  18. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry


    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  19. Bis-methionyl Coordination in the Crystal Structure of the Set up a citation RSS feed (Opens new window) Citation Feed

    Energy Technology Data Exchange (ETDEWEB)

    Aranda IV, Roman; Worley, Chad E.; Liu, Mengyao; Bitto, Eduard; Cates, M. Susan; Olson, John S.; Lei, Benfang; Phillips, Jr., George N. (UWM); (Rice); (Montana)


    Surface proteins Shr, Shp, and the ATP-binding cassette (ABC) transporter HtsABC are believed to make up the machinery for heme uptake in Streptococcus pyogenes. Shp transfers its heme to HtsA, the lipoprotein component of HtsABC, providing the only experimentally demonstrated example of direct heme transfer from a surface protein to an ABC transporter in Gram-positive bacteria. To understand the structural basis of heme transfer in this system, the heme-binding domain of Shp (Shp{sup 180}) was crystallized, and its structure determined to a resolution of 2.1 {angstrom}. Shp{sup 180} exhibits an immunoglobulin-like {beta}-sandwich fold that has been recently found in other pathogenic bacterial cell surface heme-binding proteins, suggesting that the mechanisms of heme acquisition are conserved. Shp shows minimal amino acid sequence identity to these heme-binding proteins and the structure of Shp{sup 180} reveals a unique heme-iron coordination with the axial ligands being two methionine residues from the same Shp molecule. A negative electrostatic surface of protein structure surrounding the heme pocket may serve as a docking interface for heme transfer from the more basic outer cell wall heme receptor protein Shr. The crystal structure of Shp{sup 180} reveals two exogenous, weakly bound hemins, which form a large interface between the two Shp{sup 180} molecules in the asymmetric unit. These 'extra' hemins form a stacked pair with a structure similar to that observed previously for free hemin dimers in aqueous solution. The propionates of the protein-bound heme coordinate to the iron atoms of the exogenous hemin dimer, contributing to the stability of the protein interface. Gel filtration and analytical ultracentrifugation studies indicate that both full-length Shp and Shp{sup 180} are monomeric in dilute aqueous solution.

  20. Protein- protein interaction detection system using fluorescent protein microdomains (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie


    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  1. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins. (United States)

    Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S


    We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

  2. Pregnancy and pregnancy outcome in hepatitis C type 1b.

    LENUS (Irish Health Repository)

    Jabeen, T


    A large cohort of rhesus-negative women in Ireland were inadvertently infected with hepatitis C virus following exposure to contaminated anti-D immunoglobulin in 1977-8. This major iatrogenic episode was discovered in 1994. We studied 36 women who had been infected after their first pregnancy, and compared them to an age- and parity-matched control group of rhesus-positive women. The presence of hepatitis C antibody was confirmed in all 36 by enzyme-linked immunosorbent assay and by recombinant immunoblot assay, while 26 (72%) of the cohort were HCV-RNA-positive (type 1b) on PCR testing. In the 20 years post-infection, all members of the study group had at least one pregnancy, and mean parity was 3.5. They had a total of 100 pregnancies and 85 of these went to term. There were four premature births, one being a twin pregnancy, and 11 spontaneous miscarriages. One miscarriage occurred in the pregnancy following HCV infection. There were two neonatal deaths due to severe congenital abnormalities in the PCR-positive women. Of the children born to HCV-RNA positive mothers, only one (2.3%) tested positive for the virus. Significant portal fibrosis on liver biopsy was confined to HCV-RNA-positive mothers apart from one single exception in the antibody-positive HCV-RNA-negative group. Comparison with the control group showed no increase in spontaneous miscarriage rate, and no significant difference in obstetric complications; birth weights were similar for the two groups.

  3. C-type period-doubling transition in nephron autoregulation

    DEFF Research Database (Denmark)

    Laugesen, Jakob Lund; Mosekilde, Erik; Holstein-Rathlou, Niels-Henrik


    -doubling bifurcations, mode-locking and other nonlinear dynamic phenomena in the tubular pressures and flows. Using a simplified nephron model, the paper examines how the regulatory mechanisms react to an external periodic variation in arterial pressure near a region of resonance with one of the internally generated...... mode-locked cycles. We show how the stable and unstable resonance cycles generated in this response undergo interconnected cascades of period-doubling bifurcations and how each period doubling leads to the formation of a new pair of saddle-node bifurcation curves along the edges of the resonance zone...

  4. Mosquito C-type lectins maintain gut microbiome homeostasis


    Pang, Xiaojing; Xiao, Xiaoping; Liu, Yang; Zhang, Rudian; Liu, Jianying; Liu, Qiyong; Wang, Penghua; Cheng, Gong


    The long-term evolutionary interaction between the host immune system and symbiotic bacteria determines their cooperative rather than antagonistic relationship. It is known that commensal bacteria have evolved a number of mechanisms to manipulate the mammalian host immune system and maintain homeostasis. However, the strategies employed by the microbiome to overcome host immune responses in invertebrates still remain to be understood. Here, we report that the gut microbiome in mosquitoes util...

  5. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann


    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, whereas vertebrates contain two to four genes. In cnidarians, the gene appears to encode a secreted protein, but transmembrane isoforms of the protein have also evolved, and in many species, alternative splicing facilitates the expression of both transmembrane and secreted isoforms. In most species......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...

  6. Peptide segments in protein-protein interfaces

    Indian Academy of Sciences (India)



    Sep 6, 2006 ... contact surface from the rest of the protein surface have been used to identify the interaction sites (Jones and Thornton. 1997; Neuvirth et al 2004). Protein antigenic sites (epitopes that are recognized by antibodies) could be generally confined to continuous motifs of about 8–24 amino acid residues, or may ...

  7. Surface Mediated Protein Disaggregation (United States)

    Radhakrishna, Mithun; Kumar, Sanat K.


    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  8. Physics of protein motility and motor proteins (United States)

    Kolomeisky, Anatoly B.


    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  9. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn


    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  10. Protein Data Bank (PDB) (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  11. Urine protein electrophoresis test (United States)

    Urine protein electrophoresis; UPEP; Multiple myeloma - UPEP; Waldenström macroglobulinemia - UPEP; Amyloidosis - UPEP ... special paper and apply an electric current. The proteins move and form visible bands. These reveal the ...

  12. Protein electrophoresis - serum (United States)

    ... this page: // Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  13. Statistical Properties of Protein-Protein Interfaces

    Directory of Open Access Journals (Sweden)

    Mihaly Mezei


    Full Text Available The properties of 1172 protein complexes (downloaded from the Protein Data Bank (PDB have been studied based on the concept of circular variance as a buriedness indicator and the concept of mutual proximity as a parameter-free definition of contact. The propensities of residues to be in the protein, on the surface or form contact, as well as residue pairs to form contact were calculated. In addition, the concept of circular variance has been used to compare the ruggedness and shape of the contact surface with the overall surface.

  14. Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi


    Taracena, Mabel L.; Oliveira, Pedro L.; Almendares, Olivia; Uma?a, Claudia; Lowenberger, Carl; Dotson, Ellen M.; Paiva-Silva, Gabriela O.; Pennington, Pamela M.


    Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all de...

  15. Destabilized bioluminescent proteins

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Michael S. (Knoxville, TN); Rakesh, Gupta (New Delhi, IN); Gary, Sayler S. (Blaine, TN)


    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  16. CSF total protein (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  17. Protein - Which is Best? (United States)

    Hoffman, Jay R; Falvo, Michael J


    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  18. Antimicrobial proteins : from old proteins, new tricks


    Smith, Val; Dyrynda, Elisabeth


    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. Included in the review are proteins or protein fragments ...

  19. Protein utilization in correlation to protein intake. (United States)

    Krajcovicová, M; Dibák, O


    In a 14-day experiment, weaned and adult rats were given ad libitum isocaloric diets with a mounting casein content (5, 10, 15, 25 and 40% by weight) and growth parameters of protein biological value, PER and NPR, and the utilization parameters NPU (body protein) and LPU (liver protein) were determined together with phosphoenolpyruvate carboxykinase (gluconeogenetic enzyme) and pyruvate kinase (glycolytic enzyme) activity in the animals' liver. The decrease in all the biological value parameters in weaned rats on 25% and 40% casein diets and in adult rats on 15%, 25% and 40% casein diets shows that these concentrations are too high for the organism. The decrease in PER and diminished weight and body and liver nitrogen increments in both age groups in animals with a low protein intake is evidence that 5% casein is an inadequate concentration. The optimum diet for weaned rats is thus a 15% casein diet and for adult rats a 10% casein diet, as confirmed by the linear correlation between weight increments, body and liver nitrogen and protein intake and also by gluconeogenetic enzyme activity. Under the given experimental conditions the study is a contribution to the determination of optimum physiological doses of proteins.

  20. Highly thermostable fluorescent proteins (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM


    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  1. Protein Function Prediction. (United States)

    Cruz, Leonardo Magalhães; Trefflich, Sheyla; Weiss, Vinícius Almir; Castro, Mauro Antônio Alves


    Protein function is a concept that can have different interpretations in different biological contexts, and the number and diversity of novel proteins identified by large-scale "omics" technologies poses increasingly new challenges. In this review we explore current strategies used to predict protein function focused on high-throughput sequence analysis, as for example, inference based on sequence similarity, sequence composition, structure, and protein-protein interaction. Various prediction strategies are discussed together with illustrative workflows highlighting the use of some benchmark tools and knowledge bases in the field.

  2. Highly thermostable fluorescent proteins (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM


    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  3. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan


    and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function....... Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides...

  4. Pigment-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Siegelman, H W


    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  5. Protein solubility modeling (United States)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.


    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  6. Packing in protein cores (United States)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.


    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  7. Expressed protein ligation for a large dimeric protein

    NARCIS (Netherlands)

    Karagöz, G.E.; Sinnige, T; Hsieh, O.; Rüdiger, S.G.D.


    Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic

  8. Toxic proteins in plants. (United States)

    Dang, Liuyi; Van Damme, Els J M


    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.


    Directory of Open Access Journals (Sweden)

    Michael J. Falvo


    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  10. Analysis of the hybrid proline-rich protein families from seven plant species suggests rapid diversification of their sequences and expression patterns

    Directory of Open Access Journals (Sweden)

    Fischer Lukáš


    Full Text Available Abstract Background Plant hybrid proline-rich proteins (HyPRPs are putative cell wall proteins consisting, usually, of a repetitive proline-rich (PR N-terminal domain and a conserved eight-cysteine motif (8 CM C-terminal domain. Understanding the evolutionary dynamics of HyPRPs might provide not only insight into their so far elusive function, but also a model for other large protein families in plants. Results We have performed a phylogenetic analysis of HyPRPs from seven plant species, including representatives of gymnosperms and both monocot and dicot angiosperms. Every species studied possesses a large family of 14–52 HyPRPs. Angiosperm HyPRPs exhibit signs of recent major diversification involving, at least in Arabidopsis and rice, several independent tandem gene multiplications. A distinct subfamily of relatively well-conserved C-type HyPRPs, often with long hydrophobic PR domains, has been identified. In most of gymnosperm (pine HyPRPs, diversity appears within the C-type group while angiosperms have only a few of well-conserved C-type representatives. Atypical (glycine-rich or extremely short N-terminal domains apparently evolved independently in multiple lineages of the HyPRP family, possibly via inversion or loss of sequences encoding proline-rich domains. Expression profiles of potato and Arabidopsis HyPRP genes exhibit instances of both overlapping and complementary organ distribution. The diversified non-C-type HyPRP genes from recently amplified chromosomal clusters in Arabidopsis often share their specialized expression profiles. C-type genes have broader expression patterns in both species (potato and Arabidopsis, although orthologous genes exhibit some differences. Conclusion HyPRPs represent a dynamically evolving protein family apparently unique to seed plants. We suggest that ancestral HyPRPs with long proline-rich domains produced the current diversity through ongoing gene duplications accompanied by shortening

  11. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)



    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  12. Protein flexibility as a biosignal. (United States)

    Zhao, Qinyi


    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  13. Supramolecular Chemistry Targeting Proteins. (United States)

    van Dun, Sam; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc


    The specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.

  14. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  15. [Erythrocyte membrane proteins]. (United States)

    Delaunay, J


    Proteins are important constituents of the red blood cell plasma membrane. Several important breakthroughs have occurred in their analysis over the past few years. SDS-polyacrylamide gel electrophoresis lead to the separation of the major proteins and glycoproteins. Location of most of these proteins -- either on the external, the internal or both surfaces of the membrane -- was determined. The strenght of the binding of the protein to the membrane was established. Hydrophobicity of membrane proteins has so far hindered their purification. However, the major glycoprotein (glycophorin A) was isolated and recently sequenced. The description of several membrane-associated enzyme activities has been followed by some understanding of their specific role in the red blood cell physiology. Abnormalities of glycoproteins, Ca2+-ATPase and of membrane protein phosphorylation have been reported under various conditions: sickle cell disease, hereditary spherocytoses, progressive muscular dystrophy.

  16. Algorithms for protein design. (United States)

    Gainza, Pablo; Nisonoff, Hunter M; Donald, Bruce R


    Computational structure-based protein design programs are becoming an increasingly important tool in molecular biology. These programs compute protein sequences that are predicted to fold to a target structure and perform a desired function. The success of a program's predictions largely relies on two components: first, the input biophysical model, and second, the algorithm that computes the best sequence(s) and structure(s) according to the biophysical model. Improving both the model and the algorithm in tandem is essential to improving the success rate of current programs, and here we review recent developments in algorithms for protein design, emphasizing how novel algorithms enable the use of more accurate biophysical models. We conclude with a list of algorithmic challenges in computational protein design that we believe will be especially important for the design of therapeutic proteins and protein assemblies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Mayaro virus proteins

    Directory of Open Access Journals (Sweden)

    J. M. S. Mezencio


    Full Text Available Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%. The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied.

  18. Using amino acid correlation and community detection algorithms to identify functional determinants in protein families.

    Directory of Open Access Journals (Sweden)

    Lucas Bleicher

    Full Text Available Correlated mutation analysis has a long history of interesting applications, mostly in the detection of contact pairs in protein structures. Based on previous observations that, if properly assessed, amino acid correlation data can also provide insights about functional sub-classes in a protein family, we provide a complete framework devoted to this purpose. An amino acid specific correlation measure is proposed, which can be used to build networks summarizing all correlation and anti-correlation patterns in a protein family. These networks can be submitted to community structure detection algorithms, resulting in subsets of correlated amino acids which can be further assessed by specific parameters and procedures that provide insight into the relationship between different communities, the individual importance of community members and the adherence of a given amino acid sequence to a given community. By applying this framework to three protein families with contrasting characteristics (the Fe/Mn-superoxide dismutases, the peroxidase-catalase family and the C-type lysozyme/α-lactalbumin family, we show how our method and the proposed parameters and procedures are related to biological characteristics observed in these protein families, highlighting their potential use in protein characterization and gene annotation.

  19. The LIM-only protein FHL2 attenuates lung inflammation during bleomycin-induced fibrosis.

    Directory of Open Access Journals (Sweden)

    Abdulaleem Alnajar

    Full Text Available Fibrogenesis is usually initiated when regenerative processes have failed and/or chronic inflammation occurs. It is characterised by the activation of tissue fibroblasts and dysregulated synthesis of extracellular matrix proteins. FHL2 (four-and-a-half LIM domain protein 2 is a scaffolding protein that interacts with numerous cellular proteins, regulating signalling cascades and gene transcription. It is involved in tissue remodelling and tumour progression. Recent data suggest that FHL2 might support fibrogenesis by maintaining the transcriptional expression of alpha smooth muscle actin and the excessive synthesis and assembly of matrix proteins in activated fibroblasts. Here, we present evidence that FHL2 does not promote bleomycin-induced lung fibrosis, but rather suppresses this process by attenuating lung inflammation. Loss of FHL2 results in increased expression of the pro-inflammatory matrix protein tenascin C and downregulation of the macrophage activating C-type lectin receptor DC-SIGN. Consequently, FHL2 knockout mice developed a severe and long-lasting lung pathology following bleomycin administration due to enhanced expression of tenascin C and impaired activation of inflammation-resolving macrophages.

  20. Pressure cryocooling protein crystals (United States)

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY


    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  1. Protein carbonylation in plants

    DEFF Research Database (Denmark)

    Møller, Ian Max; Havelund, Jesper; Rogowska-Wrzesinska, Adelina


    This chapter provides an overview of the current knowledge on protein carbonylation in plants and its role in plant physiology. It starts with a brief outline of the turnover and production sites of reactive oxygen species (ROS) in plants and the causes of protein carbonylation. This is followed...... by a description of the methods used to study protein carbonylation in plants, which is also very brief as the methods are similar to those used in studies on animals. The chapter also focuses on protein carbonylation in plants in general and in mitochondria and in seeds in particular, as case stories where...

  2. Engineering therapeutic protein disaggregases. (United States)

    Shorter, James


    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. © 2016 Shorter. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (

  3. Modular protein domains

    National Research Council Canada - National Science Library

    Cesareni, Giovanni


    ... encodes not only sequence, but somehow explicitly specifies folding, structure, and biological function as well. How, then, can one learn to read this 'language of proteins'? One of the most powerful approaches to 'cracking the protein code' has involved sequence comparisons between and within species, a task now greatly simplified by the ever...

  4. Advances in Protein Precipitation

    NARCIS (Netherlands)

    Golubovic, M.


    Proteins are biological macromolecules, which are among the key components of all living organisms. Proteins are nowadays present in all fields of biotech industry, such as food and feed, synthetic and pharmaceutical industry. They are isolated from their natural sources or produced in different

  5. Amino acids and proteins

    NARCIS (Netherlands)

    van Goudoever, Johannes B.; Vlaardingerbroek, Hester; van den Akker, Chris H.; de Groof, Femke; van der Schoor, Sophie R. D.


    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional

  6. Poxviral Ankyrin Proteins

    Directory of Open Access Journals (Sweden)

    Michael H. Herbert


    Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

  7. Multidomain proteins under force. (United States)

    Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel


    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  8. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either ...

  9. Stability of Hyperthermophilic Proteins

    DEFF Research Database (Denmark)

    Stiefler-Jensen, Daniel

    in the high stability of hyperthermophilic enzymes. The thesis starts with an introduction to the field of protein and enzyme stability with special focus on the thermophilic and hyperthermophilic enzymes and proteins. After the introduction three original research manuscripts present the experimental data...

  10. Protein expression-yeast. (United States)

    Nielsen, Klaus H


    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline. © 2014 Elsevier Inc. All rights reserved.

  11. MicroProteins

    DEFF Research Database (Denmark)

    Eguen, Teinai Ebimienere; Straub, Daniel; Graeff, Moritz


    MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining characterist......MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining...... can extend beyond transcription factors (TFs) to encompass different non-TF proteins that require dimerization for full function....

  12. Protein disulfide engineering. (United States)

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B


    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Artificially Engineered Protein Polymers. (United States)

    Yang, Yun Jung; Holmberg, Angela L; Olsen, Bradley D


    Modern polymer science increasingly requires precise control over macromolecular structure and properties for engineering advanced materials and biomedical systems. The application of biological processes to design and synthesize artificial protein polymers offers a means for furthering macromolecular tunability, enabling polymers with dispersities of ∼1.0 and monomer-level sequence control. Taking inspiration from materials evolved in nature, scientists have created modular building blocks with simplified monomer sequences that replicate the function of natural systems. The corresponding protein engineering toolbox has enabled the systematic development of complex functional polymeric materials across areas as diverse as adhesives, responsive polymers, and medical materials. This review discusses the natural proteins that have inspired the development of key building blocks for protein polymer engineering and the function of these elements in material design. The prospects and progress for scalable commercialization of protein polymers are reviewed, discussing both technology needs and opportunities.

  14. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott


    scientists from academia, government, and industry participated in the symposium. Experts provided overviews on known mechanisms by which proteins in food may cause sensitization, discussed experimental models to predict protein sensitizing potential, and explored whether such experimental techniques may......The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  15. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott


    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  16. [Controversies around diet proteins]. (United States)

    Cichosz, Grazyna; Czeczot, Hanna


    Critical theories regarding proteins of anima origin are still and still popularized, though they are ungrounded from scientific point of view. Predominance of soya proteins over the animal ones in relation to their influence on calcium metabolism, bone break risk or risk of osteoporosis morbidity has not been confirmed in any honest, reliable research experiment. Statement, that sulphur amino acids influence disadvantageously on calcium metabolism of human organism and bone status, is completely groundless, the more so as presence of sulphur amino acids in diet (animal proteins are their best source) is the condition of endogenic synthesis of glutathione, the key antioxidant of the organism, and taurine stimulating brain functioning. Deficiency of proteins in the diet produce weakness of intellectual effectiveness and immune response. There is no doubt that limitation of consumption of animal proteins of standard value is not good for health.

  17. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.


    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  18. Swaps in protein sequences. (United States)

    Fliess, Amit; Motro, Benny; Unger, Ron


    An important question in protein evolution is to what extent proteins may have undergone swaps (switches of domain or fragment order) during evolution. Such events might have occurred in several forms: Swaps of short fragments, swaps of structural and functional motifs, or recombination of domains in multidomain proteins. This question is important for the theoretical understanding of the evolution of proteins, and has practical implications for using swaps as a design tool in protein engineering. In order to analyze the question systematically, we conducted a large scale survey of possible swaps and permutations among all pairs of protein from the Swissport database. A swap is defined as a specific kind of sequence mutation between two proteins in which two fragments that appear in both sequences have different relative order in the two sequences. For example, aXbYc and dYeXf are defined as a swap, where X and Y represent sequence fragments that switched their order. Identifying such swaps is difficult using standard sequence comparison packages. One of the main problems in the analysis stems from the fact that many sequences contain repeats, which may be identified as false-positive swaps. We have used two different approaches to detect pairs of proteins with swaps. The first approach is based on the predefined list of domains in Pfam. We identified all the proteins that share at least two domains and analyzed their relative order, looking for pairs in which the order of these domains was switched. We designed an algorithm to distinguish between real swaps and duplications. In the second approach, we used Blast to detect pairs of proteins that share several fragments. Then, we used an automatic procedure to select pairs that are likely to contain swaps. Those pairs were analyzed visually, using a graphical tool, to eliminate duplications. Combining these approaches, about 140 different cases of swaps in the Swissprot database were found (after eliminating

  19. Anchored design of protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Steven M Lewis

    Full Text Available Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders.Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space.This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.

  20. Antimicrobial proteins: From old proteins, new tricks. (United States)

    Smith, Valerie J; Dyrynda, Elisabeth A


    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Multidomain proteins under force (United States)

    Valle-Orero, Jessica; Andrés Rivas-Pardo, Jaime; Popa, Ionel


    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91—two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins—ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  2. Protein oxidation in aquatic foods

    DEFF Research Database (Denmark)

    Baron, Caroline P.


    The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout......, and the second with oxidation in salted herring. The mechanisms responsible for initiation of protein oxidation are unclear, but it is generally accepted that free radical species initiating lipid oxidation can also initiate protein oxidation. The chapter focuses on interaction between protein and lipid...... oxidation. The protein carbonyl group measurement is the widely used method for estimating protein oxidation in foods and has been used in fish muscle. The chapter also talks about the impact of protein oxidation on protein functionality, fish muscle texture, and food nutritional value. Protein oxidation...

  3. Sound of proteins

    DEFF Research Database (Denmark)


    In my group we work with Molecular Dynamics to model several different proteins and protein systems. We submit our modelled molecules to changes in temperature, changes in solvent composition and even external pulling forces. To analyze our simulation results we have so far used visual inspection...... and statistical analysis of the resulting molecular trajectories (as everybody else!). However, recently I started assigning a particular sound frequency to each amino acid in the protein, and by setting the amplitude of each frequency according to the movement amplitude we can "hear" whenever two aminoacids...

  4. PDP: protein domain parser. (United States)

    Alexandrov, Nickolai; Shindyalov, Ilya


    We have developed a program for automatic identification of domains in protein three-dimensional structures. Performance of the program was assessed by three different benchmarks: (i) by comparison with the expert-curated SCOP database of structural domains; (ii) by comparison with a collection of manual domain assignments; and (iii) by comparison with a set of 55 proteins, frequently used as a benchmark for automatic domain assignment. In all these benchmarks PDP identified domains correctly in more than 80% of proteins.

  5. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter


    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  6. Designing microcapsules based on protein fibrils and protein - polysaccharide complexes

    NARCIS (Netherlands)

    Hua, K.N.P.


    Keywords: encapsulation, microcapsule, protein, fibril, protein-polysaccharide complex, controlled release, interfacial rheology, lysozyme, ovalbumin This thesis describes the design of encapsulation systems using mesostructures from proteins and polysaccharides. The approach was to first

  7. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Ritu Rawal

    Full Text Available In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite

  8. Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni. (United States)

    Wu, Xiao-Jun; Dinguirard, Nathalie; Sabat, Grzegorz; Lui, Hong-di; Gonzalez, Laura; Gehring, Michael; Bickham-Wright, Utibe; Yoshino, Timothy P


    Interactions between early developing Schistosoma mansoni larval stages and the hemolymph of its snail intermediate host represent the first molecular encounter with the snail's immune system. To gain a more comprehensive understanding of this early parasite-host interaction, biotinylated sporocyst tegumental membrane (Mem) proteins and larval transformation proteins (LTP) were affixed to streptavidin-agarose beads and used as affinity matrices to enrich for larval-reactive plasma proteins from susceptible (NMRI) and resistant (BS-90) strains of the snail Biomphalaria glabrata. Nano-LC/MS-MS proteomic analyses of isolated plasma proteins revealed a diverse array of 94 immune-and nonimmune-related plasma proteins. Included among the immune-related subset were pattern recognition receptors (lectins, LPS-binding protein, thioester-containing proteins-TEPs), stress proteins (HSP60 and 70), adhesion proteins (dermatopontins), metalloproteases (A Disintegrin And Metalloproteinase (ADAM), ADAM-related Zn proteinases), cytotoxins (biomphalysin) and a Ca2+-binding protein (neo-calmodulin). Variable immunoglobulin and lectin domain (VIgL) gene family members, including fibrinogen-related proteins (FREPs), galectin-related proteins (GREPs) and C-type lectin-related proteins (CREPs), were the most prevalent of larval-reactive immune lectins present in plasma. FREPs were highly represented, although only a subset of FREP subfamilies (FREP 2, 3 and 12) were identified, suggesting potential selectivity in the repertoire of plasma lectins recognizing larval glycoconjugates. Other larval-binding FREP-like and CREP-like proteins possessing a C-terminal fibrinogen-related domain (FReD) or C-type lectin binding domain, respectively, and an Ig-fold domain also were identified as predicted proteins from the B. glabrata genome, although incomplete sequence data precluded their placement into specific FREP/CREP subfamilies. Similarly, a group of FReD-containing proteins (angiopoeitin-4

  9. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut


    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  10. Electron transfer in proteins

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I


    Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkable...... specificity. The electron transfer is attained through weak electronic interaction between the active sites, so that considerable research efforts are centered on resolving the factors that control the rates of long-distance electron transfer reactions in proteins. These factors include (in addition......-containing proteins. These proteins serve almost exclusively in electron transfer reactions, and as it turns out, their metal coordination sites are endowed with properties uniquely optimized for their function....

  11. Protein Colloidal Aggregation Project (United States)

    Oliva-Buisson, Yvette J. (Compiler)


    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  12. Interactive protein manipulation

    Energy Technology Data Exchange (ETDEWEB)


    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  13. Parallel Computational Protein Design. (United States)

    Zhou, Yichao; Donald, Bruce R; Zeng, Jianyang


    Computational structure-based protein design (CSPD) is an important problem in computational biology, which aims to design or improve a prescribed protein function based on a protein structure template. It provides a practical tool for real-world protein engineering applications. A popular CSPD method that guarantees to find the global minimum energy solution (GMEC) is to combine both dead-end elimination (DEE) and A* tree search algorithms. However, in this framework, the A* search algorithm can run in exponential time in the worst case, which may become the computation bottleneck of large-scale computational protein design process. To address this issue, we extend and add a new module to the OSPREY program that was previously developed in the Donald lab (Gainza et al., Methods Enzymol 523:87, 2013) to implement a GPU-based massively parallel A* algorithm for improving protein design pipeline. By exploiting the modern GPU computational framework and optimizing the computation of the heuristic function for A* search, our new program, called gOSPREY, can provide up to four orders of magnitude speedups in large protein design cases with a small memory overhead comparing to the traditional A* search algorithm implementation, while still guaranteeing the optimality. In addition, gOSPREY can be configured to run in a bounded-memory mode to tackle the problems in which the conformation space is too large and the global optimal solution cannot be computed previously. Furthermore, the GPU-based A* algorithm implemented in the gOSPREY program can be combined with the state-of-the-art rotamer pruning algorithms such as iMinDEE (Gainza et al., PLoS Comput Biol 8:e1002335, 2012) and DEEPer (Hallen et al., Proteins 81:18-39, 2013) to also consider continuous backbone and side-chain flexibility.

  14. Protein Nitrogen Determination (United States)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  15. Disease specific protein corona (United States)

    Rahman, M.; Mahmoudi, M.


    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  16. Fast protein folding kinetics (United States)

    Gelman, Hannah; Gruebele, Martin


    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  17. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.


    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  18. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules RSAD2 CIG5 Radical S-adenosyl methionine domain-containing protein 2 Cytomegalo...virus-induced gene 5 protein, Viperin, Virus inhibitory protein, endoplasmic reticu

  19. Protein hydrolysates in sports nutrition

    Directory of Open Access Journals (Sweden)

    Manninen Anssi H


    Full Text Available Abstract It has been suggested that protein hydrolysates providing mainly di- and tripeptides are superior to intact (whole proteins and free amino acids in terms of skeletal muscle protein anabolism. This review provides a critical examination of protein hydrolysate studies conducted in healthy humans with special reference to sports nutrition. The effects of protein hydrolysate ingestion on blood amino acid levels, muscle protein anabolism, body composition, exercise performance and muscle glycogen resynthesis are discussed.


    Directory of Open Access Journals (Sweden)

    J.C.Q. Carvalho


    Full Text Available The theoretical explanation of biological concepts, associated with the use of teaching games andmodels, intensify the comprehension and increase students interest, stimulating them to participateactively on the teaching-learning process. The sta of dissemination from Centro de BiotecnologiaMolecular Estrutural (CBME, in partnership with the Centro de Divulgac~ao Cientca e Cultural(CDCC, presents, in this work, a new educational resource denoted: Protein Synthesis Game. Theapproach of the game involves the cytological aspects of protein synthesis, directed to high schoolstudents. Students are presented to day-by-day facts related to the function of a given protein in thehuman body. Such task leads players to the goal of solving out a problem through synthesizing aspecied protein. The game comprises: (1 a board illustrated with the transversal section of animalcell, with its main structures and organelles and sequences of hypothetical genes; (2 cards with thedescription of steps and other structures required for protein synthesis in eukaryotic cells; (3 piecesrepresenting nucleotides, polynucleotides, ribosome, amino acids, and polypeptide chains. In order toplay the game, students take cards that sequentially permit them to acquire the necessary pieces forproduction of the protein described in each objective. Players must move the pieces on the board andsimulate the steps of protein synthesis. The dynamic of the game allows students to easily comprehendprocesses of transcription and translation. This game was presented to dierent groups of high schoolteachers and students. Their judgments have been heard and indicated points to be improved, whichhelped us with the game development. Furthermore, the opinions colleted were always favorable forthe application of this game as a teaching resource in classrooms.

  1. Bioinformatics and moonlighting proteins

    Directory of Open Access Journals (Sweden)

    Sergio eHernández


    Full Text Available Multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. Usually, moonlighting proteins are experimentally revealed by serendipity. For this reason, it would be helpful that Bioinformatics could predict this multifunctionality, especially because of the large amounts of sequences from genome projects. In the present work, we analyse and describe several approaches that use sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. Among these approaches are: a remote homology searches using Psi-Blast, b detection of functional motifs and domains, c analysis of data from protein-protein interaction databases (PPIs, d match the query protein sequence to 3D databases (i.e., algorithms as PISITE, e mutation correlation analysis between amino acids by algorithms as MISTIC. Programs designed to identify functional motif/domains detect mainly the canonical function but usually fail in the detection of the moonlighting one, Pfam and ProDom being the best methods. Remote homology search by Psi-Blast combined with data from interactomics databases (PPIs have the best performance. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can only be used in very specific situations –it requires the existence of multialigned family protein sequences - but can suggest how the evolutionary process of second function acquisition took place. The multitasking protein database MultitaskProtDB (, previously published by our group, has been used as a benchmark for the all of the analyses.

  2. Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins. (United States)

    Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo


    Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

  3. Molecular diversity in venom proteins of the Russell's viper (Daboia russellii russellii) and the Indian cobra (Naja naja) in Sri Lanka. (United States)

    Suzuki, Mieko; Itoh, Takeshi; Anuruddhe, B M; Bandaranayake, I K; Shirani Ranasinghe, J G; Athauda, Seranath B P; Moriyama, Akihiko


    To examine the molecular diversity of the venom proteins of the Russell's viper (Daboia russellii russellii) and the Indian cobra (Naja naja) in Sri Lanka, we isolated 38 venom proteins through a combination of anion exchange chromatography followed by reversed-phase high performance liquid chromatography. From the venom of D. r. russellii we isolated 15 proteins: 5 isozymes of phospholipase A(2) (PLA(2)), 4 serine proteases, 2 C-type lectin-like proteins, 2 L-amino acid oxidases, 1 cysteine-rich secretory protein (CRISP), and 1 metalloproteinase. From the venom of N. naja we isolated 23 proteins: 10 isoforms of cytotoxins (CTX), 7 PLA(2) isozymes, 2 muscarinic toxinlike proteins, 2 CRISPs, 1 nerve growth factor, and 1 new thrombin-like serine protease. Most of these proteins contained new amino acid sequences for each species, indicating molecular diversity in venom proteins. The entire amino acid sequences of PLA(2)3 from D. r. russellii and CTX7 from N. naja were determined. Additionally, the polymorphic amino acid residues of PLA(2)3 were preferentially localized on the potential antigenic sites. While 2 types of PLA(2) (N and S types) were found in D. r. russellii (India) and D. r. siamensis (Java), all the PLA(2)s from D. r. siamensis (Burma) were N type, and those from D. r. russellii (Sri Lanka) were primarily S type.

  4. Benchtop Detection of Proteins (United States)

    Scardelletti, Maximilian C.; Varaljay, Vanessa


    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  5. Self-Assembling Protein Microarrays (United States)

    Ramachandran, Niroshan; Hainsworth, Eugenie; Bhullar, Bhupinder; Eisenstein, Samuel; Rosen, Benjamin; Lau, Albert Y.; C. Walter, Johannes; LaBaer, Joshua


    Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

  6. Changes in protein composition and protein phosphorylation during ...

    African Journals Online (AJOL)

    Changes in protein profiles and protein phosphorylation were studied in various stages of germinating somatic and zygotic embryos. Many proteins, which were expressed in cotyledonary stage somatic embryos, were also present in the zygotic embryos obtained from mature dry seed. The intensity of 22 kDa protein was ...

  7. Electrochemical nanomoulding through proteins (United States)

    Allred, Daniel B.

    The continued improvements in performance of modern electronic devices are directly related to the manufacturing of smaller, denser features on surfaces. Electrochemical fabrication has played a large role in continuing this trend due to its low cost and ease of scaleability toward ever smaller dimensions. This work introduces the concept of using proteins, essentially monodisperse complex polymers whose three-dimensional structures are fixed by their encoded amino acid sequences, as "moulds" around which nanostructures can be built by electrochemical fabrication. Bacterial cell-surface layer proteins, or "S-layer" proteins, from two organisms---Deinococcus radiodurans and Sporosarcina ureae---were used as the "moulds" for electrochemical fabrication. The proteins are easily purified as micron-sized sheets of periodic molecular complexes with 18-nm hexagonal and 13-nm square unit cell lattices, respectively. Direct imaging by transmission electron microscopy on ultrathin noble metal films without sample preparation eliminates potential artifacts to the high surface energy substrates necessary for high nucleation densities. Characterization involved imaging, electron diffraction, spectroscopy, and three-dimensional reconstruction. The S-layer protein of D. radiodurans was further subjected to an atomic force microscope based assay to determine the integrity of its structure and long-range order and was found to be useful for fabrication from around pH 3 to 12.

  8. Protein Denaturation in Foam. (United States)

    Clarkson; Cui; Darton


    The aim of this study was to elucidate the mechanism by which protein molecules become denatured in foam. It was found that damage to the protein is mainly due to surface denaturation at the gas-liquid interface. A fraction of the molecules adsorbed do not refold to their native state when they desorb. The degree of denaturation was found to correlate directly with the interfacial exposure, which, for mobile or partially mobile interfaces, is increased by drainage. Experiments with two different proteins showed that, under the conditions of the tests, around 10% of BSA molecules which had adsorbed at the surface remained denatured when they desorbed. For pepsin the figure was around 75%. Oxidation, which was previously thought to be a major cause of protein damage in foam, was found to be minimal. Neither do the high shear stresses in the liquid bulk encountered during bubble bursting cause denaturation, because energy is dissipated at a much greater length scale than that of the protein molecule. Copyright 1999 Academic Press.

  9. Protein (Cyanobacteria): 654346314 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)


  10. Protein (Cyanobacteria): 654344406 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)


  11. Polarizable protein packing

    KAUST Repository

    Ng, Albert H.


    To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full energy while maintaining tractability. We represent the polarizable packing problem for protein G as a hypergraph and solve for optimal rotamers with the FASTER combinatorial optimization algorithm. These approximate energy models can be improved to high accuracy [root mean square deviation (rmsd) < 1 kJ mol -1] via ridge regression. The resulting trained approximations are used to efficiently identify new, low-energy solutions. The approach is general and should allow combinatorial optimization of other many-body problems. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011 Copyright © 2011 Wiley Periodicals, Inc.

  12. Thermodynamics of Protein Aggregation (United States)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  13. Thermal hysteresis proteins. (United States)

    Barrett, J


    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  14. Accessory Proteins at ERES

    DEFF Research Database (Denmark)

    Klinkenberg, Rafael David

    proteins. Together these components co‐operate in cargo‐selection as well as forming, loading and releasing budding vesicles from specific regions on the membrane surface of the ER. Coat components furthermore convey vesicle targeting towards the Golgi. However, not much is known about the mechanisms...... that regulate the COPII assembly at the vesicle bud site. This thesis provides the first regulatory mechanism of COPII assembly in relation to ER‐membrane lipid‐signal recognition by the accessory protein p125A (Sec23IP). The aim of the project was to characterize p125A function by dissecting two main domains...... in the protein; a putative lipid‐associating domain termed the DDHD domain that is defined by the four amino acid motif that gives the domain its name; and a ubiquitously found domain termed Sterile α‐motif (SAM), which is mostly associated with oligomerization and polymerization. We first show, that the DDHD...

  15. Matricellular proteins and biomaterials. (United States)

    Morris, Aaron H; Kyriakides, Themis R


    Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  16. Trisulfides in Proteins

    DEFF Research Database (Denmark)

    Nielsen, Rasmus W.; Tachibana, Christine; Hansen, Niels Erik


    Trisulfides and other oligosulfides are widely distributed in the biological world. In plants, e.g., garlic, trisulfides are associated with potentially beneficial properties. However, an extra neutral sulfur atom covalently bound between the two sulfur atoms of a pair of cysteines is not a commo...... post-translational modification, and the number of proteins in which a trisulfide has been unambiguously identified is small. Nevertheless, we believe that its prevalence may be underestimated, particularly with the increasing evidence for significant pools of sulfides in living tissues...... and their possible roles in cellular metabolism. This review focuses on examples of proteins that are known to contain a trisulfide bridge, and gives an overview of the chemistry of trisulfide formation, and the methods by which it is detected in proteins....

  17. Epistasis in protein evolution (United States)

    Starr, Tyler N.


    Abstract The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions—called epistasis—within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage‐specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis—in which one mutation influences the phenotypic effect of few other mutations—is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low‐probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families. PMID:26833806

  18. The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization

    Directory of Open Access Journals (Sweden)

    Morton Daniel J


    Full Text Available Abstract Background Haemophilus influenzae requires a porphyrin source for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. This porphyrin requirement may be satisfied by either heme alone, or protoporphyrin IX in the presence of an iron source. One protein involved in heme acquisition by H. influenzae is the periplasmic heme binding protein HbpA. HbpA exhibits significant homology to the dipeptide and heme binding protein DppA of Escherichia coli. DppA is a component of the DppABCDF peptide-heme permease of E. coli. H. influenzae homologs of dppBCDF are located in the genome at a point distant from hbpA. The object of this study was to investigate the potential role of the H. influenzae dppBCDF locus in heme utilization. Findings An insertional mutation in dppC was constructed and the impact of the mutation on the utilization of both free heme and various proteinaceous heme sources as well as utilization of protoporphyrin IX was determined in growth curve studies. The dppC insertion mutant strain was significantly impacted in utilization of all tested heme sources and protoporphyin IX. Complementation of the dppC mutation with an intact dppCBDF gene cluster in trans corrected the growth defects seen in the dppC mutant strain. Conclusion The dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H. influenzae.

  19. Protein biosynthesis in mitochondria. (United States)

    Kuzmenko, A V; Levitskii, S A; Vinogradova, E N; Atkinson, G C; Hauryliuk, V; Zenkin, N; Kamenski, P A


    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

  20. Water-transporting proteins

    DEFF Research Database (Denmark)

    Zeuthen, Thomas


    Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein...... is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial...

  1. Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration. (United States)

    Simon, Jörg; Kern, Melanie


    Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

  2. Cold gelation of globular proteins

    NARCIS (Netherlands)

    Alting, A.C.


    Keywords : globular proteins, whey protein, ovalbumin, cold gelation, disulfide bonds, texture, gel hardnessProtein gelation in food products is important to obtain desirable sensory and textural properties. Cold gelation is a novel method to produce protein-based gels. It is a two step process in

  3. The Formation of Protein Structure

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren


    Dynamically induced curvature owing to long-range excitations along the backbones of protein molecules with non-linear elastic properties may control the folding of proteins.......Dynamically induced curvature owing to long-range excitations along the backbones of protein molecules with non-linear elastic properties may control the folding of proteins....

  4. A simple dependence between protein evolution rate and the number of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Hirsh Aaron E


    Full Text Available Abstract Background It has been shown for an evolutionarily distant genomic comparison that the number of protein-protein interactions a protein has correlates negatively with their rates of evolution. However, the generality of this observation has recently been challenged. Here we examine the problem using protein-protein interaction data from the yeast Saccharomyces cerevisiae and genome sequences from two other yeast species. Results In contrast to a previous study that used an incomplete set of protein-protein interactions, we observed a highly significant correlation between number of interactions and evolutionary distance to either Candida albicans or Schizosaccharomyces pombe. This study differs from the previous one in that it includes all known protein interactions from S. cerevisiae, and a larger set of protein evolutionary rates. In both evolutionary comparisons, a simple monotonic relationship was found across the entire range of the number of protein-protein interactions. In agreement with our earlier findings, this relationship cannot be explained by the fact that proteins with many interactions tend to be important to yeast. The generality of these correlations in other kingdoms of life unfortunately cannot be addressed at this time, due to the incompleteness of protein-protein interaction data from organisms other than S. cerevisiae. Conclusions Protein-protein interactions tend to slow the rate at which proteins evolve. This may be due to structural constraints that must be met to maintain interactions, but more work is needed to definitively establish the mechanism(s behind the correlations we have observed.

  5. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.


    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly...... affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among others, the following phenomena are discussed: interactions of interfacially adsorbed peptides, pore-forming amphipathic peptides, adsorption of charged proteins onto...... oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins....

  6. Protein degradation systems in platelets. (United States)

    Kraemer, B F; Weyrich, A S; Lindemann, S


    Protein synthesis and degradation are essential processes that allow cells to survive and adapt to their surrounding milieu. In nucleated cells, the degradation and/or cleavage of proteins is required to eliminate aberrant proteins. Cells also degrade proteins as a mechanism for cell signalling and complex cellular functions. Although the last decade has convincingly shown that platelets synthesise proteins, the roles of protein degradation in these anucleate cytoplasts are less clear. Here we review what is known about protein degradation in platelets placing particular emphasis on the proteasome and the cysteine protease calpain.

  7. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials

    NARCIS (Netherlands)

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; Duinkerken, Van Gert; Yu, Peiqiang


    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more

  8. Protein requirements of Penaeid shrimp.


    Kanazawa, A


    Proteins are indispensable nutrients for growth and maintenance of live of all animals. The optimum protein levels in diets for shrimps are different among the various species. Squid meal is an effective protein source for many penaeids. The effects of dietary protein, lipid, and carbohydrate levels on the growth and survival of larvae of Penaeus japonicus were examined by feeding trials using purified diet with carrageenan as a binder. As a result, the effects of protein levels on growth and...

  9. Protein oxidation and ageing

    DEFF Research Database (Denmark)

    Linton, S; Davies, Michael Jonathan; Dean, R T


    of redox-active metal ions that could catalyse oxidant formation. As a result of this decrease in antioxidant defences, and increased rate of ROS formation, it is possible that the impact of ROS increases with age. ROS are known to oxidise biological macromolecules, with proteins an important target...

  10. Thermodynamics of meat proteins

    NARCIS (Netherlands)

    Sman, van der R.G.M.


    We describe the water activity of meat, being a mixture of proteins, salts and water, by the Free-Volume-Flory–Huggins (FVFH) theory augmented with the equation. Earlier, the FVFH theory is successfully applied to describe the thermodynamics to glucose homopolymers like starch, dextrans and

  11. Protein digestion in ruminants

    African Journals Online (AJOL)

    Animal Nutrition, Animal and Dairy Science Research Institute, Irene, 1675Republic of South Africa. Although the protein requirement of domestic ruminants may be calculated from a simple one-compartment model, this approach ignores factors such as microbial fermentation in the rumen and the non-equality of feed.

  12. Protein Sorting Prediction

    DEFF Research Database (Denmark)

    Nielsen, Henrik


    Many computational methods are available for predicting protein sorting in bacteria. When comparing them, it is important to know that they can be grouped into three fundamentally different approaches: signal-based, global-property-based and homology-based prediction. In this chapter, the strengt...

  13. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

  14. Markers of protein oxidation

    DEFF Research Database (Denmark)

    Headlam, Henrietta A; Davies, Michael Jonathan


    Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed on...

  15. Protein digestion in ruminants

    African Journals Online (AJOL)

    acids absorbed into the circulation of the animal. Ideally, therefore, the biological value of a feed protein should be determined from the amount and type of amino acid appearing in the portal circulation of the animal, and not simplythe dissappearance of amino acids from the tract. Ruminant digestion may be more easily ...

  16. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: Keywords.

  17. NMR of unfolded proteins

    Indian Academy of Sciences (India)



    Jan 3, 2005 ... deposition of data and advanced search on the pattern of PDB.12. Detailed characterization of the unfolded state and consequent identification of the folding initiation sites in a given protein provide valuable insight into its folding mechanism.18 Well-formed or transient residual structures in the unfolded ...

  18. Protein Requirements during Aging

    Directory of Open Access Journals (Sweden)

    Glenda Courtney-Martin


    Full Text Available Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR and recommended dietary allowance (RDA, respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals.

  19. Protein: CAD [Trypanosomes Database

    Lifescience Database Archive (English)

    Full Text Available CAD carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotaseCAD... trifunctional proteincarbamoylphosphate synthetase 2/aspartate transcarbamylase/dihydroorotasemultifunctional protein CAD... H.sapiens 47458828 18105007 790 P27708 CAD_(gene)|| 114010 2p22-p21 hsa00250|hsa00240 ...

  20. Measuring protein breakdown in individual proteins in vivo

    DEFF Research Database (Denmark)

    Holm, Lars; Kjær, Michael


    be used to determine the breakdown rate of specific proteins and, therefore, do not keep up to the preceding methodological demands in physiological research. A newly developed approach to determine the fractional breakdown rate of single proteins seems promising. Its conceptual advantage......PURPOSE OF REVIEW: To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo. RECENT FINDINGS: None of the available methods for determining protein breakdown can...... is that the proteins of interest are the site of measurement. Hence, the application initially demands the proteins to be labeled with stable isotopically labeled amino acids. Subsequently, the loss of label from the proteins will be dependent on the protein breakdown rate when no labeled amino acids...

  1. Interaction between plate make and protein in protein crystallisation screening.

    Directory of Open Access Journals (Sweden)

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  2. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. (United States)

    Lee, Young-Ho; Stallcup, Michael R


    Endocrine regulation frequently culminates in altered transcription of specific genes. The signal transduction pathways, which transmit the endocrine signal from cell surface to the transcription machinery, often involve posttranslational modifications of proteins. Although phosphorylation has been by far the most widely studied protein modification, recent studies have indicated important roles for other types of modification, including protein arginine methylation. Ten different protein arginine methyltransferase (PRMT) family members have been identified in mammalian cells, and numerous substrates are being identified for these PRMTs. Whereas major attention has been focused on the methylation of histones and its role in chromatin remodeling and transcriptional regulation, there are many nonhistone substrates methylated by PRMTs. This review primarily focuses on recent progress on the roles of the nonhistone protein methylation in transcription. Protein methylation of coactivators, transcription factors, and signal transducers, among other proteins, plays important roles in transcriptional regulation. Protein methylation may affect protein-protein interaction, protein-DNA or protein-RNA interaction, protein stability, subcellular localization, or enzymatic activity. Thus, protein arginine methylation is critical for regulation of transcription and potentially for various physiological/pathological processes.

  3. HIV protein sequence hotspots for crosstalk with host hub proteins.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  4. Fragments of protein A eluted during protein A affinity chromatography. (United States)

    Carter-Franklin, Jayme N; Victa, Corazon; McDonald, Paul; Fahrner, Robert


    Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

  5. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Craescu Constantin T


    Full Text Available Abstract Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.

  6. Inferring protein function by domain context similarities in protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    Sun Zhirong


    Full Text Available Abstract Background Genome sequencing projects generate massive amounts of sequence data but there are still many proteins whose functions remain unknown. The availability of large scale protein-protein interaction data sets makes it possible to develop new function prediction methods based on protein-protein interaction (PPI networks. Although several existing methods combine multiple information resources, there is no study that integrates protein domain information and PPI networks to predict protein functions. Results The domain context similarity can be a useful index to predict protein function similarity. The prediction accuracy of our method in yeast is between 63%-67%, which outperforms the other methods in terms of ROC curves. Conclusion This paper presents a novel protein function prediction method that combines protein domain composition information and PPI networks. Performance evaluations show that this method outperforms existing methods.

  7. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D


    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  8. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein


    Song, Zhiqi; Zhao, Deming; Yang, Lifeng


    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  9. Shape complementarity and hydrogen bond preferences in protein-protein interfaces: implications for antibody modeling and protein-protein docking. (United States)

    Kuroda, Daisuke; Gray, Jeffrey J


    Characterizing protein-protein interfaces and the hydrogen bonds is a first step to better understand proteins' structures and functions toward high-resolution protein design. However, there are few large-scale surveys of hydrogen bonds of interfaces. In addition, previous work of shape complementarity of protein complexes suggested that lower shape complementarity in antibody-antigen interfaces is related to their evolutionary origin. Using 6637 non-redundant protein-protein interfaces, we revealed peculiar features of various protein complex types. In contrast to previous findings, the shape complementarity of antibody-antigen interfaces resembles that of the other interface types. These results highlight the importance of hydrogen bonds during evolution of protein interfaces and rectify the prevailing belief that antibodies have lower shape complementarity. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail:

  10. Dairy Proteins and Energy Balance

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist

    High protein diets affect energy balance beneficially through decreased hunger, enhanced satiety and increased energy expenditure. Dairy products are a major source of protein. Dairy proteins are comprised of two classes, casein (80%) and whey proteins (20%), which are both of high quality......, but casein is absorbed slowly and whey is absorbed rapidly. The present PhD study investigated the effects of total dairy proteins, whey, and casein, on energy balance and the mechanisms behind any differences in the effects of the specific proteins. The results do not support the hypothesis that dairy...... proteins, whey or casein are more beneficial than other protein sources in the regulation of energy balance, and suggest that dairy proteins, whey or casein seem to play only a minor role, if any, in the prevention and treatment of obesity....

  11. Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays. (United States)

    Tang, Yanyang; Qiu, Ji; Machner, Matthias; LaBaer, Joshua


    We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST. At the time of the experiment, the query protein and the target protein are produced separately through IVTT. The query protein is then applied to nucleic acid programmable protein arrays (NAPPA) that display thousands of freshly produced target proteins captured by anti-GST antibody. Interactions between the query and immobilized target proteins are detected through addition of a fluorophore-labeled HaloTag ligand. Our protocol allows the elucidation of PPIs in a high-throughput fashion using proteins produced in vitro, obviating the scientific challenges, high cost, and laborious work, as well as concerns about protein stability, which are usually present in protocols using conventional protein arrays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  12. The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae* (United States)

    Lechardeur, Delphine; Fernandez, Annabelle; Robert, Bruno; Gaudu, Philippe; Trieu-Cuot, Patrick; Lamberet, Gilles; Gruss, Alexandra


    Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a Kd of 0.5 μm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein. PMID:20332091

  13. Circular dichroism spectroscopy of fluorescent proteins

    NARCIS (Netherlands)

    Visser, N.V.; Hink, M.A.; Borst, J.W.; Krogt, van der G.N.M.; Visser, A.J.W.G.


    Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red

  14. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin


    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  15. Protein Functionalized Nanodiamond Arrays

    Directory of Open Access Journals (Sweden)

    Liu YL


    Full Text Available Abstract Various nanoscale elements are currently being explored for bio-applications, such as in bio-images, bio-detection, and bio-sensors. Among them, nanodiamonds possess remarkable features such as low bio-cytotoxicity, good optical property in fluorescent and Raman spectra, and good photostability for bio-applications. In this work, we devise techniques to position functionalized nanodiamonds on self-assembled monolayer (SAMs arrays adsorbed on silicon and ITO substrates surface using electron beam lithography techniques. The nanodiamond arrays were functionalized with lysozyme to target a certain biomolecule or protein specifically. The optical properties of the nanodiamond-protein complex arrays were characterized by a high throughput confocal microscope. The synthesized nanodiamond-lysozyme complex arrays were found to still retain their functionality in interacting with E. coli.

  16. Problems in Protein Biosynthesis (United States)

    Lengyel, Peter


    Outline of the steps in protein synthesis. Nature of the genetic code. The use of synthetic oligo- and polynucleotides in deciphering the code. Structure of the code: relatedness of synonym codons. The wobble hypothesis. Chain initiation and N-formyl-methionine. Chain termination and nonsense codons. Mistakes in translation: ambiguity in vitro. Suppressor mutations resulting in ambiguity. Limitations in the universality of the code. Attempts to determine the particular codons used by a species. Mechanisms of suppression, caused by (a) abnormal aminoacyl-tRNA, (b) ribosomal malfunction. Effect of streptomycin. The problem of "reading" a nucleic acid template. Different ribosomal mutants and DNA polymerase mutants might cause different mistakes. The possibility of involvement of allosteric proteins in template reading. PMID:5338560

  17. Accessory Proteins at ERES

    DEFF Research Database (Denmark)

    Klinkenberg, Rafael David

    distribution of mSec16B. We further dissect both mSec16A and mSec16B, and show that the region in human mSec16B encompassing residues 35‐194 and the region in human mSec16A comprising residues 1096‐1190 maintain membrane binding irrespective of the removal of membrane associating proteins by salt wash...... or proteolytic digestion. However, neither mSec16B (35‐194) nor mSec16A (1096‐1190) maintain ERES targeting. These findings support previous observations of the need for the membrane binding regions to be expressed in cis with a Central Conserved Domain (CCD) in both proteins to convey ERES targeting....

  18. Porcine prion protein amyloid


    Hammarstr?m, Per; Nystr?m, Sofie


    ABSTRACT Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat...

  19. Engineering ancestral protein hyperstability. (United States)

    Romero-Romero, M Luisa; Risso, Valeria A; Martinez-Rodriguez, Sergio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M


    Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such 'reconstructed ancestral hyperstability' can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. Immunoprecipitation-based analysis of protein-protein interactions. (United States)

    Speth, Corinna; Toledo-Filho, Luis A A; Laubinger, Sascha


    Several techniques allow the detection of protein-protein interactions. In vivo co-immunoprecipitation (Co-IP) studies are an important complement to other commonly used techniques such as yeast two-hybrid or fluorescence complementation, as they reveal interactions between functional proteins at physiological relevant concentrations. Here, we describe an in vivo Co-IP approach using either GFP affinity matrix or specific antibodies to purify proteins of interests and their interacting partners.

  1. Neutron protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Niimura, Nobuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment


    X-ray diffraction of single crystal has enriched the knowledge of various biological molecules such as proteins, DNA, t-RNA, viruses, etc. It is difficult to make structural analysis of hydrogen atoms in a protein using X-ray crystallography, whereas neutron diffraction seems usable to directly determine the location of those hydrogen atoms. Here, neutron diffraction method was applied to structural analysis of hen egg-white lysozyme. Since the crystal size of a protein to analyze is generally small (5 mm{sup 3} at most), the neutron beam at the sample position in monochromator system was set to less than 5 x 5 mm{sup 2} and beam divergence to 0.4 degree or less. Neutron imaging plate with {sup 6}Li or Gd mixed with photostimulated luminescence material was used and about 2500 Bragg reflections were recorded in one crystal setting. A total of 38278 reflections for 2.0 A resolution were collected in less than 10 days. Thus, stereo views of Trp-111 omit map around the indol ring of Trp-111 was presented and the three-dimensional arrangement of 696H and 264D atoms in the lysozyme molecules was determined using the omit map. (M.N.)

  2. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein (United States)

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng


    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases. PMID:25206608

  3. Understanding Protein Non-Folding (United States)

    Uversky, Vladimir N.; Dunker, A. Keith


    This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of specific 3-D structure. The existence of such proteins does not fit the prevailing structure-function paradigm, which states that unique 3-D structure is a prerequisite to function. Thus, the protein structure-function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: How were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases? PMID:20117254

  4. Regulation of protein function by ‘microProteins'


    Staudt, Annica-Carolin; Wenkel, Stephan


    Elegant post-translational regulation is achieved by ‘microProteins', which form homotypic dimers with their targets and act through the dominant–negative suppression of protein complex function. The recent identification of new microProteins suggests their role is general and has evolved in both the plant and animal kingdoms.

  5. Digestion of protein and protein gels in simulated gastric environment

    NARCIS (Netherlands)

    Luo, Q.; Boom, R.M.; Janssen, A.E.M.


    Despite the increasing attention to food digestion research, food scientists still need to better understand the underlying mechanisms of digestion. Most in vitro studies on protein digestion are based on experiments with protein solutions. In this study, the digestion of egg white protein and whey

  6. Molecular simulations of lipid-mediated protein-protein interactions

    NARCIS (Netherlands)

    de Meyer, F.J.M.; Venturoli, M.; Smit, B.


    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the

  7. The interface of protein structure, protein biophysics, and molecular evolution (United States)

    Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon


    Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

  8. Utilization of soya protein as an alternative protein source in ...

    African Journals Online (AJOL)

    In contrast, no significant differences were found in feed and protein utilization parameters. For carcass trait, ash, crude fat, and energy varied significantly with soya protein incorporation in fish diet. Concerning organoleptic characteristics, odour and texture in mouth were not affected by incorporation of soya protein in diet.

  9. Protein engineering techniques gateways to synthetic protein universe

    CERN Document Server

    Poluri, Krishna Mohan


    This brief provides a broad overview of protein-engineering research, offering a glimpse of the most common experimental methods. It also presents various computational programs with applications that are widely used in directed evolution, computational and de novo protein design. Further, it sheds light on the advantages and pitfalls of existing methodologies and future perspectives of protein engineering techniques.

  10. Recent excitements in protein NMR: Large proteins and biologically ...

    Indian Academy of Sciences (India)

    The advent of Transverse Relaxation Optimized SpectroscopY (TROSY) and perdeuteration allowed biomolecularNMR spectroscopists to overcome the size limitation barrier (~20 kDa) in de novo structure determination of proteins.The utility of these techniques was immediately demonstrated on large proteins and protein ...

  11. Protein stress and stress proteins: implications in aging and disease

    Indian Academy of Sciences (India)


    Apr 2, 2007 ... Environmantal stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so called stress or heat shock proteins. The stress response as well as stress proteins are ubiquitous, highly conserved ...

  12. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available feron stimulator, Mediator of IRF3 activation, Stimulator of interferon genes protein 9606 Homo sapiens Q86WV6 340061 ... ...MPA1 TLR signaling molecules TMEM173 ERIS, MITA, STING Transmembrane protein 173 Endoplasmic reticulum inter

  13. Epitope tagging of recombinant proteins. (United States)

    Brizzard, B; Chubet, R


    Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The fusion gene is cloned into an appropriate expression vector for the experimental cell type and host cells are transfected. The fusion protein can then be detected and/or purified using a monoclonal antibody specific for the epitope tag. This unit presents protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols in this unit employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG M2 affinity chromatography.

  14. Protein: FBA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA3 Atg1 kinase complex TOR1 DRR1 Serine/threonine-protein kinase TOR1 Dominant rapamycin... resistance protein 1, Phosphatidylinositol kinase homolog TOR1, Target of rapamycin kinase 1 559292

  15. Functional aspects of protein flexibility

    DEFF Research Database (Denmark)

    Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B


    Proteins are dynamic entities, and they possess an inherent flexibility that allows them to function through molecular interactions within the cell, among cells and even between organisms. Appreciation of the non-static nature of proteins is emerging, but to describe and incorporate...... this into an intuitive perception of protein function is challenging. Flexibility is of overwhelming importance for protein function, and the changes in protein structure during interactions with binding partners can be dramatic. The present review addresses protein flexibility, focusing on protein-ligand interactions....... The thermodynamics involved are reviewed, and examples of structure-function studies involving experimentally determined flexibility descriptions are presented. While much remains to be understood about protein flexibility, it is clear that it is encoded within their amino acid sequence and should be viewed...

  16. Protein Linked to Atopic Dermatitis (United States)

    ... Research Matters January 14, 2013 Protein Linked to Atopic Dermatitis Normal skin from a mouse (left) shows no ... that lack of a certain protein may trigger atopic dermatitis, the most common type of eczema. The finding ...

  17. Protein-ECE MEtallopincer Hybrids

    NARCIS (Netherlands)

    Kruithof, C.A.


    Modification of proteins with metal complexes is a promising and a relatively new field which conceals many challenges and potential applications. The field is a balance of contributions from the biological (protein engineering, bioconjugation) and chemical sciences (organic, inorganic and

  18. Leptospira Protein Expression During Infection (United States)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  19. Changes in protein expression across laboratory and field experiments in Geobacter bemidjiensis

    Energy Technology Data Exchange (ETDEWEB)

    Merkley, Eric D.; Wrighton, Kelly C.; Castelle, Cindy; Anderson, Brian J.; Wilkins, Michael J.; Shah, Vega; Arbour, Tyler; Brown, Joseph N.; Singer, Steven W.; Smith, Richard D.; Lipton, Mary S.


    Bacterial extracellular metal respiration, as carried out by members of the genus Geobacter, is of interest for applications including microbial fuel cells and bioremediation. Geobacter bemidjiensis is the major species whose growth is stimulated during groundwater amendment with acetate. We have carried out label-free proteomics studies of Geobacter bemidjiensis grown with acetate as the electron donor and either fumarate, ferric citrate, or one of two hydrous ferric oxide mineral types as electron acceptor. The major class of proteins whose expression changes across these conditions is c-type cytochromes, many of which are known to be involved in extracellular metal reduction in other, better-characterized Geobacter species. Some proteins with multiple homologues in G. bemidjiensis (OmcS, OmcB) had different expression patterns than observed for their G. sulfurreducens homologues under similar growth conditions. We also compared the proteome from our study to a prior proteomics study of biomass recovered from an aquifer in Colorado, where the microbial community was dominated by strains closely-related to G. bemidjiensis. We detected an increased number of proteins with functions related to motility and chemotaxis in the Colorado field samples compared to the laboratory samples, suggesting the importance of motility for in situ extracellular metal respiration.

  20. Yeast Interacting Proteins Database: YJL199C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...cies; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey (4) Ro...n; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies... species; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey Ro

  1. Protein: MPA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA6 Adionectin and its receptors Adipoq Acdc, Acrp30, Apm1 Adiponectin 30 kDa adipocyte complement-relate...d protein, Adipocyte complement-related 30 kDa protein, Adipocyte, C1q and collagen domain-containing, Adipocyte-specific protein AdipoQ 10090 Mus musculus 11450 Q60994 1C28, 1C3H Q60994 18446001, 19788607 ...

  2. Dipolar response of hydrated proteins


    Matyushov, Dmitry V.


    The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins. The effective dielectric constant of the solvated protein, representing the average dipole moment induced at the protein by a uniform external field, shows a remarkable variation among the proteins studied by numerical simulations. It changes from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility of ubiquitin, that is a dia-electri...

  3. Protein corona: Opportunities and challenges (United States)

    Zanganeh, Saeid; Spitler, Ryan; Erfanzadeh, Mohsen; Alkilany, Alaaldin M.; Mahmoudi, Morteza


    In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field. PMID:26783938

  4. The papillomavirus E2 proteins. (United States)

    McBride, Alison A


    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. Published by Elsevier Inc.

  5. Protein corona: Opportunities and challenges. (United States)

    Zanganeh, Saeid; Spitler, Ryan; Erfanzadeh, Mohsen; Alkilany, Alaaldin M; Mahmoudi, Morteza


    In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. A Novel Approach for Protein-Named Entity Recognition and Protein-Protein Interaction Extraction

    Directory of Open Access Journals (Sweden)

    Meijing Li


    Full Text Available Many researchers focus on developing protein-named entity recognition (Protein-NER or PPI extraction systems. However, the studies about these two topics cannot be merged well; then existing PPI extraction systems’ Protein-NER still needs to improve. In this paper, we developed the protein-protein interaction extraction system named PPIMiner based on Support Vector Machine (SVM and parsing tree. PPIMiner consists of three main models: natural language processing (NLP model, Protein-NER model, and PPI discovery model. The Protein-NER model, which is named ProNER, identifies the protein names based on two methods: dictionary-based method and machine learning-based method. ProNER is capable of identifying more proteins than dictionary-based Protein-NER model in other existing systems. The final discovered PPIs extracted via PPI discovery model are represented in detail because we showed the protein interaction types and the occurrence frequency through two different methods. In the experiments, the result shows that the performances achieved by our ProNER and PPI discovery model are better than other existing tools. PPIMiner applied this protein-named entity recognition approach and parsing tree based PPI extraction method to improve the performance of PPI extraction. We also provide an easy-to-use interface to access PPIs database and an online system for PPIs extraction and Protein-NER.

  7. Proteins: Chemistry, Characterization, and Quality

    NARCIS (Netherlands)

    Sforza, S.; Tedeschi, T.; Wierenga, P.A.


    Proteins are one of the major macronutrients in food, and several traditional food commodities are good sources of proteins (meat, egg, milk and dairy products, fish, and soya). Proteins are polymers made by 20 different amino acids. They might undergo desired or undesired chemical or enzymatic

  8. Protein: MPA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA3 NADPH oxidase regulators NOXO1 P41NOX, SH3PXD5 NOXO1 NADPH oxidase organizer 1... NADPH oxidase regulatory protein, Nox organizer 1, Nox-organizing protein 1, SH3 and PX domain-containing protein 5 9606 Homo sapiens Q8NFA2 124056 2L73 ...

  9. Protein: MPA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available 1 47 kDa autosomal chronic granulomatous disease protein, 47 kDa neutrophil oxidase factor, NCF-47K, Neutro...phil NADPH oxidase factor 1, Nox organizer 2, Nox-organizing protein 2, SH3 and PX domain-containing protein

  10. Protein: MPB1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB1 Related chemokines IL8 CXCL8 Interleukin_8 Interleukin-8 C-X-C motif chemokine... 8, Emoctakin, Granulocyte chemotactic protein 1, Monocyte-derived neutrophil chemotactic factor, Monocyte-d...erived neutrophil-activating peptide, Neutrophil-activating protein 1, Protein 3-10C, T-cell chemotactic fac

  11. Protein: FBA4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available ng kinase assembly factor MAT1 CDK7/cyclin-H assembly factor, Cyclin-G1-interacting protein, Menage a trois, RING finger 66, RING finger protein MAT1, p35, p36 9606 Homo sapiens P51948 4331 1G25 4331 P51948 ...

  12. Photoreceptor proteins from purple bacteria

    NARCIS (Netherlands)

    Hendriks, J.; van der Horst, M.A.; Chua, T.K.; Ávila Pérez, M.; van Wilderen, L.J.; Alexandre, M.T.A.; Groot, M.-L.; Kennis, J.T.M.; Hellingwerf, K.J.; Hunter, C.N.; Daldal, F.; Thurnauer, M.C.; Beatty, J.T.


    Purple bacteria contain representatives of four of the six main families of photoreceptor proteins: phytochromes, BLUF domain containing proteins, xanthopsins (i.e., photoactive yellow proteins), and phototropins (containing one or more light, oxygen, or voltage (LOV) domains). Most of them have a

  13. Protein quality of pig diets

    NARCIS (Netherlands)

    Hulshof, Tetske


    The increasing world population and per capita income imposes a risk for protein scarcity. It is, therefore, necessary to use current ingredients more efficiently which includes the accurate assessment of protein quality before inclusion in animal diets. Protein quality is defined in this thesis as

  14. Modeling complexes of modeled proteins. (United States)

    Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A


    Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Structuring high-protein foods

    NARCIS (Netherlands)

    Purwanti, N.


    Increased protein consumption gives rise to various health benefits. High-protein intake can lead to muscle development, body weight control and suppression of sarcopenia progression. However, increasing the protein content in food products leads to textural changes over time. These changes result

  16. Functional Foods Containing Whey Proteins (United States)

    Whey proteins, modified whey proteins, and whey components are useful as nutrients or supplements for health maintenance. Extrusion modified whey proteins can easily fit into new products such as beverages, confectionery items (e.g., candies), convenience foods, desserts, baked goods, sauces, and in...

  17. Protein Quantitation Using Mass Spectrometry (United States)

    Zhang, Guoan; Ueberheide, Beatrix M.; Waldemarson, Sofia; Myung, Sunnie; Molloy, Kelly; Eriksson, Jan; Chait, Brian T.; Neubert, Thomas A.; Fenyö, David


    Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples. PMID:20835801

  18. Biophysics of protein evolution and evolutionary protein biophysics (United States)

    Sikosek, Tobias; Chan, Hue Sun


    The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

  19. Protein-protein interactions and cancer: targeting the central dogma. (United States)

    Garner, Amanda L; Janda, Kim D


    Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

  20. The Proteins API: accessing key integrated protein and genome information. (United States)

    Nightingale, Andrew; Antunes, Ricardo; Alpi, Emanuele; Bursteinas, Borisas; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd; Martin, Maria


    The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to 'talk' to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at ( © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. The Proteins API: accessing key integrated protein and genome information (United States)

    Antunes, Ricardo; Alpi, Emanuele; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd


    Abstract The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to ‘talk’ to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at ( PMID:28383659

  2. Characterization of protein-protein interactions by isothermal titration calorimetry. (United States)

    Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto


    Isothermal titration calorimetry (ITC) is a powerful technique to study both protein-ligand and protein-protein interactions. This methods chapter is devoted to describing protein-protein interactions, in particular, the association between two different proteins and the self-association of a protein into homodimers. ITC is the only technique that determines directly the thermodynamic parameters of a given reaction: DeltaG, DeltaH, DeltaS, and DeltaCP. Isothermal titration calorimeters have evolved over the years and one of the latest models is the VP-ITC produced by Microcal, Inc. In this chapter we will be describing the general procedure for performing an ITC experiment as well as for the specific cases of porcine pancreatic trypsin binding to soybean trypsin inhibitor and the dissociation of bovine pancreatic alpha-chymotrypsin.

  3. Understanding Protein-Protein Interactions Using Local Structural Features

    DEFF Research Database (Denmark)

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier


    Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... (loops and domains) to comprehend the molecular mechanisms of PPIs. A paradox in protein-protein binding is to explain how the unbound proteins of a binary complex recognize each other among a large population within a cell and how they find their best docking interface in a short timescale. We use...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...

  4. Protein subcellular localization assays using split fluorescent proteins (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM


    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  5. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    DEFF Research Database (Denmark)

    Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf


    of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes......-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...

  6. Protein from methanol

    Energy Technology Data Exchange (ETDEWEB)

    Rosenzweig, M.; Ushio, S.


    The biosynthesis of proteins from methanol produced from natural gas can provide an attractive alternative to the already commercially proven technique of protein synthesis from gas oil and n-paraffin feedstocks if current pilot-plant tests in England and Japan prove successful. The methanol route also provides other advantages as a protein feedstock: it is water soluble, contains no polycyclic aromatic compounds, and requires less oxygen than methane. Its lower boiling point helps ease the separation of feedstock from the product stream. Finally, it will require lower investment costs. Both ICI and Mitsubishi Gas Chemical Co. are large methanol producers. ICI already has a 1000 ton/yr plant operating at Teeside, England, and expects to decide on a 100,000 m ton/yr plant later this year. Mitsubishi is constructing a large-scale pilot plant scheduled to come onstream this year. ICI will use a Pseudomona bacterium at 98.6/sup 0/F (37/sup 0/C) in the fermenter. Mitsubishi has not yet decided on a yeast or a bacteria, and is searching for a strain capable of withstanding up to 115/sup 0/F (46/sup 0/C). In the more advanced ICI process, methanol will be mixed with phosphoric acid, potassium sulfate, sodium chloride, and traces of iron, copper, zinc, and molybdenum; diluted with water; passed through a sterilization tank; and fermented at pH 7 in a pressure cycle fermenter. The product stream, containing a 3 percent suspension of cellular dry matter, is taken near the top of the fermenter riser, then passed through a flotation vessel and a centrifuge to pack the cell concentration to 20 percent. Water is recycled. Whatever methanol remains in the fermenter product stream is either used up by the microorganisms in subsequent processing or vaporized in the dryer. (auth)

  7. NMR Studies of Protein Hydration and Protein-Ligand Interactions (United States)

    Chong, Yuan

    Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

  8. Protein-Protein Interactions: Structurally Conserved Residues Distinguish between Binding Sites and Exposed Protein Surfaces

    National Research Council Canada - National Science Library

    Buyong Ma; Tal Elkayam; Haim Wolfson; Ruth Nussinov


    Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface...

  9. Information contained in protein shapes (United States)

    Sundaram, K.; Viswanadhan, V. N.; Macelroy, R. D.


    The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.

  10. Protein-protein interaction network-based detection of functionally similar proteins within species. (United States)

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli


    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

  11. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    NARCIS (Netherlands)

    Espinosa-Soto, C.; Immink, R.G.H.; Angenent, G.C.; Alvarez-Buylla, E.R.; Folter, de S.


    Background: MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate

  12. Discover Protein Complexes in Protein-Protein Interaction Networks Using Parametric Local Modularity

    Directory of Open Access Journals (Sweden)

    Tan Kai


    Full Text Available Abstract Background Recent advances in proteomic technologies have enabled us to create detailed protein-protein interaction maps in multiple species and in both normal and diseased cells. As the size of the interaction dataset increases, powerful computational methods are required in order to effectively distil network models from large-scale interactome data. Results We present an algorithm, miPALM (Module Inference by Parametric Local Modularity, to infer protein complexes in a protein-protein interaction network. The algorithm uses a novel graph theoretic measure, parametric local modularity, to identify highly connected sub-networks as candidate protein complexes. Using gold standard sets of protein complexes and protein function and localization annotations, we show our algorithm achieved an overall improvement over previous algorithms in terms of precision, recall, and biological relevance of the predicted complexes. We applied our algorithm to predict and characterize a set of 138 novel protein complexes in S. cerevisiae. Conclusions miPALM is a novel algorithm for detecting protein complexes from large protein-protein interaction networks with improved accuracy than previous methods. The software is implemented in Matlab and is freely available at

  13. Detection of protein complex from protein-protein interaction network using Markov clustering (United States)

    Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.


    Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.

  14. Human cancer protein-protein interaction network: a structural perspective.

    Directory of Open Access Journals (Sweden)

    Gozde Kar


    Full Text Available Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network. The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%. We illustrate the interface related affinity properties of two cancer-related hub

  15. Molecular principles of human virus protein-protein interactions. (United States)

    Halehalli, Rachita Ramachandra; Nagarajaram, Hampapathalu Adimurthy


    Viruses, from the human protein-protein interaction network perspective, target hubs, bottlenecks and interconnected nodes enriched in certain biological pathways. However, not much is known about the general characteristic features of the human proteins interacting with viral proteins (referred to as hVIPs) as well as the motifs and domains utilized by human-virus protein-protein interactions (referred to as Hu-Vir PPIs). Our study has revealed that hVIPs are mostly disordered proteins, whereas viral proteins are mostly ordered proteins. Protein disorder in viral proteins and hVIPs varies from one subcellular location to another. In any given viral-human PPI pair, at least one of the two proteins is structurally disordered suggesting that disorder associated conformational flexibility as one of the characteristic features of virus-host interaction. Further analyses reveal that hVIPs are (i) slowly evolving proteins, (ii) associated with high centrality scores in human-PPI network, (iii) involved in multiple pathways, (iv) enriched in eukaryotic linear motifs (ELMs) associated with protein modification, degradation and regulatory processes, (v) associated with high number of splice variants and (vi) expressed abundantly across multiple tissues. These aforementioned findings suggest that conformational flexibility, spatial diversity, abundance and slow evolution are the characteristic features of the human proteins targeted by viral proteins. Hu-Vir PPIs are mostly mediated via domain-motif interactions (DMIs) where viral proteins employ motifs that mimic host ELMs to bind to domains in human proteins. DMIs are shared among viruses belonging to different families indicating a possible convergent evolution of these motifs to help viruses to adopt common strategies to subvert host cellular pathways. Hu-Vir PPI data, DDI and DMI data for human-virus PPI can be downloaded from Supplementary data are

  16. Introduction to protein crystallization. (United States)

    McPherson, Alexander; Gavira, Jose A


    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  17. Bioactive proteins from pipefishes

    Directory of Open Access Journals (Sweden)

    E. Rethna Priya


    Full Text Available Objective: To screen antimicrobial potence of some pipefish species collected from Tuticorin coastal environment. Methods: Antimicrobial activity of pipefishes in methanol extract was investigated against 10 bacterial and 10 fungal human pathogenic strains. Results: Among the tested strains, in Centriscus scutatus, pipefish showed maximum zone of inhibition against Vibrio cholerae (8 mm and minimum in the sample of Hippichthys cyanospilos against Klebseilla pneumoniae (2 mm. In positive control, maximum zone of inhibition was recorded in Vibrio cholerae (9 mm and minimum in Klebseilla pneumoniae, and Salmonella paratyphi (5 mm. Chemical investigation indicated the presence of peptides as evidenced by ninhydrin positive spots on thin layer chromatography and presence of peptide. In SDS PAGE, in Centriscus scutatus, four bands were detected in the gel that represented the presence of proteins in the range nearly 25.8-75 kDa. In Hippichthys cyanospilos, five bands were detected in the gel that represented the presence of proteins in the range nearly 20.5-78 kDa. The result of FT-IR spectrum revealed that the pipe fishes extracts compriseed to have peptide derivatives as their predominant chemical groups. Conclusions: It can be conclude that this present investigation suggests the tested pipe fishes will be a potential source of natural bioactive compounds.

  18. Bioactive proteins from pipefishes

    Directory of Open Access Journals (Sweden)

    E. Rethna Priya


    Full Text Available Objective: To screen antimicrobial potence of some pipefish species collected from Tuticorin coastal environment. Methods: Antimicrobial activity of pipefishes in methanol extract was investigated against 10 bacterial and 10 fungal human pathogenic strains. Results: Among the tested strains, in Centriscus scutatus, pipefish showed maximum zone of inhibition against Vibrio cholerae (8 mm and minimum in the sample of Hippichthys cyanospilos against Klebseilla pneumoniae (2 mm. In positive control, maximum zone of inhibition was recorded in Vibrio cholerae (9 mm and minimum in Klebseilla pneumoniae, and Salmonella paratyphi (5 mm. Chemical investigation indicated the presence of peptides as evidenced by ninhydrin positive spots on thin layer chromatography and presence of peptide. In SDS PAGE, in Centriscus scutatus, four bands were detected in the gel that represented the presence of proteins in the range nearly 25.8-75 kDa. In Hippichthys cyanospilos, five bands were detected in the gel that represented the presence of proteins in the range nearly 20.5-78 kDa. The result of FT-IR spectrum revealed that the pipe fishes extracts compriseed to have peptide derivatives as their predominant chemical groups. Conclusions: It can be conclude that this present investigation suggests the tested pipe fishes will be a potential source of natural bioactive compounds.

  19. Protein Chemical Shift Prediction

    CERN Document Server

    Larsen, Anders S


    The protein chemical shifts holds a large amount of information about the 3-dimensional structure of the protein. A number of chemical shift predictors based on the relationship between structures resolved with X-ray crystallography and the corresponding experimental chemical shifts have been developed. These empirical predictors are very accurate on X-ray structures but tends to be insensitive to small structural changes. To overcome this limitation it has been suggested to make chemical shift predictors based on quantum mechanical(QM) calculations. In this thesis the development of the QM derived chemical shift predictor Procs14 is presented. Procs14 is based on 2.35 million density functional theory(DFT) calculations on tripeptides and contains corrections for hydrogen bonding, ring current and the effect of the previous and following residue. Procs14 is capable at performing predictions for the 13CA, 13CB, 13CO, 15NH, 1HN and 1HA backbone atoms. In order to benchmark Procs14, a number of QM NMR calculatio...

  20. Water-transporting proteins. (United States)

    Zeuthen, Thomas


    Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K(+)/Cl(-) and the Na(+)/K(+)/2Cl(-) cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na(+)-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.

  1. Mathematical methods for protein science

    Energy Technology Data Exchange (ETDEWEB)

    Hart, W.; Istrail, S.; Atkins, J. [Sandia National Labs., Albuquerque, NM (United States)


    Understanding the structure and function of proteins is a fundamental endeavor in molecular biology. Currently, over 100,000 protein sequences have been determined by experimental methods. The three dimensional structure of the protein determines its function, but there are currently less than 4,000 structures known to atomic resolution. Accordingly, techniques to predict protein structure from sequence have an important role in aiding the understanding of the Genome and the effects of mutations in genetic disease. The authors describe current efforts at Sandia to better understand the structure of proteins through rigorous mathematical analyses of simple lattice models. The efforts have focused on two aspects of protein science: mathematical structure prediction, and inverse protein folding.

  2. Metagenomics and the protein universe (United States)

    Godzik, Adam


    Metagenomics sequencing projects have dramatically increased our knowledge of the protein universe and provided over one-half of currently known protein sequences; they have also introduced a much broader phylogenetic diversity into the protein databases. The full analysis of metagenomic datasets is only beginning, but it has already led to the discovery of thousands of new protein families, likely representing novel functions specific to given environments. At the same time, a deeper analysis of such novel families, including experimental structure determination of some representatives, suggests that most of them represent distant homologs of already characterized protein families, and thus most of the protein diversity present in the new environments are due to functional divergence of the known protein families rather than the emergence of new ones. PMID:21497084

  3. The Papillomavirus E2 proteins

    Energy Technology Data Exchange (ETDEWEB)

    McBride, Alison A., E-mail:


    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. - Highlights: • Overview of E2 protein functions. • Structural domains of the papillomavirus E2 proteins. • Analysis of E2 binding sites in different genera of papillomaviruses. • Compilation of E2 associated proteins. • Comparison of key mutations in distinct E2 functions.

  4. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren


    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...... of proteins) are natural consequences of the suggested wring mode model. Native (folded) proteins are found to possess an intrinsic standing wring mode....

  5. Advantages of proteins being disordered. (United States)

    Liu, Zhirong; Huang, Yongqi


    The past decade has witnessed great advances in our understanding of protein structure-function relationships in terms of the ubiquitous existence of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). The structural disorder of IDPs/IDRs enables them to play essential functions that are complementary to those of ordered proteins. In addition, IDPs/IDRs are persistent in evolution. Therefore, they are expected to possess some advantages over ordered proteins. In this review, we summarize and survey nine possible advantages of IDPs/IDRs: economizing genome/protein resources, overcoming steric restrictions in binding, achieving high specificity with low affinity, increasing binding rate, facilitating posttranslational modifications, enabling flexible linkers, preventing aggregation, providing resistance to non-native conditions, and allowing compatibility with more available sequences. Some potential advantages of IDPs/IDRs are not well understood and require both experimental and theoretical approaches to decipher. The connection with protein design is also briefly discussed. © 2014 The Protein Society.

  6. Protein Adsorption in Three Dimensions (United States)

    Vogler, Erwin A.


    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  7. Protein function prediction via graph kernels

    National Research Council Canada - National Science Library

    Borgwardt, Karsten M; Ong, Cheng Soon; Schönauer, Stefan; Vishwanathan, S V N; Smola, Alex J; Kriegel, Hans-Peter


    Computational approaches to protein function prediction infer protein function by finding proteins with similar sequence, structure, surface clefts, chemical properties, amino acid motifs, interaction...

  8. Protein oxidation in aging and the removal of oxidized proteins. (United States)

    Höhn, Annika; König, Jeannette; Grune, Tilman


    Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Proteins are among the main targets for oxidants due to their high rate constants for several reactions with ROS and their abundance in biological systems. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies. Two proteolytic systems are responsible to ensure the maintenance of cellular functions: the proteasomal (UPS) and the lysosomal system. Those degrading systems provide a last line of antioxidative protection, removing irreversible damaged proteins and recycling amino acids for the continuous protein synthesis. But during aging, both systems are affected and their proteolytic activity declines significantly. Here we highlight the recent advantages in the understanding of protein oxidation and the fate of these damaged proteins during aging. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Protein-protein interaction based on pairwise similarity

    Directory of Open Access Journals (Sweden)

    Zaki Nazar


    Full Text Available Abstract Background Protein-protein interaction (PPI is essential to most biological processes. Abnormal interactions may have implications in a number of neurological syndromes. Given that the association and dissociation of protein molecules is crucial, computational tools capable of effectively identifying PPI are desirable. In this paper, we propose a simple yet effective method to detect PPI based on pairwise similarity and using only the primary structure of the protein. The PPI based on Pairwise Similarity (PPI-PS method consists of a representation of each protein sequence by a vector of pairwise similarities against large subsequences of amino acids created by a shifting window which passes over concatenated protein training sequences. Each coordinate of this vector is typically the E-value of the Smith-Waterman score. These vectors are then used to compute the kernel matrix which will be exploited in conjunction with support vector machines. Results To assess the ability of the proposed method to recognize the difference between "interacted" and "non-interacted" proteins pairs, we applied it on different datasets from the available yeast saccharomyces cerevisiae protein interaction. The proposed method achieved reasonable improvement over the existing state-of-the-art methods for PPI prediction. Conclusion Pairwise similarity score provides a relevant measure of similarity between protein sequences. This similarity incorporates biological knowledge about proteins and it is extremely powerful when combined with support vector machine to predict PPI.

  10. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    Directory of Open Access Journals (Sweden)

    Zheng Sun


    Full Text Available WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1 and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA, two integrin beta (ITGB, and one syndecan (SDC. Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp.

  11. A new protein structure representation for efficient protein function prediction. (United States)

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F


    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  12. Role for protein-protein interaction databases in human genetics. (United States)

    Pattin, Kristine A; Moore, Jason H


    Proteomics and the study of protein-protein interactions are becoming increasingly important in our effort to understand human diseases on a system-wide level. Thanks to the development and curation of protein-interaction databases, up-to-date information on these interaction networks is accessible and publicly available to the scientific community. As our knowledge of protein-protein interactions increases, it is important to give thought to the different ways that these resources can impact biomedical research. In this article, we highlight the importance of protein-protein interactions in human genetics and genetic epidemiology. Since protein-protein interactions demonstrate one of the strongest functional relationships between genes, combining genomic data with available proteomic data may provide us with a more in-depth understanding of common human diseases. In this review, we will discuss some of the fundamentals of protein interactions, the databases that are publicly available and how information from these databases can be used to facilitate genome-wide genetic studies.

  13. Profiles of carbohydrate ligands associated with adsorbed proteins on self-assembled monolayers of defined chemistries. (United States)

    Shankar, Sucharita P; Chen, Inn Inn; Keselowsky, Benjamin G; García, Andrés J; Babensee, Julia E


    Conserved protein-carbohydrate-lipid pathogen-associated molecular patterns (PAMPs) interact with cells of the innate immune system to mediate antigen recognition and internalization and activation of immune cells. We examined if analogous "biomaterial-associated molecular patterns" composed of proteins, specifically their carbohydrate modifications, existed on biomaterials, which can play a role in mediating the innate immune response to biomaterials. To probe for these carbohydrates in the adsorbed protein layer, as directed by the underlying biomaterial chemistry, self-assembled monolayers (SAMs) presenting -CH(3), -OH, -COOH, or -NH(2) were preincubated with serum/plasma, and the presence of carbohydrate ligands of C-type lectin receptors (CLRs) was investigated using lectin probes in an enzyme-linked lectin assay (ELLA). Presentation of CLR ligands was detected on control tissue culture polystyrene (TCPS). Absorbances of mannose or N-acetylglucosamine increased with decreasing incubating serum concentration, whereas absorbances of sialylated epitopes or fucose remained unchanged. Absorbances of alpha-galactose or N-acetylgalactosamine decreased with decreasing incubating serum concentration; beta-galactose was undetectable. Among SAM endgroups, preincubation with 10% serum resulted in differential presentation of CLR ligands: higher alpha-galactose on COOH SAMs than NH(2) or CH(3) SAMs, highest complex mannose on NH(2) SAMs, and higher complex mannose on OH SAMs than CH(3) SAMs. Least sialylated groups were detected on CH(3) SAMs. In summary, biomaterial chemistry may regulate protein adsorption and hence unique presentation of associated carbohydrates. The ultimate goal is to identify the effects of protein glycosylations associated with biomaterials in stimulating innate immune responses. (c) 2009 Wiley Periodicals, Inc.

  14. Crystal structure and redox properties of a novel cyanobacterial heme protein with a His/Cys heme axial ligation and a Per-Arnt-Sim (PAS)-like domain. (United States)

    Motomura, Taiki; Suga, Michihiro; Hienerwadel, Rainer; Nakagawa, Akiko; Lai, Thanh-Lan; Nitschke, Wolfgang; Kuma, Takahiro; Sugiura, Miwa; Boussac, Alain; Shen, Jian-Ren


    Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E½, of Tll0287 was -255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E½ increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Mapping Protein-Protein Interactions by Quantitative Proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy


    Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners. Quantitative mass...... spectrometry (MS)-based proteomics in combination with affinity purification protocols has become the method of choice to map and track the dynamic changes in protein-protein interactions, including the ones occurring during cellular signaling events. Different quantitative MS strategies have been used...... to characterize protein interaction networks. In this chapter we describe in detail the use of stable isotope labeling by amino acids in cell culture (SILAC) for the quantitative analysis of stimulus-dependent dynamic protein interactions....

  16. Revisiting the Voronoi description of protein-protein interfaces. (United States)

    Cazals, Frédéric; Proust, Flavien; Bahadur, Ranjit P; Janin, Joël


    We developed a model of macromolecular interfaces based on the Voronoi diagram and the related alpha-complex, and we tested its properties on a set of 96 protein-protein complexes taken from the Protein Data Bank. The Voronoi model provides a natural definition of the interfaces, and it yields values of the number of interface atoms and of the interface area that have excellent correlation coefficients with those of the classical model based on solvent accessibility. Nevertheless, some atoms that do not lose solvent accessibility are part of the interface defined by the Voronoi model. The Voronoi model provides robust definitions of the curvature and of the connectivity of the interfaces, and leads to estimates of these features that generally agree with other approaches. Our implementation of the model allows an analysis of protein-water contacts that highlights the role of structural water molecules at protein-protein interfaces.

  17. Duchenne Muscular Dystrophy (DMD) Protein-Protein Interaction Mapping. (United States)

    Rezaei Tavirani, Mostafa; OkHOVATIAN, Farshad; Zamanian Azodi, Mona; Rezaei Tavirani, Majid


    Duchenne muscular dystrophy (DMD) is one of the mortal diseases, subjected to study in terms of molecular investigation. In this study, the protein interaction map of this muscle-wasting condition was generated to gain a better knowledge of interactome profile of DMD. Applying Cytoscape and String Database, the protein-protein interaction network was constructed and the gene ontology of the constructed network was analyzed for biological process, molecular function, and cellular component annotations. Among 100 proteins related to DMD, dystrophin, utrophin, caveolin 3, and myogenic differentiation 1 play key roles in DMD network. In addition, the gene ontology analysis showed that regulation processes, kinase activity, and sarcoplasmic reticulum were the highlighted biological processes, molecular function, and cell component enrichments respectively for the proteins related to DMD. The central proteins and the enriched ontologies can be suggested as possible prominent agents in DMD; however, the validation studies may be required.

  18. On the role of electrostatics on protein-protein interactions (United States)

    Zhang, Zhe; Witham, Shawn; Alexov, Emil


    The role of electrostatics on protein-protein interactions and binding is reviewed in this article. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and basic electrostatic effects occurring upon the formation of the complex are discussed. The role of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated and indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartment. At the end, the similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity. PMID:21572182

  19. Methods for detection of protein-protein and protein-DNA interactions using HaloTag. (United States)

    Urh, Marjeta; Hartzell, Danette; Mendez, Jacqui; Klaubert, Dieter H; Wood, Keith


    HaloTag is a protein fusion tag which was genetically engineered to covalently bind a series of specific synthetic ligands. All ligands carry two groups, the reactive group and the functional/reporter group. The reactive group, the choloroalkane, is the same in all the ligands and is involved in binding to the HaloTag. The functional reporter group is variable and can carry many different moieties including fluorescent dyes, affinity handles like biotin or solid surfaces such as agarose beads. Thus, HaloTag can serve either as a labeling tag or as a protein immobilization tag depending on which ligand is bound to it. Here, we describe a procedure for immobilization of HaloTag fusion proteins and how immobilized proteins can be used to study protein-protein and protein-DNA interactions in vivo and in vitro.

  20. Manipulating protein adsorption using a patchy protein-resistant brush. (United States)

    Gon, Saugata; Bendersky, Marina; Ross, Jennifer L; Santore, Maria M


    Toward the development of surfaces for the precise manipulation of proteins, this study explores the fabrication and protein-interactive behavior of a new type of surface containing extremely small (on the order of 10 nm or less) flat adhesive "patches" or islands embedded in and partially concealed by a protein-repellant PEG (poly(ethylene glycol)) brush. The adsorption of fibrinogen, the model protein chosen to probe the biomaterial interactions of these surfaces, is very sensitive to the surface density of the adhesive patches, occurring only above a threshold. This suggests that two or more adhesive patches are needed to capture each protein. When the average spacing of the adhesive patches exceeds the fibrinogen length, no adsorption occurs because individual patches are too weakly binding for protein capture, as a result of being at least partially obstructed by the brush. The small size of the adhesive patches relative to the 47 nm fibrinogen length thus defines a limiting regime of surface design, distinct from surfaces where larger features can adhere single isolated proteins or multiple proteins together. The restricted protein-surface contact may comprise a means of preserving protein structure and function in the adsorbed state. This article demonstrates several additional interesting features of PEG brushes relevant to biomaterial design. First a moderate amount of adhesive material can be buried at the base of a brush without a measurable impact on the corona density. Second, a different amount of material at the base of a brush can be rendered ineffective to capturing adhesive proteins, despite a modest compromise of the brush corona. From this will follow insight into the design of patterned biomaterial surfaces, the bioactivity of the edges of patterned features, and an understanding of how flaws in brushes compromise protein resistance or allow access to small adhesive sites.